Establishment Of The Drug-resistant Colon Cell Line And Study Of Chemoresistance Related Genes

Objective:A major obstacle in chemotherapy is the treatment failure due to anticancer drug resistance.One of the most critical issues to be solved in regard to cancer chemotherapy is the need to establish a method for predicting efficacy of anticancer drugs for individual patient.The purpose of this study was to identify differentially expressed genes associated with resistance in human colon cancer cell,to provide clues for understanding the oxaliplatin resistant mechanisms and markers for "personalized chemotherapy".Methods:The drug sensitivities of human colon tumor cell line THC8307 to 5-FU and L-OHP were examined by MTT assay.THC8307 was continuously grown in the presence of L-OHP with the increasing concentration of 0.108μg/ml,0.144μg/ml,0.2μg/ml,0.3μg/ml,0.48μg/ml,0.6μg/ml,0.9μg/ml,1.2μg/ml,1.5μg/ml,1.8μg/ml,2.4μg/ml,3.0μg/ml,6.0μg/ml.After the cell line was cultured in the presence of the final drug concentration 6.0μg/ml for 4 months,cell proliferation and cytotoxicity were serially monitored.The long-oligomer microarray was used to identify genes differentially expressed in the parent cell line and drug resistant cell line.Semi-quantitative RT-PCR was performed to verify the expression of the genes identified in microarray experiments.Results:1.The IC₅₀ of cell line THC8307 to L-OHP was 5.334±0.576μg/ml(＜1PPC),the IC₅₀ of cell line THC8307 to 5-FU was 32.200±26.200μg/ml(＞1PPC).2.The difference of chemosensitivity to L-OHP was showed between the parental cell line THC8307 and the drug resistance cell line THC8307/L-OHP(P＜0.01),and THC8307/L-OHP was cross-resistant to 5-FU.The IC₅₀ of cell line THC8307/L-OHP to L-OHP was 172.92±54.48μg/ml,THC8307/L-OHP exhibited 30.99-fold resistant to L-OHP and the IC₅₀ of THC8307/L-OHP to 5-FU was 215.60±6.96μg/ml,6.70-fold to 5-FU.3.We identified 62 genes differentially expressed in L-OHP-resistant colon cancer cell line,among them 20 genes were up-regulated and 42 genes were down-regulated in the cell line THC8307/L-OHP.4.Seven genes(S100P,STK17A,CAâ…¨,STA15,TCF8,GDIA1 and AAMP) were selected for semi-quantitative RT-PCP and the results were in good agreement with those from the microarray data.Genes S100P,STK17A,CAâ…¨,STA15 and TCF8 were down-regulated while genes GDIA1 and AAMP were up-regulated.Conclusions:Stepwise selection in vitro resulted in the establishment of drug resistant cell line THC8307/L-OHP.These results demonstrate the potential of DNA microarrays to identify novel genes involved in mediating the response of tumor cells to chemotherapy.Genes associated with apoptosis perhaps are vital to oxaliplatin resistance in colon cancer,and maybe be the markers to predict drug sensitivities.