ONCOLOGY Days

X.
Diagnostic,
Predictive and
Experimental
ONCOLOGY
Days
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December 02 - 03, 2014
hotel NH Collection Olomouc Congress
Legionarska 21, 779 00 Olomouc,
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X.
Diagnostic,
Predictive and
Experimental
ONCOLOGY
Days
Pořadatel
Ústav molekulární a translační medicíny LF UP a FN Olomouc
Ústav klinické a molekulární patologie LF UP a FN Olomouc
MedChemBio - zájmové sdružení právnických osob
Odborná garance
Sekce diagnostické a prediktivní onkologie České onkologické společnosti ČLS JEP
Komplexní onkologické centrum Olomouc
Prezident akce
doc.MUDr. Marián Hajdúch, Ph.D.
Organizační výbor
doc. Mgr. Jiří Drábek, Ph.D., RNDr. Radek Trojanec Ph.D., MUDr. Kateřina Bouchalová, Ph.D.,
MUDr. Petr Džubák, Ph.D., MUDr. Josef Srovnal, Ph.D., doc. RNDr. Ondřej Slabý, Ph.D.,
doc. MUDr. Marek Svoboda, Ph.D.
Organizátor
Ústav molekulární a translační medicíny Lékařské fakulty Univerzity Palackého v Olomouci
Hněvotínská 5, 779 00, Olomouc
Kontaktní osoba: Mgr. Peter Vanek
mob.: +420 775 050 355, email: [email protected]
PROGRAM / PROGRAM
Program satelitního workshopu
X. dnů diagnostické, prediktivní
a experimentální onkologie
Prediktivní diagnostika nádorů
1. prosince, 2014
Část 1
(moderuje: Libor Staněk, Jiří Drábek)
10:30 – 10:50 Prediktivní genetické biomarkery na signální dráze
Ras u karcinomu kolorekta Jiří Drábek
10:50 – 11:10 Prediktivní genetické biomarkery u GIST
Alena Kalfusová
11:10 – 11:30 Biomarker HER2 u karcinomu prsu a žaludku
Magdalena Uvírová
11:30 – 12:30 Přestávka na oběd
12:30 – 12:50 Biomarker EGFR u NSCLC Libor Staněk
12:50 – 13:10 Vyšetření prediktivních markerů u maligního
melanomu Hana Vošmiková
13:10 – 13:30 Možnosti tekuté biopsie u solidních nádorů Libor
Staněk
13:30 – 13:50 Komplexní genetické biomarkery v predikci
u karcinomu prsu Marek Svoboda
13:50 – 14:25 Přestávka na kávu a exkurzi
Část 2
14:25 – 14:55 Souhrn stávajících prediktivních biomarkerů
a výhled na rok 2015 Marián Hajdúch
Část 3
(moderuje: Libor Staněk, Jiří Drábek)
14:55 – 15:55 Uzavřená sekce pro pracovníky referenčních
laboratoří Libor Staněk, Jana Stránská a další
Powered by:
Ústav molekulární a translační
medicíny, Lékařská fakulta,
Univerzita Palackého Olomouc
Hněvotínská 5, 779 00 Olomouc,
Česká republika
tašky
hl. papír
IMTM
PROGRAM / PROGRAM
X. DNY DIAGNOSTICKÉ, PREDIKTÍVNÍ A EXPERIMENTÁLNÍ ONKOLOGIE /
X. DIAGNOSTIC, PREDICTIVE AND EXPERIMENTAL ONCOLOGY DAYS
ÚTERÝ / TUESDAY - 2. prosince 2014 / December 2, 2014
9.45
Zahájení X. Dnů diagnostické, prediktivní a experimentální onkologie / Opening ceremony
10:00 – 12:00 Biomarkery nádorových onemocnění I / Cancer biomarkers I
Předsedajíci / Chairs: Marek Svoboda, Ondřej Slabý
10.00 – 10.30
Luminální/Her-2 negativní karcinom prsu a možnosti využití komplexních prognostických
systémů založených na analýze genetických molekulárních biomarkerů.
Marek Svoboda
10:30 – 11:00
11:00 – 11:15
11:15 – 11:30
11:30 – 11:45
11:45 – 12:00
12:00 – 13:00
Plasma proteomic biomarkers for early detection and characterization of colorectal cancer
Marián Hajdúch
Circulating tumor cells in pancreatic cancer
Josef Srovnal
Circulating tumor cells - a tool to monitor cancer treatment
Anna Jakabová
MiR-31-3p a miR-31-5p predikují čas do progrese u pacientů s metastatickým kolorektálním
karcinomem s nemutovaným onkogenem KRAS léčených cetuximabem
Jitka Mlčochová
MikroRNA u solidních nádorů: Od biomarkerů po terapeutické cíle
Ondřej Slabý
OBĚD / LUNCH
13:00 – 14:30 Nové modely a technologie / New models and technologies
Předsedajíci / Chairs: Viswanath Das, Karel Koberna
13:00 – 13:15
Mouse transgenic models to study tumor initiation and progression in the gut
Michaela Krausová
13:15 – 13:30
Technology of programmable nucleases and reporter mouse lines engineering for cancer research
Dominika Fričová
13:30 – 13:45
Genome editing and gene modification technologies: an overview and update
Sylvia Petrezselyová
13:45 – 14:00
Understanding biological heterogeneity with the CyTOF 2 Mass Cytometer
Mark D. Lynch
14:00 – 14:15
Three-dimensional cultures: physiologically-relevant in vitro tumor models
Viswanath Das
14:15 – 14:30
Advantages of high-throughput qPCR for gene expression profiling. Analysis and applications
Vlasta Korenková
14:30 – 15:00
PŘESTÁVKA / BREAK
15:00 – 17:00 Molekulární cíle a protinádorová léčiva I / Molecular targets and anticancer drugs I
Předsedajíci / Chairs: Marián Hajdúch, Tomáš Eckschlager
15:00 – 15:30
Lactate transport in hypoxic cancer cells is facilitated by non-catalytic function of carbonic
anhydrase IX
Holger Becker
15:30 – 15:45
Carborane - Based Carbonic Anhydrase IX Inhibitors
Jana Štěpánková
15:45 – 16:00
5-Azacytidine Nucleosides and their Derivatives: Molecular hallmarks of Drug Resistance
& Alternative Therapeutic Regimen
Kushboo Agrawal
16:00 – 16.15
The activity of disulfiram towards breast cancer
Zdeněk Škrott
PROGRAM / PROGRAM
16:15 – 16:30
16:30 – 16:45
16:45 – 17:00
19:00
Valproic Acid Increases CD133 Positive Cells that Show Stem Cell Characters in Neuroblastoma
Mohamed Khalil
Distinctive activity of ginger phenylpropanoids against lymphoblastic leukemia cells
Ermin Schadich
Tumor microenvironment: mesenchymal stromal cell contribution
Lucia Kučerová
SPOLEČENSKÝ VEČER / CONFERENCE DINNER
STŘEDA / WEDNESDAY, 3. prosince 2014 / December 3, 2014
8:00 – 9:00 Poškození a reparace DNA / DNA damage and repair
Předsedajíci / Chairs: Kateřina Bouchalová, Martin Mistrík
8:00 – 8:15
Function of cyclin-dependent kinase 12 in DNA repair pathway
Jiří Kohoutek
8:15 – 8:30
New approach to quantitative image analysis of laser induced DNA damage in live cells
Eva Veselá
8:30 – 8:45
BRCA-mutation status combined with BCL2 protein in prediction of relapse in triple-negative breast
cancer (TNBC) treated with adjuvant anthracycline-based hemotherapy.
Kateřina Bouchalová
8:45 – 9:00
2‘-deoxy-5-ethynyluridine (EdU): a thymidylate synthase inhibitor with DNA damaging activity
Anna Ligasová
9:00 – 9:30
PŘESTÁVKA / BREAK
9:30 – 11:30 Biomarkery nádorových onemocnění II / Cancer biomarkers II
Předsedajíci / Chairs: Jitka Berkovcová, Pavel Dundr
9:30 – 9:45
Možnosti predikce léčebné odpovědi na cílenou anti-EGFR a anti-VEGF terapii metastatického
kolorektálního karcinomu (mCRC)
Radim Němeček
9:45 – 10:00
Management and prognosis of childhood acute lymphoblastic leukemia on protocols ALL-BFM 90,
95 and ALL IC-BFM 2002: a retrospective single-center study
Jana Volejníková
10:00 – 10:15
Využití metody sekvenování nové generace v intratumorové heterogenitě kolorektálního karcinomu
Jitka Berkovcová
10:15 – 10:30
Introduction to RAS pyrosequencing of FFPE samples
Gabriela Gabčová
10:30 – 10:45
MiR-215 as an important tumor suppressor in colorectal cancer patients
Petra Vychytilová-Faltejsková
10:45 – 11:00
Overexpression of Filamin-A protein is associated with aggressive clinicopathological features and
poor survival outcome in NSCLC patients treated with platinum-based combination chemotherapy
Mariam Gachechiladze
11:00 – 11:15
Genetic changes in recurrent Glioblastoma - Is there any significance?
Zuzana Crlíková
11:15 – 11:30
Assay destinguishing integrated and episomal form of HPV16, 18, 31 and 58
Hana Ondryášová
PROGRAM / PROGRAM
11:30 – 12:40
OBĚD / LUNCH
12:40 – 14:10 Molekulární cíle a protinádorová léčiva II / Molecular targets and anticancer drugs II
Předsedajíci / Chairs: Petr Džubák, Jan Hraběta
12:40 – 12:55
Use of the FUCCI probe in HTS/HCA screening campaigns.
Petr Džubák
12:55 – 13:10
Validation of Cell Based Assays for High-Throughput Screening
Soňa Gurská
13:10 – 13:25
Molecular mechanisms of cell death induction by novel taxanes effective in resistant breast
cancer cells
Michael Jelínek
13:25 – 13:40
Novel triterpenoid fluoroderivatives with anticancer activity
Jiří Řehulka
13:40 – 13:55
Proteomic profiling of oxaliplatin treated cell line revealed nucleoli and ribosomal protein
downregulation
Tomáš Oždian
13:55 – 14:10
Apoferritin and its applications in nanomedicine
Simona Dostálová
14:10 – 14:30
PŘESTÁVKA / BREAK
14:30 – 15:15 Biomarkery nádorových onemocnění III / Cancer biomarkers III
Předsedajíci / Chairs: Radek Trojanec, Jiří Drábek
14:30 – 14:45
Identification of prognostic and predictive factors in non-small cell lung cancer patients treated
by Adjuvant chemotherapy
Jana Potočková
14:45 – 15:00
A workflow for the identification and validation for N-glycoprotein biomarkers in mouse
xenograft serum
Lakshman Varanasi
15:00 – 15:15
System for reliable identification of alterations using protein mass-spectrometry
Miroslav Hruška
15:15 - 15:45
PŘESTÁVKA - POSTEROVÁ SEKCE / POSTER SESSION BREAK
15:45 – 16:05 Farmakologie protinádorových léčiv a in vivo imaging /
Pharmacology of anticancer drugs and in vivo imaging
Předsedajíci / Chairs: Miloš Petřík, Josef Srovnal
15:45 – 16:00
Assessing pharmacokinetic properties in drug discovery: screening versus in silico
Alice Nová
16:15 – 16:30
68Ga labelled peptides for glioblastoma imaging
Miloš Petřík
16:30 – 16:45
Imaging of glioblastoma multiforme in mice using microPET/CT systém
Zbyněk Nový
16:45 – 17:00
UKONČENÍ KONFERENCE/CLOSING CEREMONY
A8
Abstract book
Postery / Posters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Exprese HNF-1β v karcinomech děložního čípku
Kristyna Nemejcova
Temperature uniformity of the thermocyclers
tested using melting temperature of the same
amplicon
Eva Hruskova
Inhibition of proliferation by inhibitors of histone
deacetylases
Tomas Groh
Multi-walled carbon nanotubes as a promising
doxorubicin nanocarrier and their effects on neuroblastoma cell lines
Tereza Cerna
Prognostický a prediktivní význam sérových onkomarkerů u pacientů s pokročilým neskvamózním NSCLC léčených pemetrexedem
Ondrej Fiala
Sérové onkomarkery jako prediktivní biomarker
u pacientů s pokročilým NSCLC léčených EGFR-TKI
Ondrej Fiala
Influence of quercetin and taxifolin on microRNA
375 expression in HepG2 cell line and human
hepatocytes.
Zdenek Dostal
PHAGE λ as a suitable doxorubicin nanocarrier
Simona Dostalova
Fluorescenční charakterizace zlatem modifikovaného liposomu s uzavřenými protinádorovými
léčivy a s připojenou antisense N-myc DNA uchycenou k magnetickým částicím
Sylvie Skalickova
SLEDOVÁNÍ INTERAKCE DOXORUBICINU A
ELIPTICINU S BIOLOGICKY ODBOURATELNOU
SLOUČENINOU - ALBUMINEM
Sylvie Sklickova
STUDY OF AFFINITY OF COORDINATION COMPLEXES OF METAL IONS TO DNA BASED ON
THE CHANGE OF FLUORESCENCE INTENSITY
OF INTERCALATION LABELS (DOXORUBICIN
AND ETHIDIUM BROMIDE)
Sylvie Skalickova
Nanotools in microRna isolation and detection
Marketa Vaculovicova
Charakterizace dlouhé nekódující RNA MALAT-1
u buněk karcinomu prsu
Jaroslav Juracek
DEREGULACE PIWIL PROTEINŮ A VYBRANÝCH
PIRNA U RCC A CRC
Robert Iliev
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Navýšení exprese miR-215 in vivo ovlivňuje velikost nádoru v animálním modelu kolorektálního
karcinomu
Jana Merhautova
Molecular genetic analysis of pheochromocytomas and paragangliomas in Czech patients
Musil Zdenek
Skp2 associates with Slug and androgen receptor in patients with high Gleason score and
lymph node metastasis of prostate cancer
Gvantsa Kharaishvili
Ubiquitin specific peptidase 7 (USP7) regulates
DNA damage bypass pathway through stabilizing
Cdc7 kinase and RAD18 ubiquitin ligase.
Zsofia Turi
Is Epithelial to Mesenchymal Transition Followed
by Global DNA Methylation Changes?
Bozena Smolkova
The effect of acquired resistance to paclitaxel on
expression of ABC transporters at breast cancer
cells: role of ABCB1
Matej Behounek
Vliv rentgenového záření na genovou expresi
v lidských chlupových folikulech
Hanus Slavik
Sada 6-ti mikroRNA predikuje celkové přežívání
u pacientů s multiformním glioblastomem
Jiri Sana
The comparison of 2-D and 3-D model of gene
therapy mediated by genetically modified mesenchymal stem cells on human ovarian carcinoma cells SKOV-3.
Lenka Toro
MESENCHYMAL STROMAL CELLS PLAY AN
IMPORTANT ROLE IN MIGRATION AND CHEMOSENSITIVITY OF BREAST CANCER CELL
LINES
Svetlana Skolekova
A novel non-laborious and cost-effective approach for mammalian cell synchronization
Martin Liptay
MIF: A prognostic marker in Glioblastoma multiforme?
Nato Narsia
Ultra deep amplicon sequencing of RAS genes
and its use for mCRC predictive diagnostics
Jana Stranska
Výhody vysokokapacitního qPCR pro genově
expresní profilování Analýza a aplikace
Vlasta Korenková
Abstract book
Biomarkery nádorových onemocnění I
Cancer biomarkers I
Předsedajíci / Chairs: Marek Svoboda, Ondřej Slabý
úterý / 2. prosince 2014 / Tuesday / December 2, 2014 / 10.00 - 12.00 hod.
Luminální/Her-2 negativní
karcinom prsu a možnosti
využití komplexních
prognostických systémů
založených na analýze
genetických molekulárních
biomarkerů.
úterý / 2. prosince
10.00 - 10.30 hod.
2014
/
Marek Svoboda
Klinika komplexní onkologické
péče
Masarykův onkologický ústav, Žlutý
kopec 7, 656 53 Brno
Karcinom
prsu
tvoří
skupinu
jednotlivých podtypů s odlišným
biologickým
chováním,
jejichž
charakterizace je založena na
histologických
a
molekulárních
znacích, vycházejících z jejich
rozdílného
genetického
profilu.
V současnosti tak rozlišujeme 5
hlavních podtypů karcinomu prsu, a
to: 1. luminální – typ „A“, 2. luminální
– typ „B“, 3. Her-2 pozitivní/luminální,
4. Her-2 pozitivní/neluminální, 5.
triple-negativní, jejichž stanovení je
již rutinní součástí klinické praxe,
neboť se sebou přináší prognostické
a prediktivní informace nezbytné
k určení
správného algoritmu
onkologické léčby.
Nejvyšší
prevalence
mezi
pacientkami s karcinomem prsu
dosahují luminální (A i B)/Her-2
negativní karcinomy. V populaci
českých pacientek tvoří 65 - 70
% (data MOÚ z let 2005 – 2009).
Vyznačují se zejména expresí
estrogenového receptoru alfa (ERalfa) a přítomností nemutované
formy genu Her-2. Jsou sice
obecně prognosticky příznivější,
nicméně i mezi nimi existuje značná
heterogenita ve vývoji onemocnění,
a to i v těch případech, kdy jsou
zachyceny v časném stádiu, tj. kdy je
nádor omezen pouze na prsní žlázu.
Ve srovnání s ostatními podtypy
u nich častěji dochází k pozdním
relapsům.
V dosavadní klinické praxi jsou
pacientky
s
luminálním/Her-2
negativním typem karcinomu prsu
členěny do skupin s odlišným rizikem
relapsu na základě klasických a
molekulárních
prognostických
biomarkerů, jako jsou: věk, velikost
tumoru, postižení lymfatických uzlin,
grading, zastoupení nádorových
buněk exprimujících estrogenový
receptor alfa a progesteronový
receptor, markery proliferace (např.
Ki-67) a případně další. Pacientky
ve zvýšeném riziku relapsu pak
podstupují intenzivnější adjuvantní
systémovou léčbu, a to v podobě
sekvenční
a/nebo
prodloužené
hormonoterapie
nebo
aplikace
chemoterapie
před
hormonální
léčbou. V těchto případech však
současně dochází k navyšování
toxicity
léčby,
při
podávání
chemoterapie významnou měrou,
a s tím souvisejících přímých i
nepřímých nákladů, tj. spojených
s vlastní protinádorovou léčbou
nebo léčbou její toxicity. Z tohoto
pohledu největší problém činí
stanovení prognózy, a tedy i
indikace intenzivnější adjuvantní
systémové léčby, u pacientek s
luminálním/Her-2 negativním typem
karcinomu prsu, jejichž onemocnění:
a) prokazatelně nedisseminovalo
do regionálních lymfatických uzlin
(N0), přitom jejich tumor přesahuje
2 cm, b) postihlo sentinelové a/
nebo maximálně 3 lymfatické uzliny,
c) má přítomny jiné prognosticky
nepříznivé parametry (např. vyšší
grading,
zvýšená
proliferační
aktivita, nižší – ale stále pozitivní exprese estrogenové receptoru).
Přestože určitého konsensu v užívání
a interpretaci výše uvedených
biomarkerů
bylo
dosaženo
prostřednictvím
prognostických
schémat a systémů, které vychází z
doporučení odborných panelů (např.
závěry z konferencí v St. Gallen,
NCCN a ASCO doporučení) nebo z
klinických registrů (např. Adjuvant!
Online), problém správného určení
strategie léčby ve výše uvedených
případech tímto způsobem zcela
vyřešen není. Důvodů omezenosti
uvedených
prognostických
schémat je několik, mezi hlavní
patří i ta skutečnost, že stanovení
gradingu, exprese estrogenového,
progesteronového receptoru a Ki-67
podléhá do značné míry technické
a interindividuální variabilitě, a dále,
že výše uvedené biomarkery u
takto vymezené skupiny pacientek
(luminální/Her-2 negativní karcinom)
již nejsou schopny blíže diferencovat
biologickou povahu onemocnění.
Zcela zásadní změnu v určení
prognózy těchto pacientek přinesly
prognostické systémy založené
na metodicky jednotném, vysoce
přesném stanovení a interpretaci
několika biomarkerů pomocí metod
molekulární
genetiky.
Několik
těchto komplexních molekulárních
prognostických systémů se již
dostalo do užívání v klinické praxe
a další se tomu blíží Jejich použití
u indikované skupiny pacientek
s
luminálním/Her-2
negativním
karcinomem prsu vede ke změně
postupu léčby v rozmezí 27 – 74 %
případů, a to v závislosti na použitém
a srovnávavném prognostickém
systému, vždy však s převahou
případů,
kdy
jsou
pacientky
adjuvantní chemoterapie ušetřeny
(20 – 40 % pacientek). Cílem naší
přednášky bude shrnout stávající
situaci v oblasti komplexních
molekulárních
prognostických
systémů, se zaměřením se na jejich
dostupnost, přednosti, případně
slabá místa. Diskutovány budou
testy Breast Cancer Index, Clarient
Insight Dx, EndoPredict, eXagenBC,
MammaPrint, Oncotype DX® Breast
A9
A10
Abstract book
Cancer, Prosigna Breast cancer
prognostic gene signature assay a
další.
Plasma proteomic biomarkers
for early detection and
characterization of colorectal
cancer
úterý / 2. prosince
10.30 - 11.00 hod.
2014
/
S.Surinova1*,M.
Dziechciarková2,A.
Sethi1,Ch.-Y.Chang3,T.
Clough3,L.Radova2,M.
Skrovina4, K. Vyslouzil5,M.
Okoniewski6,H.Weisser1,E.
Cattaneo7,O.Vittek3,G.Marra7,
R.Aebersold1, M.Hajdúch2
Institute of Molecular Systems
Biology, ETH Zurich, Switzerland;
2
Institute of Molecular and
Translational Medicine, Palacký
University and University Hospital
in Olomouc, Czech Republic
3
Department of Statistics, Purdue
University, West Lafayette IN, USA
4
J.G.Mendel Oncology Centre,
Novy Jicin, Czech Republic
5
Department of Surgery, University
Hospital in Olomouc, Czech
Republic,
6
Functional Genomics Center, and
7
Institute of Molecular Cancer
Research, University of Zurich,
Switzerland
1
The current management
of colorectal cancer (CRC)
would greatly benefit from
non-invasive prognostic and
predictive biomarkers indicative
of clinical and molecular tumor
characteristics. Here we employed
targeted proteomic profiling of 80
glycoprotein biomarker candidates
across a well annotated patient
cohort with comprehensive CRC
characteristics. Clinical data
included 8-year overall survival,
tumor staging, histological grading,
regional localization, and molecular
pathway state of microsatellites
and the KRAS gene. The obtained
quantitative proteomic data set
was subjected to the development
of biomarker signatures predicting
the respective clinical endpoints.
Protein candidates were selected
into the signatures based on
significance testing and a stepwise
protein selection, each within
10-fold cross validation. A sixprotein biomarker signature of
patient outcome could predict
survival beyond clinical stage,
and was able to stratify patients
into groups of better and worse
prognosis. Additional signatures
predicting colon versus rectal tumor
localization, mutation in the KRAS
gene, and metastatic disease were
also identified. The integration
of rich clinical data, quantitative
proteomic technologies, and
tailored computational modeling
facilitated the characterization
of these signatures in patient
circulation. These findings propose
the promise of a simultaneous
assessment of important
prognostic and predictive disease
characteristics within a single
measurement.
Circulating tumor cells in
pancreatic cancer
úterý / 2. prosince
11.00- 11.15 hod.
2014
/
Josef Srovnal1, Andrea
Prokopova1, Martin Lovecek2,
Roman Havlik2, Dusan Klos2,
Jana Vrbkova1, Marian
Hajduch1
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
2
Department of Surgery, University
Hospital Olomouc, Olomouc,
Czech Republic
Introduction
The aim of this ongoing study was
to evaluate the hypothesis that
presence of circulating tumor cells
(CTCs) is a negative prognostic factor
in pancreatic cancer. Pancreatic
cancer is one of the most aggressive
malignancies with a poor prognosis.
Assessment of CTCs could prevent
aggressive surgery in patients with
advanced systemic dissemination.
Materials/methods
1
This was a prospective study to test
the presence of CTCs in systemic
blood, tumor draining blood, bone
marrow and peritoneal lavage of
175 pancreatic carcinoma patients
at the time of surgery using realtime RT-PCR for carcinoembryonic
antigen (CEA), epidermal growht
factor receptor 1 (EGFR1) and
human telomerase (hTERT). Gene
expression of tested markers was
correlated with clinical/pathological
characteristics and overall survival.
Results and conclusions
Overall, 151 of 175 (86.3%)
pancreatic
carcinoma
patients
died, 81 of 175 (46.3%) of patients
underwent curative surgery (R0
surgery). A significant association
between EGFR expression levels in
the tumor draining blood and clinical
stage was found, where patients
with advanced disease have a
higher expression of EGFR in the
tumor draining blood, than patients
with lower clinical stage (p<0.001).
In addition, significant association
between CEA expression levels in
the peritoneal lavage and surgical
radicality was found (p<0.01). The
pancreatic cancer patients with
the presence of occult tumor cells
detected in the peritoneal lavage
using CEA had significantly shorter
overall survival (p<0.006). It enables
identify patients with advance
disease for whom radical surgery is
of small benefit. However, we didn‘t
find any significant correlation of
CTC presence in blood and bone
marrow and overall survival. It
seems, that CTCs play minimal role
in pancreatic cancer prognosis due
to high agressivness of the disease.
Acknowledgements:
Supported
by grants IGA UP LF_2014_019,
CZ.1.07/2.3.00/30.0004,
NPU
LO1304 and TAČR TE02000058.
Circulating tumor cells - a tool
to monitor cancer treatment
úterý / 2. prosince
11.15 - 11.30 hod.
2014
Anna Jakabová1,2, Michael
Pinkas1,2, Petra Eliášová3,
Zuzana Ušiaková4,
Robin Strnad5, Katarína
/
Abstract book
Kološtová1,2, Robert Gürlich3,
Vladimír Bobek1,2
Department of Tumor Biology,
Third Faculty of Medicine, Charles
University in Prague, Prague,
Czech Republic,
2
Department of Laboratory
Genetics, University Hospital
Kralovske Vinohrady, Prague,
Czech Republic,
3
Department of Surgery, University
Hospital Kralovske vinohrady,
Prague, Czech Republic,
4Oncology Clinic, General
University Hospital Prague, Prague,
Czech Republic, 5Department of
Surgery, Thomayer’s Hospital,
Prague, Czech Republic
Introduction
Introduction: Circulating tumor cells
(CTCs) are shed from tumor to blood
circulation of patients with cancer
diseases and it is believed that they
are responsible for the formation of
metastasis. Monitoring of them can
be useful for determination of disease
progression and treatment response.
CTCs isolation from peripheral blood
is in comparison to tumor tissue
biopsy less invasive and can be
repeated. Circulating tumor cells
(CTCs) are shed from tumor to blood
circulation of patients with cancer
diseases and it is believed that they
are responsible for the formation
of metastasis. Monitoring of them
can be useful for determination of
disease progression and treatment
response. CTCs isolation from
peripheral blood is in comparison
to tumor tissue biopsy less invasive
and can be repeated.
Materials/methods
Methodology:
Patients
with
colorectal carcinoma (CRC) (n=84)
and breast carcinoma (BC) (n=14)
were included into the study report.
CTCs were tested in a prospective
manner and the peripheral blood
was taken in parallel with clinical
examination (every 3 months). All of
the CRC patients were undergoing
primary surgery, if possible blood
from tumor draining vein has been
withdrawn, too. Within the surgery
peritoneal washings were taken
and analyzed for disseminated
1
tumor cells (DTC) presence. In the
group of BC patients CTC-presence
was tested to monitor the effect of
neoadjuvant therapy.
CTCs were enriched from unclothed
peripheral blood (4-8 ml) by sizebased separation (MetacellTM).
CTC- presence is confirmed by
cytomorphological
evaluation
in parallel with gene expression
analysis of tumor associated genes.
Fluorescent dyes for labelling both
nucleus (NucBlue®) and cytoplasm
(CellTrackerTM) in viable cells were
used. Partly, CTCs were stained
by May-Grünwald or Diff. Quick
protocol.
Gene
expression
of
tumor
associated genes (e.g. KRT7,
KRT18, KRT19, KRT20, EpCAM,
MUC1, EGFR, MIF...) was tested
in an enriched fraction of CTCs
immediately after separation and/
or after in vitro culture (3-5days).
The gene expression profiles were
compared to the profiles obtained
in whole peripheral blood. CD45,
CD68 and -actin were used as
normalizers. If CTCs were present
the gene expression analysis of
chemoresistance-associated genes
followed. Results were analyzed by
GENEX software 6.0 (MultiD).
Results and conclusions
Results: Based on cytomorphological
evaluation CTCs were found in 41%
(24/59) of CRC patient samples of
peripheral blood, and DTCs in 31%
(14/46) of peritoneal washings.
Interestingly, in 27% of the patients
CTCs were found in peripheral
blood and in tumor blood in parallel.
Subsequently, gene expression
profiles of CTCs and DTCs were
compared to primary tumor tissue.
The results have shown, that the
gene expression profiles of CTCs
are closely related to DTCs profile,
additionally,
DTCs
expression
profiles are related to primary tumor
tissue.
In the group of BC patients 78%
(11/14) were CTC-positive before
the therapy starts. CTCs gene
expression profiles have changed
during the therapy. The most
interesting case studies of CRC and
A11
BC patients will be reported.
