穿心莲内酯对骨肉瘤143B细胞的抑制作用及其机制

Effect of andrographolide on osteosarcoma 143B cells and its mechanism

目的:探究穿心莲内脂(AG)对人骨肉瘤143B细胞的抑制作用及其潜在的机制。方法:体外培养人骨肉瘤143B细胞,使用不同浓度(0~20μmol/L)的AG处理143B细胞,分别用结晶紫染色、MTT法和集落形成实验来检测AG对143B细胞增殖的影响。划痕愈合实验检测骨肉瘤143B细胞的迁移能力。Transwell小室实验检测骨肉瘤143B细胞的侵袭能力。Hoechst 33258染色实验和流式细胞术检测AG对骨肉瘤细胞凋亡的影响。不同浓度AG处理后,使用Western blot检测骨肉瘤细胞增殖、迁移侵袭和凋亡相关蛋白的水平。应用Western blot进一步检测Wnt信号通路中的β-catenin及其相关分子c-Myc的表达量。结果:与空白组相比,AG处理组中143B骨肉瘤细胞的增殖、迁移和侵袭能力明显受到抑制(P<0.05),且呈浓度依赖性。迁移侵袭相关蛋白基质金属蛋白酶9(MMP-9)、波形蛋白(vimentin)和Snail蛋白的水平均下调(均P<0.05),抗凋亡蛋白Bcl-2表达量升高,凋亡相关蛋白caspase-3的蛋白水平下降而cleaved caspase-3的蛋白水平升高,同时Wnt信号通路相关蛋白β-catenin和c-Myc的表达水平均明显下调(P<0.01)。结论:穿心莲内酯可能通过抑制Wnt信号通路的活性,从而达到抑制人骨肉瘤143B细胞增殖、迁移和侵袭能力,并且促进其凋亡。

AIM:To investigate the inhibitory effect of andrographolide(AG)on human osteosarcoma 143B cells and its underlying molecular mechanism.METHODS:Osteosarcoma 143B cells were cultured in vitro and treated with AG at different concentrations(0~20μmol/L),and the effect of AG on the proliferation of 143B cells was determined by crystal violet staining,MTT assay and colony formation assay.The wound-healing assay was performed to detect the migration ability of osteosarcoma 143B cells.Transwell assay was performed to analyze the invasive capacity of osteosarcoma143B cells.The effect of AG on the apoptosis of osteosarcoma 143B cells was detected by Hoechst 33258 staining and flow cytometry.After treatment with of AG at different concentrations,the protein levels of the molecules related to proliferation,migration,invasion and apoptosis of osteosarcoma 143B cells were determined by Western blot.The expression ofβ-catenin and its related molecule c-Myc in the Wnt signaling pathway was analyzed by Western blot.RESULTS:Compared with blank group,the proliferation,migration and invasion of osteosarcoma 143B cells in AG treatment group were significantly inhibited(P<0.05)in a concentration-dependent manner.The expression levels of invasion-and migrationrelated proteins matrix metalloproteinase-9(MMP-9),vimentin and Snail were all down-regulated(P<0.05).AG also increased the expression of antiapoptotic protein Bcl-2,and the levels of apoptosis-related protein caspase-3 was decreased but cleaved caspase-3 was increased.At the same time,the expression levels of Wnt/β-catenin signaling pathway related proteinsβ-catenin and c-Myc were significantly decreased(P<0.05).CONCLUSION:Andrographolide may inhibit the proliferation,migration,and invasion of osteosarcoma 143B cells and promote their apoptosis by inhibiting the activity of Wnt signaling pathway.