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J Cancer Metastasis Treat 2016;2 Suppl 1

Abhijeet Deshmukh , Frank Arfuso , Philip Newsholme , Invitrogen). Results: The MTT assays conducted
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Arun. M. Dharmarajan 1 showed the chemo-sensitisation effect of sFRP4 when
used in combination with tumour-specific drugs. The
1 Stem Cell and Cancer Biology Laboratory, School of Biomedical Sciences, post-transcription data (Gene-Expression) collected
Curtin Health Innovation Research Institute, Curtin University, Perth, Australia;
2 School of Biomedical Sciences, Curtin Health Innovation Research Institute, from CSCs that have undergone combinatorial
Curtin University, Perth, Australia treatment with sFRP4 and chemotherapeutic drugs
suggests there is downregulation of drug transporters
Background: Cancer stem cells (CSCs) are the and upregulation of angiogenic/apoptotic/cell death
unipotent cell population present within the tumour markers. The post-translational modification (protein
mass. CSCs are known to be highly chemo-resistant expression) of CSCs shows the chemo-sensitisation
and, in recent years, have gained intense interest as effect of sFRP4, when used in combination with tumour-
key tumour-initiating cells that play an integral role specific drugs, by downregulating the cell-survival and
in cancer recurrence following chemotherapy. Aim: oncogenes signals and upregulation pro-apoptotic
The study investigates molecular signals essential signals. In tumour cell lines, sFRP4 in combination
to sustain CSCs and target their activity using with doxorubicin/Cisplatin, reduced the proliferative
secreted frizzled-related protein 4 (sFRP4) alone or in capacity of CSC population in vitro. Conclusion:
combination with chemotherapeutic drugs. Methods: Wnt/β-catenin signalling is important for proliferation
Cancer stem cells isolation: CSCs isolated from and self-renewal of CSCs in association with human
Breast (MDA231/MCF7), Ovary (A2780 P/ADR/Cis), tumorigenesis. The silencing of this signalling pathway
and Prostrate (PC3/LnCap) tumour cell lines in serum- by the application of sFRP4 suggests potential for
free conditions and enriched with growth factors (EGF/ improved in vivo chemo-responses.
FGF/B-27). Chemo-sensitisation/drug treatment:
Sensitisation with sFRP4 was performed by adding Key words:
sFRP4 to the CSCs culture alone or in combination with Cancer stem cells, chemo-sensitisation, Wnt signalling
chemotherapeutic agents (Doxorubicin/Cisplatin) for pathway, Wnt antagonist, secreted frizzled related
24 h. Viability assay: MTT based was used according protein 4
to the manufacturer’s protocol to measure cell
metabolic viability. Cell surface markers: To assist in A30
determining their identity, cell surface markers (CD44 / Induction of radioresistance and cisplatin
+
CD24 /CD133 ) were examined in both monolayers resistance in HNSCC cell line after ionizing
+
-
and CSCs by flow cytometry, using CellQuest data radiation
acquisition and analysis software. RNA isolation and
cDNA synthesis: Total RNA was isolated from cells Vesna Todorovic , Ajda Prevc , Martina Niksic Zakelj ,
1
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1
using TRIzol reagent followed by chloroform extraction, Blaz Groselj , Primoz Strojan , Maja Cemazar , Gregor
1,2
1
1
isopropanol precipitation, and a 75% (v/v) ethanol Sersa 1
wash and further transcribed into cDNA using a High
Capacity cDNA kit. Western blotting: Total proteins 1 2 Institute of Oncology Ljubljana, Ljubljana, Slovenia;
University of Primorska, Faculty of Health Sciences, Izola, Slovenia
were extracted from cells using RIPA denaturing buffer.
The protein extracts were estimated using BCA Kit Head and neck squamous cell carcinoma (HNSCC)
and 20 μg of proteins were separated by 12% SDS- constitutes approximately 6% of all cancers
PAGE and transferred onto a nitrocellulose membrane. worldwide. The risk of HNSCC is strongly associated
Immunoblotting was performed by blocking the to habitual exposure to tobacco or alcohol. In
membrane in 5% Non-Fat Dry Milk (NFDM) solution addition, oropharyngeal SCC (OPSCC) can arise
and incubating the membrane in 5% NFDM/BSA also from infection with human papilloma virus
containing primary antibodies overnight at 4°C. The (HPV). Management of HNSCC is complex and is in
membranes were incubated in 3% NFDM containing part correlated to risk factors. Namely, HPV-positive
secondary antibodies for 1 h at RT after three washes OPSCC has a greater response to radiation or
with PBS containing 0.1% Tween 20. Signals were chemoradiation than tobacco/alcohol related HNSCC.
detected on a Chemi-Doc imaging analyser using ECL In addition, a significant problem of HNSCC is its
Western Blotting Substrate. Caspase assay: The recurrence, specifically in previously irradiated areas,
intracellular levels and activation of caspase-8 and due to induced radioresistance and radiation tolerance
caspase-3 were followed by Western blotting using limits of already irradiated normal tissues.
antibodies specific for the proenzymes and activated
species. Caspase-3 activity was measured using the The aim of our study was to investigate the response
EnzChek Caspase-3 Assay Kit II (Molecular Probes, of three HNSCC cell lines to ionizing radiation and
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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ November 16, 2016