Conclusion:
CTCs analysis could be used within
regular clinical examination to
monitor possible recurrence and
therapy developed resistance.
MiR-31-3p a miR-31-5p
predikují čas do progrese u
pacientů s metastatickým
kolorektálním karcinomem
s nemutovaným onkogenem
KRAS léčených cetuximabem
úterý / 2. prosince
11.30- 11.45 hod.
2014
/
Jitka Mlcochova2, Petra
Vychytilova-Faltejskova1,2,
Hana Mlcochova1,2,
Radim Nemecek1, Jana
Nekvindova3, Lenka Radova2,
Manuela Ferracin4, Barbara
Zagatti4, Rostislav Vyzula1,
Massimo Negrini4, Ondrej
Slaby1,2
Klinika komplexní onkologické pece
MOU, Brno, Czech Republic,
2
CEITEC, Masarykova Univerzita,
Brno, Czech Republic,
3
Ústav klinické biochemie a
diagnostiky, FN Hradec Králové,
Hradec Králové, Czech Republic,
4
Laborator experimentální medicíny
a diagnostiky, Ferrara, Italy
Introduction
Terapie
metastatického
kolorektálního karcinomu (mCRC)
se rozšířila o anti-EGFR terapii
(cetuximab, panitumumab), která cílí
a blokuje receptor pro epidermální
růstový faktor (EGFR). Úspěšnost
léčby je podmíněná nemutovaným
onkogenem KRAS (wt-KRAS). I
přesto má klinický benefit z léčby
jen u přibližně pětina pacientů s
wt-KRAS. To je důvodem nutnosti
nalézt nové biomarkery schopné
predikovat odpověď k anti-EGFR
terapii, která v případě neúčinnosti
zbytečně oddaluje možnost podání
jiné potenciálně efektivní terapie.
Těmito biomarkery by mohly být
miRNA zapojené do klíčových
signálních drah a regulující také
jednotlivé složky signální dráhy
EGFR.
1
A12
Abstract book
Materials/methods
Do studie bylo zařazeno 71 klinicky
charakterizovaných
pacientů
s mCRC s wt-KRAS léčených
cetuximabem
a
25
léčených
panitumumabem.
Pacienti
byli
rozděleni na základě odpovědi na
anti-EGFR terapii.
Ze 41 FFPE
vzorků odebraných před zahájením
léčby byla vyizolována celková RNA
obohacená o frakci krátkých RNA,
která byla následně použita pro
stanovení expresních profilů miRNA
pomocí technologie Agilent miRNA
MicroArrays. Následovala validace
na nezávislé kohortě 30 pacientů
léčených cetuximabem a 25 pacientů
léčených panitumumabem pomocí
qRT-PCR.
Results and conclusions
Výsledky:
Po
vyhodnocení
profilů
exprese
723
miRNA
bylo identifikováno 9 miRNA s
nejsignifikantnějším
rozdílem
v
expresi mezi pacienty s dobrou
a špatnou odpovědí na terapii
cetuximabem (P ≤ 0,01). Těchto 9
miRNA bylo dále validováno a bylo
zjištěno, že hladiny exprese miR-315p, miR-31-3p (P < 0,001) korelují s
časem do progrese (TTP) u pacientů
léčených cetuximabem (miR-31-3p
– 43 vs. 14 týdnů, miR-31-5p, 44
vs. 14 týdnů), ale nikoliv pacientů
léčených panitumumabem (miR-313p – 20 vs. 29 týdnů, miR-31-5p, 20
vs. 30 týdnů).
Závěr: Na základě získaných
výsledků se zdá, že miR-31-5p,
miR-31-3p by mohly sloužit jako
nové prediktivní biomarkery k
predikci odpovědi na cetuximab u
pacientů s mCRC s wt-KRAS, což
by přispělo k vyšší individualizaci
léčby. Následné studium miRNA a
jejich zapojení do regulace signální
dráhy EGFR by mohlo poodhalit
další z molekulárních mechanizmů
rezistence k anti-EGFR terapii.
Práce byla podpořena grantovým
projektem IGA MZČR NT 138604/2012.
MikroRNA u solidních
nádorů: Od biomarkerů po
terapeutické cíle
úterý / 2. prosince
11.45- 12.00 hod.
2014
/
Ondřej Slabý1,2
Masarykův onkologický ústav,
Klinika komplexní onkologické
péče, Brno;
2
CEITEC – Středoevropský
technologický institut, Masarykova
univerzita, Brno;
1
MikroRNA (miRNA) jsou krátké
nekódujcí RNA dlouhé 18-25
nukleotidů, které post-transkripčně
regulují genovou expresi v průběhu
rozličných buněčných procesů jako
jsou apoptóza nebo diferenciace,
ale maligní transformace. Změny
v expresních profilech miRNA již
byly pozorovány u většiny solidních
nádorů. Mechanistické studie v
nádorové buňce prokázaly schopnost
jednotlivých miRNA vykazovat funkci
nádorových supresorů a onkogenů.
Nejnovější
pozorování
navíc
popisují potenciál jedné miRNA
vykazovat v závislosti na kontextu
jak funkci nádorového supresoru
tak onkogenu. Tato pozorování
zásadním
způsobem
rozšířila
koncept molekulární patogeneze
nádorových onemocnění a naznačila
potenciál
miRNA
nejen
jako
diagnostických biomarkerů, ale také
jako potenciálních terapeutických
cílů.
Sdělení zahrnuje novinky z oblasti
biogeneze a funkce miRNA, izomiRs,
koncept kompetujících endogenních
RNA (ceRNAs), význam miRNA v
nádorové biologii a jejich zapojení
do hlavních znaků maligního
nádoru, biologie nádorové kmenové
buňky či autofagie. V kontextu
našich výsledků bude diskutována
schopnost
vybraných
miRNA
složit jako tkáňové biomarkery
(prognostické a prediktivní), sérové
a močové biomarkery (diagnostické)
a potenciální terapeutické cíle u
kolorektálního karcinomu, renálního
karcinomu a glioblastomu.
Specifické expresní profily miRNA
byly u pacientů se solidními nádory
úspěšně využity ke stanovení
prognózy, k predikci léčebné
odpovědi na vybrané terapeutické
režimy nebo upřesnění diagnostiky
u metastáz neznámého původu.
Přítomnost miRNA byla prokázána
v krevním séru a plazmě, ale také
moči nebo mozkomíšním moku,
kde vykazovaly nejen vysokou
míru stability, ale u vybraných
solidních nádorů rovněž velice
dobré analytické vlastnosti. V
současné době je kromě možného
diagnostického využití cirkulujících
miRNA intenzivně studován jejich
původ a příčiny jejich extrémně
vysoké stability. MiRNA jsou také
velice
slibnými
terapeutickými
cíli, přičemž první protinádorová
terapie na bázi miRNA vstupuje do
klinického hodnocení začátkem
příštího roku.
Výzkum byl podpořen granty IGA
MZ ČR NT13549-4/2012, NT13860
4/2012,
NT-13547
-04/2012,
NT13514-4/2012
a
NT112144/2010.
obchodní a servisní zastoupení firem
Abstract
book
A13
obchodní
a servisní
zastoupení
firem
THERMO Scientific
– divize
laboratorní
techniky
THERMO
Scientific
–
divize
laboratorní
techniky
BIOQUELL . ERLAB . HMC EUROPE . LANCER . RUSKINN
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boxy pro PCR
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bezodtahové digestoře
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boxy pro laboratorní zvířata
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gel—imaging a analýza inkubátory - termostaty
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software pro 1D i 2D analýzu
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transiluminátory
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elektroforézy
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zdroje pro elfo
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mrazicí a chladící boxy, kryoboxy mikrodestičková instrumentace
mrazicí a chladící boxy, kryoboxy mikrodestičková instrumentace
spektrofotometry, fotometry
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fluorometry, luminometry
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dávkovače
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promývačky
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třepací termostaty pro mikrodestičky
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KingFisher
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chladící boxy 0°C až 15°C
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čidla pro validaci autoklávů
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vakuové centrifugační koncentrátory
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stolní lyofilizátory
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ochranné a přestýlací boxy
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steppery a dávkovače
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spotřební plastik Nalgene, Nunc
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stripy a destičky
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drobné laboratorní přístroje příprava čisté vody
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A14
Abstract book
Nové modely a technologie
New models and technologies
Předsedajíci / Chairs: Viswanath Das, Karel Koberna
úterý / 2. prosince 2014 / Tuesday / December 2, 2014 / 13.00 - 14.30 hod.
Mouse transgenic models
to study tumor initiation and
progression in the gut
addition, currently utilized mouse
models of intestinal cancer will be
presented.
úterý / 2. prosince 2014 / Results and conclusions
13.00 - 13.15 hod.
The talk will illustrate how genetic
Michaela Krausova, Bohumil manipulations in mouse deepened
our knowledge about cellular
Fafilek, Vladimir Korinek
architecture of adult gut tissue. The
Institute of Molecular Genetics AS
contribution of various signaling
CR, v.v.i., Prague, Czech Republic pathways to normal stem cell
physiology and to tumor initiation and
Introduction
The epithelia of the gastrointestinal progression will also be discussed.
tract represent one of the most Technology of programmable
rapidly self-renewing tissues in the nucleases and reporter mouse
adult mammalian body. The single- lines engineering for cancer
layer epithelium of the small intestine
research
is formed by microscopic projections
úterý / 2. prosince 2014 /
into the intestinal lumen called villi,
13.15 - 13.30 hod.
and invaginations into underlying
mesenchyme, so-called crypts of Dominika Fricova, Maja
Lieberkühn. Tissue homeostasis Sabol, Petr Kaspar, Petr
is maintained by intestinal stem
Kasparek, Trevor Epp, Inken
cells residing in the bottom part of
the crypts. The stem cells divide Beck, Radislav Sedlacek
asymmetrically, giving rise to fast- Laboratory of Transgenic Models
cycling transit amplifying cells of Diseases and Czech centre for
located above the stem cell zone. Phenogenomics, BIOCEV, Institute
At the crypt orifice these cells of Molecular Genetics of the ASCR,
differentiate to generate specialized Prague, Czech Republic
cell lineages of the gut. Differentiated Introduction
cells continue their movement
TALE (Transcription-activator like
up to the top of the villus, where
effector) and CRISPR (Clustered
they are extruded to the intestinal
Regularly
Interspaced
Short
lumen. The process of epithelial
Palindromic Repeats) associated
self-renewal is completed in 3-5
nucleases are able to generate genetic
days. The tract lining is concurrently
modifications by introduction of
exposed to chemical stress and
double-stranded breaks. Subsequent
mechanical tensions. Consequently,
activation of repair pathways and
the combination of high proliferation
homologous recombination (HR)
rate and environmental stress results
represent molecular basis for knockin the accumulation of somatic
in strategies. This approach allows
mutations hitting tumor suppressor
genomic insertions (1) within the
genes or proto-oncogenes leading to
locus of the gene of interest or (2) in
the tumor formation.
unrelated, well described genomic
Materials/methods
region as ROSA26, which provides
The talk will be focused on recent ubiquitous transgene expression with
methodologies employed for the no adverse consequences on mouse
identification of specific markers phenotype. In the order to facilitate
of the intestinal stem cells and for the analysis of the function(s) of genes
cell tracking in transgenic mice. In involved in senescence (p16Ink4a
and p21) and DNA damage response
(Fancd2 and 53BP1), we employed
programmable nucleases mediated
targeting systems to generate
mouse lines expressing reporters as
luciferase and/or fluorescent protein
inserted in desired genomic region.
Materials/methods
Programmable nucleases including
TALENs and CRISPR/Cas9 system
enable
generation
of
genetic
modifications in cultured cells, as
well as in whole animals. In order to
create reporter mouse lines we used
different strategies. We generated
targeting
constructs
containing
reporters and specific homology
arms for targeting specific genomic
region. In Fancd2 and 53BP1
targeting strategy, gene encoding for
citrine was inserted behind the start
codon using TALENs and CRISPRs
in two different approaches. The
donor vectors were assembled
using ligation-independent cloning,
and contained homology arms from
region surrounding the ATG site. For
p16Ink4a and p21 reporter models
we prepared constructs containing
genes encoding for luciferase and
tdTomato fluorescent protein in
tandem fusion under p16Ink4a or
p21 promoter control. After in vitro
confirmation of its specific activity
in senescent cells, we cloned
reporter construct into donor vector
containing homology arms from
first intron of the ROSA26 locus.
TALENs or CRISPRs combined with
the linearized donor construct were
injected into mouse zygotes.
Results and conclusions
Reporter gene mouse lines represent
crucial tool for in vivo studies in variety
of scientific fields from functional
genomics, proteomics, cell biology
to cell-based drug screenings. In this
study we designed and used both
TALENs and CRISPR/Cas9 assisted
targeting for creation of reporter
mouse lines for genes involved in DNA
Abstract book
damage response (Fancd2, 53BP1)
and senescence (p16Ink4a and
p21). To prepare models expressing
citrine-tagged versions of protein
products of Fancd2 and 53BP1
we targeted directly genes loci of
these genes. In p16Ink4a and p21
reporter mice strategy we inserted
genes encoding for luciferase and
tdTomato under p16Ink4a (or p21)
promoter control into ROSA26 locus.
Mouse embryonic fibroblasts were
developed from created mouse lines
and the activity of tagged proteins
was examined. Our models could be
used for deeper analysis of the role
of senescence and DNA damage
response in context of cancer
development and its treatment.
Genome editing and gene
modification technologies: an
overview and update
úterý / 2. prosince
13.30 - 13.45 hod.
2014
/
Silvia Petrezselyova, Radislav
Sedlacek
Laboratory of Transgenic Models
of Diseases and Czech centre
for Phenogenomics, BIOCEV,
Institute of Molecular Genetics of
the ASCR, v.v.i., Prague, Czech
Republic
Introduction
Functional gene studies require
precise and efficient genome
engineering
technologies
and
gene targeting via homologous
recombination (HR) has been
extensively used to generate
specific mutant species for decades.
However, the use of this technique
has been hampered because of
several limitations: low HR rate in
mammalian cells, labor-intensive and
time-consuming selection/screening
strategies and construction of large
targeting vectors. In recent years,
discovery of new genome-editing
technologies, such as ZFN (zinc finger
nucleases), TALENs (transcription
activator-like effector nucleases)
and
CRISPR
(the
Clustered
Regularly
Interspaced
Short
Palindromic Repeats) associated
with Cas9 (CRISPR-associated
9) protein, has revolutionized
generation of targeted mutations in
many model organisms. All these
technologies work on the same
principle, i.e. sequence-guided DNA
endonucleases induce DNA doublestrand breaks that subsequently
stimulate either non-homologous
end joining (NHEJ) recombination
and/or homology-directed repair
(HDR) system at targeted loci. The
NHEJ recombination is an errorprone event forming small insertion
or deletion (indels) and therefore
results in a frame-shift mutation.
Because the HDR system requires
a DNA template to repair a DSB, it
is used to introduce a desired point
mutation or insert a missing gene/
reporter gene.
Materials/methods
In our laboratory, we employ
both TALEN and CRISPR/Cas9
approaches to create site specific
modifications in the mouse genome.
Knock-out mice are prepared by
targeting one site or two sites
simultaneously that allows frame-shift
and deletion mutations, respectively.
Double-stranded
constructs
or
single-stranded oligonucleotide are
introduced via HR to generate point
mutation changes, reporter genes
or conditional alleles. TALEN and
CRISPR/Cas9-related
constructs
are microinjected into mouse
zygotes to generate gene knockout or knock-in mice or transfected
into embryonic stem (ES) cells for
the creation of recombinant ES cell
clones.
Results and conclusions
Transgenic and knock-out mice are
valuable models for understanding
of physiological functions of proteins
and their mechanisms. Our model
mice will be applied in many human
disease studies, including cancer.
Several examples (single point
mutation mice, simple/conditional
knock-outs and reporter mice) out
of about 39 finalized and 30 ongoing
projects will be presented.
Understanding biological
heterogeneity with the CyTOF
2 Mass Cytometer
úterý
/
2.
prosince
2014
/
A15
13.45- 14.00 hod.
Mark D. Lynch
Fluidigm Europe BV, Les Ulis,
France
Introduction
Single-cell biology endeavors to
unravel biological heterogeneity
using multidimensional approaches.
Cells use DNA, RNA, and proteins to
power biological networks.
Materials/methods
The C1TM Single-Cell Auto Prep
System delivers an easy, complete
workflow that captures and prepares
individual cells for gene expression
and sequencing, while the CyTOF®
2 Mass Cytometer allows highspeed acquisition of multiparametric
single-cell proteins.
Results and conclusions
With
these
innovative
tools,
multidimensional single-cell biology
is providing profound insights into
the dynamic elements of biological
heterogeneity.
Three-dimensional cultures:
physiologically-relevant in
vitro tumor models
úterý / 2. prosince
14.00 - 14.15 hod.
2014
/
Viswanath Das, Marta
Zbožínková, Marián Hajdúch
Institute of Molecular and
Translational Medicine, Olomouc,
Czech Republic
Introduction
Three-dimensional (3D) cultures of
cancer cells are increasingly being
recognized
as
physiologicallyrelevant in vitro tumor models for
effective selection of anticancer
drugs in preclinical studies. Herein,
we present a comparative study
of the effect of a few selected
anticancer agents that are clinically
used in the treatment of various solid
tumors in two-dimensional (2D) and
3D cultures of colorectal HCT116
and HT29 carcinoma cells.
Materials/methods
Methods
Cytotoxic effects of anticancer
agents in 2D and 3D cultures of
A16
Abstract book
HCT116 and HT29 cells were
examined using MTT cell proliferation
assay and bright-field imaging.
Cell cycle alteration in 3D cultures
was monitored by flow cytometry.
Changes in the expression of
proteins in 2D and 3D cultures were
detected by Western blot analyses.
Results and conclusions
Anticancer drugs that are highly
cytotoxic in 2D cultures show
low cytotoxicity in 3D cultures
of HCT116 and HT29 cells. 3D
cultures of HCT116 and HT29 cells
showed G0/G1 cell cycle arrest
and an increase in the expression
of carbonic anhydrase IX. Our
results illustrate relative closeness
of three-dimensional cultures to
primary tumors and provide further
support for the potential use of
3D cell cultures for the screening
of promising anticancer agents in
preclinical screens.
Výhody vysokokapacitního
qPCR pro genově expresní
profilování. Analýza a
aplikace. (Advantages of highthroughput qPCR for gene
expression profiling. Analysis
and applications.)
úterý / 2. prosince
14.15 - 14.30 hod.
2014
/
Vlasta Korenková1, Lucie
Langerová1, Vendula
Novosadová1, Mikael
Kubista1,2
Biotechnologický ústav AV CR,
v.v.i., Praha, Czech Republic,
2
TATAA Biocenter, Goteborg,
Sweden
Introduction
Expresní profilování představuje
měření exprese více genů najednou
za účelem vytvoření obrazu o
buněčné funkci. Profily genové
exprese mohou být použity například
pro
diagnostické
monitorování
odpovědi pacienta na léčbu a tím k
optimalizaci individuální terapie.
Materials/methods
Vysokokapacitní zařízení BioMark
(Fluidigm),
které
je
umístěno
1
v
qPCR
servisním
pracovišti
Biotechnologického
ústavu
AV
ČR, umožňuje současnou analýzu
exprese až 96 genů v 96 vzorcích
(9216 reakcí) v rámci jediného
experimentu.
Měření
probíhá
pomocí kvantitativní polymerázové
řetězové reakce (qPCR), která je
jednou z nejcitlivějších metod pro
kvantifikaci mRNA či mikroRNA.
Možnost analyzovat větší množství
genů najednou vybízí k použití
multivariantních statistických analýz.
Results and conclusions
Využití vysokokapacitního qPCR
přístroje BioMark poskytuje množství
výhod. 1. Efektivita: Výrazná úspora
vzorku (5 µl pro kompletní analýzu),
detekční chemie, mastermixu a
spotřebního materiálu. 2. Rychlost:
Celý vysokokapacitní experiment lze
provést v rámci několika hodin, ne dnů
či týdnů. 3. Automatizace a vysoká
kapacita: Díky zautomatizovanému
pracovnímu
postupu
je
minimalizována možnost lidské
chyby. Není potřeba mezidestičkové
kalibrace. 4. Sensitivita a flexibilita
qPCR
systému.
5.
Možnost
dalších aplikací: vysokokapacitní
genotypování a digitální PCR.
Abstract book
Molekulární cíle a protinádorová léčiva I
Molecular targets and anticancers drugs I
Předsedajíci / Chairs: Marián Hajdúch, Tomáš Eckschlager
úterý / 2. prosince 2014 / Tuesday / December 2, 2014 / 15.00 - 17.00 hod.
Lactate transport in hypoxic
cancer cells is facilitated
by non-catalytic function of
carbonic anhydrase IX
úterý / 2. prosince
15.00- 15.30 hod.
2014
/
Somayeh Jamali1, Michael
Klier1,2, L. Felipe Barros3,
Robert McKenna4, Joachim
W. Deitmer2, Holger M.
Becker1
Division of Zoology / Membrane
Transport, University of
Kaiserslautern, Kaiserslautern,
Germany,
2
Division of General Zoology,
University of Kaiserslautern,
Kaiserslautern, Germany,
3
Centro de Estudios Científicos
(CECS), Valdivia, Chile,
4
Department of Biochemistry and
Molecular Biology, University of
Florida, Gainesville, USA
Introduction
The most aggressive and invasive
tumor cells, which often reside
in hypoxic environments, rely on
extensive glycolysis to meet their large
demand for energy and biosynthetic
precursors. Thereby they release
vast amounts of lactate and protons
via monocarboxylate transporters
(MCTs),
which
exacerbates
extracellular
acidification
and
supports the formation of a hostile
environment. In the present study
we investigated the mechanisms that
regulate lactate transport in cancer
cells during the switch from normoxia
to hypoxia.
Materials/methods
Changes in intracellular pH and
lactate concentration in single MCF7 breast cancer cells were monitored
by pH imaging and with the lactatesensitive FRET nanosensor Laconic,
respectively.
Hypoxia-induced
changes in the expression levels
of various enzymes and transport
1
Translational Medicine, Olomouc,
Czech Republic,
2
Institute of Organic Chemistry
and Biochemistry ASCR, v.v.i.,
Structural Biology, Prague, Czech
Republic,
3
Institute of Inorganic Chemistry of
the AS CR, v.v.i., Department of
Syntheses, Husinec Rez, Czech
Republic
Introduction
Carbonic anhydrase IX (CA IX) is
highly overexpressed in different
solid tumors. It is a transmembrane
isoform of carbonic anhydrase with
an extracellular-facing catalytic site
and therefore is well positioned
to act in the control of tumor pH.
Function of CA IX can be inhibited
by CA IX-selective sulphonamides.
Inhibition its function perturbs in vitro
survival under hypoxia conditions.
The aim of this study is to evaluate
the effect of new carboranes with
functional sulfonamide residues
on CA IX function. Carboranes,
icosahedral
clusters
containing
boron, carbon and hydrogen replace
various hydrophobic structures in
biologically active molecules.
Materials/methods
The most interesting drugs were
Carborane - Based Carbonic
chosen based on enzymatic assay.
Anhydrase IX Inhibitors
úterý / 2. prosince 2014 / To characterize their CA IX inhibition
mode in cellular level, several cell
15.30- 15.45 hod.
biology methods were used. Drug
Jana Stepankova1, Pavlina
cytotoxicity
and
consequently
extracellular pH of carboranes
Rezacova2, Brynda Jiri2,
treated cell lines under hypoxia
Harvanova Monika1, Masek
conditions was evaluated. Data
Vlastimil1, Nova Alice1,
shows reversed hypoxia-induced
Schiller Michal1, Das
decline of extracellular pH in treated
Viswanath1, Dolezal Dalibor1, HT-29 and 4T1 cell lines. Moreover,
we set up a new method of Raman
Grüner Bohumir3, Sicha
3
1
spectroscopy locating carboranes
Vaclav , Konecny Petr ,
Znojek Pawel1, Dzubak Petr1, distribution in cells. To extend
this study, pharmacokinetics and
Hajduch Marian1
pharmacology
profile
describe
1
Faculty of Medicine and Dentistry
ADME methods and experiments on
Palacky University Olomouc,
animal model.
Institute of Molecular and
proteins were analyzed in MCF-7
cells by qRT-PCR and western blot.
Functional interactions between
MCTs and carbonic anhydrase
CAIX were determined in Xenopus
oocytes by single-site mutation
and subsequent measurements of
intracellular pH with ion-sensitive
microelectrodes.
Results and conclusions
Under hypoxia, expression of MCT1
and MCT4 in MCF-7 breast cancer
cells remained unchanged, while
expression of carbonic anhydrase IX
(CAIX) was greatly enhanced. Realtime measurements of intracellular
pH and lactate concentration
show that CAIX augments lactate
flux via MCT1 by a non-catalytic
interaction. Mutation studies in
Xenopus oocytes indicate that CAIX,
via its intramolecular H+-shuttle
His200, functions as a „protoncollecting/distributing antenna“ to
facilitate rapid lactate flux via MCT1.
Knockdown of CAIX significantly
reduced proliferation of MCF-7
cancer cells, suggesting that rapid
efflux of lactate and H+, as enhanced
by CAIX, contributes to cancer cell
survival under hypoxic conditions.
A17
A18
Abstract book
Results and conclusions
The results shows the ability of
these compounds inhibit CA IX
in enzymatic level and cellular
level. Thus, novel carboranes with
functional sulfonamide residues
indicate new selective CA IX
inhibitors as potential anticancer
drugs.
Acknowledgement: ProMedChem,
reg. n.: CZ.1.07/2.3.00/30.0060,
BIOMEDREG,
reg.
n.:
CZ.1.05/2.1.00/01.0030
5-Azacytidine Nucleosides and
their Derivatives: Molecular
Hallmarks of Drug Resistance
& Alternative Therapeutic
Regimen
úterý / 2. prosince
15.45- 16.00 hod.
2014
/
Khushboo Agrawal1,
Dušan Holub1, Petr Vojta1,
Ivo Frydrych1, Zuzana
Maceckova1, Miroslav
Otmar2, Petr Džubák1, Marián
Hajdúch1
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
2
Institute of Organic Chemistry and
Biochemistry, ASCR v.v.i., Prague,
Czech Republic
Introduction
Aberrant DNA methylation turning
‘off’ the gene expression remains
the consistent hallmark due to its
frequent involvement in all types
of cancer. The prototypal DNA
methylation inhibitors, 5-azacytidine
and 2-deoxy-5-azacytidine, are
currently one of the most effective
epigenetic drugs, for the treatment
of blood malignancies. However,
the chemotherapeutic resistance
to these medicines is the major
obstacle, forefending the successful
epigenetic therapy.
Materials/methods
We developed several HCT116
p53 wild-type cell clones, resistant
towards
2‘-deoxy-5-azacytidine.
1
Principal methods used to study
the molecular alterations during the
development of resistance included,
flow cytometry based analyses,
high throughput RNA sequencing
based transcriptomics, and mass
spectrometry based proteomics,
utilizing stable isotope labelling of
amino acids in cell culture (SILAC).
Further, we used MTT cytotoxicity
assays to determine the crossresistance or sensitivity of the
resistant clones towards other
epigenetic inhibitors.
Results and conclusions
Flow cytometry based studies
revealed significant up-regulation of
DNA and RNA synthesis. Molecular
profiling of resistant clones unveiled
8010 genes and 3352 proteins,
which were differentially expressed
(ANOVA p<0.05) compared to
parental cell line. The major affected
cellular pathways were (i) Cell cycle:
nucleocytoplasmic transport of CDK/
cyclins, role of 14-3-3 proteins in cell
cycle regulation, G1/S transition and
initiation of mitosis (ii) Apoptosis and
survival: granzyme A signaling, BAD
phosphorylation, p53 dependent
apoptosis (iii) Transcription: role of
heterochromatin protein I family in
transcriptional silencing (iv) DNA
damage: role of SUMO in p53
regulation. During MTT cytotoxicity
assays, resistant clones exhibited
cross-resistance
towards
all
the tested epigenetic inhibitors,
however,
significant
sensitivity
was exceptionally observed for
bromodomain inhibitors, which was
further validated by down-regulation
of BET bromodomain, BRD4 gene, in
all the resistant cell clones. Validation
of relevant genes and/or proteins
as biomarkers of drug resistance,
and bromodomains as alternative
therapeutic target, for re-sensitizing
the cancer patients, resistant to DNA
methylation inhibitors is currently
ongoing.
The present study will aid to the
understanding of the molecular
basis of acquired tumor resistance
to 2‘-deoxy-5-azacytidine and help
in predicting its clinical response,
as well as in designing alternative
treatment regimens for overcoming
resistance, hence furthering clinical
development.
The activity of disulfiram
towards breast cancer
úterý / 2. prosince
16.00- 16.15 hod.
2014
/
Zdenek Skrott1, Martin
Mistrik1, Boris Cvek2, Pavla
Pouckova3, Jiri Bartek1,4
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
2
Department of Cell biology and
Genetics, Faculty of Science,
Palacky University, Olomouc,
Czech Republic,
3 st
1 Medical Faculty, Charles
University, Prague, Czech
Republic,
4
Danish Cancer Society Research
Canter, Copenhagen, Denmark
Introduction
Drug repurposing is a new viable
approach of drug development.
