萝卜硫素通过调节Bax表达降低结肠癌细胞株对5-氟尿嘧啶的耐药性

Effect of Sulforaphane on Decrease of Drug Resistance of Colon Cancer Cells to 5-FU via Regulating Bax Expression

目的:研究Bax在萝卜硫素(SFN)逆转结肠癌细胞5-氟尿嘧啶(5-FU)化疗耐药中的作用及其机制。方法:体外培养结肠癌SW-480细胞,CCK8法分析了SFN对5-FU抑制SW-480细胞增殖的影响;流式细胞仪Annexin V-FITC/PI法分析了SFN对5-FU作用下的SW-480细胞凋亡水平的影响;实时荧光定量PCR(RT-PCR)技术检测了SW-480细胞中凋亡相关基因Bax、Suvivin、Bcl-2、p53及caspase-3表达量;Western Blot分析SW-480细胞中Bax蛋白表达,采用pcDFNA-WEE1质粒沉默技术对SW-480细胞中Bax蛋白进行低表达。结果:SFN能明显增强5-FU对结肠癌SW-480细胞增殖的抑制效应;凋亡实验结果提示,在SW-480细胞中,单药5-FU组中的细胞凋亡率为(36.76±4.29)%、SFN+5-FU联合组为(51.10±6.34)%,两组比较差异有统计学意义(P<0.05);RT-PCR实验提示,Bax可能介导SFN促使SW-480细胞凋亡;Western Blot结果显示,SFN+5-FU联合组Bax的表达量显著高于单药5-FU组,差异有统计学意义(P<0.05);pcDFNA-Bax质粒转染能明显下调SW-480细胞中Bax蛋白表达,并且显著降低了SFN联合5-FU对结肠癌SW-480细胞增殖的抑制效应。结论:SFN通过上调Bax表达增强5-FU对结肠癌细胞化疗耐受的能力,并促使结肠癌细胞凋亡。

Objective: To study the chemotherapy resistance reversal in colon cancer cells with 5-FU,screen the target gene Bax associated with the above reversal process and study its mechanism. Methods: SW-480 cells were cultured in vitro ,the cell viability was determined by CCK8 assay.The impact of SFN on the apoptosis of 5-FU-based colon cancer cells was measured by Annexin V-FITC/PI method.The expression levels of Bax,Suvivin,Bcl-2,p53 and caspase-3 genes in SW-480 cells were determined by using RT-PCR.The expression level of Bax protein was analyzed by Western Blot method.The down-expression of Bax in SW-480 cells was performed by the transfection of pcDFNA-Bax plasmid. Results: SFN could significantly induce the inhibitory effects of 5 -FU on the proliferation of SW-480 cells.The apoptosis experiment displayed that the apoptotic rates of SW-480 cells in 5-FU group and SFN+5-FU group were (36.76±4.29 )% and (51.10±6.34 )%,and there was a significant difference among them ( P <0.05 ).A RT-PCR experiment results displayed that Bax might mediate SFN in promoting the apoptosis of colon cancer cells.Western Blot results showed that the Bax expression in SW-480 cells treated with the combination of SFN and 5-FU was greatly increased compared with 5-FU alone ( P <0.05 ).The trasfection of pcDFNA-Bax plasmid could greatly down-regulate the Bax expression in SW-480 cells,and significantly increase the inhibitory effects of the combination of SFN and 5-FU on the proliferation of SW-480 cells. Conclusion: SFN by down-regulated the Bax gene expression enhanced ability of the resistance of 5-FU to colon cancer cells to chemotherapy,and promote the colon cancer cells apoptosis.