表没食子儿茶素没食子酸酯对人骨肉瘤细胞143B的抑制作用及其机制探讨

Inhibitory effect of epigallocatechin-3-gallate on human osteosarcoma cell line 143B and its underlying mechanism

目的:研究表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)对人骨肉瘤细胞143B的抑制作用,并初步探讨其可能的分子机制。方法:分别用0~50μmol/L的EGCG处理骨肉瘤143B细胞,然后采用结晶紫染色法观察细胞增殖能力的变化,Hoechst染色法分析细胞凋亡情况,细胞划痕愈合实验和Transwell小室法分析细胞迁移能力,蛋白质印迹法检测细胞凋亡及迁移相关蛋白的表达水平。同时,采用荧光素酶报告基因系统分析细胞中Wnt/β-catenin信号通路的活化情况,再用蛋白质印迹法检测Wnt/β-catenin信号通路中关键因子β-catenin及其下游靶分子c-Myc和cyclin D1的表达情况。结果:EGCG处理后,骨肉瘤143B细胞的增殖和迁移能力均受到明显抑制(P值均<0.05),而晚期凋亡细胞数明显增加(P<0.05);细胞中凋亡蛋白caspase-3及其剪切体的表达水平均明显上调(P值均<0.05),而Bcl-2、金属基质蛋白酶2(matrix metalloproteinase-2,MMP-2)、β-catenin、c-Myc和cyclin D1的蛋白表达水平均明显下调(P值均<0.05)。此外,EGCG处理组的荧光素酶活性较未处理组明显下降(P<0.05)。结论:EGCG能抑制骨肉瘤143B细胞的增殖和迁移,促进细胞凋亡;这一作用可能与其阻断Wnt/β-catenin信号通路有关。

Objective： To investigate the inhibitory effect of epigallocatechin-3-gallate （EGCG） on human osteoscarcoma cell line 143B, and its possible molecular mechanism.Methods： Human osteoscarcoma 143B cells were treated with 0-50 μmol/L EGCG. Then the proliferation, apoptosis and migration of 143B cells were measured by crystal violet staining, hoechst staining, cell wound-healing and Transwell chamber assays, respectively. The expression levels of apoptosis-and migration-related proteins were detected by Western blotting. The activity of Wnt/β-catenin signaling pathway was detected by luciferase reporter assay. Furthermore, the protein expression levels of β-catenin and its downstream target molecules c-Myc and cyclin D1 were detected by Western blotting.Results： After treatment with EGCG at highly concentration, the proliferation and migration of 143B cells were significantly inhibited （both P 〈 0.05）, but the proportion of apoptotic cells was markedly increased （P 〈 0.05）. Compared with the EGCG-untreated group, EGCG up-regulated the expressions of caspase-3 and its cleaved protein （both P 〈 0.05）, and down-regulated the expressions of Bcl-2, matrix metalloproteinase-2 （MMP-2）, β-catenin, c-Myc and cyclin D1 proteins （all P 〈 0.05）. In addition, the luciferase activity reflecting Wnt/β-catenin signaling pathway activity in EGCG-treated group was significantly lower than that in EGCG-untreated group （P 〈 0.05）. Conclusion： EGCG can inhibit the proliferation and migration of osteoscarcoma 143B cells, and promote apoptosis, which may be related to the blocking of Wnt/β-catenin signaling pathway.