Disulfiram (Antabuse), as an old drug
used for decades in alcohol aversion
therapy, shows promising anticancer
activity. The potent disulfiram´s
antitumor effect is attributed to the
complex formed from the reaction
between disulfiram and copper
in the human body. It is assumed
that disulfiram-copper complex
kills cancer cells by inhibition of
ubiquitin-proteasome system, which
is responsible for degradation of
cellular proteins.
Materials/methods
The toxicity of the main metabolites
of disulfiram was evaluated in vitro
with various breast cancer cell lines
(MDA MB231, MCF7, HS578T,
T47D, Cal51) and in vivo using MDA
MB231 xenografts. The cellular
effect of disulfiram-copper complex
was measured in selected breast
cancer cell lines or Ub-(G67V)-GFP
expressing Hela cell line by standard
methods (Western blot, fluorescent
microscopy).
Results and conclusions
1
Abstract book
Disulfiram complexed with copper
supress cancer cell viability and
ubiquitin-dependent
protein
degradation. In contrast to previous
report, the complex does not exhibit
any inhibition of 20S core particle
of the proteasome. On the other
hand, disulfiram-copper complex
induces notable accumulation of
ubiquitinated proteins, indicating
that the degradation of proteins is
impaired. Moreover, the complex
stabilizes
both,
physiological
proteasome substrate IκBα, and
artificial Ub(G76V)-GFP reporter.
Collectively, these data indicate
that disulfiram´s anticancer activity
is linked to the inhibition of the
ubiquitin-proteasome system by
a mechanism that is distinct to the
conventional proteasome inhibitors.
Valproic Acid Increases
CD133 Positive Cells that
Show Stem Cell Characters in
Neuroblastoma
úterý / 2. prosince
16.15- 16.30 hod.
2014
/
Mohamed Ashraf Khalil1, Jan
Hraběta1, Tomáš Groh1,2,
Pavel Procházka3, Helena
Doktorová1, Šimon Cipro4,
Tomáš Eckschlager1
Department of Pediatric
Hematology and Oncology, 2nd
Medical Faculty, Charles University
and University Hospital Motol,
Prague, Czech Republic,
2
Department of Biochemistry,
Faculty of Science, Prague, Czech
Republic,
3
Institute of Experimental Medicine,
Academy of Sciences, Prague,
Czech Republic,
4
Department of Pathology and
Molecular Medicine, 2nd Medical
Faculty, Charles University and
University Hospital Motol, Prague,
Czech Republic
Introduction
Recently, Cancer stem cell model
proposes that tumor consists
of variety of cells with different
proliferative
and
tumorigenic
capacities. According to this theory,
a small population of cancer stem
1
cells (CSCs) was suggested to drive
tumor growth and be responsible
for its resistance, recurrence and
metastasis. CSCs can be identified
by the presence of a single or
combination of markers. CD133 is
one of the most important markers
for CSCs in neuroblastoma (NB)
and solid tumors. It is obvious that
epigenetic modifications such as
histone acetylation and promoter
methylation
regulate
CD133
transcription. Valproic acid (VPA), a
well known antiepileptic drug, is a
histone deacetylase (HDAC) inhibitor
that
demonstrates
antitumor
activities. Hence, we evaluated
the effect of VPA on expression of
CD133 in NB cell lines.
Materials/methods
Cell lines: neuroblastoma UKF-NB3,
IMR 32, SH-SY5Y and UKF-NB4.
Western blot: detection of CD133/1
and acetylated histone H3 & H4.
Flow Cytometry: detection of
CD133/2, cell cycle and cleaved
caspase 3.
Colony formation assay.
Bisulfite conversion.
drugs used: Valproic acid ,
Valpromide
(VPA
analogue)
and
5-aza-2’-deoxycytidine
(demethylating agent).
Results and conclusions
Flow cytometric and western blot
analysis showed increased CD133+
population to several folds as far
as VPA is included in the culture of
UKF-NB3 and SH-SY-5Y cells. The
increase of CD133 was accompanied
by increase of histones H3 and
H4 acetylation level. CD133+ cells
were mainly present in S and G2/M
phases of cell cycle and showed
less cleaved caspase-3 compared
to CD133- cells when treated with
VPA and cytostatics. VPA treated
UKF-NB3 cells have acquired higher
colony formation capacity, unlike
IMR-32 and UKF-NB4 which were
not expressing CD133 protein and
had no increase in colony formations
in VPA pretreated samples. CD133
has been reactivated in IMR-32 and
UKF-NB4 after using demethylating
agent
5-aza-2’-deoxycytidine.
A19
Reappearance of CD133 protein
was in parallel with demethylation of
CD133 promoter P1. In conclusion,
we proved that VPA can induce
CD133+ cells which show CSCs
features in some NB cell lines. In
addition, we confirm the epigenetic
regulation of CD133 expression in
NB.
This work was support by GACR,
grant No. 14-18344S.
Distinctive activity of ginger
phenylpropanoids against
lymphoblastic leukemia cells
úterý / 2. prosince
16.30- 16.45 hod.
2014
/
Ermin Schadich1, Marek
Handl2, Soňa Křupková1, Ivo
Ivo Frydrich1, Petr Džubák1,
Marián Hajdúch1
Faculty of Medicine and Dentistry,
Palacký
University
Olomouc,
Olomouc, Czech Republic,
2
Faculty of Science, Palacký
University, Olomouc, Czech
Republic
Introduction
Plant
phenylpropanoids
are
considered as useful source for
development of novel drugs due to
their anti-oxidant, anti-inflammatory
and chemopreventive properties.
Specifically,
previous
studies
showed that some of them might
also have the pro-oxidant effect
associated with distinct cytotoxic
activity only in the certain cancer
lines. The aim of our studies was
to assess the cytotoxic activity of
phenylpropanoids from rhizome of
ginger (Zingiber officinale) against
three different cancer cell lines,
hepatocellular carcinoma (HepG2)
cells, glioblastoma (U-87 MG) and
acute T lymphoblastic leukemia
(CCRF-CEM
(CRM-CCL-119))
cells, and fibroblasts (CRL-2522) ,
immortalized keratinocytes (HaCat)
and human embryonic kidney
(HEK293) cells.
Materials/methods
The
phenylpropanoids
from
methanol extract of ginger rhizome
were tested for components
1
A20
Abstract book
and cytotoxic activity against
all seven cell lines using liquid
chromatography (LC-MS) and MTT
assay respectively. Their property
to modulate the antioxidant and
anti-inflammatory pathway was
assessed using three different
luciferase reporter cell lines, HepG2
and HaCat cell line with antioxidant
response element reporter and
rat glioma (CCL-107) cell line with
NF-κB (nuclear factor kappa-lightchain-enhancer of activated B cells)
reporter.
Results and conclusions
The results LC-MS analysis showed
that methanol extract of ginger
rhizome contained both two major
phenylpropanoids, 6-gingerol and
6-shogaol and their derivatives.
These phenylpropanoids increased
the signaling of all of the three
reporter cells lines showing their
antioxidant and anti-inflammatory
property. Significantly, they had only
the cytotoxic activity against CCRFCEM cells. Ginger phenylpropanoids
of ginger rhizome have the
distinct cytotoxic activity against
CCRF-CEM cells indicating their
potential for development of novel
chemotherapeutic drugs against
lymphoblastic leukemia.
Tumor microenvironment:
mesenchymal stromal cell
contribution
úterý / 2. prosince
16.45- 17.00 hod.
2014
/
Lucia Kucerova
Molecular Oncology Department,
Cancer Research Institute SAS,
Bratislava, Slovakia
Introduction
Tumor
microenvironment
substantially
affects
tumor
progression and tumor cell behavior.
The extrinsic factors and signals
coming from the surrounding
stromal tissue alter numerous
processes in the tumor cells. It
represents a complex structure
composed of various non-malignant
cell types and secreted molecules
including extracellular matrix, growth
factors, chemokines and cytokines.
Mesenchymal stromal cells (MSCs)
significantly contribute to tumor
microenvironment and a complex
cross talk between heterogeneous
malignant cells and the MSC
together with the extracellular
matrix determines also the tumor
therapeutic responses.
Materials/methods
MSCs represent a population of
cells with multilineage differentiation
capability, which secrete wide array
of chemokines and growth factors.
The MSCs are defined by their ability
to differentiate into osteoblasts,
adipocytes
and
chondrocytes,
but they can also differentiate
into other cell types; pericytes;
cancer stem cell niche cells; and
tumor associated fibroblasts in
the tumor microenvironment.We
have evaluated the interactions by
live-cell kinetic imaging, viability
and apoptotic assays, expression
analysis and functional migration
assays.
Results and conclusions
The MSCs can function in promoting
or inhibiting tumor initiation and
progression. The MSCs may directly
promote tumor cell proliferation
and inhibit cell death. The MSCs
can exert their effect on tumor also
indirectly by promoting angiogenesis
or affecting some immune functions.
Tumor
metastasis
might
be
supported at multiple steps (from
initial dissemination to formation
of novel niches in distal tissues) by
the MSCs. They can also regulate
cancer stem cell self-renewal
and differentiation. The detailed
understanding of the interactions
in tumor microenvironment could
unravel novel intervention points
for anticancer therapies. Our
recent data will demonstrate data
multilevel nature of the interaction
between MSC and breast cancer
cells with the focus on the biological
relevance. The MSCs represent a
valuable cell source for regenerative
therapies therefore it is of critical
importance to examine the safety
of MSCs clinical application in the
patients with cancer predisposition
or history.
Abstract book
Poškození a reparace DNA
DNA damage and repair
Předsedajíci / Chairs: Martin Mistrík, Kateřina Bouchalová
středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 8.00 - 9.00 hod.
Function of cyclin-dependent
kinase 12 in DNA repair
pathway
středa / 3. prosince
8.00 - 8.15 hod.
2014
/
Hana Paculová1, Marta
Dzimková1, David Vrábel1,
Matija Peterlin2, Dalibor
Blažek3, Jiří Kohoutek1
Veterinary Research Institute, Brno,
Czech Republic,
2
School of Medicine, University
of California San Francisco, San
Francisco, USA,
3
Central European Institute of
Technology, Masaryk University,
Brno, Czech Republic
Introduction
In many aspects, genome stability is
essential for protection from various
diseases, including cancer. The DNAdamage-response (DDR) pathway
is a cellular mechanism which has
evolved to protect cellular integrity by
detection and repair of DNA lesions.
The DDR is orchestrated by diverse
type of proteins and among them,
transcriptional
cyclin-dependent
kinase (Cdk) complexes, and
phosphorylation of the C-terminal
domain of RNA polymerase II were
identified to play a key role.
Materials/methods
We have demonstrated that the cyclindependent kinase 12 (CDK12), and
its binding partner cyclin K, maintains
genome stability via regulation of
expression of DDR genes. Depletion
of the CycK/CDK12 complex from
cells led to aberrant expression
of several critical regulators of
genome
stability,
specifically,
BRCA1, RAD51, ATR, FANCI and
FANCD2
proteins.
Importantly,
down regulation of the CycK/CDK12
complex caused induction of the
53BP1 and γH2AX foci, markers of
spontaneous DNA damage signaling
followed by accumulation of cells in
1
Copenhagen, Denmark
Introduction
Many
fluorescence
microscopy
techniques
enable
powerful
combination of single cell analysis
and high-content (HC) and highthroughput (HT) screening. With the
use of reporter cell lines expressing
fluorescently tagged proteins, live
cell analysis can be implemented
to reduce costs and obtain even
more valuable data such as various
dynamic studies. Here we focused
on laser scanning microscopy (LSM)
which usage for HC/HT screening is
rather limited due to low speed of
image acquisition. On the other hand,
LSM mode offers, apart from simple
visualization, several approaches
for photo-manipulation techniques,
which are only poorly exploited in HC/
HT protocols. We optimized dynamic
readouts of protein translocation to
sites of laser-induced DNA lesions
and fluorescence recovery after
photo-bleaching (FRAP). This novel
approaches could be implemented
into automated HC/HT mode for
screening of compounds and/or
factors interfering with important
cellular processes involved in cancer
New approach to quantitative
biology.
image analysis of laser induced Materials/methods
DNA damage in live cells
Two reporter cell lines with
středa / 3. prosince 2014 /
fluorescently
tagged
proteins,
8.15 - 8.30 hod.
U2OS-MDC1-GFP
and
U2OS1
1
53BP1-GFP
were
used
to
address
Eva Veselá , Martin Mistrík ,
two
different
DNA-damage
Tomáš Fürst1,2, Hana
repair
pathways,
homologous
Hanzlíková3, Ivo Frydrych1,
recombination and non-homologous
Jirí Bártek1,4
end-joining.
Cell
visualization
1
and DNA damage induction was
Institute of Molecular and
performed on AxioObserver Z.1
Translational Medicine, Olomouc,
inverted microscope combined with
Czech Republic,
2
LSM780 confocal module (Zeiss).
Department of Mathematical
Quantitative image analysis was
Analysis and Applications of
done by specialized software which
Mathematics, Olomouc, Czech
was developed and optimized as a
Republic,
3
part of this study.
Institute of Molecular Genetics,
ASCR, Prag, Czech Republic,
4
Danish Cancer Society,
the G2-M phase of the cell cycle.
Finally, the loss of the CycK/CDk12
complex rendered cell sensitivity
to various DNA damaging agents,
including camptothecin, etoposide
and mitomycin. Of note, mutations in
Cdk12 have been identified in several
malignancies, among them ovarian
cancer with defective homologous
recombination in half of the tumors
was characterized with recurrent
mutations in kinase domain of CDK12
gene. Importantly, detail analyses
of mRNA expression in tumors with
mutated CDK12 revealed aberrant
expression of several DDR genes
and impaired DNA damage repair.
Results and conclusions
In summary, we believe that
understanding the physiological role
of CDK12 will help us in the future to
dissect precise function of CDK12
in the pathophysiology of the tumor.
We propose that CDK12 is a new
tumor suppressor.
The project is supported by the
grant from the Ministry of Agriculture
MZE002716202 and the Internal
Grant Agency of the Ministry of
Health, NT14599-3/2013.
A21
Rakovina věc veřejná
N A D A C E
P R O
V Ý Z K U M
R A K O V I N Y
Počet nově diagnostikovaných nádorů roste v České republice každých deset let o
čtvrtinu. I proto byla Proto byla v roce l997 založena nadace pro výzkum rakoviny
Olomouc, jejímž cílem je podpora výzkumu a vývoje v oblasti nádorových onemocnění, v
oblasti primární a sekundární prevence zhoubných novotvarů a jiných závažných
civilizačních nemocí.
Nadace Rakovina věc veřejná založila v roce 2012
stejnojmennou
obecně
prospěšnou
společnost,
pomocí níž chce zvýšit povědomí o problematice
infekce HPV virem (Human Papillomavirus) mezi
ženami. Hodlá je také motivovat k molekulárnímu
vyšetření HPV infekce, a to zejména infekce
nejrizikovějšími genotypy tohoto viru, takzvanými
hrHPV (high-risk HPV). V případě pozitivního výsledku
na hrHPV infekci klientky vyhledají svého gynekologa,
který na základě cytologických nebo histologických
vyšetření doporučí další kroky.
- V rámci projektu „Pošli příběh“ se uskutečnilo divadelní představení s Vandou
Hybnerovou ve studiu Alta nebo veřejné čtení veršů Tomáše Tajchnera, které četl Saša
Rašilov
- Výstava fotografií Pavly Hodkové za podpory Jana Saudka
- Dražba darů sportovců, účastníků Olympijských her
- Každoroční nadační kalendáře, v letošním roce se fotilo s dětmi, které prodělaly
náročnou onkologickou léčbu
- Tradiční benefice, spojené s módní přehlídkou v Praze a Olomouci
Děkujeme všem partnerům, známým osobnostem i široké veřejnosti, kteří se v roce 2014
2013
zapojili do benefičních projektů nadace Rakovina věc veřejná.
Přejeme hodně úspěchů v roce 2015
2014.
Renata Hilšer Grmolenská
ředitelka nadace
A24
Abstract book
Results and conclusions
The presented method is based
on atypical microscope settings
and specialized image analysis
software. It is possible to follow
DNA damage recognition and repair
in live cells in several time-points
after localized UV DNA damage.
Compared to commonly used handbased techniques which usually
analyze about dozen of cells, this
approach enables to evaluate tens
to hundreds cells per sample in
an automatic mode. The method
was tested on two different LSM
systems and several experimental
settings (various concentrations of
pre-sensitizer, compounds directly
interfering with observed proteins).
It may become a useful tool for
characterization of chemicals and
factors interfering with DNA damage
response, which is a general principle
of multiple existing or tested anticancer treatment strategies.
The work was supported by Ministry
of the interior of the Czech Republic,
grant nr. VG20102014001 and
GACR, grant no. P305/11/P683.
BRCA-mutation status
combined with BCL2 protein
in prediction of relapse
in triple-negative breast
cancer (TNBC) treated with
adjuvant anthracycline-based
chemotherapy.
středa / 3. prosince
8.30 - 8.45 hod.
2014
/
K. Bouchalova1,5,
M. Svoboda2, G. Kharaishvil3,
J. Vrbkova1, L. Radova1, J.
Bouchal3, R. Trojanec1, V.
Koudelakova1, K. Cwiertka4,
M. Hajduch1, Z. Kolar3
Laboratory of Experimental
Medicine, Institute of Molecular
and Translational Medicine,
Faculty of Medicine and Dentistry,
Olomouc, Czech Republic,
2
Masaryk Memorial Cancer Institute,
Brno, Czech Republic,
3
Laboratory of Molecular Pathology,
Faculty of Medicine and Dentistry,
Palacky University, Olomouc,
1
Czech Republic,
Department of Oncology, Faculty
of Medicine and Dentistry and
Faculty Hospital, Olomouc, Czech
Republic,
5
Department of Pediatrics, Faculty
of Medicine and Dentistry and
Faculty Hospital, Olomouc, Czech
Republic
Introduction
TNBC is aggressive and the
prognosis is poor. Patients cannot
benefit from targeted treatment
based on hormonal or HER2
receptors. For this reason, there
is an urgent need for markers
that will predict the outcome of
chemotherapy (Bouchalova et al.
Current Drug Targets 2014). The
role of BRCA mutation status as
a predictor in TNBC is unclear.
Data show an association of high
BCL2 expression and resistance to
anthracyclines. BCL2, size and nodal
status are independent predictors
for both relaps and death in TNBC
treated with adjuvant anthracyclines
(Bouchalova et al. ASCO 2012).
The objective of this study was to
determine whether combination of
BCL2 and BRCA1 status predicts
outcome in TNBC patients treated
with adjuvant anthracycline-based
therapy.
Materials/methods
The study included 187 patients with
TNBC, 178 of whom were treated
with adjuvant chemotherapy (164
had anthracyclines). BCL2 analysis
was performed using IHC. BRCA1
status was obtained from patients
records: mutation (mut), wildtype
(wt) and unknown status were
present in 21.39, 19.25, and 59.36
%, respectively. The data were
analysed with software Statistica
and R.
Results and conclusions
Among six BCL2/BRCA1 TNBC
subtypes,
BCL2high/BRCA1wt
predicts the worst, while BCL2low/
BRCA1mut the best RFS (logrank
p<0.05).
BCL2high
protein
expression predicts poor relapse
free survival (RFS) in
BRCA1wt TNBC patients treated
4
with adjuvant anthracycline-based
regimens (logrank p=0.007, hazard
ratio, HR 13.24, 95%CI 1.19147.93). Interestingly, there was no
significant difference in RFS between
BCL2low/BRCA1mut and BCL2high/
BRCA1mut, but between BCL2low/
BRCA1mut and BCL2high/BRCA1wt
(logrank p=0.009) TNBC patients
treated with adjuvant anthracyclinebased therapy. BCL2high/BRCA1wt
predicts trend to the worst overall
survival (OS) analyzed together with
other subtypes treated with adjuvant
anthracycline-based
regimens
(logrank p=0.081).
Conclusions: Dividing TNBC into
subtypes according BCL2 protein
expression and BRCA1 mutation
status predicts good, vs. poor
outcome in patients treated with
adjuvant
anthracycline-based
chemotherapy. BCL2 expression
together
with
BRCA1
status
could facilitate decision making
on adjuvant therapy. Underlying
mechanisms could be revealed by
further research. In patients with
BCL2high/BRCA1wt other types
of adjuvant therapy should be
considered.
The study was presented at ASCO
Annual Meeting 2014 [Bouchalova
et al., J Clin Oncol 32, 2014 (suppl;
abstr 1132)].
Grants: IGA NT14599-32013 and
IGA NS10286-3. Infrastructural part
of this project (Institute of Molecular
and Translational Medicine) was
supported from the Operational
Programme
Research
and
Development for Innovations (project
CZ.1.05/2.1.00/01.0030), National
Sustainability Programme (LO1304),
and Czech Technology Agency
(Center of competence for molecular
diagnostics
and
personalized
medicine,TE02000058).
2‘-deoxy-5-ethynyluridine
(EdU): a thymidylate synthase
inhibitor with DNA damaging
activity
středa / 3. prosince
8.45 - 9.00 hod.
2014
Anna Ligasová1, Dmytro
Strunin2, David Friedecký1,
/
Abstract book
Tomáš Adam1, Karel
Koberna1
Institute of Molecular and
Translational Medicine, Faculty
of Medicine, Palacký University,
Olomouc, Czech Republic,
2
Institute of Organic Chemistry
and Biochemistry ASCR, Prague,
Czech Republic
Introduction
The use of 2‘-deoxy-5-ethynyluridine
(EdU) as an anti-viral substance
was already studied in the nineteen
seventies [1,2]. Although this
analogue of 2‘-deoxyuridine evinced
an anti-HSV-1 and HSV-2 effect and
also an impact against the vaccinia
virus, the effective concentration also
inhibited the growth and metabolism
of non-infectious cells [1]. Similar
results were also obtained in 2007
in the case of cytomegalovirus [3].
EdU was also successfully tested
as a possible inhibitor of the cell
growth of human breast cancer cells
[4]. The mechanism of the inhibition,
however,
remained
unknown,
although some of the data indicated
that EdU can act as an inhibitor
of thymidylate synthase [5]. The
interest in EdU was greatly revived in
2008 when this nucleoside analogue
was used as a marker of cellular
replicational activity [6]. Due to its
simple and fast visualization, EdU
immediately became very strong
competitor of the most frequently
used marker to date nucleoside 5-bromo-2‘-deoxyuridine (BrdU). In
contrast to BrdU detection based
on the use of specific antibodies, the
reaction between the azido group
of the tag molecule and the ethynyl
group of EdU is employed in EdU
detection [6]. Due to the renewed
interest in EdU and the high number
of cell lines used in various studies,
new findings about the impact of EdU
on cell metabolism were obtained
indicating that EdU incorporation
can lead to DNA breaks followed
by cell death, suppression of in
vitro population expansion and in
vivo tumour progression [7]. On the
bases of immunolocalisation studies
of the proteins γH2AX and p53BP1
it was suggested that EdU induces
1
double-stranded DNA breaks as
well [8]. Although it is evident that
EdU toxicity is highly dependent
on the cell line used [3,4,8,9], the
reason for the different effect of
EdU in various cell lines remained
unknown. In the study presented,
we have focused on the possibility
that the different cytotoxic effect of
EdU could be related to the different
rate of EdU incorporation in DNA.
We also studied (i) the changes in
the rate of DNA replication and cell
cycle progression, (ii) the possibility
that EdU can generate interstrand
crosslinks and (iii) the role of the
metabolism of 2‘-deoxythymidine
(dT) in EdU-mediated toxicity.
Materials/methods
Cells lines used
HeLa cells, 143B PML BK TK and
HCT116 cells.
MTT assay
The MTT assay was performed
according to [10].
Microscopic analysis
The incorporated EdU was visualised
by means of a click reaction, the
nuclear DNA was stained by DAPI.
Nucleotide pools analysis by HPLCMS
The nucleotide pools were performed
according to [11-13].
Doubling time and the relative length
of S phase
The doubling were calculated using
wide-field microscopy. The relative
length of the S phase was determined
on the basis of the determination of
the fraction of EdU-labelled cells
after 1-hour labelling with EdU as
compared to the overall number of
the cells.
Analysis of the cell cycle and BrdU
detection
The analysis of the cell cycle was
performed using DAPI labelling
followed by image cytometry. In
the case of the analysis of the DNA
replication, the cells were labelled
with BrdU followed by its revelation
using copper(I) ions [14] and indirect
immunofluorescence.
Reverse comet assay
The comet assays were performed
according
to
[15]
with
the
modification of Dr. Dušínska
(http://www.cometassayindia.org/
Dusinska-protocol.PDF).
Data analysis
The data of particular experiments
were analysed using following
software: CellProfiller, Microsoft
Excel, GraphPad Prism 6 and
CometScore software.
Results and conclusions
a) EdU toxicity positively correlates
with the efficiency of its incorporation
and this efficiency is different in
different cell lines.
b)
The
incorporation
of
EdU
is
dependent
on
the
intracellular
concentrations
of dT and 2‘-deoxythymidine
5‘-monophosphate (dTMP).
c) EdU incorporation in DNA leads to
the deceleration and deformation of
the cell cycle including the slowdown
of the S phase accompanied by
a decrease in the DNA synthetic
activity.
d) Although the in vivo inhibitory
effect of EdU on the activity of
thymidylate synthase is substantially
lower when compared to 5-fluoro2‘-deoxyuridine (FdU), this effect
contributes to the high toxicity
of EdU especially at higher EdU
concentrations. It results in a
lowering of the dTMP, dTDP and
dTTP pools and subsequently in the
higher incorporation of EdU in DNA.
e)
EdU
induces
interstrand
crosslinks.
f) The use of non-toxic concentrations
of EdU (less than 1% cells die using
a standard cytotoxicity test) for
labelling replicated DNA results in
a substantial decrease of the signal
when compared to the maximal
signal or does not allow any labelling
at all. The non-toxic concentration is
lower than 0.501 µM, 0.044 µM and
0.47 µM in HeLa, 143B PML BK and
HCT116 cells, respectively.
References
1. De Clercq E, et al (1979) PNAS 76:
2947-2951.
2. De Clercq E, et al. (1980) J Infect
A25
A26
Abstract book
Dis 141: 563-574.
3. Cristofoli WA, et al. (2007) J Med
Chem 50: 2851-2857.
4. Meneni S, et al. (2007) Bioorg Med
Chem 15: 3082-3088.
5. De Clercq E, et al. (1978) Mol
Pharmacol 14: 422-430.
6. Salic A, Mitchison TJ (2008) PNAS
105: 2415-2420.
7. Ross HH, et al. (2011) J Neurooncol
105: 485-498.
8. Zhao H, et al. (2013) Cytometry A
83: 979-988.
9. Diermeier-Daucher S, et al. (2009)
Cytometry A 75: 535-546.
10. Freshney RI (2005) In: Freshney
RI, editor. 5th. ed. New Jersey: John
Wiley and Sons, Inc. pp. 365-369.
11. Bennett BD, et al. (2008) Nat
Protoc 3: 1299-1311.
12. Ivanisevic J, et al. (2013) Anal
Chem 85: 6876-6884.
13. Yuan M, et al. (2012) Nat Protoc
7: 872-881.
14. Ligasová A, et al. (2012) PLoS
One 7: e52584.
15. Singh NP, et al. (1988) Exp Cell
Res 175: 184-191.
16. This work was supported by the
Technology Agency of the Czech
Republic [TA03010598, TA03010719
and TE02000058] and the Ministry
of Education, Youth and Sports
[LO1304].
Abstract book
Biomarkery nádorových onemocnění II
Cancer biomarkers II
Předsedajíci / Chairs: Jitka Berkovcová, Pavel Dundr
středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 9.30 - 11.30 hod.
Možnosti predikce léčebné
odpovědi na cílenou antiEGFR a anti-VEGF terapii
metastatického kolorektálního
karcinomu (mCRC)
středa / 3. prosince
9.30 - 9.45 hod.
2014
/
Radim Nemecek1,2
Masarykuv onkologicky ustav,
Brno, Czech Republic,
2
Lekarska fakulta MU, Brno, Czech
Republic
Introduction
Systémová léčba mCRC je založena
na použití chemoterapie v kombinaci
s monoklonálními protilátkami proti
VEGF a EGFR. Na tuto léčbu však
pozitivně odpoví jen část pacientů,
je proto vyvíjena intenzivní snaha
o nalezení faktorů predikujících
odpověď či rezistenci ještě před
zahájením
onkologické
terapie.
Zatímco v případě dráhy pro VEGF
predikce dosud selhává, v případě
receptoru EGFR je prediktorů známo
hned několik a některé z nich se již
používají v běžné klinické praxi.
Materials/methods
Mezi „pozitivní prediktory“ odpovědi
na anti-EGFR terapii se řadí zvýšený
počet kopií genu pro EGFR a hladina
exprese mRNA dvou hlavních ligandů
EGFR - epiregulinu a amphiregulinu.
Z tzv. „klinických“ prediktorů je
diskutována
role
akneiformního
exantému,
hypomagnesémie
a
tzv. „časné regrese nádoru“, resp.
hloubky nádorové odpovědi. V
klinické praxi se tyto prediktory
dosud nepoužívají.
„Negativních prediktorů“ je známo
více. Vychází z jednotlivých článků
signálních kaskád vedoucích od
EGFR k jádru buňky (RAS-RAFMAP2K-ERK a PI3K-AKT-mTOR).
Mutace v onkogenu RAS (mRAS)
je nejrozšířenější a jako jediná se
používá v rutinní klinické praxi.
