ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

P75. 5-FLUOROURACIL-SELECTED TUMOUR CELLS EXERT CROSS-RESISTANCE WHICH
CAN BE MODULATED BY PHARMACOLOGICAL AND MOLECULAR INHIBITION OF
ALDEHYDE DEHYDROGENASE
Miroslava Matuskova, Erika Durinikova, Lucia Kucerova, Zuzana Kozovska
Cancer Research Institute of Slovak Academy of Sciences, Bratislava Slovak Republic
One of the obstacles of effective cancer treatment is selection for chemo-resistant subpopulations
of malignant cell during chemotherapy. Innovative approaches need to be
developed to affect malignant cells resistant to conventional treatment. Chemo-resistant
tumour cells use more mechanisms to escape cytotoxic effect of drugs. Increased
expression of aldehyde dehydrogenase (ALDH), particularly 1A1 isoform (ALDH1A1) is
also involved in drug resistance (Januchowski et al., 2013).
We aimed to evaluate the sensitivity of 5-Fluorouracil (5-FU) resistant tumour cells to
panel of drugs, and prove the effect of inhibition of ALDH to evaluate its role in response
to chemotherapy.
Model chemo-resistant tumour cell line HT-29/EGFP/FUR proliferating in clinically
relevant plasma concentration of 5-FU was derived from human colon carcinoma cells
HT-29 by long-term cultivation in sequentially increasing 5-FU concentration. Tumour
cells were retrovirally transduced with enhanced green fluorescent protein (EGFP) for
unequivocal identification in cytometric experiments and evaluation of sensitivity to
chemotherapy treatment by fluorimetric assay. Immunophenotype, ALDH activity and
apoptosis were determined by flow cytometry. Kinetic experiments were performed
using Incucyte device. Gene expression was evaluated by qPCR, siRNA was delivered
by nucleofection. Athymic mice were used for experiments in vivo.
We demonstrated altered expression of enzymes involved in nucleotide metabolism
and transporter protein ABCC4. Changes correlated with their suggested role in 5-FU
resistance. Differences in expression of stem cells` markers ALDH1A1 and CD133 were
observed. We expected increased expression of ALDH1A1 enzyme in chemo-resistant
derivate of HT-29 cells. The functional assay ALDEFLUOR confirmed increased activity
of ALDH however ALDH1A1 – specific qPCR showed decreased expression of 1A1 isoform.
Lower expression of ALDH1 was also confirmed on protein level by Western-blotting.
Other ALDH isoforms have probably increased expression. HT-29/EGFP/FUR cells were
cross-resistant to Cis-Platin, Paclitaxel, Doxorubicine and Cyclofosamide, .which are
structurally and functionally unrelated to 5-FU. HT-29/EGFP/FUR derived xenografts
maintained their chemo-resistance for several months without selection pressure
of 5-FU, but exerted lower tumorigenicity. MSC co-injection had tumour-favouring
potential on the HT-29/EGFP/FUR xenograft growth. Immunomagnetically separated
CD133-positive subpopulation showed decreased sensitivity to chemotherapy.
ALDH activity was pharmacologically inhibited by diethylaminobenzaldehyde (DEAB)
or by RNA interference using set of ALDH1A1 specific small interfering RNA (siRNA).
Nucloefection of ALDH1A1 siRNA has short term effect on target genes’ expression.
Decreased level of mRNA was observed up to 72 hours post transfection. Tumour cells with
inhibited ALDH activity exerted altered sensitivity to 5-FU, Cis-platin and oxidative stress.
178 Analytical Cytometry VII