茶多酚对人膀胱癌BIU-87细胞间隙连接蛋白43表达和细胞间隙连接通讯功能的影响

Effects of EGCG on the Expression of Connexin43 and Gap Junction Intercellular Communication of the Bladder Cancer Cell Line BIU-87

背景与目的:研究茶多酚的主要植物化学物质成分(-)-epigallocatechin-3-gallate(EGCG)对人膀胱癌BIU-87细胞间隙连接蛋白43(Cx43)的表达和细胞间通讯功能的影响,探讨EGCG防治膀胱肿瘤的机制。材料与方法:分别采用四甲基偶氮唑蓝(MTT)法、膜联蛋白/碘化丙锭双标流式细胞术、RT-PCR、Western印迹和划痕标记荧光染料传输技术,体外观察不同浓度的EGCG(0、5、10、20mg/L)对BIU-87细胞的生长抑制率、凋亡率、Cx43mRNA及其蛋白的表达、细胞间间隙连接通讯功能(GJIC)的变化。结果:10mg/L和20mg/L的EGCG均能明显抑制BIU_87细胞的生长和诱导细胞凋亡,其抑制率分别为(15.67±1.15)%、(18.33±1.53)%,凋亡率分别为(42.00±4.34)%、(27.33±3.21)%。与对照组和5mg/LEGCG相比,EGCG10mg/L和20mg/L组能显著上调Cx43mRNA及其蛋白的表达,并增强细胞间的GJIC功能(P<0.05)。EGCG在10～20mg/L范围内对BIU_87细胞的生长的抑制作用、诱导细胞凋亡和上调Cx43表达的效应均随浓度的增加而增强,呈剂量依赖性(P<0.05)。结论:EGCG(10、20mg/L)能通过有效上调BIU_87细胞Cx43转录水平的表达、增强细胞间隙连接所介导的GJIC功能,从而诱导膀胱肿瘤细胞凋亡,抑制其生长。

BACKGROUND ＆ AIM： To investigate the effects of （-） -epigallocatechin-3-gallate （EGCG, the major phytochemical in green tea） on the expression of connexin43 （Cx43） gene and the intercellular communication of the hmnan bladder cancer cell line BIU-87, and to explore its possible mechanisms of prevention and cure of the bladder tumor. MATERIALS AND METHOVS： The methyl thiazolyl tetrazolium and Annexin-V/ PI double-labeled flow cytometry methods were used to examine the growth inhibitory rate （IR） and apoptosis rate （AR） of BIU-87 cells treated by EGCG at different concentrations （0, 5, 10, 20 mg/L） . The reverse transcfiption-polymerase chain reaction （RT-PCR） and Western Blot analysis were used to measure the relative expression levels of the Cx43 mRNA and its protein. The scrape-loading fluorescence dye transfer method was used to assess the gap junction intercellular communication （GJIC） through fluorescence microscopy. RESULTS： EGCG at concentrations of 10 mg/L and 20 mg/ L could both significantly inhibit the proliferation and induce the apoptosis of BIU-87 cells. The IR and AR were （15.67 ± 1.15）%, （18.33 ± 1.53）% and （42.00±4.34）%, （27.33± 3.21）%, respectively. And EGCG could significantly up-regulate the expression of Cx43 mRNA and its protein, enhance the GJIC of BIU-87 cells compared with groups of 0 and 5 mg/L （P〈0.05） . The effects showed significant correlation with the dose of EGCG. CONCLUSION： EGCG （10, 20 mg/L） could effectively up-regulate Cx43 expression and enhance the GJIC of BIU-87 cells, thereby inducing bladder tumor cells apoptosis and inhibiting its growth. This provides experimental evidence for the mechanism of chemical prevention and cure of the bladder tumor by EGCG.