Nachází se u cca 50% pac. s CRC
1
a je zodpovědná za trvalou aktivaci
kinázy RAS vysílající stimulační
signály k jádru bez ohledu na stav
EGFR. Nicméně i po vyloučení
této mutace (a tedy potvrzení
nemutovaného genu RAS - wt RAS)
odpoví na anti-EGFR terapii jen cca
polovina pacientů. Hledají se tedy
další prediktory s cílem léčbu ještě
více „personalizovat“. Mutace v
onkogenu BRAF (V600E) se nachází u
cca 5-15% pac. s CRC. Nevyskytuje
se nikdy současně s mRAS. Kromě
evidentně negativně prognostického
vlivu byl prokázán i její vliv negativně
prediktivní, obzvláště u předléčených
pacientů.
Mutace onkogenu PIK3CA se
vyskytuje u 15-18% pacientů s
mCRC. Byly identifikovány 2 varianty
- mutace v exonu 9 (60-65% pac.) a
v exonu 20 (20-25% pac.). Negativně
prediktivní vliv byl prokázán pouze u
druhé z nich. Dráha PI3K-AKT-mTOR
je inhibována kinázou PTEN, jejíž
mutace vedou k nedostatečnému
efektu anti-EGFR terapie u mCRC.
Exprese PTEN souvisí s mírou
mikrosatelitové nestability a je
rozdílná v primárním nádoru a
metastázách. Existují rovněž data
o prediktivním vlivu statutu HER2 a
mutace p53.
Results and conclusions
Personalizovaná onkologická léčba
založená na genovém profilování
nádorů je považována za léčebnou
strategii budoucnosti. Se současnou
úrovní znalostí však stojíme na
samém začátku této nesmírně
zajímavé kapitoly.
Jana Volejníková1,2, Jana
Vrbková2, Marián Hajdúch2,
Vladimír Mihál1,2
Department of Pediatrics, Palacky
University and University Hospital,
Olomouc, Czech Republic,
2
Institute of Molecular and
Translational Medicine, Olomouc,
Czech Republic
Introduction
Great progress has been made
in the diagnostics and treatment
of childhood acute lymphoblastic
leukemia (ALL) over the past decades.
The vast majority of children are
cured, but there is still room for
improvement, especially in specific
patient subgroups. In this study, we
retrospectively evaluated treatment
and prognosis of children with
ALL enrolled in three consecutive
treatment protocols ALL-BFM 90,
95 and ALL IC-BFM 2002 between
years 1990 and 2007.
Materials/methods
We compared characteristics and
survival data of 97 patients treated for
ALL at University Hospital Olomouc
with records of 924 children from
7 remaining centers in the Czech
Republic.
Results and conclusions
Within the entire Czech Republic,
successive improvement in both
event-free (EFS) and overall survival
(OS) among protocols were observed
(RFS: 81.1±2.2%, 78.6±2.2% and
88.2±1.9%, p=0.01; EFS: 70.5±2.4%,
70.4±2.7%
and
84.5±2.1%,
p=8.4e-05;
OS:
76.3±2.3%,
Management and prognosis of 79.0±2.5% and 91.3±1.7%, p=6echildhood acute lymphoblastic 06, respectively). There was also a
trend to better survival in a single
leukemia on protocols ALLcenter on ALL-BFM 95 trial than in
BFM 90, 95 and ALL IC-BFM
the rest of the Czech Republic (EFS:
2002: a retrospective single83.3±6.2% vs. 70.4±2.7%, p=0.11;
center study
OS: 91.7±4.6% vs. 79.0±2.5%,
středa / 3. prosince 2014 / p=0.07). This fact might be explained
9.45 - 10.00 hod.
by a significantly lower proportion of
1
A27
A28
Abstract book
boys and an inclusion of only one high
risk patient in our center. However,
multiple minor changes among
protocols (regarding diagnostics,
patient enrollment and risk group
stratification, cranial irradiation and
course of chemotherapy) might
have influenced the mentioned
results and will be discussed in
detail. Cytogenetic examination in
our center showed an exceptionally
high yield rate, but its result was not
decisive for the risk stratification.
In conclusion, we demonstrate a
gradual progress and a high quality
of ALL diagnostics and treatment
in the Czech Republic in general,
which is possible to maintain in
a single center setting. The given
outcomes surpassed international
results of the ALL IC-BFM 2002
trial (5-year EFS 74%, OS 82%)
and were comparable with worldś
leading childhood leukemia trials
- e.g. a 7-year EFS 80.4% and OS
91.8% for AIEOP-BFM ALL 2000;
5-year OS 90.4% for the Childrenś
Oncology Group.
Due to advances in molecular
diagnostics, excellent collaboration
on both national and international
level and high overall standards of
care, Czech Republic has survival
rates comparable with leading
international study groups for
childhood ALL. The future of ALL
treatment lies in its individualization,
including pharmacogenetics. One
of the most important predictors
of outcome and risc stratification
criteria is the level of minimal residual
disease (MRD), which quantification
is highly specialized, laborious and
expensive. Due to our collaborative
effort, the Czech Republic has
since 2010 joined the MRD-based
international „state of the art“
AIEOP-BFM ALL 2009 protocol for
childhood ALL.
Supported by: NPU LO1304
Využití metody sekvenování
nové generace
v intratumorové heterogenitě
kolorektálního karcinomu
středa / 3. prosince
10.00 - 10.15 hod.
2014
/
Jitka Berkovcová1, Lenka
Radová2, Šárka Rathouzská1,
Pavel Fabian1
Masarykuv onkologický ústav,
odd. onkologické patologie, Brno,
Czech Republic,
2
CEITEC, Výzkumná skupina
Lékarská genetika, Brno, Czech
Republic
Introduction
Nádorová heterogenita je dobře
známý jev (Kinjn et al. 2011),
stejně jako teorie multiklonáního
modelu progrese nádorů v čase
(Marusyk et al. 2010). Rozdíl ve
stanovení mutačního stavu genu
KRAS bývá popisován asi ve 4 %
případů kolorektálních karcinomů.
Metody sekvenování nové generace
(NGS) díky svojí vysoké senzitivitě
umožňují zachytit minoritní molekuly
DNA v analyzovaném vzorku. Je
to tedy vhodný nástroj ke studiu
heterogenity nádorové populace.
Cíle: Přesná mutační analýza genů
s klinickým významem u pacientů
s
kolorektálním
karcinomem.
Porovnání vzdáleně oddálených
odběrů rozsáhlých tumorů, případně
morfologicky odlišných klonů u
jednoho pacienta.
Materials/methods
Do
souboru
bylo
zařazeno
22
pacientů
s
operovaným
kolorektálním karcinomem v roce
2014. Jako hlavní kriterium byla
zvolena velikost nádoru minimálně
40mm. Celkem byla izolována
DNA z 51 vzorků s vysokým
zastoupením nádorové populace
(40-70 %). Příprava DNA knihovny
byla provedena pomocí TruSight
Tumor kitu (illumina). Tento kit je
designován tak, že obsahuje exony
26 vybraných genů s „Hot Spots“
oblastmi (celkem 175 amplikonů).
Základní analýza sekvencí byla
provedena
pomocí
softwarů
illumina dostupných v BaseSpace,
detekce variant byla provedena
pomocí softwaru VariantStudio. K
následným datovým úpravám byly
použity balíky R/Bioconductor.
Results and conclusions
Naše izlovaná DNA z tkáňových
1
bloků dosahuje velice dobré kvality
a ze všech vzorků bylo možné
připravit kvalitní DNA knihovny. Q
Skore 30 dosahujeme u více jak 96
% sekvencí. Průměrná coverage
dosahuje 4000 čtení. Sekvenace
a analýza dat zatím proběhla u
12 pacientů. U 7 pacientů jsme
detekovali mutaci v genu KRAS, u 1
mutaci v genu BRAF, u 7 pacientů
mutaci v genu TP53, u 2 pacientů
mutaci v genu APC, u 1 pacienta
mutaci v genu PIK3CA a u 1 pacienta
mutaci v genu FBXW7. U 1 pacienta
byly detekovány rozdílné genetické
klony v odlišných odběrech tumoru.
Vedle toho zaznamenáváme řadu
změn na úrovni subpopulací.
Očekává se, že NGS napomůže
zodpovědět řadu otázek souvisejících
s heterogenitou nádorových buněk.
Zároveň předpokládáme, že NGS
povede k upřesnění predikce cílené
protinádorové terapie.
Podpořeno MZ ČR - RVO (MOÚ,
00209805)
Introduction to RAS
pyrosequencing of FFPE
samples
středa / 3. prosince
10.15 - 10.30 hod.
2014
/
Jana Stranska, Gabriela
Gabcova, Rastislav
Slavkovsky, Veronika
Holinkova, Miroslava
Rabcanova, Jiri Drabek
Institute of Molecular and
Translational Medicine, Palacky
University, Olomouc, Czech
Republic
Introduction
KRAS and NRAS (RAS) are
components of the EGFR signaling
network. EGFR regulates cancer-cell
proliferation, apoptosis and tumorinduced angiogenesis. RAS links
growth-promoting signals from the
cell surface to the nucleus by acting
as a molecular switch. The switch
normally occurs transiently when
growth factor receptors (such as
EGFR) are activated. When specific
mutations in codons 12, 13, 59, 61,
117 and 146 of RAS genes occur,
Abstract book
the resulting KRAS/NRAS protein
is constitutively active. Anti-EGFR
monoclonal antibodies (cetuximab
and panitumumab) treat only RAS
wildtype patients with metastatic
colorectal cancer (mCRC). The high
number of tested mutations makes
probe-based qPCR genotyping
methods
problematic and while
sequencing techniques can identify
all mutations.
In this project, we test feasibility of
pyrosequencing for RAS genotyping
in mCRC.
Materials/methods
Primers for detection of mutations
of KRAS and NRAS exons 2, 3
and 4 were designed according to
the literature and PyroMark Assay
Design Software 2.0. Three different
PCR mixes (separate reagents
(Thermo Scientific), HotStarTaq
Plus Master Mix Kit (Qiagen),
PyroMark PCR Kit (Qiagen)) were
compared. Concentration of MgCl2
(1.5 - 3 mM) and PCR profile for all
mutation mixes analyzed in one run
were optimized. Volumes of 10 and
15 μl with or without purification
of amplicons (QIAquick PCR
Purification Kit, Qiagen) were tested.
Pyrosequencing was performed
with PyroMark® Q96 ID (Qiagen)
according to the machine manual.
Results and conclusions
All primer sets provided amplicons
of corresponding length. The best
amplification and pyrosequencing
results
were
obtained
with
HotStarTaq Plus Master Mix
Kit, 3 mM MgCl2, 15 μl, without
purification step. All read sequences
corresponded to their design.
Further experiments for sensitivity
evaluations will continue.
The work was supported by
grants
CZ.1.05/3.1.00/14.0307,
CZ.1.07/2.3.00/30.0060
and
CZ.1.07/2.3.00/30.0041.
MiR-215 as an important
tumor suppressor in colorectal
cancer patients
středa / 3. prosince
10.30 - 10.45 hod.
Petra VychytilovaFaltejskova1,2, Jitka
2014
/
Mlcochova2, Jana
Merhautova3, Lenka Radova2,
Marek Svoboda1,2, Rostislav
Vyzula1, Ondrej Slaby1,2
Masaryk Memorial Cancer Institute,
Brno, Czech Republic,
2
CEITEC, Masaryk University, Brno,
Czech Republic, 3Department
of Pharmacology, Faculty of
Medicine, Masaryk University,
Brno, Czech Republic
Introduction
Colorectal cancer (CRC) is one
of the most common types of
cancers worldwide with the high
incidence and mortality. Therefore,
several efforts have been made to
find new diagnostic, prognostic
and predictive biomarkers, but
also therapeutic targets. One of
the promising approaches is the
characterization of the solid tumors
using microRNAs (miRNAs). MiRNAs
are small, non-coding RNAs that
post-transcriptionally regulate gene
expression. They regulate many
biological processes such as cell
cycle, apoptosis, proliferation or
invazivity and they can also affect
anti-cancer
therapy
efficiency,
therefore, their deregulation leads to
the origin and progression of many
severe diseases including CRC.
However, only little is known about
miRNAs‘ target molecules and
signaling pathways in which they are
involved.
Materials/methods
We have analyzed expression
profiles of 667 miRNAs in 8 patients
diagnosed for CRC and 8 paired
adjacent non-tumoral tissues using
TaqMan Low Density miRNA arrays.
We have found miR-215 to be one
of the most deregulated miRNAs,
therefore
its
expression
was
further validated on independent
cohort of 250 paired samples and
correlated with clinicopathological
features of the patients. Afterwards,
we investigated its involvement
in CRC pathogenesis. We have
overexpressed miR-215 in CRC
stable cell lines (DLD-1, HCT 116,
HT-29, SW620) and analyzed the
effect on the viability (MTT test),
proliferation (cell counting), cell cycle
1
A29
and apoptosis (flow cytometry),
migration of the cells (scratch assay,
transwell migration assay) and the
ability to found new colonies (colony
forming
assay).
Subsequently,
several targets of miR-215 have
been identified using quantitative
real time PCR, and effects of this
miRNA have been also analyzed in
vivo on mouse model.
Results and conclusions
We have observed significantly
decreased levels of miR-215 in
primary CRC tissues and also in
metastatic tissue. Moreover, we
have found correlation between low
expression of this miRNA, clinical
stage, lymph node metastasis and
disease free survival of the patients.
In vitro analyses proved that higher
levels of miR-215 lead to the arrest of
cell cycle, increased apoptosis and
reduced migration and proliferation
of the cells. Using qRT-PCR we
have identified several potential
targets of miR-215 including XIAP,
CD164 or HOXB9. The results of in
vivo analyses will be the part of the
message.
The reduced expression of miR-215
has been observed in several tumor
diseases, therefore we presume that
this miRNA functions as an important
tumor suppressor. The results of our
study also indicated that miR-215
could serve as a new diagnostic
and prognostic biomarker and also
potential therapeutic target for the
treatment of CRC patients.
This work has been supported by
IGA MZČR NT13549-4/2012 and
NT13860-4/2012.
Overexpression of Filamin-A
protein is associated with
aggressive clinicopathological
features and poor survival
outcome in NSCLC patients
treated with platinum-based
combination chemotherapy
středa / 3. prosince
10.45 - 11.00 hod.
2014
Mariam Gachechiladze1,2,
Josef Škarda1,2, Maria
Janikova1,2, Gvantsa
Kharaishvili1,2, Vítězslav
/
A30
Abstract book
Kolek3, Jiří Klein4, Alžběta
Poprachová5
Department of Clinical and
Molecular Pathology, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
2
Institute of Molecular and
Tranlational Medicine, Olomouc,
Czech Republic,
3
Department of Tuberculosis and
Respiratory Diseases, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
4
Laboratory of Experimental
Medicine, Departments of
Pediatrics and Oncology, Faculty
of Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
5
Department of Normal Anatomy,
Faculty of Medicine and Dentistry,
Palacky University, Olomouc,
Czech Republic
Introduction
An actin-binding protein Filamin A
connects the actin filament network
to cell membrane receptors, and
acts as a scaffold for various
signaling pathways related to cancer
growth and progression. Recently,
it has been reported that Filamin A
is required for efficient regulation of
early stages of DNA repair process.
Moreover, some in vitro studies
showed that the overexpression of
Filamin A determines resistance to
various cytotoxic drugs, including
cisplatin. We aimed to analyse the
expression of Filamin A protein in
resected NSCLC specimens, to
investigate the association of the
level of Filamin A protein expression
and
other
clinicopathological
features, and possible relationship
between the expression of Filamin
A and survival outcome in NSCLC
patients, treated with platinumbased combination chemotherapy.
Materials/methods
We performed FLNA protein
immunohistochemistry on formalinfixed
and
parrafin-embedded
tissue sections from 134 NSCLC
patients, using EP2405Y antibody
against C-terminus of FLNA (LSBio).
1
Cytoplasmic, membranous and
nuclear staining were evaluated semiquantitatively, scored according to
Histoscore method, and correlated
with all available clinicopathological
factors. Patients were divided in two
groups for survival analysis (I group
- patients with adjuvant platinum
based chemotherapy, II group patients with surgical treatment
only).
Results and conclusions
We found significant positive
correlation
between
Filamin-A
protein expression and NSCLC
stage (r=.249; p<0,05), lymph
node metastases (N) (r=0.205;
p<0,05) and distant metastases (M)
(r=0.332; p<0.01). The increased
expression of Filamin A protein
was also positively associated with
primary tumor size (T), grade (G),
and with lymphovascular invasion
(LVI). Multivariate Cox proportional
hazards regression analysis showed
that Filamin A overexpression
>90, represents a risk factor for
disease relapse together with
tumor stage, size and metastases
(HR=1.723, 95%CI [1.021:2.909],
p<0.05). Increased Filamin A protein
expression has been significantly
assosiated with poor survival
outcomes in patients with adjuvant
platinum-based chemotherapy: OS
(HR=1.005,
95%CI[1.000;1.010],
p=0.037), DFS (HR=1.004, 95%CI
[1.001:1.008], p=0,017). Our study
results suggest the important role
of Filamin A protein in NSCLC
porgression and it might represent
not only a prognostic marker, but
also one of the additional predictive
markers
for
platinum-based
treatment outcome in patients with
NSCLC.
GENETIC CHANGES
IN RECURRENT
GLIOBLASTOMA-IS THERE
ANY SIGNIFICANCE?
středa / 3. prosince
11.00 - 11.15 hod.
2014
/
Zuzana Crlikova1, Magdalena
Megova1, Jana Vrbkova1,
Radek Trojanec1, Lucie
Tuckova2, Jana Potockova1,
Sona Mlcochova1, Marian
Hajduch1
Laboratory of Experimental
Medicine, Institute of Molecular
and Translational Medicine,
Faculty of Medicine and Dentistry,
Palacky University, Olomouc,
Czech Republic,
2
Laboratory of Molecular Pathology,
Faculty of Medicine and Dentistry,
Palacky University, Olomouc,
Czech Republic
Introduction
Glioblastoma multiforme belongs
to the most aggressive tumors with
very short-time survival. Treatment
of this disease is very limited and
only temozolomide and radiation
is widely used in clinical practice.
New biomarkers could allow deeper
insight in gliomagenesis and also
can help to facilitate possible targets
in prognosis and prediction which
may lead to development of new
therapy regimes.
Materials/methods
In our study, status of genes and
chromosomes
(MDM2/chr.12,
EGFR1/chr.7, BCR/chr.22, P53/
chr.17, RB1/chr.13, C-MET/chr.7,
PTEN/10p, 19q/19p, 1p/1q, 9p/
chr.9) was investigated, using FISH
assay. We examined 25 patients
with recurrent glioblastoma. Control
group comprising of 111 patients
with glioblastoma was used for
comparison (both groups with
treatment regime 54Gy and 40 days
of temozolomide).
Results and conclusions
Statistical
analysis
revealed
significant
difference
in
age
(P=0.0002) and loss of p53
(P=0.093) between control group
and recurrent glioblastoma group.
Other significant relevances of this
study are summarized and clinical
relevance of our findings will be
associated with intended additional
study.
Support:
NT
13581;
TE02000058;
LF_2014_019;
CZ.1.05/2.1.00/01.0030.
1
Abstract book
Assay destinguishing
integrated and episomal form
of HPV16, 18, 31 and 58
středa / 3. prosince 2014 /
11.15 - 10.30 hod.
Hana Ondryasova, Vladimira
Koudelakova, Rastislav
Slavkovsky, Veronika
Venskova, Marian Hajduch
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic
Introduction
Cervical cancer is the third most
common malignancy in women.
Early detection screening is in Czech
Republic available and covered by
insurance since 2008. This screening
detect precancerous states caused
by aggressive subtypes of human
papillomavirus (mainly HPV16 and
18). The problem of the screening,
which standardly uses cytology, is
a high false negative rate, which is
reported in up to 20% of cases. Only
ambiguous cases with abnormal
cytology (ASC-US, LSIL) are sent to
test at level of DNA (HPV DNA). HPV
DNA diagnostics are much more
sensitive than standard cytology
which was demonstrated by large
studies. HPV DNA diagnostics
should be therefore a part of primary
screening However, commercially
available HPV DNA diagnostics
can also mis HPV positive cases
(paradoxically the serious ones).
Low viral load below the detection
limit of the method is the first reason.
The fragmentation of the gene
(which detection kit is based on)
occurs during the integration of the
virus into the human genome, may
be another reason. The paradox is
that just tumors with lower viral load
and related degradation of some
viral genome fragments are more
aggressive with worse prognosis
and response to treatment. For
proper diagnosis of cervical cancer
is therefore necessary to capture
also tumors in whom conventional
diagnostic kits fail. From this reason
we developed method for semiquantification and differentiation of
free-episomal from integrated HPV
form in cervical cells.
Materials/methods
Cervical smears have been tested
by Cobas 4800 system (Roche) for
presence of HPV16/18 and other
hrHPV genotypes (12 hrHPV). All
samples have been also analyzed
by PapilloCheck HPV- Screening
system (Greiner Bio-one) detecting
18 hrHPV and 6 lrHPV genotypes.
Samples positive for HPV16, 18,
31 and 56 have been analyzed for
presence of episomal, integrated
or mixed form of HPV infection
by multiplex real-time PCR assay.
GAPDH has been used as internal
control.
Results and conclusions
Our assay is able to distinguish
integrated and episomal form of
HPV16, 18, 31 and 58. Detection
limit of this assay is 4 copies of HPV
per reaction. We collect clinicopathological data from HPV positive
patients in these days. We have
analysed 116 HPV positive samples.
It is necessary to analyze large
set of HPV positive samples with
known cytology/histology results
to prove that this assay could help
to recognize patients in real risk of
cervical cancer.
(Supported
by:
CZ.1.05/3.1.00/14.0307
,
CZ.1.05/2.1.00/01.0030.)
A31
A32
Abstract book
Molekulární cíle a protinádorová léčiva II
Molecular targets and anticancer drugs II
Předsedajíci / Chairs: Petr Džubák, Jan Hraběta
středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 12.40 - 14.10 hod.
Use of the FUCCI probe
in HTS/HCA screening
campaigns.
středa / 3. prosince
12.40 - 12.55 hod.
2014
/
Petr Dzubak, Pawel Znojek,
Sona Gurska, Marian
Hajduch
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacký
University in Olomouc, Olomouc,
Czech Republic
Introduction
High content screening (HCS) is
a method that uses automatic
microscopy and image analysis
techniques to extract multiple
physiologically
relevant
measurements at cellular level.
Our goal was to use HCS to
develop robust, homogeneous and
inexpensive assay for cell cycle
phase detection as alternative to
FACS.
Materials/methods
In our endeavors we used HeLa
cells stably expressing FUCCI
(Fluorescent Ubiquitination-based
Cell-Cycle Indicator) probe. The
FUCCI probe was generated by
fusing red and green fluorescent
proteins with ubiquitination domains
of Cdt1 and Geminin respectively.
As a means of tracking cell cycle
progression FUCCI exploits cyclical
expression and degradation of the
ubiquitination oscillators Cdt1 and
Geminin to specifically mark cell
cycle phases in living cells. Cell
cycle perturbation can be evaluated
by measuring mean fluorescence
intensity of Cdt1-Red and GemininGreen in single cell and eventually
based on fluorescence intensity
range cells can be classified as
one of G1, G1/S, S/G2/M and M
cell cycle phase. The assay was
designed for full automatization of
cell plating, addition of compounds,
cytotoxicity measurement and high
content microscopy on our triple
robotic system. For setting up
and validation of the assay, dose
response curve was generated for
8 reference compounds with known
activity on cell cycle to choose
desired concentrations for which
there is evident block of cell cycle
progression in specific phase.
Results and conclusions
Chosen compounds were used to
assess assay reproducibility over
plates and experimental days by
evaluation of Z‘ calculated for each
phase of cell cycle. Overall we
obtained good assay reproducibility
and robustness with an overall Z‘
value of above 0.5 for G1, S/G2/M
and M phases. Finally, we tested
our approach against commercially
available LOPAC library which
includes 90 drug-like molecules
used as model drugs in the fields
of cell signaling and neuroscience.
The results of cytotoxicity assay
allowed to validate 26 compounds
as active based on dose response
curve fitting. High content analysis
of FUCCI probe showed that several
agents among these can be identified
as possible cell cycle modulators.
Results demonstrated here shows
that this newly developed assay is
simple to set up, robust, sensitive and
inexpensive. Project was supported
by the Ministry of Education of
the Czech Republic (LO1304 and
CZ.1.07/2.3.00/30.0041).
Validation of Cell Based
Assays for High-Throughput
Screening
Univesity, Olomouc, Czech
Republic
Introduction
High-Throughput Screening (HTS) is
an important step in the discovery of
new drugs. It allows the assaying of
a large number of potential biological
modulators against a chosen set
of defined targets. The chemical
compounds are tested in the primary
screen firstly. Only compounds
with positive results (HITs) are
investigated in the secondary screen
and the IC50 values are calculated.
This testing is performed by
biological and biochemical tests.
Materials/methods
For validation of our HTS, the LOPAC
Library (Library of Pharmacologically
Active Compounds) was used. This
library contains 90 compounds
with known biological activity. Their
cytotoxic effects were testing on the
group of 10 cell lines by MTT and
XTT methods. The IC50 value for
each compound was calculated.
Results and conclusions
To quantify the suitability of the
used cytotoxic assays in a HTS the
Z-factor was determined for each
plate. Obtained results and some
troubles and errors will be presented
and discussed.
Molecular mechanisms of
cell death induction by novel
taxanes effective in resistant
breast cancer cells
středa / 3. prosince
13.10 - 13.25 hod.
2014
/
Michael Jelínek, Kamila
Balušíková, Adéla Kábelová,
středa / 3. prosince 2014 / Jan Kovár
12.55 - 13.10 hod.
Sona Gurska, Pawel Znojek,
Petr Džubák, Marián Hajdúch
Institute of Molecular and
Translational Medicine, Palacky
Department of Biochemistry, Cell
and Molecular Biology, Division
of Cell and Molecular Biology,
Third Faculty of Medicine, Charles
University, Prague, Czech Republic
Abstract book
Introduction
The classical taxanes paclitaxel
(Taxol®) and docetaxel (Taxotere®)
are routinely used in therapy of
solid tumors. Unfortunately, cancer
cells often become resistant to
taxanes. Therefore, new taxanes
were prepared and tested for
using in the therapy of resistant
cancer cells. As for mechanisms of
cell death induction, taxanes are
known to block depolymerization of
tubulin and to induce programmed
cell death. However, the exact
mechanisms of cell death induction
are still not understood. To help to
elucidate this issue, we investigated
breast cancer cells after application
of classical and novel taxanes.
Materials/methods
We studied taxane-induced cell
death in two breast cancer cell
lines SK-BR-3 and MCF-7. We
used paclitaxel and novel taxanes
SB-T-1216 and SB-T-12854 (being
more efficient against resistant
cancer cells) at a death inducing
concentration. We also employed
western blot analysis, specific
siRNAs and cell fractionation
followed by western blot anylysis.
For testing of mitochondria functions
we made imunoflorescence and also
FACS analysis.
Results and conclusions
Programmed
cell
death
was
observed in both tested cell lines
after application of classical as well
as novel taxanes. Next we studied
the role of proteins of Bcl-2 family.
We confirmed the importance of
proteins Bad and Bim in cell death
induction described previously and
in addition we found that proteins Bik
and Bok also seem to function in cell
death induction. The translocation
of cytochrome C and decrease of
mitochondrial membrane potential
were observed only in SK-BR-3 cells
and not in MCF-7 cells, so the role of
mitochondria is questionable. On the
other hand, caspase -2, -8, -9, -7 and
-3 (caspase-3 only in SK-BR-3 cells)
were activated in SK-BR-3 as well
as MCF-7 cells 36 h after application
of taxanes. As previously reported,
caspase-2 seems to be the most
apical caspase in both cell lines.
Using of specific siRNA showed that
also caspase-3 and caspase-7 had
also important function in apoptotic
cascade induced by novel taxanes.
We found that classical and novel
taxanes induced cell death very
similarly in breast cancer cells.
Proteins from Bcl-2 family, (Bad,
Bim, Bik and Bok) and caspase-2,
-3 and -7 probably play the most
important role in cell death induction.
In summary, new taxanes could be
used in future anticancer therapy of
resistant breast cancer cells
Novel triterpenoid
fluoroderivatives with
anticancer activity
středa / 3. prosince
13.25 - 13.40 hod.
2014
/
Jiri Rehulka1, Milan Urban1,
Lucie Borkova2, Ivo
Frydrych1, Jan Sarek2, Petr
Dzubak1, Marian Hajduch1
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
University and University Hospital,
Olomouc, Czech Republic,
2
Department of Organic Chemistry,
Institute of Molecular and
Translational Medicine, Faculty
of Science, Palacky University,
Olomouc, Czech Republic
Introduction
Pentacyclic
triterpenes
are
secondary
metabolites
usually
found in plants that have a variety
of biological activities. Some of
their semisynthetic derivatives are
extensively studied for anti-cancer,
anti-HIV, and various protective
effects. The aim of this study was
to evaluate the newly synthesised
betulinic acid fluoroderivatives,
which were designed to enhance
cytotoxic effect towards cancer cell
lines.
