Modulation of ABCC2-Mediated Oxaliplatin Resistance in Colorectal Cancer Caco-2 Cell Line
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Cancer has been a great concern world-wide among people and has raised great challenges to oncologists and scientists. In New Zealand, colorectal cancer has the world’s highest cancer incidence and is the second-highest cause of cancer death. It is reported to be one of the highest causes of cancer deaths in the world. Statistics say that in 2011, 3030 people were diagnosed with bowel cancer and out of which only 1,839 survived. There has been strong evidence reported by various clinical scientists that oxaliplatin-based chemotherapy has been considered as the preferred regimen for the treatment of colonrectal cancers (CRC), both in the adjuvant and metastatic settings. However, its dose-limiting toxicities and tumour resistance are major limitations for many patients in clinical practice. In a New Zealand observational patterns-of-care study, more than 40% of a total of over 500 patients receiving chemotherapy including oxaliplatin stopped that chemotherapy early due to either severe toxicity or lack of efficacy. Given oxaliplatin is an important compound to treat CRC, it is important to increase the efficacy of the anti-cancer drug. The efficacy is found to be low in the tumour cells with over expression of the ABCC2 gene. The ABCC2 gene encodes for the membrane protein ABCC2 (aka MRP2 protein) which is known for the efflux of the drugs. Hence the efficacy of oxaliplatin based chemotherapy is less efficient. In this study we hypothesize that modulation of ABCC2 protein by using the CRISPR Cas9 gene-editing tool or by a model ABCC2 inhibitor could increase the efficacy of oxaliplatin. In this study, we have first used the CRISP Cas-9 gene-editing tool to knockout the ABCC2 gene that encodes for MRP2. The efficiency of the gene editing genomic cleavage assay was confirmed to be 24% by using the T7 endonuclease I method. A substrate based accumulation study was done using 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) on both wild type (WT) and knockout (KO) Caco-2 cells. Oxaliplatin sensitivity was determined using the MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Finally a model MRP2 inhibitor benzbromarone was used to inhibit the function of MRP2 and enhance oxaliplatin efficacy. This study demonstrates that the modulation of ABCC2 protein that causes multidrug resistance can increase oxaliplatin efficacy in a colorectal cancer Caco-2 cell line.