表没食子儿茶素没食子酸酯对两株不同肝癌耐药细胞株基因表达谱的影响

Impact of Epigallocatechin Gallate on Gene Expression Profiles of Human Hepatocellular Carcinoma Cell Lines BEL7404/ADM and BEL7402/5-FU

背景与目的:绿茶中的儿茶素单体表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)在体内外能逆转肝癌细胞多药耐药性。鉴于多药耐药机制的复杂性,本研究拟采用高通量基因芯片技术进一步探讨EGCG逆转人肝癌细胞BEL7404/ADM、BEL7402/5-FU多药耐药的可能机制。方法:MTT法检测药物敏感性,基因芯片技术分析EGCG作用于两株不同肝癌耐药细胞的基因表达谱差异,RT-PCR和Western blot法分别检测相同处理条件下两株肝癌耐药细胞的基因MDR1、LRP和Cyclin G1蛋白表达变化以验证基因芯片技术结果。结果:EGCG对BEL7404/ADM、BEL7402/5-FU细胞的IC10分别为24.76mg/L、20.60mg/L。20mg/L EGCG联合0.05mg/L ADM、100μmol/L 5-FU对BEL7404/ADM、BEL7402/5-FU的逆转倍数分别为9.66、2.36倍。EGCG作用BEL7404/ADM双荧光通路显著差异基因210个,上调表达38个,下调表达172个。与耐药机制相关的可能基因有ABCB10(MDR/TAP)、TOP2A、TOP2B、CCNG1上调,ABCB1、MVP、ARHD、HDAC5、GSS、GSTPI、HSPA1B、HSPB7、CDKN1A、RAB11B、RAB9P40下调。作用BEL7402/5-FU双荧光通路显著差异基因179个,上调表达31个,下调表达148个。与耐药机制相关的可能基因有ABCG(BCRP)、CCNG2、GADD34、RB1、RBBP4上调,DTYMK、GPX1、USP5、BAX、BAK1、HSPA1L下调等。相同处理条件下两组肝癌耐药细胞的MDR1、LRP基因表达均减少,CyclinG1蛋白表达均增加。结论:EGCG对肝癌多药耐药细胞BEL7404/ADM、BEL7402/5-FU具逆转作用,但作用于不同肝癌多药耐药细胞的基因表达谱变化不完全相同。

BACKGROUND ＆ OBJECTIVE= Epigallocatechin gallate （EGCG） from green tea could reverse multidrug resistance （MDR） in human hepatocellular carcinoma （HCC） in vitro and in vivo. This study was to investigate the mechanism of reversing effect of EGCG on MDR of human hepatocelluar carcinoma cell lines BEL7404/ADM and BEL7402/5-FU. METHODS： Drug sensitivity of BEL7404/ADM and BEL7402/5-FU cells was tested by MTT assay. The different gene expression profiles of BEL7404/ ADM and BEL7402/5-FU cells were detected by cDNA microarray before and after treatment of EGCG. The expression of MDR1 and LRP genes was detemined by reverse transcription-polymerase chain reaction （RT-PCR）; the expression of Cyclin G1 protein was detected by Western blot to confirm the results of cDNA microarray. RESULTS= The 10% inhibitory concentration （IC10） of EGCG was 24.76 mg/L for BEL7404/ADM cells and 20.60 mg/L for BEL7402/5-FU cells. When treated with 0.05 mg/L adriamycin （ADM） and 100 μmol/L 5-fluorouracil （5-FU） in combination, 20 mg/L EGCG reversed the MDR by 9.66 folds in BEL7404/ADM cells and by 2.36 folds in BEL7402/5-FU cells. After treatment of EGCG, 210 differentially expressed genes were identified in BEL7404/ADM cells. 38 were up-regulated and 172 were down-regulated; the potential MDR-related genes included the up- regulated ABCB10 （MDR/TAP）, TOP2A, TOP2B, CCNG1, and down- regulated ABCB1, MVP, ARHD, HDAC5, GSS, GSTPI, HSPA1B, HSPB7, CDKN1A, RAB11B, RAB9P40. After treatment of EGCG, 179 differentially expressed genes were identified in BEL7402/5-FU cells= 31 were upregulated and 148 were down-regulated; the potential MDR-related genes included the up-regulated ABCG （BCRP）, CCNG2, GADD34, RB1, RBBP4, and down-regulated DTYMK, GPX1, USP5, BAX, BAK1, HSPA1L. The downregulation of MDR1 and LRP expression was confirmed by RT-PCR; the upregulation of Cyclin G1 expression was confirmed by Western blot. CONCLUSION. EGCG could reverse the MDR of BEL7404/ADM and BEL7402/5-FU cells, but the changes of gene expression profiles of these two HCC cell lines are different.