GCS通过影响bcl-2的表达介导结肠癌细胞的多药耐药

Glucosylceramide synthase induces multidrug resistance by regulating bcl-2 expression in human colon cancer cells

目的:本文旨在通过检测在不同条件下结肠癌耐药细胞HCT-8/VCR及其敏感株细胞HCT-8中葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)、bcl-2及细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)的表达,探讨其相互关系及可能的作用机制.方法:将细胞分为3组,A组:结肠癌敏感细胞HCT-8常规培养;B组:耐药细胞HCT-8/VCR加VCR 1μg/mL培养,维持其耐药性;C组:GCS抑制剂PPMP处理耐药细胞HCT-8/VCR.Western blot检测各组细胞中GCS、bcl-2及ERK蛋白水平的表达.实时定量PCR检测(real-time quantitative PCR,qRT-PCR)检测GCS、ERK、bcl-2基因表达情况.结果:与敏感细胞相比,多药耐药(multidrug resistance,MDR)细胞中GCS与bcl-2的mRNA和蛋白表达量明显升高(P<0.05),PPMP处理HCT-8/VCR细胞,GCS蛋白和mRNA的表达受到明显抑制(P<0.05),同时bcl-2蛋白及mRNA的表达也明显下降(P<0.05),ERK蛋白的表达量在加入PPMP后也较前下降(P<0.05).结论:GCS通过影响抗凋亡蛋白bcl-2的表达,从而介导结肠癌细胞MDR,这一过程可能是通过ERK信号通路完成的.加入GCS抑制剂PPMP,可抑制bcl-2的表达,逆转细胞耐药.

AIM： To investigate the relationship between glucosylceramide synthase（GCS） and bcl-2 in human colon cell line HCT-8 and multidrug resistant（MDR） cell line HCT-8/VCR, in order to explore their role in multidrug resistance of colon cancer cells.METHODS： The study contained three groups：normally cultured HCT-8 cells（group A）,HCT-8/VCR cells treated with VCR to maintain the drug resistance（group B）, and HCT-8/VCR cells treated with VCR and inhibitor of GCS（PPMP）（group C）. Expression of GCS, bcl-2,and extracellular regulated protein kinases（ERK） proteins was detected by Western blot.Expression of GCS, bcl-2, and ERK mRNAs was tested by real-time quantitative PCR（qRTPCR）.RESULTS： Compared with HCT-8 cell line,expression of GCS, ERK and bcl-2 proteins and mRNAs was higher in MDR cell line. After treatment with PPMP, expression of those proteins and mRNAs were obviously restrained in HCT-8/VCR cell line.CONCLUSION： GCS induces multidrug resistance by regulating the expression of bcl-2,and this process may involve ERK signaling pathway. The inhibitor of GCS（PPMP） can inhibit the expression of bcl-2 and reverse multidrug resistance in human colon cancer cell line.