Establishment Of An Oxaliplatin-Induced Human Colon Carcinoma Drug-Resistant Cell Line And Its Reversion

Human colon carcinoma is one of most common maligmant tumors. Chemotherapy is an important step in treatment of colon carcinoma, and have important effect on prolong of exist and improving life's quality. Oxaliplatin (L-OHP) is one of the most important anticancer agents now used to colon carcinoma. Despite its broad clinical application, however, the resistance of cancer cells to oxaliplatin often culminates in chemotherapeutic failure and becomes one of the urgent questions to resolve in clinical chemotherapy.In order to explore its resistant mechanisms, a resistant cell line—HT29/L-OHP was established by stepwise increasing dose of L-OHP and intermittent administration. The eukaryotic expression plasmids of TSG101-siRNA was constructed successfully by RNA interference technology. The eukaryotic expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP and increases the sensitivity to chemotherapy. PART ONE ESTABLISHMENT OF OXALIPLATIN-INDUCED RESISTANT HUMAN COLON CANCER CELL MODEL AND PRELIMINARY STUDY ON THE RESISTANT MECHANISMSObjective Oxaliplatin(L-OHP) is one of the most important anticancer agents now used. Despite its broad clinical applications, however, the resistance of cancer cells to L-OHP often culminates in chemotherapeutic failure. In order to explore its resistant mechanisms, a resistant cell line—HT29/L-OHP was established. Methods A resistant cell line—HT29/L-OHP was established by stepwise increasing dose of L-OHP and intermittent administration. The growth curves,multidrug resistance and resistance index of HT29/L-OHP cell line to anticancer agents were detected by MTT assay. The changes of its biological characteristics were determined by light microscope, electron microscope, flow cytometry etc. The expression of glutathione S-transferase-Ï€(GST-Ï€) and Bcl-2 etc were analyzed and compared by immunohistochemical technique. The expression of tsg101mRNA and TSG101 protein in HT29 and HT29/L-OHP cell line was detected by RT-PCR and Western-blot. Results HT29/L-OHP cell line was established after 3 moths with stable resistance to L-OHP and resistance index 10.6430; HT29/L-OHP cells exhibited cross resistance to many other chemotherapeutic agents. As compared with parental cells, the morphological and chromatosome number of HT29/L-OHP changed; its doubling time prolonged; and the number of cells in S phase and G₀/G₁ phase decreased while in G₂/M phase increased by cell cycle analysis. The expression of tsgmRNA and TSG101 protein was decreased in the HT29 cell line. The expression of GST-Ï€, MRP, NF-kB, INOS, HSP70, MTP53, Bcl-2 and E-cd was enhanced, but the expression of Bax and AE1/AE3 protein kept stable in the HT29/L-OHP cell line. Conclusion HT29/L-OHP cell line showed the typical multidrug resistant phenotype and might to serve as an ideal model for studying the mechanisms of resistance to L-OHP and filtrating reversing drug. HT29/L-OHP cell line possessed the basic characteristics of resistant cells. The primary factors resulting in HT29/L-OHP resistance to L-OHP were higher expression of GST-Ï€, MRP, NF-kB, INOS, HSP70, MTP53, Bcl-2 and TSG101. PART TWO Construction and effect of TSG101-siRNA eukaryotic expression vectors with oxaliplatin on proliferation of HT29/L-OHPObjective To construct the eukaryotic expression plasmids of TSG101-siRNA and investigate the effect of RNA interference targeting tsg101 gene with oxaliplatin on colon cancer resistant cell line HT29/L-OHP. Methods The targeting fragments specifically against TSG101 were designed according to the principle of small interfering RNA designation using computer software. The sequences were cloned into siRNA expression plasmids through DNA recombinant technology. The expression of tsg101mRNA after transfected TSG101-siRNA was detected by RT-PCR. The expression levels of TSG101 after transfected TSG101-siRNA was detected by Western blotting. MTT test were applied to measure the inhibition combined with oxaliplatin on proliferation of HT29/L-OHP strain. Distribution of cell cycle was analyzed using flow cytometry after RNA interference HT29/L-OHP. Results The expected fragments were obtained by digestion identification and further confirmed by DNA sequencing. These TSG101-siRNA inhibited the expression of tsg101mRNA , and the expression of TSG101 protein, and proliferation of HT29/L-OHP obviously when combined with oxaliplatin. The analysis of cell cycle indicated that cells of G₂/M phases reduced and of G₀/G₁ and S phases increased. Conclusion The eukaryotic expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP and increases the sensitivity to chemotherapy.