Reversing ABCC2-Mediated Oxaliplatin Resistance in Human PANC-1 Cells Using the CRISPR_cas9 System
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Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly aggressive malignancy that often shows minimal symptoms, early signs of the disease are difficult and often impossible to identify until the later stages of the disease due to the location of the pancreas. Oxaliplatin is a critically important treatment for not only pancreatic cancer but also many other gastrointestinal cancers, it plays a key role in improving the disease and increasing the life expectancy of patients. However, due to acquired drug resistance and accumulated toxicities the efficiency of oxaliplatin in treating PDAC is limited. High doses of the drug can cause a variety of toxic effects in patients, while low doses of the drug are less effective at eradicating the tumor and can lead to resistance to the drug. Previous studies have indicated that the membrane transporter protein (MRP2) works as an efflux pump to move cytotoxic compounds out of the cell and plays a role in the cellular accumulation of cytotoxic platinum drugs. It is suggested that the MRP2 protein is over expressed in pancreatic cancer cells leading to the increased efflux of oxaliplatin out of the cell limiting its efficiency. From this we have hypothesized that resistance could be reduced by knocking out the ABCC2 gene (encoding MRP2). The CRISPR-Cas9 gene editing system can be used to knock out your gene of interest in an in vivo cell model. We hypothesized that the CRISPR-Cas9 system can be used to knock out the ABCC2 gene in Panc1 cells. Panc1 cells were transfected with sgRNA and Cas9 protein using the CRISPRMAX transfection kit, the efficiency of the ABCC2 knockout was assessed using a T7 endonuclease cleavage assay. An accumulation study of the ABCC2 substrate 5-carboxy-2',7'-dichlorofluorescein (CDCF) was used to distinguish the difference in accumulation between the knockout cells and the wildtype. MTT assay was undertaken to compare the differences of oxaliplatin sensitivity between MRP2 knockout and the wildtype PANC1 cells. The results show the feasibility of the CRISPR-Cas9 system in knocking out the ABCC2 gene. The genomic cleavage assay yields a cleavage efficiency of 42.43%. We were able to see an increase in CDCF accumulation and oxaliplatin cytotoxicity in the MRP2 knockout cells in comparison to the wildtype. The results show that the use of CRISPR-Cas9 to silence the ABCC2 gene does reduce the function of the MRP2 protein and improves the oxaliplatin sensitivity in PANC1 cells.