Materials/methods
MTT assay, Flow cytometric
analyses
Results and conclusions
We prepared a set of fluorinated
derivatives of betulinic acid and
1
A33
allobetulin in order to increase the
cytotoxic activity of the parent
compounds. Previously unknown
2,2-difluorodihydrobetulinic acid and
its esters had significant cytotoxicity
on several cancer cell lines with
somewhat insufficient selectivity in
comparisson with normal human
fibroblasts. Derivatives of allobetulin
were inactive. In the context of
other
triterpenoid
derivatives
prepared earlier, we may conclude
that the introduction of an electron
withdrawing substituent to the
position 2 of betulinic acid enhanced
its potency. In the case of fluorinated
compounds, however, more studies
need to be done in order to lower the
toxicity on non-cancer cells.
The biological part of this work
(MTT tests, cell cycle analysis
etc.) was supported by grant from
the Ministry of Education, Youth
and Sports of the Czech Republic
(No.
CZ.1.07/2.3.00/30.0041)
and internal grant of Palacky
University
IGA_LF_2014_010.
The infrastructural part (Institute
of Molecular and Translational
Medicine) was supported from the
Operational Program Research
and Development for Innovations
(project
CZ.1.05/2.1.00/01.0030)
and NPUII project LO1304. The
chemical part was supported by
Technology Agency of the Czech
Republic (TE01020028) and by
the Ministry of Education, Youth
and Sport of the Czech Republic
and by the European Social Fund
(CZ.1.07/2.3.00/30.0060).
Proteomic profiling of
oxaliplatin treated cell
line revealed nucleoli
and ribosomal protein
downregulation
středa / 3. prosince
13.40 - 13.55 hod.
2014
/
Tomáš Oždian, Dušan
Holub, Gabriela Rylová, Petr
Džubák, Marián Hajdúch
Laboratory of Experimental
Medicine, Institute of Molecular
and Translational Medicine, Faculty
of Medicine and Dentistry, Palacký
A34
Abstract book
University in Olomouc,, Olomouc,
Czech Republic
Introduction
Oxaliplatin is a third generation of
platinum drug, which shows no
cross resistance with cisplatin.
Nowadays the oxaliplatin is used
in the monotherapy of colorectal
canceror in combination with
5-fluorouracil and leucovorin where
all drugs shows synergic effect.
Although oxaliplatin is a powerful
drug, patients suffers from several
toxicities,
with
most
serious
neuropathy. Its known mechanism of
action is binding of reactive platinum
species to DNA and forming intraand inter-strand crosslinks with
guanines and adenines leading to
the DNA damage. The aim of our
study was to analyse response to
oxaliplatin at complex protein level.
Materials/methods
This has been achieved by
influencing CCRF-CEM cell line (1
mil. cells / ml) by 5xIC50 (29.3 μM)
for time corresponding to half-time
to apoptosis (4h). Treated cells were
mixed with SILAC labeled control
cells, lyzed, digested and analyzed
on
LC/MS-MS.
Approximately
3200 proteins were identified in
each of three biological replicates.
By applying Max Planck Institute‘s
significance B statistical test, 74
proteins changed significantly in at
least two replicates were identified.
We have identified 26 ribosomal
proteins with average ratio (treated/
control) 0.85, 21 nucleolar and RNA
processing associated proteins and
26 proteins with different localization
and function. Nucleolar toxicity
of oxaliplatin was confirmed by
Smetana toluidine blue staining and
western blotting.
Results and conclusions
Up to date, there are only few studies
describing nucleolar toxicity and
ribosomal stress as the response to
oxaliplatin treatment. The novelty of
our work is to show down regulation
of ribosomal and nucleolar proteins
in response to oxaliplatin. However
the direct mechanism leading to
nucleolar toxicity was not identified.
This work was supported by
grants LF_2014_010, TE02000058,
CZ.1.05/3.1.00/14.0307. Institutional
part of the work was supported
by an EU Operational Program
Research and Development for
Innovations infrastructure grant
project CZ.1.05/2.1.00/01.0030
APOFERRITIN AND
ITS APPLICATIONS IN
NANOMEDICINE
středa / 3. prosince
13.55 - 14.10 hod.
2014
/
Zbynek Heger1,2, Simona
Dostalova1,2, Ondrej
Zitka1,2, Vojtech Adam1,2,
Tomas Eckschlager3, Marie
Stiborova4, Rene Kizek1,2
Central European Institute of
Technology, Brno University of
Technology, Brno, Czech Republic,
2
Department of Chemistry
and Biochemistry, Laboratory
of Metallomics and
Nanotechnologies, Mendel
University in Brno, Brno, Czech
Republic,
3
Department of Paediatric
Haematology and Oncology,
2nd Faculty of Medicine, Charles
University, and University Hospital
Motol, Prague, Czech Republic,
4
Department of Biochemistry,
Faculty of Science, Charles
University, Pragu, Czech Republic
Introduction
Nanomedicine as a continuously
evolving discipline is still looking for
a structure with perfect properties
usable
as
a
multifunctional
transporter. Ferritins as the proteins
naturally occurring in most living
organisms throughout evolution
show that they are the possible
choice. These proteins show perfect
biodegradability,
biocompatibility
and non-toxicity highly important for
nanotransporters [1-3]. The structure
of apoferritin is remarkably stable
and robust, and it is able to withstand
biologically extreme temperatures
(up to 70 °C) and wide pH range (pH
2.0 - 10.0) for an appreciable period
of time without significant disruption
of their quaternary structure. Twenty1
four ferritin subunits form a spherical
cage-shaped protein shell folded in
a bundle of four long parallel and
antiparallel α-helices (A, B, C, and D)
with a fifth shorter C-terminal helix
E, inclined at 60° to the major helix
bundle [4]. Apoferritin structure has
six 2-fold symmetry axes, four 3-fold
symmetry axes and three 4-fold
symmetry axes. It is known that there
are narrow hydrophilic channels
along the 3-fold symmetry axes
consisting of negatively charged
amino acids (glutamic and aspartic
acid) and hydrophobic channels
along the 4-fold symmetry axes. The
protein shell forms an inner cavity
with inner and outer diameters of
7-8 and 12-13 nm, respectively
[4]. The inner cavity with an 80 Å
diameter is capable to store up to
4500 Fe(III) atoms. Protein cage
may be reversibly disassociated in
unfavourable environment and after
changing of environment conditions
may be reconstituted backwards
holding
therapeutic
agent
in
its cavity, protecting it against
unwanted interactions. Moreover,
a surface of protein cage may be
easily functionalized with antibodies,
aptamers, etc. to achieve highly
specific nanoparticles.
Materials/methods
APOFERRITINS IN NANOMEDICINE
As a drugs or contrast agents
carriers, apoferritins could protect
its cargo against degradation and
pre-releasing causing undesired
side effects. The first mention of
the ability of ferritins to encapsulate
the anti-cancer therapeutics was
published in 2005 by Simsek and
Kilic in the paper entitled „Magic
ferritin: A novel chemotherapeutic
encapsulation bullet“ [5]. As time
goes by after seven years in 2012,
Kilic et al. formed the apoferritin
complex
with
doxorubicin
commonly used cytostatic drug.
The doxorubicin encapsulation was
carried out using direct and stepwise change of pH of the solution
from 2.5 to 7.4. It was found that
up to 28 molecules of doxorubicin
can be capsulated per apoferritin
protein and no significant drug
Abstract book
leakage occurs during several days‘
long storage [6]. Although apoferritin
exhibits great attributes to serve
as a platform for nanomedicine,
the possible undesired immune
response of patients still exists.
The ideal nanotransporter has to
go through a body undetected
by
immune
system.
Despite
evidence that excessive amount
of apoferritin administered for long
period can trigger immune complex
glomerulonephritis in mice [7], there
is still lack of evidence about immune
response in human.
Another
way
of
apoferritin
utilization for medical purposes is
photodynamic therapy in cancer
treatment.
The
photodynamic
therapy (PDT) is s a new therapeutic
modality that is emerging as a
powerful tool against malignant
tumors [8]. Apoferritin nanocage,
used as a delivery system for
photosensitizers to the intracellular
space, provides unique transporter
protecting loaded photosensitizers
from reactive biomolecules in the
cell membranes. This enables further
targeting of singlet oxygen upon
specific light irradiation to tumour
cells only.
The gene therapy includes the
insertion, removal or modification
of the defective gene(s) for the
treatment of genetically inherited
diseases. The commonly used
transporters for gene delivery are
viral vectors, liposomes, peptides
and
cationic
polymers.
The
main requirements of the gene
delivery vector are the protection
of delivered nucleic acid against
nucleases, targeting, and ability to
disrupt the endosomal membrane,
and thus the DNA is delivered to
the nucleus [9, 10]. Among the main
obstacles of gene delivery vectors,
aggregation, instability, toxicity and
their propensity to be captured by
the mononuclear phagocyte system
(MPS) are the most significant.
Although the usage of apoferritin as a
gene vector has not been published
yet, it exhibits few advantages, but
it is necessary to study apoferritins
due to their colloidal behaviour,
charge, possessing an electrostatic
repulsion as well as the stability of
encapsulated DNA.
Results and conclusions
One of the main goals of nanomedicine
is to create a nanocarrier that can
efficiently and specifically deliver
therapeutic agents to target sites
in the body. Moreover, to enable
efficient and specific delivery, a
nanocarrier needs to have an ability
to be easily modified. Replacement
of synthetic materials such as
porous hollow silica nanoparticles,
single-wall nanotubes, fullerenes,
etc. with natural materials that are
more acceptable to many people
has become an attractive way of this
field of research.
The authors gratefully acknowledge
financial support from the Grant
Agency of the Czech Republic
(NANOCHEMO GA CR 14-18344S).
REFERENCES
1.
Shevchenko, E.V., et
al., Structural diversity in binary
nanoparticle superlattices. Nature,
2006. 439(7072): p. 55-59.
2.
Roney, C., et al., Targeted
nanoparticles for drug delivery
through the blood-brain barrier for
Alzheimer‘s disease. Journal of
Controlled Release, 2005. 108(2-3):
p. 193-214.
3.
Agnihotri, S.A., N.N.
Mallikarjuna, and T.M. Aminabhavi,
Recent advances on chitosanbased micro- and nanoparticles in
drug delivery. Journal of Controlled
Release, 2004. 100(1): p. 5-28.
4.
Uto, K., et al., Characterization
of stable, electroactive protein cage/
synthetic polymer multilayer thin
films prepared by layer-by-layer
assembly. J. Nanopart. Res., 2013.
15(4): p. 1-11.
5.
Simsek, E. and M.A.
Kilic, Magic ferritin: A novel
chemotherapeutic
encapsulation
bullet. J. Magn. Magn. Mater., 2005.
293(1): p. 509-513.
6.
Kilic, M.A., E. Ozlu, and
S. Calis, A Novel Protein-Based
Anticancer Drug Encapsulating
Nanosphere:
ApoferritinDoxorubicin Complex. Journal of
Biomedical Nanotechnology, 2012.
8(3): p. 508-514.
7.
Li, M., et al., CD100 enhances
dendritic cell and CD4(+) cell
activation leading to pathogenetic
humoral responses and immune
complex glomerulonephritis. Journal
of Immunology, 2006. 177(5): p.
3406-3412.
8.
Saboktakin, M.R. and R.M.
Tabatabaee, The novel polymeric
systems for photodynamic therapy
technique. International Journal of
Biological Macromolecules, 2014.
65: p. 398-414.
9.
Mahato, R.I., Non-viral
peptide-based approaches to gene
delivery. J. Drug Target., 1999. 7(4):
p. 249-268.
10.
Tan, P.H., C.L.H. Chan,
and A.J.T. George, Strategies
to improve non-viral vectors potential applications in clinical
transplantation. Expert Opinion on
Biological Therapy, 2006. 6(6): p.
619-630.
A35
A36
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Biomarkery nádorových onemocnění III
Cancer biomarkers III
Předsedajíci / Chairs: Radek Trojanec, Jiří Drábek
středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 14.30 - 15.15 hod.
IDENTIFICATION OF
PROGNOSTIC AND
PREDICTIVE FACTORS
IN NON-SMALL CELL
LUNG CANCER PATIENTS
TREATED BY ADJUVANT
CHEMOTHERAPY
středa / 3. prosince
14.30 - 14.45 hod.
2014
/
Jana Potockova1, Radek
Trojanec1, Jiri Drabek1, Jana
Stranska1, Jana Vrbkova1,
Ivona Grygarkova2, Vladimira
Koudelakova1, Zuzana
Crlikova1, Sona Mlcochova1,
Marian Hajduch1
Laboratory of Experimental
Medicine, Institute of Molecular
and Translational Medicine, Faculty
of Medicine and Dentistry, Palacky
University, Olomouc, Hnevotinska
5, 779 00, Czech Republic,
2
Department of Pulmonary Diseases
and Tuberculosis, University
Hospital in Olomouc, Olomouc,
I.P. Pavlova 6, 779 00, Czech
Republic
Introduction
Lung carcinomas represent one
of the most common types of
all tumors and can be classified
into several groups according to
various criteria such as biological
behavior, histological composition,
or localization. In practice, however,
it is most useful to divide them into
small-cell lung carcinoma SCLC
(20-25%) and non-small-cell lung
carcinoma
NSCLC
(75-80%).
In comparison, NSCLC usually
exhibits slower growth, so that in
practice surgical removal becomes
a favorable possibility, assuming
that the tumor has not metastasized.
NSCLC also responds less favorably
to anti-tumor cytotoxic therapy
and radiotherapy than SCLC. It is
therefore that NSCLC has become
the subject of our work.
1
Materials/methods
In our study, we investigated
a group of 124 patients with
NSCLC who were treated with an
adjuvant regimen consisting of a
combination of platinum derivatives
and vinorelbine. For the material,
formalin-fixed paraffin embedded
(FFPE) tissue samples were used.
Using the FISH assay, the status of
EGFR, FGFR, C-MET, the presence
of rearrangements in ALK and ROS1
genes, and the number of copies
of chromosomes 7 and 8 were
determined. Concurrently, the qPCR
method was used to investigate the
mutational status in the K-RAS and
B-RAF genes.
Results and conclusions
Statistical analyses of the clinical and
laboratory results were performed;
the control group comprised NSCLC
patients without adjuvant therapy.
Some interesting correlations were
revealed. For example, patients
with the higher copy number of
chromosomes 7 or 8 and higher copy
number of the genes EGFR and MET
(located on these chromosomes),
exhibited shorter relapse free
survival (RFS). Other correlations will
be also presented.
Acknowledgement: This work was
supported by grant project IGA MZ
ČR NT/13569 and BIOMEDREG
CZ.1.05/2.1.00/ 01.0030
A workflow for the
identification and validation for
N-glycoprotein biomarkers in
mouse xenograft serum
středa / 3. prosince
14.45 - 15.00 hod.
2014
/
Lakshman Varanasi1,
Miroslav Hruska2, Dusan
Holub1, Petr Dzubak1, Marian
Hajduch1
1
Institute of Molecular and
Translational Medicine, Palacky
University, Olomouc, Czech
Republic,
Department of Computer Science,
Palacky University, Olomouc,
Czech Republic
Introduction
Serum is one of several types of
patient samples routinely analyzed
in the clinic and is often preferred
because it can be obtained noninvasively in sufficient quantities.
Serum proteins that indicate a
condition or disease, its severity, the
patient‘s chances of recovery and
of the patient‘s possible response
to therapy are of clinical value.
Glycosylation of cellular proteins
post-translation has been reported
as an indicator of altered cellular
physiology. We describe here the
optimization of a workflow for the
identification of such N-glycosylated
proteins using the HCT116 mouse
xenograft model system. The
premise for this approach is that
human proteins observed in mouse
serum originate in the tumor
xenograft.
Materials/methods
Hydrazide slurry was purchased
from BioRad. All other reagents
were
obtained
from
SigmaAldrich. Samples were processed
by SPEG (Solid-phase extraction
of glycoproteins). Tandem massspectra were acquired using an
Orbitrap Elite mass-spectrometer
(Thermo).
Acquired
tandem
mass spectra were matched with
corresponding peptides by Proteome
Discoverer (Thermo) in a database
of human and mouse peptides.
The peptide spectral matches
were classified as uniquely human,
uniquely murine or as peptides
common to the two species, using a
program developed in-house.
Results and conclusions
Altogether 30 uniquely human
peptides were identified in the
discovery
phase.
These
are
2
Abstract book
candidates for the validation phase
of the study viz quantification in
a colorectal cancer patient serum
sample cohort by the single-reaction
monitoring (SRM) technique.
System for reliable
identification of alterations
using protein massspectrometry
středa / 3. prosince
15.00 - 15.15 hod.
2014
/
Miroslav Hruska1,2, Jiri
Voller1, Lakshman Varanasi1,
Petr Vojta1, Dusan Holub1,
Khushboo Agrawal1, Petr
Dzubak1, Marian Hajduch1
Institute of Molecular and
Translational Medicine, Olomouc,
Czech Republic,
2
Department of Computer Science,
Olomouc, Czech Republic
Introduction
Typical cancer cells carry mutations
in up to hundreds of genes.
Knowledge of mutation profiles
allows us to understand which
processes are altered in the cancer
cells of individual patients and
select the therapy accordingly.
Modern high-throughput methods
like SNP arrays or next-generation
sequencing (NGS) are used for
mutation screening. However, the
mass spectrometry of proteome,
another method with a great
potential in personalized medicine,
is not used for this purpose yet. The
limiting factor is insufficient coverage
of mutant variants in proteome
databases and incompleteness of
fragmentation spectrum for peptide
de novo reconstruction.
Materials/methods
To advance the current state, we
have developed a system for the
identification of mutant, polymorphic
proteins on protein level. The core
consists of dedicated mutation/
SNPs
database
with
unique
coverage (number of both mutation
and integrated information sources).
The system, Decryptor, enables
users to search for alterations in their
MS/MS spectra. The identification
1
process itself is performed using
Dymka, in-house developed, multiengine, cluster-powered system
built on top of open-source system
OpenMS. Decryptor works both with
peptide database search engines
and tag-based de novo systems.
Obtained results are subjected to
careful artefact analysis to separate
relevant findings from potential
artefacts. For example, one class
of artefacts emerges because
of peptide homeometricity, i.e.,
existence of different peptides with
highly similar fragmentation spectra.
Final results are then annotated from
multiple data sources and presented
in a form amenable to further
investigations.
Results and conclusions
Performance of Decryptor was
evaluated on colorectal cancer line
HCT116. The necessity of artefact
analysis was shown to be especially
important for reliable identification of
rare sequence variants. In such case
peptide homeometric to mutant
peptide is often found among posttranslationally modified reference
peptides.
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A38
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Farmakologie protinádorových léčiv a in vivo imaging
Pharmacology of anticancer drugs and in vivo imaging
Předsedajíci / Chairs: Miloš Petřík, Josef Srovnal
středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 15.45 - 16.30 hod.
Assessing pharmacokinetic
properties in drug discovery:
screening versus in silico
středa / 3. prosince
15.45 - 16.00 hod.
2014
/
Alice Nová, Martina
Michalová, Monika
Harvanová, Milan Urban
Institute of Molecular and
Translational Medicine Faculty of
Medicine and Dentistry Palacky
University Olomouc, Olomouc,
Czech Republic
Introduction
ADME (absorbtion, distribution,
metabolism and excretion) in silico
modeling can be a useful tool in
drug design and development to
help in finding the most promising
compounds,
preferably
prior
synthetizing them; however in silico
prediction has its pitfalls.
Materials/methods
Here, we compare in silico ADME
predictions gained from free ADME
prediction softwares to in vitro
experimental data obtained from
testing three groups of compounds.
We also compare the ability and
accuracy of in silico ADME tools to
predict ADME properties depending
on the complexity of the molecule
structure of tested compounds. In
order to predict ADME properties we
have chosen three online software
tools:
ACD/I-lab,
admetSAR
and online BBB (blood brain
barrier) predictor. We have tested
compounds from the groups of
aminopyrimidines (anti-inflammatory
compounds), triterpenes (potential
antitumor agents) and carboranes
(potential inhibitors of carbonic
anhydrase IX, which is selectively
expressed in hypoxic tumors).
Results and conclusions
Aminopyrimidines
can
be
characterized as small and relatively
simple molecules. Both ACD/I-lab
and admetSAR predicted accurately
GIT permeability, however active
efflux was not correctly predicted.
All three used software tools
predicted brain penetration as
sufficient for CNS activity and these
results were in accordance with
the data obtained from the in vitro
BBB model using MDR1-MDCK
cell line. Only ACD/I-lab is able to
predict plasma protein binding and
all tested compounds were generally
satisfactorily predicted.
Compounds from the group of
triterpens represent more complex
structures and larger molecules
compared to aminopyrimidines. In
silico prediction of triterpene GIT
permeability achieved 75% reliability
against Caco-2 cell line testing,
however both predictors failed in
active efflux prediction. In case of
prediction of the brain penetration,
in silico tools have diverged among
themselves in BBB prediction. In vitro
MDR1-MDCK permeability testing of
CNS permeability of triterpenes is
currently in the process.
Carborane-based
compounds
are examples of such complex
structures, that they cannot be
processed by previously selected in
silico predictors.
In conclusion, GIT permeability
and brain penetration of relatively
uncomplicated
molecules
are
possible to predict in silico with
sufficient reliability. Metabolism and
plasma stability are not possible to
reliably predict using free in silico
modeling at all. Here we wanted to
point out, that the ADME in silico
modeling has its place in the drug
development, but it is necessary to
take into account that it can never
outperform the experimental data.
Ga labelled peptides for
glioblastoma imaging
68
středa / 3. prosince
16.00 - 16.15 hod.
2014
/
Milos Petrik, Jana
Stepankova, Zbynek Novy
Institute of Molecular and
Translational Medicine, Olomouc,
Czech Republic
Introduction
Glioblastoma multiforme (GBM)
is the most aggressive type of
primary brain tumors in humans with
more than 90% 5-year mortality.
Early diagnosis may be the key
factor to improving patient survival
rates through the prevention of
tumor growth. Positron emission
tomography (PET) is a useful imaging
tool allowing early stage tumors
identification, monitoring of treatment
efficacy and follow up for disease
recurrence. 18F-fluorodeoxyglucose
(18F-FDG) is the most common
clinically utilized PET tracer for
imaging of GBM, although it has
several limitations based on high
glucose uptake within the brain.
Here we report on the preclinical
evaluation of two 68Ga labelled
peptides as potential agents for
GBM imaging and their comparison
with 18F-fluorothymidine (18F-FLT),
another PET tracer widely used in
clinical practice.
Materials/methods
68
Ga labelling of studied peptides
(NODAGA-conjugated
RGD
dimer
and
DOTA-conjugated
Substance P) was optimized
concerning temperature, peptide
amount and reaction time. For in
vitro
characterization,
partition
coefficient,
protein
binding
properties and stability values in
different media were determined.
Ex vivo biodistribution and animal
imaging was studied in Balb/c mice.
Abstract book
Results and conclusions
Both 68Ga labelled peptides were
prepared with high radiochemical
purity (> 97%) under various
reaction conditions. 68Ga-NODAGARGD dimer showed hydrophilic
properties and was stable in human
serum, iron and DTPA solutions as
well as at pH = 7. In contrast to 68Ga
labelled RGD peptide, 68Ga-DOTASubstance P displayed much lower
stability, especially in iron and DTPA
solutions. Protein-bound activity
of 68Ga-NODAGA-RGD dimer after
120 min incubation (3.9% ± 1.9)
was found to be 7-fold lower than
for 68Ga-DOTA-Substance P. In
mice, both 68Ga labelled tracers
revealed mainly renal elimination
with low blood values 90 min p.i.,
while 18F-FLT showed much slower
kinetics and not only renal, but also
gastrointestinal excretion.
Studied peptides bound 68Ga with
high affinity, and 68Ga-NODAGARGD dimer, especially, displayed
high in vitro stability and excellent
pharmacokinetics. Both 68Ga labelled
tracers showed superior in vivo
properties compared to clinically
used 18F-FLT. 68Ga-NODAGA-RGD
dimer, in particular, holds promise for
PET imaging of glioblastoma, which
needs to be confirmed in U87MG
xenografts. Animal experiments
in U87MG xenograft tumors are
currently ongoing.
Imaging of glioblastoma
multiforme in mice using
microPET/CT system
středa / 3. prosince
16.15 - 16.30 hod.
2014
/
Zbynek Novy1, Milos Petrik1,
Hana Adamkova2, Marian
Hajduch1
Faculty of Medicine and Dentistry,
Palacky University in Olomouc,
Olomouc, Czech Republic,
2
Faculty of Science, Palacky
Universtity in Olomouc, Olomouc,
Czech Republic
Introduction
Glioblastoma multiforme is a primary
brain tumor with very poor prognosis.
Early diagnosis is essential key
1
point for successful therapy of this
disease.
Non-invasive
imaging
techniques play very important
role in the diagnosis and staging of
glioblastoma multiforme. To facilitate
preclinical in vivo research in the
field of glioblastoma multiforme we
have compared the biodistribution
and the imaging potential of three
clinically used radiopharmaceuticals
for positron emission tomography in
a mouse tumor model.
Materials/methods
Tumor
mouse
model
was
established using SCID mouse
strain and U-87 MG tumor cells
xenografted
subcutaneously.
Visualizations of tumors were
performed by employing three PET
radiopharmaceuticals administered
already routinely in human medicine
- 18F-fluorodeoxyglucose (FDG),
18F-fluorothymidine
(FLT)
and
18F-fluorocholine (FCH). Healthy
non-tumorous mice were used as
a negative control. MicroSPECT/
PET/CT system (Albira, Carestream,
USA) was used to acquire images
of tested mice after the application
of the tracers. The mice in general
anesthesia were PET-scanned during
90 minutes after administration of
the radiopharmaceuticals to reveal
the tracer´s biodistribution and the
best time point for visualization of
the tumor. The acquired data were
quantified in PMOD software.
Results and conclusions
The
biodistribution
of
three
mentioned tracers in SCID mouse
was determined using output
data from PMOD software. As
well as the tumor to brain rations
were calculated to find out the
specificity of the tracers. The most
appropriate biodistribution (fast
renal excretion) and in the same
time the highest tumor to brain
ratio (4,4) was observed in case
of 18F-fluorothymide. The other
two tested radiopharmaceuticals
(FDG and FCH) have not showed
satisfactory results for imaging
of glioblastoma multiforme in
mice. In conclusion, the benefits
of
used
radiopharmaceuticals
for imaging the studied tumor
were evaluated and as the most
appropriate radiopharmaceutical for
this examination was determined
18F-flurothymidine.
A39
A40
Abstract book
Posterová sekce /
Poster section
vyskytuje u většiny adenokarcinomů LightCycler 480 II, Roche).
Exprese HNF-1β
v karcinomech děložního čípku děložního hrdla, oproti tomu u Materials/methods
Kristyna Nemejcova, Pavel
Dundr, Libor Stanek, Ctibor
Povysil
General Facuty Hospital, Prague,
Czech Republic
Introduction
HNF-1β je maker běžně využívaný
v
diferenciální
diagnostice
světlobuněčných karcinomů ovária
a endometria. Recentní studie
prokázaly expresi HNF-1β v menším
rozsahu i u dalších ovariálních a
endometriálních tumorů. Ohledně
děložního čípku byla exprese HNF-1β
pouze okrajově zmíněna u některých
typů adenokarcinomů. Nicméně
systematická analýza exprese HNF1β u cervikálních karcinomů dosud
nebyla provedena.
Materials/methods
Cílem studie bylo stanovit expresi
HNF-1β v různých typech invazivních
karcinomů čípku a zhodnotit přínos
imunohistochemické
analýzy
exprese
tohoto
proteinu
pro
diferenciální diagnostiku jednotlivých
histologických typů nádorů.
Studie zahrnovala 155 případů
invazivních karcinomů děložního
čípku
(86
dlaždicobuněčných
(SCC), 56 adenokarcinomů (ACAs),
13 nediferencovaných karcinomů.
Exprese HNF-1β byla určována
imunohistochemicky a výsledky byly
korelovány s expresí ER, PR, CEA,
p16, p63, p40 a D2-40.
Results and conclusions
HNF-1β byl exprimován v 42/56
ACAs (75 %). Ve skupině SCC,
byly pozitivní pouze 2/86 (2,32%)
případy, které exprimovaly HNF1β v 70 % a ve 30 % nádorových
buněk. Nediferencované karcinomy
vykazovaly expresi HNF-1β ve 2/13
případů.
Závěr ukázal, že exprese HNF-1β se
dlaždicobuněčných
karcinomů
jsme ji zastihli pouze raritně.
Tento marker by tedy mohl být
potenciálně využitelný jako pomocný
marker v diferenciální diagnostice
málo diferencovaných karcinomů
děložního hrdla, jejichž odlišení je na
morfologické úrovni někdy obtížné až
nemožné. Samotnou expresi tohoto
markeru však nelze nadřadit nad jiné
rutinně používané markery jako je
např. p63, p40 a D2-40.
Acknowledgement: Práce byla
podporována IGA MZ CR NT140013/2013
In the present study, we investigated
if there is a possibility to check
the performance of thermocycler
regarding temperature uniformity
over the heating block using
PCR reagents readily available
in the laboratory. We tried to find
correlations
between
melting
temperature, Tm, and location
of the well by comparing melting
temperature of the same amplified
DNA sequence across the thermal
block. We supposed that differences
in Tm across the block are caused
not only by small temperature
Temperature uniformity of the heterogeneity of the thermal block
but also by other factors (i.e.
thermocyclers tested using
imprecission of pipetting).
melting temperature of the
Results and conclusions
same amplicon
We tested several possibilities to
Eva Hruskova, Jiri Drabek
alleviate the sources of unwanted
Institute of Molecular and
variability with the aim to increase the
Translational Medicine of the
reproducibility of Tm measurements.
