摘要
目的:探讨绿茶提取物(green tea extract,GTE)对人卵巢癌SKOV3细胞株生长增殖的影响及其作用途径。方法:应用MTT法观察SKOV3细胞生长增殖的情况;光学显微镜观察SKOV3细胞的形态学变化;流式细胞术(FCM)检测SKOV3细胞周期及凋亡的情况;DAPI标记法观察细胞凋亡形态变化;Western blotting检测p-ERK1/2、ERK1/2、Caspase-3及Bcl-xl等相关蛋白的表达水平。结果:随GTE浓度的增加及作用时间的延长,SKOV3细胞存活率逐渐降低(P<0.01),SKOV3细胞数目逐渐减少,细胞受损逐渐增多;经GTE(80μg/L)处理24h、48h、72h的SKOV3,G0/G1期细胞比例与对照组比均显著减少(P<0.01),S期细胞比例及sub-G0期细胞比例与对照组比均显著升高(P<0.01),而添加GTE24h后,G2/M期细胞比例与对照组比明显升高(P<0.05);DAPI标记示有典型的凋亡小体;经GTE(40μg/L、80μg/L)作用24h后与对照组比,SKOV3细胞中p-ERK1/2、Bcl-xl蛋白表达下调,p-ERK1/2分别为0.334±0.030、0.053±0.140(P<0.01);Bcl-xl分别为0.410±0.026、0.267±0.020(P<0.01);Caspase-3蛋白分别为0.128±0.090、0.287±0.028,与对照组相比显著升高(P<0.01),而ERK1/2蛋白则未见明显变化(P>0.05)。结论:GTE在体外能有效地抑制SKOV3细胞的生长,其作用途径可能是通过抑制ERK信号通路的转导,导致细胞周期阻滞,并下调Bcl-xl、上调Caspase-3,最终导致SKOV3细胞凋亡。
Objective： To investigate the anticancer effect and related molecular-level mechanism of green tea extract （GTE） on human ovarian cancer cells. Methods： The human ovarian cancer cell line SKOV3 cells were used. The cytotoxic effect of GTE on human ovarian cancer SKOV3 cells was determined by MTT assay. The cell cycle arrest effect of GTE on SKOV3 cells was determined with indicated concentration of GTE treatment by flow cytometry. To determine whether GTE-mediated loss of SKOV3 cell viability was the result of the induction of apoptosis, the effect of GTE on cell morphology was observed by phase-contrast photomicrographs and DAPI staining assay. Expression levels of phosphorylated ERK1/2 proteins, p-ERK1/2, Caspase-3 and Bcl-xl were determined by Western blotting. Results： Incubation of SKOV3 cell with GTE resulted in the marked inhibition of cellular proliferation and cell viability （P0.01）. Phase-contrast photomicrographs taken 24 h after GTE treatment revealed a dose-dependent decrease in cell density. Cell flow cytometric analysis 24 h, 48 h, and 72 h after 80 μg/L GTE treatment revealed a dramatic increase of the cell population in S phase （P0.01） and sub-G0 phase （P0.01）. There was also a dramatic increase in G2/M phase 24 h after GTE treatment compared with the control （P0.05）, while in G0/G1, there was a dramatic decrease of the cell population. Simultaneously, the presence of chromatin condensation and nuclear fragmentation which were characteristics of apoptosis were also evaluated by fluorescence microscope. GTE （40 μg/L, 80 μg/L, respectively） treatment of SKOV3 resulted in downregulation of the protein expression of pERK, Bcl-xl, which was 0.334 ± 0.030, 0.053 ± 0.140; 0.410 ± 0.026, 0.267 ± 0.020, respectively, a dramatic decrease compared with controls （P0.01）, and upregulation of Caspase-3, which was 0.128 ± 0.090, 0.287±0.028, a dramatic increase compared with the control （P0.01）, while there was no significant change of ERK protein expression. Conclusion： GTE can inhibit ovarian cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of apoptotic-related proteins. Thereby, the GTE-mediated apoptosis can be applied to an advanced strategy in the development of a potential drug against ovarian cancer.
出处
《生殖与避孕》
CAS
CSCD
北大核心
2010年第4期245-252,共8页
Reproduction and Contraception
