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The role of copper transporters in in vitro cytotoxicity of oxaliplatin and their expression in colorectal cancer
thesisposted on 2023-05-26, 01:15 authored by Cui, H
Colorectal cancer (CRC) is a major health problem and cause of morbidity and mortality worldwide. The clinical efficacy of oxaliplatin (OXL), a commonly used chemotherapy agent, is limited by tumor resistance largely due to reduced drug accumulation. The role of copper (Cu) transporters in the transport and pharmacology of OXL is unclear, including Cu uptake transporter 1 (CTR1), and efflux transporter Cu+-transporting P-type ATPase ATP7A and ATP7B. The project aimed to investigate the contribution of Cu transporters to the uptake and cytotoxicity of OXL, and their expression in CRC. CRC cells were engineered to overexpress hCTR1 gene (hCTR1/DLD-1 cells) or the empty vector as mock control cells to study the uptake and cytotoxicity of OXL. A fluorescent probe FDCPt1 was used to visualize the cellular uptake of OXL-derived monofunctional platinum bio-transformed products. The expression profile of Cu transporters was characterized in colorectal cancer cell lines before evaluating the synergism between Cu chelators and OXL in colorectal cancer cells based on their regulation on hCTR1 protein. The expression of copper transporters was also investigated in paired patient tumor biopsies. In addition, a colitis model of Winnie mouse carrying point mutation of Muc2 gene and a dextran sodium sulphate (DSS)-induced colonic dysplasia model were used to determine the correlation of Cu transporters with these precancerous conditions. Overexpression of hCTR1 contributes to OXL cytotoxicity and uptake in recombinant colorectal cancer DLD-1 cells, with increased sensitivity and stronger FDCPt1-derived fluorescent signals than mock cells. The mRNA of Cu transporters was detected at constantly high levels in colorectal cancer cell lines with different origins. hCTR1 protein was expressed abundantly with the expression intensity higher than ATP7A or ATP7B across cell lines. Cu chelators, ammonium tetrathiomolybdate, and D-penicillamine, and bathocuprione disulphonate up regulated hCTR1 protein levels and enhanced the cell-killing capacity of OXL in some of CRC cells. Human colonic tissues were stained positive for Cu transporters with varying percentage of staining between individual patients. Semi-quantitative analysis using de-convolution method shows hCTR1 staining seems to be similar between tumor and the matched normal tissues. ATP7B expression exhibits a trend towards up regulation in tumor tissues compared to that of normal tissues, suggesting this protein may be involved in the development of colonic malignancy. Chronic intestinal colitis and dysplasia were confirmed histologically in colonic tissues of Winnie mice, and DSS-treated Winnie mice, respectively. Mouse Cu transporter 1 (mCTR1) was detected in colonic tissues of these animals, but with varying localization and intensity. The percentage of mCTR1 staining increased in colonic tissues of Winnie mice, but decreased in tissues of dysplasia model compared to that of the normal colon tissues. ATP7A and ATP7B expression did not change in colitis tissues but decreased significantly in dysplasia tissues. In conclusion, our work demonstrated for the first time the positive involvement of hCTR1 in the uptake and cytotoxicity of OXL in some colorectal cancer cells genetically or pharmacologically modified to overexpress hCTR1 protein. The abundant expression of hCTR1 in human colorectal cancer cells and tumor tissues highlighted again the importance of copper in the development of colorectal adenocarcinoma. The altered expression of copper transporters in the precancerous colonic tissues of Winnie mice suggests copper homeostasis may be disturbed during the chronic progress of colorectal carcinogenesis.