GCS Induces Multidrug Resistance By Regulating The Expression Of BCL-2 In Human Colon Cancer Cell Lines

Background and objectives At the present stage, chemotherapy is also the main therapeutic method of advanced colorectal cancer. Multidrug resistance is the main cause of the failure of tumor chemotherapy, lead to a poor prognosis. Multidrug resistance(MDR) refers to when tumor cell line resistance to a kind of chemotherapy drug, it will resistance to other chemotherapy drugs that have different chemical structure and principles. Ceramide play an important role in inhibiting proliferation and inducing apoptosis. While, glucosylceramide synthase(GCS) transfers glucose from UDP-glucose to ceramide, thereby producing Glc Cer. In this way, cells escape ceramide-induce apoptosis. More and more evidences have been proved, multidrug resistance is closely associated with increased glucosylceramide symthase levels. In addition, high levels of GCS also cause the overexpression of Bcl-2 gene, an apoptosis suppressor. The levels of Bcl-2 protein could be improved by overexpression of ERK protein and then act on the common downstream of apoptosis, play a role of anti-apoptosis. Anti-apoptotic effect of Bcl-2 has been reported to participate in multidrug resistance formation of tumor cells. It has been reported, high levels of GCS protein and m RNA, as well as Bcl-2, have been detected in leukemia MRD cell lines. However, the mechanisms and relationship between GCS and Bcl-2 in colon cancer are poorlyreported. In order to explore the mechanism and relationship between GCS and Bcl-2 in human colon cell line HCT-8 and MDR cell line HCT-8/VCR, we detected the expression of GCS, Bcl-2, ERK protein and m RNA under different conditions.Methods 1 Cell culture 1)HCT-8: human colon cancer drug sensitive cell line. Normally cultured in RPMI-1640 complete culture medium containing 10% fetal bovine serum at 37 in ℃5%CO2. The following experiments were performed after 2 weeks culture. 2)HCT-8/VCR: human colon cancer drug resistance cell line. Cultured in complete RPMI-1640 culture medium with 1.0μl/ml of vincristine(VCR) to maintain drug resistance. and cultured in drug-free medium for 2 weeks before experiment. Other condition the same as HCT-8 cell line. 3)HCT-8/VCR+PPMP: After HCT-8 cell line cultured in drug-free medium, added 20μmol/L PPMP and culturing 48 h. 2 Western Blot We collected cells in HCT-8, HCT-8/VCR and HCT-8/VCR+PPMP, respectively, and extract total protein. Detect GCS, Bcl-2, ERK protein expression in each cell lines by Western Blot method. 3 RT-q PCR We collected cells in HCT-8, HCT-8/VCR and HCT-8/VCR+PPMP, respectively, and extract total RNA. Detect GCS, Bcl-2, ERK m RNA expression in each cell lines by RT-q PCR method.Results 1.GCS and Bcl-2, an anti-apoptotic protein, expression in drug sensitive cell lineHCT-8 as well as drug resistance cell line HCT-8/VCR. The levels of GCS and Bcl-2 protein in MDR cell line are more higher than drug sensitive cell line, shown obvious difference(P<0.05). 2. Compared HCT-8 to HCT-8/VCR, the expression of GCS protein was significantly suppression after added GCS inhibitor PPMP(P<0.05). While, Bcl-2 and ERK protein down-regulate in HCT-8/VCR+PPMP cell line compared with HCT-8/VCR(P<0.05). 3.Detect the expression of target genes by RT-q PCR method. The result showed, expression of GCS, Bcl-2 and ERK m RNA in drug resistance cell line are significantly higher than those in drug sensitive cell line(P<0.05). 4.Compared HCT-8/VCR to HCT-8/VCR+PPMP, the expression of GCS m RNA in HCT-8/VCR+PPMP was obviously lower than HCT-8/VCR cell line(P<0.05); after added GCS inhibitor PPMP, Bcl-2 and ERK gene expression in cells were significantly down-regulate compared with drug resistance cells.Conclusions 1. In human colon cancer cell line HCT-8 and drug resistance cell line HCT-8/VCR, the expression of anti-apoptotic protein Bcl-2 was in accordance with GCS. 2.GCS induces multidrug resistance by regulating the expression of Bcl-2, this process worked via ERK signaling pathway. 3.The expression of GCS was significantly inhibited after added GCS inhibitor PPMP in cells. And then down-regulated Bcl-2 gene and protein expression.