Faculty of Medicine and Dentistry
In this contribution, we present
of Palacký University, Olomouc,
our findings and suggestion for
Czech Republic
practical use of Tm for thermal block
temperature uniformity testing.
Introduction
Supported
Real-time PCR is one of the most Acknowledgement:
widely used method in clinical by grants LO1304, NT13569 and
diagnostic laboratories. Because CZ.1.05/3.1.00/14.0307.
even a small change of the
Inhibition of proliferation
temperature can lead to insufficient
denaturation
or
hybridization by inhibitors of histone
resulting in loss of specificity, deacetylases
sensitivity and reproducibility, the Tomas Groh1, Mohamed
temperature needs to be precise Asraf Khalil2, Jan Hrabeta2,
and uniform. To confirm the proper
Blanka Jedlickova3, Tomas
operation of thermocyclers and
2
fulfill ISO17025 and ISO15189 Eckschlager , Marie
accreditation requirements, regular Stiborova1
calibration of thermal cyclers is 1 Department of Biochemistry,
needed. Calibration is usually Faculty of Science, Charles
performed by accredited calibration University, Prague, Czech Republic,
laboratory
using
metrologically 2 Department of Pediatric
traceable
temperature
probes. Hematology and Oncology, 2nd
However, sometimes such calibration Medical School, Charles University
is not possible because the cycler is and University Hospital Motol,
inaccessible for thermoprobes (i. e. Prague, Czech Republic,
Abstract book
Department of Chemistry and
Clinical Biochemistry, 2nd Medical
School, Charles University and
University Hospital Motol, Prague,
Czech Republic
Introduction
N-myc protein is a member of Myc
family transcription factors. Different
alterations in gene or protein
function of Myc family members
are connected to variety of human
cancers. Myc family members are
responsible for fast proliferation
of cells, chemoresistance and
metabolic adaptation of cancer
cells. N-myc has been studied
intensively since it was discovered
as the most important prognostic
factor in neuroblastoma (NB). NB
is a most common extracranial
solid tumor in infancy and some
its forms has a very bad curability.
N-myc amplification is related to a
worse prognosis and poor survival
of patients suffering from NB.
Inhibitors of histone deacetylases
(HDAC) are promising drugs for
cancer treatment. Some of them are
in clinical testing, for their ability to
supress cancer cells growth, to stop
cell cycle, differentiate cancer cells
and ability to induce programmed
cell death. Inhibitors of HDAC act
as epigenetic modifiers of histone
tails or change acetylation status of
different proteins.
Materials/methods
Western blot was used for detection
of N-myc and HIF-1a transcription
factors. Flow cytometry was used to
measure cell cycle distribution.
Results and conclusions
We studied mechanism of inhibition
of cell growth of NB cells induced by
inhibitor of HDAC valproate (VPA).
We tested three different NB cell
lines that amplify N-myc gene (IMR32, UKF-NB-3, UKF-NB-4). N-myc
protein
expression
decreased
after 24h and 48h treatment with
nontoxic dose of VPA (1mM). That
decrease was even under hypoxic
conditions (1% O2). Cells stopped
proliferate mostly under hypoxic
conditions after treatment with 1mM
VPA, that correlate with decrease
3
of both N-myc trascription factor
and hypoxia inducible factor 1
alpha (HIF-1a). As we observed
earlier, inhibition of HDAC by VPA
increased acetylation of histone
H3 and histone H4. Simultaneously
VPA induced cell cycle arrest and
increased doubling time of cells. We
hypothesize that, decrease of N-myc
transcription factor induced by VPA
plays an important role in inhibition
of proliferation NB cells.
The study was supported by the
Charles University in Prague, project
GA UK No. 635712 and No. 620612.
Multi-walled carbon nanotubes
as a promising doxorubicin
nanocarrier and their effects
on neuroblastoma cell lines
Tereza Cerná1, Jan Hrabeta2,
Tomáš Groh1, Hoai Viet
Nguyen3, Amitava Moulick3,
Tomáš Eckschlager2, Marie
Stiborová1
Department of Biochemistry,
Faculty of Science, Charles
University, Prague, Czech
Republic,
2
Department of Pediatric
Hematology and
Oncology,2nd Medical Faculty,
Charles University and University
Hospital Motol, Prague, Czech
Republic,
3
Laboratory metallomics and
nanotechnology, Mendel University
in Brno and Central European
Institute of Technology, Brno
University of Technology, Brno,
Czech Republic
Introduction
Neuroblastoma is the most common
extracranial solid tumor of childhood.
Despite advances in cancer diagnosis
and therapy, gradually developing
tumor cell chemoresistance is one
of the major causes of treatment
failure of high grade neuroblastoma.
Currently nanocarriers are promising
agents to improve drug therapeutic
index,
divert
ABC-transporter
mediated drug efflux mechanism
and selectively target tumor cells.
Selective nanocarrier targeting to
tumor overcomes dose-limiting
1
side effects, lack of selectivity,
tissue toxicity and limited drug
access to tumor tissues, moreover
it allows application of higher doses
of cytostatics. Carbon nanotubes
(CNTs) are emerging as a family of
nanovectors for drug delivery into
biological systems.
Materials/methods
To evaluate potential application of
this technology for neuroblastoma
therapy, the aim of this pilot study
was to compare the cytotoxic
effects of doxorubicin loaded
multi-walled CNTs (MWCNTs) and
free doxorubicin on sensitive and
resistant neuroblastoma cell lines
UKF-NB-4 in vitro in normoxic and
hypoxic conditions. Cell viability
was assessed using the MTS
cytotoxicity assay and DNA doublestrand breaks were detected by flow
cytometry as phosphorylation of
histone H2A variant.
Results and conclusions
We show here that effect of
doxorubicin
loaded
MWCNTs
on human neuroblastoma cell
lines is very similar to the effect
of free doxorubicin. There were
also detected hypoxia-induced
resistance to doxorubicin both free
and in MWCNTs . These results
seem promising to begin animal
experiments, where it is hoped that
in vivo studies will demonstrate
higher effectivity of MWCNTs.
This work was supported by GACR,
grant No. 14-18344S.
Prognostický a prediktivní
význam sérových
onkomarkerů u pacientů s
pokročilým neskvamózním
NSCLC léčených
pemetrexedem
Ondřej Fiala1, Miloš Pešek2,
Jindřich Fínek1, Vít Martin
Matějka1, Luboš Holubec1,
Zbyněk Bortlíček3, Ondřej
Topolčan4
Onkologická a radioterapeutická
klinika LF UK a FN Plzeň, Plzen,
Czech Republic,
2
Klinika pneumologie a ftizeologie
1
A41
A42
Abstract book
LF UK a FN Plzeň, Plzen, Czech
Republic,
3
Institut biostatistiky a analýz, LF
MU, Brno, Brno, Czech Republic,
4
Oddělení nukleární medicíny –
imunoanalytická laboratoř LF UK
a FN Plzeň, Plzen, Czech Republic
Introduction
Pemetrexed
je
antifolátové
cytostatikum
nové
generace,
jehož
účinnost
i
bezpečnost
byla
prokázána
v
několika
randomizovaných studiích fáze III. V
současné době standardně užíván
pro léčbu pacientů s pokročilým
stádiem NSCLC (st. IIIB nebo IV),
neskvamózního histologického typu,
v kombinaci s platinovým derivátem
v první linii a v monoterapii v linii
druhé. Recentní studie rovněž
pokázaly
efektivitu
udržovací
léčby pemetrexedem. Cílem této
práce bylo zjištění prediktivního
a
prognostického
významu
hladin
sérových
onkomarkerů
karcinoembryonálního
antigenu
(CEA),
fragmentu
cytokeratinu
19 (CYFRA 21-1), Monototalu,
chromograninu A, thymidin kinázy
(TK) a antigenu skvamózních
karcinomů (SCCA) u pacientů s
pokročilým stadiem NSCLC, kteří
byli léčeni pemetrexedem.
Materials/methods
Soubor pacientů čítá celkem 114
pacientů s pokročilým stadiem
NSCLC (IIIB, IV) neskvamózního
histologického typu, kteří byli
léčeni pemetrexedem v kombinaci
s platinovým derivátem nebo v
monoterapii. U všech pacientů byla
před zahájením léčby stanovena
sérová
hladina
zkoumaných
onkomarkerů.
Přežití
pacientů
bylo
hodnoceno
metodikou
podle Kaplana-Meiera, srovnání
bylo provedeno log-rank testem.
Prognostický a prediktivní význam
onkomarkerů byl dále verifikován
pomocí Coxova vícerozměrného
modelu.
Results and conclusions
Mediány
přežití
bez
známek
progrese (PFS) a celkového přežití
(OS) u pacientů s vysokou hladinou
CEA činily 2,4 a 12,8 vs. 3,1 a
11,5 měsíce u pacientů s nízkou
hladinou CEA (p=0,137 a p=0,866).
Mediány PFS a OS u pacientů s
vysokou hladinou CYFRA 21-1 činily
2,4 a 10,3 vs. 2,7 a 23,4 měsíce u
pacientů s nízkou hladinou CYFRA
21-1 (p=0,899 a p<0,001). Mediány
PFS a OS u pacientů s vysokou
hladinou Monototalu činily 3,1 a 12.4
vs. 2,9 a 22,3 měsíce u pacientů
s nízkou hladinou Monototalu
(p=0,543 a p=0,451). Mediány PFS
a OS u pacientů s vysokou hladinou
chromograninu A činily 1,4 a 7,9
vs. 2,4 a 13,5 měsíce u pacientů s
nízkou hladinou chromograninu A
(p=0,196 a p<0,224). Mediány PFS a
OS u pacientů s vysokou hladinou TK
činily 2,2 a 11,3 vs. 2,8 a 23,4 měsíce
u pacientů s nízkou hladinou TK
(p=0,163 a p=0,003). Mediány PFS
a OS u pacientů s vysokou hladinou
SCCA činily 2,7 a 8,7 vs. 2,9 a 16,6
měsíce u pacientů s nízkou hladinou
SCCA (p=0,557 a p=0,510). Coxův
vícerozměrný
model
prokázal,
že sérové hladiny CYFRA 21-1
(HR=2,26; p=0,001) a TK (HR=2,09;
p=0,003) představují významný,
nezávislý faktor predikující OS.
Výsledky naší studie prokázaly,
že vysoké předléčebné hladiny
sérových
onkomarkerů
CYFRA
21-1 a TK představují významný,
nezávislý faktor predikující krátké
OS u pacientů s pokročilým NSCLC.
Žádný ze zkoumaných onkomarkerů
se
neukázal
jako
významný
prediktivní biomarker pro odhad
efektu léčby pemetrexedem.
Podpořeno MZ ČR - FNPl,
00669806.
Sérové onkomarkery jako
prediktivní biomarker u
pacientů s pokročilým NSCLC
léčených EGFR-TKI
Ondřej Fiala1, Miloš Pešek2, Jindřich Fínek1, Marek Minárik3, Lucie
Benešová3, Zbyněk Bortlíček4, Ondřej Topolčan5
1
Onkologická a radioterapeutická
klinika LF UK a FN Plzeň, Plzeň,
Czech Republic,
2
Klinika pneumologie a ftizeologie
LF UK a FN Plzeň, Plzeň, Czech
Republic,
Centrum aplikované genomiky
solidních nádorů (CEGES),
Genomac výzkumný ústav, Praha,
Praha, Czech Republic,
4
Institut biostatistiky a analýz, LF
MU, Brno, Brno, Czech Republic,
5
Oddělení nukleární medicíny –
imunoanalytická laboratoř LF UK a
FN Plzeň, Plzeň, Czech Republic
Introduction
Zavedení inhibitorů tyrozinkináz
receptoru
pro
epidermální
růstový faktor (EGFR-TKI) do
léčby nemalobuněčného plicního
karcinomu
(NSCLC)
přispělo
k prodloužení přežití pacientů
s pokročilým stádiem tohoto
onemocnění.
Aktivační
mutace
genu EGFR představují spolehlivý
prediktor dobrého efektu léčby
EGFR-TKI u pacientů s pokročilým
NSCLC. Výskyt těchto mutací v
evropské populaci je nízký (cca
10-15%), navíc je stále mnoho
pacientů, u kterých vyšetření na
přítomnost aktivační mutace genu
EGFR není možné provést z různých
důvodů. Z tohoto důvodu je hledání
dalších prediktivních biomarkerů
nutné. Cílem této práce bylo zjištění
významu sérových onkomarkerů
CEA, CYFRA 21-1, NSE a TK pro
predikci efektu léčby EGFR-TKI.
Materials/methods
Soubor pacientů, u kterých jsme
hodnotili význam onkomarkerů CEA
a CYFRA 21-1 čítá 144 pacientů
a soubor pacientů, u kterých jsme
hodnotili význam onkomarkerů NSE
a TK čítá 163 pacientů s pokročilým
stadiem NSCLC (IIIB, IV), kteří byli
léčeni EGFR-TKI (erlotinib, gefitinib).
U všech pacientů byla před zahájením
léčby stanovena sérová hladina
zkoumaných onkomarkerů. Přežití
pacientů bylo hodnoceno metodikou
podle Kaplana-Meiera, srovnání
bylo provedeno log-rank testem.
Prognostický a prediktivní význam
onkomarkerů byl dále verifikován
pomocí Coxova vícerozměrného
modelu.
Results and conclusions
Mediány
přežití
bez
známek
progrese (PFS) a celkového přežití
(OS) u pacientů s vysokou hladinou
3
Abstract book
CEA činily 1,9 a 8,6 vs. 2,9 a 16,1
měsíce u pacientů s nízkou hladinou
CEA (p=0,046 a p=0,116). Mediány
PFS a OS u pacientů s vysokou
hladinou CYFRA 21-1 činily 1,9 a
6,1 vs. 3,4 a 23,8 měsíce u pacientů
s nízkou hladinou CYFRA 21-1
(p<0.001 a p<0.001). Mediány PFS
a OS u pacientů s vysokou hladinou
NSE činily 1,1 a 3,7 vs. 2,6 a 11,6
měsíce u pacientů s nízkou hladinou
NSE (p=0,002 a p=0,003). Mediány
PFS a OS u pacientů s vysokou
hladinou TK činily 2,1 a 8,5 vs. 2,9
a 17,4 měsíce u pacientů s nízkou
hladinou TK (p=0.026 a p=0.020).
Coxův vícerozměrný model prokázal,
že sérové hladiny CEA (HR=1,72;
p=0,007), CYFRA 21-1 (HR=2,17;
p<0,001) a NSE (HR=2,36; p=0,003)
představují významný, nezávislý
faktor predikující PFS a hladina
CYFRA 21-1 (HR=2,74; p<0,001)
představuje významný, nezávislý
faktor predikující OS.
Výsledky naší studie prokázaly,
že vysoké předléčebné hladiny
sérových onkomarkerů CEA, CYFRA
21-1 a NSE představují významný,
nezávislý faktor predikující nízký
efekt léčby EGFR-TKI a dále
potvrdily negativní prognostický
význam onkomarkeru CYFRA 21-1.
Podpořeno MZ ČR - FNPl,
00669806.
Influence of quercetin and
taxifolin on microRNA 375
expression in HepG2 cell line
and human hepatocytes.
Zdenek Dostal1, Martin
Sebera2, Josef Srovnal3,
Michaela Sedlackova3, Lenka
Radova4, Katerina Staffova3,
Martin Modriansky1, Jitka
Ulrichova1
Department of Medical Chemistry
and Biochemistry, Faculty of
Medicine, Palacky University,
Olomouc, Czech Republic,
2
Department of Kinesiology,
Faculty of sport studies, Masaryk
University, Brno, Czech Republic,
3
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
1
University and University Hospital
Olomouc, Olomouc, Czech
Republic,
4
Central European Institute of
Technology, Research Group
Medical Genomics, Masaryk
University, Brno, Czech Republic
Introduction
Taxifolin and quercetin belong to
a group of polyphenols that are
natural compounds present in
fruits, vegetables, beverages such
as tea, and in several other foods
and natural products. In general
these substances display positive
biological effects and due to a
relatively high consumption are they
considered beneficial. According to
many studies the daily dose of these
substances may reach more than 1
g/day. One possible mode of action
is an influence of these compounds
on microRNA expression and the
associated change in amount of
target proteins. The change may
reflect in cell phenotype and this
affects its behavior.
Materials/methods
Quercetin and taxifolin were tested
for their ability to influence the
expression of microRNA 375 and its
potential impact on the expression
of one potential target gene,
procaspase 3. The influence of both
substances was estimated from
chip array and verified by RT-PCR
method. The impact on procaspase
3 expression was studied by
western blot. Because liver is the
first organ possibly affected by both
substances, we selected HepG2
cell line as it is derived from human
hepatoma and primary human
hepatocytes. The dose of both
substances was 1 mM based on
toxicity studies. The treatment time
period was established as 24 hours.
Total RNA was isolated by phenolchloroform method.
Results and conclusions
Firstly, we compared the data
obtained from microRNA chips with
those obtained by RT-PCR. Our
data show slight but stable increase
in microRNA 375 expression by
taxifolin in HepG2 cell line. On the
other hand, expression of microRNA
375 in human hepatocytes differed
significantly among six individual
samples. Quercetin causes an
increase in expression of the
microRNA of interest in both cell
types. As both tested substances
are perceived as cytoprotective, we
selected to monitor the expression
of the protein procaspase 3, which
is one of several possible target
genes of microRNA 375. However,
western blot experiments showed no
significant influence on procaspase
3 expression even though a trend
toward attenuation of its expression
in dose dependent manner was
observed. Our data suggest that
beneficial biological activity of
taxifolin and quercetin may proceed
through microRNA modulation.
Acknowledgements : This work
was supported by grants IGA_
LF_2014_14 and LO1304
PHAGE λ as a suitable
doxorubicin nanocarrier
Simona Dostalova1,2,
Dita Munzova2, Marketa
Vaculovicova1,2, Tomas
Eckschlager3, Marie
Stiborova4, Vojtech Adam1,2,
Rene Kizek1,2
Central European Institute of
Technology, Brno University of
Technology, Brno, Czech Republic,
2
Department of Chemistry
and Biochemistry, Laboratory
of Metallomics and
Nanotechnologies, Mendel
University in Brno, Brno, Czech
Republic,
3
Department of Paediatric
Haematology and Oncology,
2nd Faculty of Medicine, Charles
University, and University Hospital
Motol, Prague, Czech Republic,
4
Department of Biochemistry,
Faculty of Science, Charles
University, Prague, Czech Republic
Introduction
One of frequently used cytotoxic drug
for cancer treatment is doxorubicin.
It is an anthracycline antibiotic drug.
Doxorubicin can block enzyme
topoisomerase II, resulting in
1
A43
A44
Abstract book
disruption of newly replicated DNA.
It also intercalates in DNA, which
leads to DNA breaks and defects
during replication and transcription;
and forms free radicals. It is used in
treatment of breast, lung, ovarian,
bladder, thyroid gland, testicle or
head and neck carcinomas, as well
as neuroblastomas, leukaemia,
lymphomas and many others.
However, the administration of
doxorubicin during the treatment
leads to many severe side effects
for healthy cells, lowering the wellbeing of patients. The side effects
include nausea and vomiting, sores
in the mouth and on the lips, hair
loss, diarrhea, darkening of the
soles, palms or nails, blood in the
urine, unusual bleeding and bruising
or swelling of feet. The most severe
is its cardiotoxicity which can lead to
patient‘s death.
Encapsulation
of
doxorubicin
in
suitable
nanocarrier
can
eliminate the negative side effects.
Nanocarriers can be composed
of many different materials, either
inorganic or organic. Protein-based
nanocarriers, including viral capsids,
are more suitable for administration
in patients in comparison with
inorganic materials.
Materials/methods
Cultivation and purification of
phage λ
Phage
λ-producing
strain
of
Escherichia coli was cultivated in
Luria-Bertani broth (1 % tryptone,
0.05 % yeast extract and 1 %
sodium chloride) with 0.2 % maltose
for 24 h at 37 °C and 600 rpm. After
cultivation, the culture was mixed
with chloroform at 6:1 ratio and
incubated for 1 h at 25 °C to kill the
producing E. coli. The samples were
centrifuged at 5200 g and 4 °C for
10 min to remove E. coli and then at
10000 g and 4 °C for 6 min to remove
remaining contaminants. Next the
supernatant containing phage was
ultracentrifuged at 130000 g and
4°C for 3 h. The pellet containg
phage was resuspended in PBS at
protein concentration of 15 μg/ml
and stored at 4 °C.
Encapsulation of doxorubicin in
phage λ
The doxorubicin was encapsulated
in purified phage by infusion
method. 80 μl of phage was mixed
with 80 μl of doxorubicin at different
concentrations (200; 100; 50; 25;
12.5 and 0 μg/ml). Incubation was
conducted for 2 h at 25 °C in dark.
Free doxorubicin was subsequently
dialyzed using Amicon 3K (MerckMillipore, MA, USA) for 15 min at
7000 g and 20 °C and phage was
twice rinsed with water. The volume
was then filled to original volume.
Encapsulation of doxorubicin in
apoferritin
The doxorubicin was encapsulated
in apoferritin by opening and
closing of apoferritin in various pH.
80 μl of apoferritin (15 μg/ml) was
mixed with 80 μl of doxorubicin at
different concentrations (200; 100;
50; 25; 12.5 and 0 μg/ml). 1 μl of
1M hydrochloric acid was added
to sample to lower the pH to 2 and
open the apoferritin. The samples
were mixed for 15 min at 25 °C
and 600 rpm. 1 μl of 1M sodium
hydroxide was added to higher the
pH to 7 and encapsulate doxorubicin
in apoferritin. Free doxorubicin was
subsequently dialyzed using Amicon
3K (Merck-Millipore, MA, USA) for 15
min at 7000 g and 20 °C and phage
was twice rinsed with water. The
volume was then filled to original
volume.
Verification of doxorubicin
encapsulation in nanocarriers
Absorption spectra of 50 μl of
nanocarriers with encapsulated
doxorubicin were measured from
200 to 800 nm. Emission spectra
of samples were measured with
excitation at 480 nm (absorption
maximum of doxorubicin) and
emission from 515 to 815 nm.
Results and conclusions
In this work, well-studied coliphage
λ was used as a nanocarrier for
doxorubicin, using the infusion
method of encapsulation. The
successful
encapsulation
was
proven
by
absorbance
and
fluorescence measurement of the
whole phage. The absorbance of
phage λ at 480 nm increased from
0.06 to 0.22 after encapsulation
of 200 μg/ml doxorubicin. The
fluorescence of phage λ at 600
nm (the emission maximum of
doxorubicin) increased from 1000 to
22000 after encapsulation of 200 μg/
ml doxorubicin.
Encapsulation by phage λ was
compared
with
well-studied
nanocarrier apoferritin. At the same
concentration,
apoferritin
was
able to encapsulate 4 times lower
amount of doxorubicin than phage.
Undesired release of doxorubicin
from apoferritin was 10 times higher
in comparison with phage. It can be
concluded, that phage λ can serve as
a suitable nanocarrier for anticancer
drug doxorubicin.
The authors gratefully acknowledge
financial support from the Grant
Agency of the Czech Republic
(NANOCHEMO GA CR 14-18344S).
Fluorescenční charakterizace
zlatem modifikovaného
liposomu s uzavřenými
protinádorovými léčivy
a s připojenou antisense
N-myc DNA uchycenou
k magnetickým částicím
Lukas Nejdl1,2, Sylvie
Skalickova1,2, Jiri Kudr1,2, Ana
Maria Jimenez Jimenez1,
Branislav Ruttkay-Nedecky1,2,
Tomas Eckschlager3, Marie
Stiborova4, Vojtech Adam1,2,
Rene Kizek1
Mendelova univerzita v Brne;,
Agronomicka fakulta, Ustav
chemie a biochemie, Brno, Czech
Republic,
2
Stredoevropsky technologicky
institut CEITEC VUT v Brne, Brno,
Czech Republic,
3
Ustav pediatricke hematologie
a onkologie, 2. lekarska fakulta
Karlova univerzity, a univerzitni
nemocnice Motol, Praha, Czech
Republic,
4
Ustav biochemie, Prirodovedecka
fakulta, Karlova univerzita, Praha,
Czech Republic
1
Abstract book
Introduction
Onkogen N-myc hraje důležitou
roli během vývoje a diferenciace
neuroektodermu. Jeho nadměrná
exprese výrazně přispívá k malignímu
potenciálu
buňky.
Nadbytečná
exprese tohoto genu je spojena se
vznikem několika nádorů, převážně
s neuroblastomem. Neuroblastom
je maligní embryonální nádor
dětského věku odvozený z nezralých
a nediferencovaných buněk neurální
lišty. V současné době mají v léčbě
rakoviny velký potenciál liposomy
pro cílený transport léčiv a genovou
terapii. Cílem této studie bylo
vyvinout nanokonstrukt skládající
se z oligonukleotidu (AAA-ODNSH) značeného na jedné straně
adeninovými
repeticemi
pro
vazbu na tyminem modifikovanou
magnetickou částici a na straně
druhé thiolovou skupinou pro vazbu
zlatem modifikovaného liposomu
s uzavřenými léčivy (etoposidem,
elipticinem
a
doxorubicinem).
Námi navržený nanokonstrukt nesl
specifickou sekvenci anti-sense
oligonukleotidu
(ODN
sonda),
který je schopen blokovat expresi
N-myc genu. Doxorubicin, elipticin
a etoposid a jejich enkapsulované
varianty
byly
charakterizovány
pomocí fluorescenční spektroskopie.
Materials/methods
Fluorescenční spektra byly získány
pomocí automatického fluorimetru
Tecan Infinite 200 PRO (TECAN,
Švýcarsko).
Excitační
vlnové
délky pro elipticin, doxorubicin a
etoposid byly 420, 480 and 250 nm.
Fluorescenční sken pro sledované
látky byl v rozmezí 450 - 850 nm pro
elipticin, 510 - 850 pro doxorubicin
a 280 - 850 nm pro etoposid.
Citlivost detektoru byla nastavena
na 100%. Absorbance ssDNA byla
měřena v rozmezí vlnových délek
260 - 280 nm. Vzorky objemu 2 µl
byly dávkovány na 16 jamkovou
nanodestičku Tecan NanoQuant
plate (TECAN, Švýcarsko). Všechna
měření byla provedena při 30 °C.
Results and conclusions
Účinnost uzavření léčiv do liposomu
byla
sledována
fluorescenční
analýzou léčiv a léčiv uzavřených do
liposomu. Z rozdílů fluorescence byla
vypočítaná účinnost enkapsulace
50 %. Dále byl sledován výtěžek
hybridizace
nanokonstruktu,
ukotveného na magnetické částici,
stanovením koncentrace ssDNA.
Po každém hybridizačním kroku
byly nenavázané oligonukleotidy
odstraněny promýváním ukotveného
nanokonstruktu
na
magnetické
částici. Pro oligonukleotid navázaný
na magnetickou částici a liposom
(AAA-ODN-SH) byl výtěžek 8 %,
pro ODN sondu s cílovou sekvencí
pro N-myc gen byl 6 % a konečný
výtěžek
hybridizace
N-myc
genu k nanokonstruktu byl 4%.
Koncentrace enkapsulovaných léčiv
v nanokonstruktu byla 0,8 µg/ml pro
doxorubicin, 10 µg/ml pro etoposid
a 60 µg/ml pro elipticin. V naší
studii jsme uvedli možný způsob
využití nanokonstruktu pro genovou
terapii, tak jako pro značení cílových
neuroblastomových buněk.
Autoři
děkují
za
finanční
podporu grantové agentuře ČR
(NANOCHEMO GA CR 14-18344S).
SLEDOVÁNÍ INTERAKCE
DOXORUBICINU
A ELIPTICINU S BIOLOGICKY
ODBOURATELNOU
SLOUČENINOU - ALBUMINEM
Sylvie Sklickova1,2, Lukas
Nejdl1,2, Martina Omastova1,
Branislav Ruttkay-Nedecky1,2,
Tomas Eckschlager3, Marie
Stiborova4, Vojtech Adam1,2,
Rene Kizek1,2
Department of Chemistry and
Biochemistry, Mendel University in
Brno, Brno, Czech Republic,
2
Central European Institute of
Technology - Brno University of
Technology, Brno, Czech Republic,
3
Department of Paediatric
Haematology and Oncology,
2nd Faculty of Medicine, Charles
1
University, and University Hospital
Motol, Prague, Czech Republic,
4
Department of Biochemistry,
Faculty of Science, Charles
University, Pregue, Czech Republic
Introduction
Doxorubicin (hydroxyldaunorubicin)
je
antracyklinové
cytostatikum
s vysokou účinností. Jde o
kardiotoxickou látku, proto jej
lze podávat jen v omezených
dávkách. Elipticin a jeho deriváty
jsou účinné protinádorové látky,
které
fungují
prostřednictvím
různých mechanismů účastnících
se zastavení buněčného cyklu a
zahájení apoptózy. Interakcí s DNA
však dochází k jejímu poškození.
Bovinní sérový albumin (BSA)
obsahuje 2 tryptofany (Trp-134, Trp212). Lidský sérový albumin (HSA)
má naproti tomu jen 1 tryptofan
(Trp-214). Tryptofan je velmi citlivý
na změny okolního prostředí. Při
jeho zkoumání se proto využívá
vlastní fluorescence. Princip zhášení
fluorescence je charakterizován
bimolekulárními
deaktivačními
mechanismy, které zahrnují přenos
energie z jedné molekuly na druhou.
