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|a Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid tumours. gemcitabine-based regimens have been used as front-line treatments for patients with advanced pancreatic cancer. gemcitabine may initially show a response, but most often multi-drug resistance (MDR) rapidly develops. Several processes are responsible for drug resistance in pancreatic cancer cells; one of the major processes is the decreased accumulation of drugs/active moieties within cancer cells because of increased drug efflux. Accumulating evidence suggests multidrug resistance protein 5 (MRP5, encoded by ABCC5) is over-expressed in pancreatic cancer and is associated with gemcitabine resistance. However, the mechanistic mechanisms of MRP5- gemcitabine interactions remain unclear. This research proposes to better understand the roles of MRP5 in conferring gemcitabine resistance by using two MRP5 overexpressing pancreatic cancer cell lines. Two different approaches for the modulation of MRP5 were used - transient knockdown of ABCC5 using siRNA (Chapter 4) and genome editing using the CRISPR-CAS9 system (Chapter 5). To assure that an observed gene silencing effect on protein level or gene function is specific to siRNA sequence(s) transfected, the mRNA level of ABCC5 is essential to be measured and off-target effects should be simultaneously assessed. Accordingly, a robust, sensitive and precise real-time PCR (qPCR) assay has been developed and validated to evaluate the mRNA level of genes associated with gemcitabine transport and metabolism (Chapter 3). qPCR conducted also confirmed literature reports that both MIA PaCa-2 and PANC-1 cells express MRP5. After transfection with three different siRNA sequences, the percentage of transient knock-down was calculated using ΔΔCp method (Livak's method) with GAPDH as a house-keeping gene, which showed stable expression in the cell lines treated with transfection reagents and ABCC5 and scramble siRNA sequences. A maximum of 85.18 ± 1.50 % and 80.94 ± 3.26 % of ABCC5 mRNA knockdown (10 pmol) was achieved, respectively, in MiaPaca-2 and PANC-1 cells transfected with ABCC5 siRNAs. Off-target effects of siRNA transfection were analysed and compared for 7 different ABC transporters and for the enzymes involved in gemcitabine metabolism. The MRP5 functional assay was performed by measuring the accumulation of a specific ABCC5 substrate, 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein (BCECF) in control and knockdown cells by using flow cytometry. Cellular accumulation of BCECF in MRP5-silencing cells increased by 80.75 ± 1.97% and 97.93% ± 7.00% (p<0.0001) for MIA PACa-2 and PANC-1 cells, respectively. MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was undertaken to determine gemcitabine sensitivity, and half-inhibitory concentration (IC50) was calculated. A significant increase in the sensitivity to gemcitabine was observed in ABCC5-transfected cells as compared with the control-siRNA transfected cells. The mean IC50 values in MIA PaCa-2 cells with control-siRNA, ABCC5-siRNA-1 and ABCC5-siRNA-2 were 31.64 ± 0.85 nM, 16.48 ± 1.5 nM and 13.56 ± 1.6 nM, respectively. The IC50 values in PANC-1 cells for the control, ABCC5-siRNA-1 and ABCC5-siRNA-2 transfected cells were: 5.84 ± 1.80 µM, 0.90 ± 0.04 µM and 1.81 ± 0.28 µM, respectively. The percentage of apoptosis was measured using the flow cytometer based annexin V and PI staining. Silencing ABCC5 significantly increased gemcitabine-induced apoptotic population in both MIA PaCa-2 cells and PANC-1 cells. For direct and efficient genome editing, CRISPR (clustered regularly interspaced short palindromic repeats)-CAS9 (CRISPR associated protein 9) system was used to target ABCC5 gene at the DNA level. The PANC-1 cells were transfected with CAS9 protein/ABCC5 guide-RNA ribonucleoprotein complexes through liposome-mediated delivery (Chapter 5). From the mixed population of cells, single cell knock-out clones were selected using the limiting dilution method. The efficiency of ABCC5 gene disruption was then assessed by flow cytometry analysis of cell surface MRP5 immunostaining. MTT assay was undertaken to determine gemcitabine sensitivity, and half-inhibitory concentration (IC50) of gemcitabine was calculated. Knocking out ABCC5 significantly decreased MRP5 surface staining by 88.14 ± 4.87 % (P < 0.0001). For functional studies of MRP5, cellular accumulation of BCECF in ABCC5-knock out clones increased by 90.36 ± 2.24% (p<0.0001) compared with those in wild-type. The 72-hr IC50 values for the control, Clone 1, 2 and 3 are 12.25 ± 2.32 µM, 3.20 ± 0.06 µM, 4.92 ± 0.55and 5.43 ± 0.82 µM, respectively, indicating that knocking out ABCC5 significantly increased the sensitivity of PANC-1 cells to gemcitabine. Taken together, our results confirm that MRP5 confers resistance to gemcitabine and modulation of ABCC5 gene expression increased the sensitivity of MiaPaca-2 and PANC-1 cells to gemcitabine and modulation of ABCC5 activity may represent a novel strategy to reverse gemcitabine resistance in pancreatic cancer cells. Screening tumour MRP5 expression levels to select patients for treatment with gemcitabine-based regimen alone or in combination with MRP5 modulation, could improve outcomes of pancreatic cancer treatment.
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