茶多酚联合表柔比星对人膀胱癌T24细胞增殖和凋亡的影响及机制

Effects of tea polyphenols combined with epirubicin on T24 bladder cancer cell line and its mechanism

摘要:

目的：研究茶多酚（polyphenols，TP）对表柔比星（epirubicin，EPI）诱导人膀胱癌T24细胞增殖及凋亡的影响，探讨两者联合对T24细胞生长影响的机制。方法：不同浓度TP和EPI作用膀胱癌T24细胞24 h，MTT试验选择药物作用浓度TP（88.8 μmol/L）和EPI（4.6 μmol/L）。试验分空白对照组、TP组（88.8 μmol/L）、EPI组（4.6 μmol/L）、联合组（TP 88.8 μmol/L +EPI 4.6 μmol/L）共4组，处理T24细胞24 h后，Annexin V-PI双流式细胞术检测各组细胞凋亡率；处理T24细胞8 h后，透射电镜观察各组细胞内自噬体的变化；处理稳定表达GFP-LC3的T24细胞8 h后，荧光显微镜下观察绿色荧光蛋白LC3荧光斑的形成与变化；处理T24细胞不同时间，Western blot技术检测自噬标志蛋白LC3-Ⅱ（8 h）、底物蛋白P62（8 h）、凋亡相关蛋白CASP3、 PARP（16 h）表达变化。结果：TP（88.8 μmol/L）联合EPI（4.6 μmol/L）处理组较单药组引起的细胞增殖抑制率（F=730.411，P=0.000）和细胞凋亡率（F=161.945，P=0.000）明显提高。EPI（4.6 μmol/L）单药处理T24细胞后引起明显自噬，透射电镜下细胞质内自噬体双层膜结构形成；荧光显微镜下胞质内有明显点状荧光斑聚集形成，且含荧光斑的细胞数量也明显增加，差异有统计学意义（F=41.944，P=0.000）；Western blot结果显示EPI组自噬相关蛋白LC3-Ⅱ的表达增多，P62表达降低。联合TP作用后自噬明显减弱，自噬相关蛋白表达降低，而凋亡相关蛋白活性显著增高。结论：TP可以增强EPI对膀胱癌T24细胞的致凋亡敏感性，其机制与TP抑制EPI化疗过程中诱导的保护性自噬有关。

Abstract:

Objective：To investigate the effect of tea polyphenols（TP） on epirubicin（EPI）-induced cell proliferation and cell apopto－sis of human bladder cancer cells T24 in vitro and its mechanism. Methods：MTT assay was applied to investigate the effects of differ－ent levels of tea polyphenols or epirubicin on the T24 cells. The experiment was divided into 4 groups：control group（PBS），TP group（88.8 μmol/L），EPI group（4.6 μmol/L），and TP（88.8 μmol/L） plus EPI（4.6 μmol/L） group. The proliferation inhibition rate was as－sayed by MTT；Annexin V-FITC/PI double staining flow cytometry was used to analyze apoptosis. The ultra-structural cellular changes were observed by transmission electron microscope（TEM）；The formation of MAP1LC3-Ⅱ puncta was examined after transfection of a GFP-LC3 plasmid. The levels of LC-3-Ⅱ，P62，CASP3，PARP proteins were detected by Western blot，respectively. Results：Treatment with TP（88.8 μmol/L） and EPI（4.6 μmol/L） leaded to a significant increase of inhibiting rate（P=0.000） and a dramatic improvement in apoptotic changes compared to single EPI or TP group（P=0.000）. Autophagy was observed obviously in bladder cancer cells T24 treated with EPI after 8 h. TEM examination revealed the appear－ance of autophagosome with double-membrane structure. Im－munofluorescence showed that EPI strongly augmented the number of cells with increased GFP-LC3 puncta and GFP-LC3 puncta formation within the cell. Western blot showed the ex－pression of LC-3-Ⅱ was increased in T24 cells after treated with EPI，while the expression of P62 was decreased. TP com－bined with EPI and single groups both induced the expression of active apoptosis proteins，and the effect was more obvious in TP plus EPI group. Conclusion：TP combined with EPI can synergisti－cally inhibit the proliferation and promote cell apoptosis，which may be associated with the reduction of EPI-induced autophagy by TP.