Jde o proces, který snižuje kvantový
výtěžek fluorescence, vyvolaný
molekulárními
interakcemi
se
zhášecí molekulou. Snímání zhášení
fluorescence tryptofanu BSA a
HSA lze tedy využít pro sledování
interakce doxorubicinu a elipticinu
se sérovými proteiny.
Materials/methods
Fluorescenční spektra byla naměřena
pomocí fluorimetru Tecan Infinite
200 PRO (TECAN, Švýcarsko).
Excitační vlnová délka pro albumin
byla 280 nm. Fluorescenční sken
byl v rozsahu 280 - 430 nm. Citlivost
detektoru byla nastavena na 100%.
Vzorky byly měřeny 2x na 96 jamkové
mikrotitrační destičce Costar (Fisher
Scientific, USA). Všechna měření
probíhala při 30 °C .
Results and conclusions
Interakci sérových proteinů s
doxorubicinem a elipticinem lze
sledovat prostřednictvím zhášení
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A46
Abstract book
fluorescence tryptofanu bovinního
sérového
albuminu
(BSA,
2
tryptofany) a lidského sérového
albuminu (HSA, 1 tryptofan). Tímto
zhášením fluorescence se snižuje
kvantový výtěžek fluorescence,
který je vyvolaný molekulárními
interakcemi se zhášecí molekulou.
Cílem práce byla charakterizace
interakce albuminu s doxorubicinem
a elipticinem v různém pufračním
prostředí
pomocí
UV/VIS
spektrofotometrie. Jako prostředí
pro interakci byl zvolen fyziologický
roztok (0,9 % NaCl). Pro interakci
BSA a HSA s doxorubicinem byly
stanoveny rovnice regrese y =
-49,41x + 5033,9 a y = -30,459x +
2638,1 s R² = 0.9594 a 0.8956. Pro
interakci BSA a HSA s elipticinem
byly stanoveny rovnice regrese y =
-622.61x + 103876 a y = -314.38x
+ 47559 s R² = 0.9203 a 0.8925. Z
výsledků vyplývá, že se stoupajícími
koncentracemi
doxorubicinu
a elipticinu se lineárně snižuje
emitované záření albuminu. Strmější
pokles intenzity fluorescence byl
pak zjištěn u vzorků, které neprošly
hodinovou inkubací.
Autoři děkují za finanční podporu
grantové agentuře ČR (GA CR
CYTORES P301/10/0356).
STUDY OF AFFINITY OF
COORDINATION COMPLEXES
OF METAL IONS TO DNA
BASED ON THE CHANGE OF
FLUORESCENCE INTENSITY
OF INTERCALATION LABELS
(DOXORUBICIN AND
ETHIDIUM BROMIDE)
Lukas Nejdl1,2, Jiri Kudr1,2,
Sylvie Skalickova1,2, Branislav
Ruttkay-Nedecky1,2, Tomas
Eckschlager3, Marie
Stiborova4, Vojtech Adam1,2,
Rene Kizek1,2
Central European Institute of
Technology, Brno University
of Technology, Brno, Czech
Republic,
2
Department of Chemistry
and Biochemistry, Laboratory
of Metallomics and
Nanotechnologies, Mendel
1
University in Brno, Brno, Czech
Republic,
3
Department of Paediatric
Haematology and Oncology,
2nd Faculty of Medicine, Charles
University, and University Hospital
Motol, Prague, Czech Republic,
4
Department of Biochemistry,
Faculty of Science, Charles
University, Prague, Czech Republic
Introduction
Metals are ubiquitous and essential
for cellular processes in all living
organisms [1, 2]. Metal ions
coordination to the nucleic acids is
critical for their structural properties
and proper function [3]. Given that
the
cytostatic
platinum-based
drugs are successful in a number
of cancer diseases, enormous
research developing metal-based
anticancer agents and to elucidating
the mechanisms involved in the
action of these compounds. The
success of platinum complexes
in cancer therapy is due to their
ability to bind with coordination
bond to the DNA bases [4, 5]. The
formation of these bonds results in
influencing the secondary structure
of DNA and thereby blocking of
important cellular processes such
as replication and transcription [6].
Platinum compounds are still the
most effective cytostatic drugs,
although a lot of Ru(II), Os(II) and/or
Ir(II) compounds have a quite similar
properties [7]. In this work affinity
of cobalt [Co3(tet)3(m-btc)](ClO4)3
and nickel [Ni2(tet)2(m-tda)](ClO4)2
coordination complexes, cisplatin
and Hg(II) ions with neuroblastoma
cell line SIMA was studied.
Materials/methods
All chemicals cisplatin, Hg(NO3)2
and ACS water were supplied from
Sigma Aldrich.
Preparation
of
coordination
complexes
[Co3(tet)3(m-btc)](ClO4)3
Triethylenetetramine hydrate (tet)
(0.2 g, 1 mmol) was added to a stirred
solution of Co(ClO4)2.6H2O (0.36 g,
1 mmol) in water (50 mL). Solution
of
1,3,5-benzenetricarboxylic
acid (btcH3) (0.07 g, 0.33 mmol)
neutralized with NaOH (0.04 g, 1
mmol) in water (5 mL) was added.
Finally, pH was adjusted to 7 with
addition of perchloric acid and
solution was diluted with water to
final volume of 100 mL.
[Ni2(tet)2(m-tda)](ClO4)2
Tet (0.2 g, 1 mmol) was added to a
stirred solution of Ni(ClO4)2.6H2O
(0.36 g, 1 mmol) in water (50 mL).
Solution of thiodiacetic acid (tdaH2)
(0.075 g, 0.5 mmol) neutralized with
NaOH (0.04 g, 1 mmol) in water (5 mL)
was added. Finally, pH was adjusted
to 7 with addition of perchloric acid
and solution was diluted with water
to final volume of 100 mL.
UV / Vis spectrophotometry and
fluorescence analysis
Fluorescence and absorption
spectra were acquired by a
multifunctional microplate reader
Tecan Infinite 200 PRO (TECAN,
Switzerland). Wavelength of 545
nm was used as an excitation
wavelength and the fluorescence
scan was measured within the range
from 575 to 800 nm per 2-nm steps.
The detector gain was set to 100. 50
μl of sample was placed to each well.
All measurements were performed at
30°C controlled by the Tecan Infinite
200 PRO (TECAN, Switzerland).
Cyclic voltammetry
Cyclic voltammetry was measured
in the range of potentials -1 to 1 V.
Screen-printed electrodes was used
[8]. Changes in electrochemical
signals were recorded with a
potentiostat PGSTAT 101 (Metrohm,
Herisau, Switzerland) and the results
were evaluated by the Software
NOVA 1.8 (Metrohm, Herisau,
Switzerland).
Results and conclusions
Synthesis, electrochemistry and
spectroscopic
techniques
are
described for cobalt [Co3(tet)3(mbtc)](ClO4)3 and nickel [Ni2(tet)2(mtda)](ClO4)2
coordination
complexes, cisplatin and Hg(II)
ions. Their stability in water have
been studied by electrochemical
methods (cyclic voltammetry) using
the screen printed electrode. It was
electrochemically demonstrated that
Abstract book
the studied complexes and Hg(II)
ions in comparison with cisplatin
are more stable in water, because
cisplatin showed after 48 hours a
significant change in reduction signal
at potential -0.13 V. The affinity of
these substances with DNA isolated
from the neuroblastoma cell line
SIMA was also studied and assessed
by fluorescence spectroscopy. The
change in fluorescence intensity of
doxorubicin and ethidium bromide
(EtBr) was monitored. EtBr is a
typical mutagen and intercalating
dye for DNA and it has been studied
for many years [9]. This method
was investigated by Zhu et al. when
studying interactions of DNA with
5-hydroxymethyl-2-furfural [10]. In
contrast with doxorubicin can be
observed decrease in fluorescence
intensity upon intercalation into
DNA. For all the studied substances
higher affinity to DNA as to cisplatin
was demonstrated.
Acknowledgements
The authors gratefully acknowledge
financial support from the Grant
Agency of the Czech Republic (GA
CR CYTORES P301/10/0356).
References
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Gitlin J, Lill R (2006)
Biochim. Biophys. Acta-Mol. Cell
Res. 1763:577-577. DOI 10.1016/j.
bbamcr.2006.06.011
[2]
Florea AM, Büsselberg D
(2011) Cancers 3:1351-1371.
[3]
Park KS, Park HG (2014)
Curr. Opin. Biotechnol. 28:17-24.
DOI 10.1016/j.copbio.2013.10.013
[4]
Chang CL, Lando DY,
Fridman AS, Hu CK (2012)
Biopolymers 97:807-817. DOI
10.1002/bip.22077
[5]
Brabec V, Kasparkova
J (2005) Drug Resist. Update
8:131-146. DOI 10.1016/j.
drup.2005.04.006
[6]
Theile D, Detering JC,
Herold-Mende C, Dyckhoff G,
Haefeli WE, Weiss J, Burhenne
J (2012) J. Pharmacol. Exp.
Ther. 341:51-58. DOI 10.1124/
jpet.111.189621
[7]
Dhahagani K, Mathan
KS, Chakkaravarthi G, Anitha K,
Rajesh J, Ramu A, Rajagopal G
(2014) Spectroc. Acta Pt. A-Molec.
Biomolec. Spectr. 117:87-94. DOI
10.1016/j.saa.2013.07.101
[8]
Prasek J, Trnkova L,
Gablech I, Businova P, Drbohlavova
J, Chomoucka J, Adam V,
Kizek R, Hubalek J (2012) Int. J.
Electrochem. Sci. 7:1785-1801.
[9]
Bi SY, Zhang HQ, Qiao CY,
Sun Y, Liu CM (2008) Spectroc.
Acta Pt. A-Molec. Biomolec.
Spectr. 69:123-129. DOI 10.1016/j.
saa.2007.03.017
[10]
Zhu J, Chen L, Dong Y, Li J,
Liu X (2014) Spectrochimica acta.
Part A, Molecular and biomolecular
spectroscopy 124:78-83. DOI
10.1016/j.saa.2013.12.091
Nanotools in microRna
isolation and detection
Kristyna Smerkova1,2,
Marcela Vlcnovska2,
Veronika Vlahova2, Marketa
Vaculovicova1,2, Rene Kizek1,2
Central European Institute of
Technology, Brno University of
Technology, Brno, Czech Republic,
2
Department of Chemistry
and Biochemistry, Laboratory
of Metallomics and
Nanotechnologies, Mendel
University in Brno, Brno, Czech
Republic
Introduction
The microRNA (miRNA) molecules
belong to small, non-coding RNAs
that regulate gene expression. The
miRNAs significantly affect different
biological
processes
including
pathological processes such as the
cancer origin and development.
The aberrant miRNA expressions
were found at different tumor types
and moreover the various tumors
have specific expression profiles.
Because of their importance as
diagnostic and prognostic cancer
biomarkers the rapid, low-cost and
sensitive detection technique is
required. The magnetic particles
ensure specific miRNA isolation and
in combination with electrochemistry
detection it is possible to achieve
nanomolar detection limit. The
other nanoparticles used for miRNA
determination in this work were
1
quantum dots (QDs), providing
specific labeling.
Materials/methods
Magnetic particles-based miRNA
isolation
The magnetic microparticles (MPs)
Dynabeads M-270 Streptavidin (Life
Technologies) were used for miRNA
isolation. After washing of MPs
the biotinylated complementaryDNA probe immobilization on MPs
surface was done using 500 µg of
MPs and 3 µl of 100 µM biotinylated
complementary-DNA probe. The
mixture was incubated for 10
minutes. Further, the hybridization
with the target miRNA was
performed according to Huska et al.
(Talanta, 2009). Subsequently, the
MPs were resuspended in 50 µl of
the elution solution (0.2 M NaCl, 0.1
M Na2HPO4 and 0.1 M NaH2PO4).
During the elution, the sample was
heated to 70°C for 5 minutes and
this caused double-stranded RNA
(dsRNA) denaturation and the miRNA
was released from MPs surface.
Conjugation of quantum dots with
streptavidin, biotinylated probe and
microRNA
CdTe
QDs
capped
with
mercaptopropionic acid precipitated
by isopropanol (0.1 mg/ml in
water) were mixed with 10 µl of
carbodiimidazole (10 mM in 100
mM PBS pH=7.4) and the solution
was kept at 25°C for 30 min. The
mixture was mixed with streptavidin
solution (final concentration of
streptavidin was 0.1 mg/ml) and
the reaction took place at 25°C for
2 hours. Subsequently, streptavidincoated QDs were conjugated
with biotinylated complementaryDNA probe (25°C for 1 hour). QDs
modified by probe were hybridized
with target miRNA at 25°C for 1 hour.
Capillary electrophoresis with laserinduced fluorescence detection (CELIF)
CE-LIF analysis was carried out
using
PACE/MDQ
instrument
(Beckman Coulter). Argon ion laser
with wavelength 488 nm was used
as an excitation light source and
emission was measured at 510 nm.
A47
A48
Abstract book
20 mM sodium borate buffer was
used as separation electrolyte and
separation voltage was 10 kV. The
sample was injected by pressure 5
psi for 5 seconds into the capillary
with 75 μm internal diameter, 63.5
cm total length and 53.5 cm effective
length.
Results and conclusions
Isolation based on streptavidincoated
MPs
is
thanks
to
their surface modification by
biotinylated probe highly specific.
Optimal amount of biotinylated
complementary-DNA probe was
found to be 1.02 µg per 500 µg of
MPs to cover to MPs surface. The
magnetic separation selectivity was
verified using non-complementary
oligonucleotides.
The
noncomplementary
oligonucleotides
signal was approximately 15 times
lower in comparison with target
complementary
miRNA.
This
significantly higher isolation yield of
target miRNA confirms the optimized
magnetic separation specificity.
The coupling of QDs with microRNA
caused the fluorescence signal
change of both the migration time
and peak shape. The electrophoretic
mobilities for QDs modified with
streptavidin,
QDs-streptavidin
modified with probe and QDsstreptavidin-probe with hybridized
miRNA were -16.84×10-9 m2/V/s,
-20.83×10-9 m2/V/s and -21.66×109 m2/V/s, respectively.
The authors gratefully acknowledge
financial support from the Grant
Agency of the Czech Republic
(NanoBioTECell P102/11/1068).
Charakterizace dlouhé
nekódující RNA MALAT-1
u buněk karcinomu prsu
Jaroslav Juracek1, Alexandra
Kondelova2, Marek
Svoboda1,2, Ondrej Slaby1,2
Masarykuv onkologický ustav,
Klinika komplexni onkologicke
pece, Brno, Czech Republic,
2
CEITEC, Masarykova univerzita,
Brno, Czech Republic
Introduction
Přesto že strukturní geny představují
1
u člověka méně než 2 % lidského
genomu,
celogenomové
studie
odhalily, že až 90 % z celkového
počtu genů je aktivně transkribováno.
Tyto RNA, kterým však není
přisuzována žádná protein-kódující
kapacita, byly původně považovány
za odpadní molekuly nahromaděné
během evoluce. Nedávný výzkum
však poukazuje na jejich důležitost
ve fyziologických i patologických
buněčných procesech. Na základě
délky transkriptu jsou nekódující RNA
děleny na krátké a dlouhé. Zatímco
krátké nekódující RNA, mezi které
se řadí např. mikroRNA, jsou dnes
již velmi dobře charakterizovány,
dlouhé nekódující RNA (lncRNA) byly
dosud značně přehlíženy. Do této
obsáhlé skupiny přitom spadá velké
množství 200-10000 bp dlouhých
RNA, které hrají klíčové role jak v
transkripční, tak v post-transkripční
regulaci genové exprese. Kromě
toho byla u mnoha onemocnění
včetně nádorových pozorována
deregulovaná hladina lncRNA a
proto lze předpokládat jejich zapojení
do procesu maligní transformace.
Většina lncRNA vystupuje jako
onkogeny, mezi něž patří také
MALAT-1, jejíž zvýšená exprese
byla potvrzena i u karcinomu prsu.
Právě u tohoto onemocnění je i přes
snižující se mortalitu nutné hledat
nové terapeutické cíle, zejména z
důvodu častého rozvoje rezistence
na stávající léčbu. Studium dlouhých
nekódujících RNA v tomto kontextu
představuje
významný
zdroj
potenciálních cílových molekul.
Materials/methods
Z
důvodu
bližší
identifikace
působení dlouhé nekódující RNA
MALAT-1 na buňky karcinomu prsu
byly pomocí qRT-PCR stanoveny
hladiny exprese této lncRNA u
pěti stabilních buněčných liniích
karcinomu prsu. Následná in vitro
charakterizace byla provedena na
dvou buněčných liniích, u kterých byl
pozorován vliv utlumení MALAT-1
pomocí sekvenčně homologní siRNA
na proliferaci, apoptózu, migraci a
klonogenicitu.
Results and conclusions
Po umělém snížení hladiny MALAT-1
pomocí transfekce siRNA nebyl
pozorován významný vliv této
onkogenní lncRNA na proliferaci,
apoptózu ani migraci. Snížení
exprese MALAT-1 však mělo
významný inhibiční účinek na tvorbu
kolonií nádorových buněk u obou
testovaných linií, což by mohlo
naznačovat zapojení MALAT-1 do
rozvoje metastáz.
DEREGULACE PIWIL
PROTEINŮ A VYBRANÝCH
PIRNA U RCC A CRC
Robert Iliev1, Petra
Vychytilova1, Michal Stanik3,
Jan Dolezel3, Michal
Fedorko4, Dalibor Pacik4,
Marek Svoboda2, Ondrej
Slaby1,2
CEITEC, Laborator molekularni
onkologie II - solidni nadory, Brno,
Czech Republic,
2
MOU, Klinika komplexni
onkologicke pece, Brno, Czech
Republic,
3
MOU, Oddeleni urologicke
onkologie, Brno, Czech Republic,
4
FN Brno, Urologicka klinika, Brno,
Czech Republic
Introduction
RNA interagující s PIWIL proteiny
jsou genetické a epigenetické
buněčné regulační faktory, které
udržují genomovou stabilitu a jsou
začleněny v umlčování a regulaci
genové exprese. PiRNA jsou krátké
jednořetězcové RNA dlouhé 26 až
31 nukleotidů vázající se vážou na
PIWIL proteiny. Jedná se podrodinu
proteinů typu Argonaut. U lidí
se rodina Piwi proteinů skládá z
proteinů PIWIL1, PIWIL2, PIWIL3 a
PIWIL4. Fyziologická exprese PIWIL
proteiů byla nejdříve pozorována
hlavně v zárodečných a kmenových
buňkách. Současné studie ukazují,
že deregulovaná exprese PIWIL
proteinů je společná mnoha typům
nádorů. Je prokázáno, že exprese
PIWIL proteinů koreluje s klinickými
parametry a s horší prognózou u
pacientů s nádorem prsu, cervixu,
vaječníku, střev a dalších. V
prvních studiích bylo pozorováno,
že se podílejí na umlčování
1
Abstract book
transponovatelných
elementů.
Změněná exprese piRNA byla však
v poslední době zjištěna i u nádorů
prsu a žaludku.
Materials/methods
Pro náši pilotní studii byly k analýzám
vybrány kromě PIWIL proteinů i
piR-X a piR-Y, jejichž deregulovaná
exprese byla zjištěna u různých
typů nádorů. V experimentu jsme
použili nádorovou a příslušnou
nenádorovou tkáň 56 pacientů s
renálním karcinomem (RCC). Z
tkání jsme izolovali celkovou RNA
a pomocí metody RT-qPCR byla
stanovena exprese genů PIWIL,
piR-X a piR-Y. Pro analýzu sérových
piRNA jsem byl použit soubor 125
pacientů s RCC, 100 pacientů s
kolorektálním karcinomem (CRC)
a 75 kontrolních sér od zdravých
jedinců.
Results and conclusions
Po porovnání hladin RNA jsme
objevili
významný
rozdíl
v
expresních hladinách piR-X, piR-Y
a PIWIL1 u párových vzorků mezi
nenádorovou a nádorovou tkání. U
hladin exprese PIWIL2, PIWIL3 a
PIWIL4 jsme neprokázali statisticky
významné rozdíly. Hladiny PIWIL2 a
PIWIL4 u pacientů s RCC významně
korelovaly se stagem a gradem.
Identifikovali jsme také korelaci mezi
vyšší hladinou PIWIL1, piR-X a piR-Y
a delším celkovým přežitím. U piR-X
byla zjištěna signifikantně snížená
hladina v krevním séru pacientů s
RCC. Po ROC analýze jsme odlišili
pacienty a kontrolní jedince se
sensitivitou 77 % a specificitou 72
%. U analyzovaných hladin sérové
piR-Y v séru pacientů s RCC a
CRC jsme zjistili signifikantní rozdíly
v expresi oproti nenádorovým
kontrolním
vzorkům,
přičemž
pacienti s RCC a CRC měli sníženou
hladinu exprese (RCC - p<0.0001,
CRC – p<0.0001). Pacienty s RCC
jsme po ROC analýze dokázali
odlišit od kontrolních jedinců s 97%
senzitivitou a 99% specificitou.
Skupinu pacientů s CRC jsme od
kontrolních jedinců dokázali odlišit s
94% senzitivitou a 91% specificitou.
Po dalším detailním výzkumu role
piR-Y v patogenezi RCC a CRC by
mohla sloužit nejen jako diagnostický
prostředek, ale také jako potenciální
terapeutický cíl.
Navýšení exprese miR-215
in vivo ovlivňuje velikost
nádoru v animálním modelu
kolorektálního karcinomu
Jana Merhautová1,2, Petra
Vychytilová2, Regina
Demlová1, Ondřej Slabý2
Farmakologický ústav, Lékařská
fakulta, Masarykova univerzita,
Brno, Czech Republic,
2
Skupina Molekulární medicína
II – solidní nádory, CEITEC,
Masarykova univerzita, Brno, Czech
Republic
Introduction
MikroRNA jsou krátké nekódující
RNA, které působí jako regulátory
exprese
většiny
genů.
Byla
publikována řada prací prokazující
dysregulaci mikroRNA u nádorových
onemocnění, mimo jiné také u
kolorektálního karcinomu (CRC),
v jehož incidenci zaujímá ČR
celosvětově přední příčky. V
přecházející
sérii
experimentů
jsme identifikovali miR-215 jako
specifickou pro tkáň CRC. Tato
mikroRNA v in vitro testech prokázala
tumorově supresorové vlastnosti.
Prezentované in vivo experimenty
ověřují její působení v kontextu
celého organismu. Posloužily také
k zavedení a optimalizaci techniky
subkutánní xenotransplantace na
našem pracovišti.
Materials/methods
a) Transfekce a selekce klonů: Buňky
linie HCT-116+/+ byly lipofekcí
transfekovány CMV vektorem s
klonovanou sekvencí pre-miR-215 a
kontrolním mock-vektorem (OriGene
Technologies, Rockville, MD, USA).
Stabilně transfekované buňky byly
selektovány pomocí G-418 a za
fluorescenční kontroly exprese GFP.
Po limitním naředění bylo provedeno
namnožení 14 jednotlivých klonů
buněk a jejich charakterizace
(morfologie, rychlost proliferace,
exprese miR-215 a genů XIAP,
EREG, TYMS, HOXB9 a CD164).
Vybrán byl klon s nejpříznivějším
1
expresním profilem a nejnižší
rychlostí proliferace.
b) Subkutánní xenotransplantace:
1. pilotní experiment - 3 samicím
NSG myši bylo na dorzální stranu
těla vpravo subkutánně aplikováno
5×106 buněk klonu 33 miR-215HCT-116+/+. Jako kontrola byl
aplikován vlevo stejný počet buněk
mock-HCT-116+/+. 27. den po
aplikaci byl pokus ukončen, nádory
byly změřeny a zváženy a byl
proveden odběr vzorku nádorové
tkáně do RNAlateru a neutrálního
formalínu. 2. pilotní pokus - 5
samcům NSG myši bylo analogicky
aplikováno 2,5×106 buněk klonu
33 miR-215-HCT-116+/+. Jako
kontrola byl aplikován vlevo stejný
počet buněk mock-HCT-116+/+. 24.
den po aplikaci byl pokus ukončen,
nádory byly změřeny a zváženy a byl
proveden odběr vzorku nádorové
tkáně do RNAlateru a neutrálního
formalínu.
Results and conclusions
V
předkládaném
experimentu
byly úspěšně připraveny stabilně
transfekované buňky linie odvozené
od CRC s navýšenou experesí miR215. Selekcí klonů s následnou
charakterizací
byl
vybrán
a
namnožen klon s optimálními
vlastnostmi (změna exprese miR215, genů XIAP, EREG a dalších).
Namnožený klon byl subkutánně
aplikován NSG myším. Po 1012 dnech byly v podkoží hmatné
tumory, experimenty byly ukončeny
27. resp. 24. den po aplikaci.
Odebraná nádorová tkáň byla
histologicky zhodnocena, nádorové
buňky byly nízce diferencované
a
vysoce
mitoticky
aktivní.
Makroskopicky i mikroskopicky
vykazovaly kontrolní tumory vyšší
míru nekróz než tumory vzniklé z
buněk s navýšenou expresí miR-215.
Velikost nádorů byla signifikantně
menší (p < 0,05) u tumorů vzniklých
z buněk s navýšenou expresí miR215 než u kontrolních tumorů.
Nebyly pozorovány mezipohlavní
rozdíly v růstu nádorů u zvířat. MiR215 ovlivňuje proliferaci nádorů – v
závislosti na stavu p53 indukuje
zástavu buněčného cyklu a zvyšuje
míru apoptózy nádorových buněk.
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A50
Abstract book
Molecular genetic analysis
of pheochromocytomas and
paragangliomas in Czech
patients
Musil Zdenek1,2, Stanek
Libor3, Vícha Aleš2, Vosecka
Tatiana2,4, Krepelova Anna4,
Zelinka Tomas5, Hamplova
Barbora5, Widimsky Jiri5,
Vocka Michal6, Turkova
Hana6, Puchmajerova Alena4,
Simandlova Martina4, Frysak
Zdenek7, Vaclavik Jan7,
Kohoutova Milada1
Institute of Biology and Medical
Genetics of the 1st Faculty of
Medicine and General Teaching
Hospital, Charles University,
Prague, Czech Republic,
2
Department of Paediatric
Haematology and Oncology,
University hospital Motol and
2nd Faculty of Medicine, Charles
University, Prague, Czech
Republic,
3
Institute of Pathology of the 1st
Faculty of Medicine and General
Teaching Hospital, Charles
University, Prague, Czech
Republic,
4
Department of Biology and Medical
Genetics, University hospital
Motol and 2nd Faculty of Medicine,
Charles University, Prague, Czech
Republic,
5 rd
3 Medical Department - Clinical
Department of Endocrinology
and Metabolism of the 1st Faculty
of Medicine, Charles University,
Prague, Czech Republic,
6
Department of Oncology of
the 1st Faculty of Medicine and
General Teaching Hospital,
Charles University, Prague, Czech
Republic,
7 rd
3 Clinical Department of Faculty
of Medicine, Olomouc, Czech
Republic
Introduction
Pheochromocytomas are tumors
arising from adrenal gland, whereas
paragangliomas are tumors arising
from
extra-adrenal
chromaffin
tissue. Functional paragangliomas
1
are secreting tumors which can
be found mainly in the abdomen
region, pelvis and chest. Contrarily,
paragangliomas located in the skull
region and neck are non-functional
in most cases. Most of these tumors
occur as sporadic tumors but up to
24% of them can be familial. The
prevalence of pheochromocytoma
can be estimated to lie between
1:1450 and 1:1700, with the annual
incidence of 3-8 cases out of a
million inhabitants in general public.
Up to 10% of PHEO/PARA is
malignant (they are able to produce
distant metastases or they are
tumors that recur). Genetic factors
have a significant role in the PHEO/
PARA development. Currently, 18
genes (SDHA, SDHB, SDHC, SDHD,
VHL, RET, NF1, MAX, TMEM 127,
SDHAF2, KIF 1B, HIF 2A, H-RAS,
K-RAS, IDH 1, PHD 2/ EGLN 1,
FH, BAP 1) participating in PHEO/
PARA development are described.
Pheochromocytomas
and
paragangliomas are also associated
with genetic syndromes (MEN2, VHL,
PGL 1 - 4, Pacak-Zhuang). Many
mutations can be found as somatic
mutations in cancerous tissues.
Materials/methods
Genetic testing, which should be
indicated in every patient, is guided
by the clinical presentation as well
as by the secretory phenotype
and
the
immunohistochemical
characterization of the tumours.
Genetic examinations may also
have prognostic significance, since
for example SDHB gene mutations
are connected with significantly
higher probability of malignancy.
Mutation detection can be helpful in
diagnostics of other manifestations
of
life-endangering
genetic
syndromes (for example MEN2related medullary carcinoma, or
VHL-related central nervous system
or abdominal tumors).
Results and conclusions
PHEO/PARA can be divided into two
groups according to the expression
change in genes being present in
various signaling pathways: the
first group leeds to the activation
of pseudohypoxic pathway (VHL,
SDHA, SDHB, SDHC, SDHD,
probably also HIF2A and FH), the
second group includes receptor
signaling kinases and protein
translation pathways (NF1, RET,
KIF1B, TMEM127 and MAX).
