The Relationship Beteewn Glucosylceramide Synthase Gene And Drug Resistance In HCT-8Cells

ObjectiveDrug resistance is one of the chief impediments to the successful treatment and prognosis in patients with colon cancer. It is a phenomenon that human tumours which obtain resistance to a kind of drugs are resistant to several other drugs. Its mechanisms of colon cancer are complicated, one of our understanding of MDR came with the discovery of P-glycoprotein and other transporters which expressed in some cancer cells and could strengthen the efflux of diverse chemotherapeutic agents from cells. GCS which can reduce the level of ceramide that allows cellular escape from ceramide-induced cell apoptosis was deem to be related with multidrug resistance (MDR). However, the mechanism under it is still unclear.In order to explore the role of GCS in the MDR of colon cancer, we designed this research. The proliferation and chemosensitivity of cells were measured with Cell Counting Kit-8(CCK-8). The mRNA levels of GCS, Caspase-3and MDR1were detected by semiquantitative reverse transcription-PCR amplification, The protein levels of GCS and P-gp proteins were indicated by Western blotting, The protein levels of caspase-3were measured by immunocytochemistry. The apoptosis rates of cells were measured with flow cytometry. According to this experiment, we investigate the relation between GCS gene and drug resistance in HCT-8cells.Methods1The sensitive colon cancer cell line HCT-8and the cell line HCT-8/VCR were bought from Xiangya Medical University of Hunan. HCT-8cells were cultured in RPMI-1640culture medium (Hyclone) containing10%FBS at37℃in humidified atmosphere containing5%CO2, and the medium of HCT-8/VCR cells containing10%FBS and2μg/ml vincristine.The cells were cultured another two weeks in the medium without vincristine before experiment.2The cells were seeded in6well plate with antibiotic-free medium.48hours after transfection, aspirate the medium and replace it with fresh medium containing100μg/ml puromycin. The medium was changed every3days. The following experiments were performed after20days culture.3The proliferation and chemosensitivity of cells were measured with Cell Counting Kit-8(CCK-8).4The mRNA levels of GCS, caspase-3and MDR1were detected by semiquantitative reverse transcription-PCR amplification. The protein levels of GCS and P-gp proteins were indicated by Western blotting, The protein levels of caspase-3and bcl-2were measured by immunocytochemistry.5The apoptosis rates of cells were measured with flow cytometry.Results1We measured the growth of the cells every24h, for7days. The cells were full in the well at the fourth day. There were no significant differences among the cells. And the IC50of Cis-platinum complexes became lower after trasfection.2There were lower expression of GCS mRNA and protein after transfection. It means that the GCS gene was knocked down by UGCG shRNA.3The expression level of MDR1mRNA and protein (P-gp) also became lower after transfection. And the apoptosis-related gene Caspase-3expressed lower in the transfected cells.4The apoptosis rate was higher in the non-transfected cells than the transfected cells.Conclusions It plays an important role in multidrug resistance mechanisms of HCT-8cells with high expression of GCS gene. And the suppression of GCS restore sensitivity of multidrug resistance HCT-8/V cells to drug treatment.