This work was supported by the
research projects PRVOUK-P27/
LF1/1, SVV-260023/2014 and the
research organisation 00064203
Skp2 associates with Slug and
androgen receptor in patients
with high Gleason score and
lymph node metastasis of
prostate cancer
Gvantsa Kharaishvili1, Jan
Bouchal1, Milan Kral2
Laboratory of Molecular Pathology,
Institute of Molecular and
Translational Medicine, Faculty of
Medicine and Dentistry, Palacky
University, Olomouc, Czech
Republic,
2
Department of Urology, University
Hospital Olomouc, Olomouc,
Czech Republic
Introduction
The Skp2 F-box protein is the
substrate recruiting component of
the SCF (Skp1-Cullin 1-F-box) type
of E3 ubiqutin-ligase complexes.
Skp2 plays important role in prostate
tumorigenesis which needs further
elucidation.
Materials/methods
Prostate cancer patients cohort
(N=101)
was
analyzed
by
immunohistochemistry
for
the
following proteins (Skp2, AR, Ki67, Slug, E-cadherin, beta-catenin
and PPAR gamma) and statistically
evaluated with clinico-pathological
parameters.
Results and conclusions
High Gleason score (>8, N=30) was
significantly associated with higher
nuclear Skp2 and lower E-cadherin
expression (p<0.001 and 0.011,
respectively) and with a trend for
higher androgen receptor (p=0.062).
Patients with metastasis in lymph
nodes (N=29) had lower E-cadherin
and cytoplasmic Skp2, (p<0.001 and
0.018, respectively), while nuclear
1
Abstract book
Skp2 and PPAR gamma, Slug (both
nuclear and cytoplasmic), Ki-67 and
androgen receptor showed a trend
(p=0.176, 0.179, 0.103, 0.032, 0.079
and 0.12, respectively) towards
higher expression in the prostate
cancer cells. Similar changes of the
mentioned proteins were observed
also in risk categorization (based
on serum PSA, pT classification
and Gleason score). Nuclear Skp2
slightly correlated with AR in the
whole patients cohort (Rs 0.37).
In patients with high Gleason
score, nuclear Skp2 potently
correlated with AR, nuclear Slug
and cytoplasmic PPAR gamma (Rs
0.53, 0.56 and 0.37, respectively).
In patients with metastasis into
lymph nodes, nuclear Skp2 similarly
correlated with nuclear Slug and AR
(Rs 0.56 and 0.37, respectively) while
androgen receptor further correlated
with Ki-67 (Rs 0.50).
In summary, Skp2 correlates
with
androgen
receptor
and
Slug which might contribute to
agressive prostate cancer. Further
mechanistical
elucidation
and
clinical validation is needed.
Ubiquitin specific peptidase 7
(USP7) regulates DNA damage
bypass pathway through
stabilizing Cdc7 kinase and
RAD18 ubiquitin ligase.
Zsofia Turi1, Marketa
Senkyrikova1, Jiri Bartek1,2,
Masayuki Yamada1
Laboratory of Genome Integrity,
Institute of Molecular and
Translational Medicine, Palacky
University, Olomouc, Czech
Republic,
2
Danish Cancer Society Research
Center, Copenhagen, Denmark
Introduction
Cdc7 kinase is essential to replicate
genomic DNA. However, its role in
DNA repair and recombination, as
well as its interplay with ubiquitinproteasome system, are poorly
understood. We recently described
a novel pathway that stabilizes the
human Cdc7-ASK (also called Dbf4),
its regulation, and its function in
1
cellular responses to compromised
DNA replication.
Stalled DNA replication evoked
stabilization of the Cdc7-ASK
complex in a manner dependent
on ATR-Chk1-mediated checkpoint
signaling and its interplay with the
anaphase-promoting
complex/
cyclosome(Cdh1)
(APC/CCdh1)
ubiquitin ligase. Mechanistically,
Chk1 kinase inactivates APC/CCdh1
through degradation of Cdh1 upon
replication block, thereby stabilizing
APC/CCdh1 substrates, including
Cdc7-ASK. Furthermore, motif C of
ASK interacts with RAD18, a ubiquitin
ligase essential for DNA damage
bypass pathway, and this interaction
is required for chromatin binding
of RAD18. Impaired interaction
of ASK with RAD18 disables foci
formation by RAD18 and hinders
chromatin loading of translesion
DNA polymerase , suggesting that
Cdc7 kinase plays a pivotal role in
DNA damage bypass pathway.
In this workshop, we will present new
data which strongly suggest that
USP7 is involved in DNA damage
bypass pathway through stabilizing
both Cdc7-ASK and RAD18.
Materials/methods
The protein abundance of Cdc7ASK and RAD18 as well as
monoubiquitylated PCNA, which is
catalysed by RAD18, were examined
in USP7-depleted U2OS cells under
replication stress by Western blotting.
HCT116-derived USP7 knockout
cell line was also used for the same
analysis. Moreover, foci formation
of RAD18 in USP7 knockdown cells
was examined by immuostaining
and subsequent quantitative data
analysis with ScanR software.
Results and conclusions
We found a significant decrease of
the protein levels of Cdc7-ASK and
RAD18 in USP7 knockdown cells
and USP7 knockout cells. We also
found that monoubiqutylation of
PCNA under replication stress was
impaired in these cells. In addition,
foci formation of RAD18 caused by
replication stress was disturbed by
knockdown of USP7. These data
indicate that USP7 regulates protein
stability of Cdc7-ASK and RAD18,
therefore plays an important role in
DNA damage bypass pathway.
Our new findings will define a
novel mechanism that orchestrates
ubiquitin-proteasome
machinery
and DNA damage bypass pathway
to guard against replication collapse
under conditions of replication
stress.
Is Epithelial to Mesenchymal
Transition Followed by Global
DNA Methylation Changes?
Svetlana Skolekova1,
Naouale El Ymani2, Mária
Dusinska2, Viera Kajabova1,
Tatiana Sedlackova3,
Iveta Zmetakova1, Tomas
Krivulcik1, Ivana Fridrichova1,
Miroslava Matuskova1,
Bozena Smolkova1
Cancer Research Institute of
Slovak Academy of Sciences,
Bratislava, Slovakia,
2
Health Effects Laboratory MILK,
NILU- Norwegian Institute for Air
Research, Kjeller, Norway,
3
Department of Molecular Biology,
Faculty of Natural Sciences,
Comenius University, Bratislava,
Slovakia
Introduction
Deregulation of the epigenetic
mechanisms, which developmentally
influence gene expression via
modifications to DNA, histone
proteins, and chromatin, have been
hypothesized to play a key role in
many human diseases including
cancer.
Tumour
cells
before
intravasation can undergo partial
or total epithelial mesenchymal
transition (EMT). Recent investigation
of the mechanisms controlling
EMT
revealed
a
significant
epigenetic regulatory impact. It
was shown, that soluble factors
in the tumour microenvironment
can orchestrate EMT and induce
cancer stem cells (CSC) formation
from more differentiated tumour
cells. We hypothesize, that cells
in their epigenetically plastic
1
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A52
Abstract book
state could be programmed by
the microenvironment, to acquire
epigenetic changes associated with
tumourigenesis and EMT induction.
Materials/methods
The aim of our study was to induce
EMT in in vitro model system and
simultaneously investigate changes
in global DNA methylation. As EMT
is one of the main mechanisms
underlying development of cancer
metastasis, which induces stem
like properties and confers drug
resistance, reversing of EMT process
could be a unique therapeutic
approach. Taking into account
reversible character of epigenetic
processes, epigenetic therapy offers
a potential way to influence those
pathways directly.
Results and conclusions
After induction of EMT by
mesenchymal stromal cells in
primary breast cancer cell culture
we
evaluated
EMT-associated
changes in global and gene
specific DNA methylation (CDH1
and TWIST promoter methylation).
Understanding of the epigenetic
regulation of EMT processes can
contribute to the new approaches in
epigenetic drugs discovery.
This work was supported by the
European Commission FP7 projects
NANoREG
[NMP.2012.1.3-3],
Contract no. 310584; QualityNano
[INFRA-2010-1.131] Contract no:
214547-2, NILU-TAF-279; VEGA
2/0169/14 grant and RFL2009
program founded by the Slovak
Cancer Research Foundation.
The effect of acquired
resistance to paclitaxel
on expression of ABC
transporters at breast cancer
cells: role of ABCB1
Dana Kopperová1, Matej
Behounek1, Kamila
Balušíková1, Vlasta
Nemcová1, Veronika
Brynychová2, Viktor Hlavác2,
Pavel Soucek2, Jan Kovár1
1
Department of Biochemistry, Cell
and Molecular Biology, Division
of Cell and Molecular Biology,
Third Faculty of Medicine, Charles
University in Prague, Prague,
Czech Republic,
2
Department of toxicogenomic,
National Institute of Public Health,
Prague, Czech Republic, Prague,
Czech Republic
Introduction
Acquired resistance of cancer
cells to chemotherapeutics is very
spread phenomenon that prevent
successful therapy of patients.
The increased expression of ABC
transporters represents one of main
suspect mechanisms of acquirement
of cancer cell resistance.
Materials/methods
We studied molecular mechanism of
resistance of cancer cells to taxanes
in breast cancer cell lines SKBR-3 and MCF-7 and in paclitaxelresistant sublines (derivate from the
original lines by cultivation in medium
with increasing concentration of
paclitaxel). We determined the
expression of all 49 human ABC
transporters at mRNA level by realtime PCR in paclitaxel-sensitive
and resistant sublines in SK-BR-3 a
MCF-7 cells. Moreover, the role of
transporter ABCB1 in resistance to
paclitaxel was tested at protein level
by western blot and by silencing of
its expression by specific siRNA.
Results and conclusions
We studied molecular mechanism of
resistance of cancer cells to taxanes
in breast cancer cell lines SKBR-3 and MCF-7 and in paclitaxelresistant sublines (derivate from the
original lines by cultivation in medium
with increasing concentration of
paclitaxel). We determined the
expression of all 49 human ABC
transporters at mRNA level by realtime PCR in paclitaxel-sensitive
and resistant sublines in SK-BR-3 a
MCF-7 cells. Moreover, the role of
transporter ABCB1 in resistance to
paclitaxel was tested at protein level
by western blot and by silencing of
its expression by specific siRNA.
Conclusions: Taken together, we
suppose increased efflux of paclitaxel
from cell in result from induction of
transporter ABCB1 expression. This
is probably significant but not the
only one mechanism responsible for
acquired resistance of tested breast
cancer cells cancer.
Vliv rentgenového záření na
genovou expresi v lidských
chlupových folikulech
Hanuš Slavík, Pavlína
Dušková, Laura Ewerlingová,
Karolína Burdová, Jiří
Drábek, Martin Mistrík
UMTM, Olomouc, Czech Republic
Introduction
Vlasové a chlupové folikuly jsou lehce
dostupným zdrojem genomické DNA
a RNA. Jejich odběr je méně invazivní
než biopsie a venopunkce a má
potenciál poskytovat diagnostické
biomarkery
vlivu
rentgenového
záření. Cílem našeho projektu je
zavést metodiku, která by umožnila
kvantifikaci exprese genů na dráze
ATM/CHEK2/p53, modifikovaných
vlivem záření.
Materials/methods
Chlupy byly odebrány pracovníkům
laboratoře
pomocí
speciální
vakuové pistole, která zamezuje
kontaminaci a degradaci vzorku a
standardizuje množství odebíraného
materiálu. Vzorky byly izolovány a
analyzovány ihned po odběru nebo
po předchozí 3 h či 24 h inkubaci v
buněčném médiu, kdy část z nich
byla vystavena záření dávkou 6,32
Gy. Celková RNA byla izolována
pomocí miRNase Mini Kit a eluována
do 30 µl. Množství a kvalita RNA byly
analýzovány na Agilent Pico RNA
Chipu, popřípadě fluorometricky.
Následovala dvou-kroková RTqPCR pro geny SESN1, CDKN1A a
MDM2, s výsledky normalizovanými
na expresi provozního genu HPRT1.
Reakce probíhaly jednotlivě (ve
čtyřech zkumavkách) s detekcí
signálu z interkalovaného fluoroforu
SYBR Green I.
Results and conclusions
Izolovali jsme dostatečné množství
RNA v dobré kvalitě i po 24 h
inkubaci ve vhodně zvoleném
buněčném médiu. Optimalizovali
Abstract book
jsme qPCR pro dané geny pro
dosažení specifické amplifikace.
Prezentujeme předběžné výsledky
optimalizace metodiky.
Práce na projektu byly prováděny
s podporou grantů LO1304 a
CZ.1.05/3.1.00/14.0307
Sada 6-ti mikroRNA predikuje
celkové přežívání u pacientů s
multiformním glioblastomem
Jiri Sana1,2, Lenka Radova1,
Radek Lakomy2, Leos
Kren4, Pavel Fadrus3, Jana
Nekvindova5, Rostislav
Vyzula2, Ondrej Slaby1,2
Stredoevropsky technologicky
institut - CEITEC, Masarykova
univerzita, Brno, Czech Republic,
2
Klinika komplexni onkologicke
pece, Masarykuv onkologicky
ustav, Brno, Czech Republic,
3
Oddeleni neurochirurgie, Fakultni
nemocnice Brno, Brno, Czech
Republic,
4
Oddeleni patologie, Fakultni
nemocnice Brno, Brno, Czech
Republic,
5
Ustav klinicke biochemie a
diagnostiky, Fakultni nemocnice
Hradec Kralove, Hradec Kralove,
Czech Republic
Introduction
Multiformní glioblastom (GBM) je
nejčastěji se vyskytující maligní
nádor mozku astrocytálního původu
s mediánem celkového přežívání
přibližně 13 měsíců od stanovení
diagnózy. Ačkoliv je prognóza
jednotlivých
pacientů
značně
rozdílná, histologické profily tohoto
nádorového onemocnění jsou si
navzájem velmi podobné. Proto je
velmi důležité najít takové molekulární
markery, které by onkologům v
klinické praxi umožnily s co největší
přesností
stanovit
prognózu
pacienta a predikovat odpověď na
standardně podávanou adjuvantní
konkomitantní chemoradioterapii s
temozolomidem (RT/TMZ) a případně
se tak rozhodnout pro intenzivnější
či alternativní způsoby léčby, jež se
v současné době začínají v léčbě
GBM testovat. Z tohoto pohledu jsou
nyní velmi diskutované mikroRNA
1
(miRNA), krátké nekódující RNA,
které
posttranskripčně
regulují
genovou expresi a jejich deregulace
tak výrazně ovlivňuje biologii mnoha
nádorových onemocnění, nevyjímaje
GBM.
Materials/methods
Pomocí technologie TaqMan Low
Density Array byla provedena
globální expresní analýza miRNA
u 58 vzorků FFPE tkáně GBM a 10
FFPE vzorků nenádorové mozkové
tkáně získané z temporálních laloků
resekovaných u pacientů trpících
epilepsií.
Results and conclusions
Porovnáním exprese miRNA v
nádorové a nenádorové tkáni
bylo identifikováno 28 významně
deregulovaných miRNA, které byly
zpětně schopny všechny zkoumané
vzorky správně klasifikovat. Navíc
korelace s klinickými daty pacientů
s GBM identifikovala sadu 6-ti
miRNA (miR-31, miR-224, miR-432*,
miR-454, miR-672 a miR-885-5p),
která byla významně asociována
jak
s
celkovým
přežíváním,
tak s přežíváním bez progrese
onemocnění pacientů s GBM. Na
základě dosažených výsledků se
tedy domníváme, že zmíněné miRNA
by mohly být slibnými diagnostickými
a prognostickými markery u GBM.
Následné in-vitro funkční analýzy
navíc ukázaly, že zvýšená exprese
miR-31 vedla k zástavě buněčného
cyklu, což se projevilo na snížené
proliferaci u GBM buněčných linií.
Současně došlo u těchto buněk k
významnému poklesu schopnosti
migrovat.
Práce byla podpořena grantovým
projektem
NT13514-4/2012
MZČR a projektem „CEITEC Středoevropský
technologický
institut” (CZ.1.05/1.1.00/02.0068).
The comparison of 2-D and
3-D model of gene therapy
mediated by genetically
modified mesenchymal
stem cells on human ovarian
carcinoma cells SKOV-3.
Lenka Toro, Lucia Kucerova
Cancer Research Institute of
Slovak Academy of Sciences,
Bratislava, Slovakia
Introduction
Adipose
tissue-derived
mesenchymal stem cells (AT-MSC)
may serve as vehicles carrying
genes capable of conversion of
non-toxic substances into their toxic
derivates to destroy the tumor. We
engineered AT-MSC to express
bacterial cytosine deaminase (BCD)
or yeast cytosine deaminase::uracil
phosphoribosyltransferase
(CD::UPRT), capable of converting
the prodrug 5-fluorocytosine (5-FC)
into highly toxic 5-fluorouracil (5FU). Synthetised toxic metabolites
induce apoptosis and together
with bystander effect increase the
elimination of tumor cells. SKOV3 represents a human ovarian
carcinoma cell line resistant to a
variety of chemotherapeutics.
Materials/methods
2-D model: Cell line SKOV-3/GFP
was cultured in concentration
gradient of 5-FU and the effect of
chemotherapeutic was evaluated
by
using
fluorimetric
assay,
chemoluminescent assay and cell
kinetic imaging. Also we tested the
chemosensitivity of SKOV-3 cells
alone or co-cultured with AT-MSC,
BCD-MSC or CD::UPRT-MSC in
the presence of 5-FC. The cytotoxic
effect of therapeutical AT-MSC was
evaluated using fluorimetric assay.
3-D model: SKOV-3/GFP cells were
seeding acording to CellPlayerTM
96-well Kinetic 3-D Spheroid
Protocol (Essen BioScience) with
different ratio of CD::UPRT-MSC
in the absence/presence of 5-FC.
Evaluation was performed by
chemoluminescence assay and cell
kinetic imaging.
Results and conclusions
SKOV-3
cells
showed
chemosensitivity upon exposure
to 5-FU. The treatment using
CD::UPRT-MSC is more efficient
than bacterial BCD-MSC under the
same conditions. Remarkably the
ratio 1:160 of CD::UPRT-MSC to
SKOV-3 cells was efficient enough
to eliminate 60-80% of tumor cells.
A53
A54
Abstract book
3-D cultivation of SKOV-3 cells with
CD:UPRT-MSC in the presence of
5-FC showed decreased response
to the treatment in comparison to
2-D culture. Nevertheless, the 40%
elimination of SKOV-3 cells was
obtained, when ratio of CD::UPRTMSC to SKOV-3 cells was 1:10 in
the presence of 5-FC.
MESENCHYMAL
STROMAL
CELLS PLAY AN IMPORTANT
ROLE IN MIGRATION AND
CHEMOSENSITIVITY
OF
BREAST CANCER CELL LINES
Svetlana Skolekova, Lucia
Kucerova
Cancer Research Institute of
Slovak Academy of Sciences,
Bratislava, Slovakia
Introduction
Mesenchymal stromal cells (MSC)
are an important component of
tumor microenvironment and can
directly interact with tumor cells
or alter the behaviour of the tumor
cells in paracrine manner. MSC are
capable of altering the behavior of
tumor cells on multiple levels. Their
preference for injured, inflamed
and tumor tissue is exploited in
regenerative medicine and also as
a promising therapeutic approach
in gene-directed enzyme prodrug
therapy. However, it is very important
to understand the crosstalk between
MSC and tumor cells for their safe
use.
Materials/methods
The tumor cells were cultivated in
conditioned medium from adipose
tissue-derived mesenchymal stromal
cells (CM-MSC) or in chemotherapy
pretreated CM-MSC. We analysed
chemosensitivity, migration and
proliferation of breast cancer cell
lines by fluorimetric, mammosphere
culture assay and IncuCyte ZoomTM
Kinetic Imaging System. The
expression of specific genes was
analysed by PCR.
Results and conclusions
AT-MSC-secreted factors are able
to increase migration of Sk-Br-3 and
MCF-7 breast cancer cell lines. We
propose the role of SCF/cKit signaling
in MSC-mediated migration of tumor
cells as the expression of both
increased in tumor cells cultivated in
CM-MSC. Chemotherapy pretreated
AT-MSC secreted factors protecting
tumor cells against apoptosis and
increasing resistance of tumor cells
to cisplatin and doxorubicin in 2D
and also 3D conditions.
We have shown that AT-MSC
express a wide scale of cytokines,
chemokines and growth factors,
which
influence
tumor
cells
depending on the context and are
able to confer increased metastatic
potential and resistance of breast
cancer cells. Thus, there is a need to
consider intrinsic properties of MSCs
during their application in cancerrelated diseases and potential
interaction that might be important
for the therapeutic efficiency.
A novel non-laborious and
cost-effective approach
for mammalian cell
synchronization
Martin Liptay, Kamila
Jahodíková, Zuzana
Loubalová, Juraj Kramara,
Iva Protivánková, Martin
Mistrík, Jiří Bártek
Institute of Molecular and
Translational Medicine, Olomouc,
Czech Republic
Introduction
In the study of molecular and
biochemical events and their
consequences during the cell
division, it is crucial to use a
population of cell cycle synchronized
cells. Many methods have been
established
to
synchronize
mammalian cells at specific phases
of the cell-cycle. Commonly used
cell synchronization techniques
include pharmacological inhibition of
processes that are essential for the
cell cycle progression (e.g. thymidine
block, mitotic poisons), serum
starvation, elutriation, sorting and
mitotic shake-off. These approaches
differ in many essential aspects
like the yield of synchronized cells,
toxicity, cost- and labor demands.
Choosing the appropriate technique
is then always a trade-off among
these parameters.
Materials/methods
The method combines the principle
of mitotic shake-off with so-called
anchorage dependency. Constant
vibration causes the loosely attached
mitotic cells to detach from the
support and remain in suspension.
Anchorage dependency prevents
the detached cells from finishing
cell division and arrests them in the
post-mitotic, bi-nuclear stage.
Results and conclusions
We will introduce a cell synchronization
method that overcomes some of the
above mentioned limitations and
provides a simple means of getting
synchronized cells from adherently
growing mammalian cultures in
sufficient amount and purity.
MIF: A prognostic marker in
Glioblastoma multiforme?
Nato Narsia, Mariam
Gachechiladze, Gvanca
Kharaishvili
Department of Clinical and
Molecular Pathology, Institute
of Molecular and Tranlational
Medicine, Faculty of Medicine
and Dentistry, Palacky University,
Olomouc, Czech Republic
Introduction
Glioblastoma Multifome (GBM, WHO
grade IV astrocytic tumour) is one
of the most aggressive malignant
tumors in humans. Macrophage
migration inhibitory factor (MIF) is
a pluripotent cytokine which plays
an important role in inflammation,
immune response and in cancer
development. Role of MIF in tumor
growth and malignant behavior
appears to be complex. Contrasting
reports on MIF functions were
published where in some cancers
MIF acts as a tumor suppressor
through the induction of various
cytokines from macrophages, and
in other cancers it functions as a
promoting factor when neutralized by
anti-MIF antibody. These conflicting
results suggest that MIF exerts dual
functions with regard to tumor cell
growth and behavior, possibly due
Abstract book
to localization of the MIF protein.
In the current study MIF protein
expression as a prognostic factor
in different grades of gliomas is
elucidated.
Materials/methods
FFPE tissue of 87 gliomas were
included in this study out of which
15 were diffuse astrocytomas,
14 were anaplastic astrocytomas
and 58 were glioblastomas. MIF
protein expression was detected
by imunohistochemistry. Nuclear
and cytoplasmic expression was
assessed
semiquantitaively
by
histoscore.
Logistic
regression
analysis was done to evaluate
prognostic importance.
Results and conclusions
MIF protein expression was present
diffusely in the cytoplasm of
tumor cells in all tumor specimens
examined. An increase in MIF
expression by every 10 units resulted
in 5% decrease of risk of death in
GBM group (n=58). A significant
association
between
cancer
related death and MIF cytoplasmic
expression was observed (p=0.039),
indicating MIF as a good prognostic
factor in GBM. However, studies on
larger patient cohorts is necessary.
Ultra deep amplicon
sequencing of RAS genes and
its use for mCRC predictive
diagnostics
Rastislav Slavkovsky, Jana
Stranska, Veronika Venskova,
Veronika Holinkova,
Miroslava Rabcanova, Jiri
Drabek
Introduction
EGFR pathway regulates cancercell proliferation, apoptosis and
tumor-induced
angiogenesis.
Tumor DNA testing of KRAS and
NRAS (RAS) genes,from the EGFR
signaling network, is a prerequisite
for proper personalized biological
treatment using anti EGFR drugs
(panitumumab and cetuximab) in
mCRC. The anti-EGFR treatment
is prescribed only in wildtype RAS
patients. Detection of increasing
number of possible mutations with
probe-based qPCR is cumbersome
while amplicon ultra deep nextgeneration sequencing (NGS) has
a potential to be suitable method
for simultaneous direct detection of
all somatic mutation within tested
regions and with a defined detection
limit.
Aim of the study is to optimize
and verify KRAS and NRAS NGS
sequencing of tumor DNA samples
for detection of mutations at codon
12, 13, 59, 61, 117, and 146 of NRAS
and KRAS genes.
Materials/methods
DNA samples of mCRC patients
with sufficient quality of DNA were
used for testing. Two methods of
NGS RAS assays with longer (189
– 295 bp) or shorter (70 - 150 bp)
amplicons were introduced into
laboratory. Both RAS assays consist
of qPCR with a control of amplicons
by melting curves and purification
of the samples. “Long RAS assay”
continues with tagmentation and
index amplification, otherwise “short
RAS assay” procedures consist
of end-repair, adapter ligation,
purification, and index amplification.
After size selection of the products
and their purification, samples
from both assays were normalized,
pooled, and applied to platform
Illumina MiSeq. Results with at
least 5 % mutation frequency were
concluded as “mutation detected”,
according to consensus of the
Czech Society of Pathology.
Results and conclusions
In every RAS run, KRAS G13D (461
%) or NRAS Q61L (473 %) mutation
standards were correctly found,
confirming reproducibility of the
method. The first pilot study of 24
samples was in 100 % concordance
with therascreen® KRAS RGQ
PCR Kit (KRAS codons 12 and 13,
CE-IVD, Qiagen), in 96 % in last 50
samples. Interlaboratory comparison
of 12 samples with CGB Ostrava
yielded one discrepant sample that
will be retested in both laboratories.
External quality assessments yielded
one result differing from consensus
out of ten. Upon investigating the
A55
cause of this discrepance with EQA
organizers, we found that sample
was artificially prepared cell line
that did not cover sequence of our
primers.
During almost one year, more than
237 samples were tested. So far we
have detected mutations in 11 out of
12 investigated codons.Thirty six out
of 40 samples that were not possible
to be analyzed by “long RAS assay”
due to DNA quality, were succesfully
analyzed by “short RAS assay”. 4
samples (10 %, 1.7 % from total
number of tested samples) remained
untestable.
Taken together we show that it is
crucial to understand the topology of
DNA around the studied mutations.
Especially in case of FFPE derived
samples the shortening of the
sequencing area could be very
beneficial for improving the quality
of the results.
The work was supported by grants
CZ.1.07/2.3.00/30.0060
and
CZ.1.07/2.3.00/30.0041.
Výhody vysokokapacitního
qPCR pro genově expresní
profilování. Analýza a
aplikace. (Advantages of highthroughput qPCR for gene
expression profiling. Analysis
and applications.)
úterý / 2. prosince
14.15 - 14.30 hod.
2014
/
Vlasta Korenková1, Lucie
Langerová1, Vendula
Novosadová1, Mikael
Kubista1,2
Biotechnologický ústav AV CR,
v.v.i., Praha, Czech Republic,
2
TATAA Biocenter, Goteborg,
Sweden
Introduction
Expresní profilování představuje
měření exprese více genů najednou
za účelem vytvoření obrazu o
buněčné funkci. Profily genové
exprese mohou být použity například
pro
diagnostické
monitorování
odpovědi pacienta na léčbu a tím k
optimalizaci individuální terapie.
Materials/methods
1
A56
Abstract book
Vysokokapacitní zařízení BioMark
(Fluidigm),
které
je
umístěno
v
qPCR
servisním
pracovišti
Biotechnologického
ústavu
AV
ČR, umožňuje současnou analýzu
exprese až 96 genů v 96 vzorcích
(9216 reakcí) v rámci jediného
experimentu.
Měření
probíhá
pomocí kvantitativní polymerázové
řetězové reakce (qPCR), která je
jednou z nejcitlivějších metod pro
kvantifikaci mRNA či mikroRNA.
Možnost analyzovat větší množství
genů najednou vybízí k použití
multivariantních statistických analýz.
Results and conclusions
Využití vysokokapacitního qPCR
přístroje BioMark poskytuje množství
výhod. 1. Efektivita: Výrazná úspora
vzorku (5 µl pro kompletní analýzu),
detekční chemie, mastermixu a
spotřebního materiálu. 2. Rychlost:
Celý vysokokapacitní experiment lze
provést v rámci několika hodin, ne dnů
či týdnů. 3. Automatizace a vysoká
kapacita: Díky zautomatizovanému
pracovnímu
postupu
je
minimalizována možnost lidské
chyby. Není potřeba mezidestičkové
kalibrace. 4. Sensitivita a flexibilita
qPCR
systému.
5.
Možnost
dalších aplikací: vysokokapacitní
genotypování a digitální PCR.
Poznámky / Notes:
Ústav molekulární a translační medicíny
Lékařské fakulty Univerzity Palackého
děkuje všem partnerům, kteří se zúčastnili
a podpořili konferenci
X. Dny diagnostické, prediktivní a experimentální
onkologie
Generální partner:
Partneři:
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INSTITUTE OF MOLECULAR AND
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FACULTY OF MEDICINE AND DENTISTRY
Palacký University in Olomouc
Hněvotínská 5, 779 00 Olomouc
Czech Republic
5
barevná varianta logotypu