紫云英苷通过抑制HIF-1α诱导的糖酵解途径发挥抗卵巢肿细胞作用的研究

Study of the effect of astragalin on proliferation of ovarian cancer cells by inhibiting the glycolytic pathway induced Via HIF-1α

目的探讨紫云英苷在2 D及3 D细胞培养条件下对人卵巢癌OVCAR-8细胞的增殖作用及其可能机制。方法采用CCK-8试剂盒检测紫云英苷在2 D培养水平对OVCAR-8细胞增殖活力的作用;采用3 D细胞增殖活性检测试剂盒检测紫云英苷在3 D培养水平对OVCAR-8细胞增殖活力的作用;采用划痕实验检测紫云英苷对OVCAR-8细胞迁移能力的作用;采用凋亡试剂盒检测细胞凋亡水平;采用Western blot法在2 D及3 D培养条件下检测紫云英苷对OVCAR-8细胞细胞凋亡相关蛋白Bcl2(B-cell lymphoma 2)、Bax(Bcl-2-associated X protein)和cleaved-caspase-3以及糖酵解相关蛋白Glut(Glucose transporter) 1、Glut3、HK2(Hexokinase 2)、PDK(Pyruvate dehydrogenase lipoamide kinase) 1、PDK3以及HIF(Hypoxia-inducible factor)-1α的蛋白表达水平的影响;采用ELISA试剂盒检测HK2活性。结果 4～100μmol/L紫云英苷在2 D培养条件下对OVCAR-8细胞增殖均有抑制作用,且呈剂量-时间依赖效应关系(P <0. 05);紫云英苷可在3 D培养条件下明显抑制OVCAR-8细胞增殖(P <0. 05);同时,紫云英苷可明显抑制OVCAR-8细胞迁移能力。此外,紫云英苷处理后细胞凋亡水平明显增高,且紫云英苷处理在2 D及3 D培养条件下均可抑制Bcl2、Glut1、Glut3、HK2、PDK1和PDK3蛋白的表达,增加Bax及cleaved-caspase-3蛋白水平并抑制3 D培养诱导的HIF-1α的蛋白表达,此外,紫云英苷在2 D培养条件下可抑制HK2活性,均呈一定的剂量依赖效应关系(P <0. 05)。结论紫云英苷在2 D及3 D培养条件下对卵巢癌OVCAR-8细胞增殖均具有抑制作用。且可能通过抑制HIF-1α诱导的糖酵解通路以及激活线粒体凋亡通路。

Objective The objective of this study was to explore the effect of astragalin on human ovarian cancer OVCAR-8 cells in 2 D and 3 D culture conditions and its possible mechanism.Methods CCK-8 assay was use to detect the effect of astragalin on the proliferation of OVCAR-8 cells in 2 D culture conditions.The 3 D cell proliferation activity assay kit was used to detect the effect of astragalin on OVCAR-8 cells in 3 D culture conditions.Cell apoptosis kit was used to detect the cell apoptosis rate after astragaline treatment.In addition,Western blot was used to detect the levels of apoptosis related proteins such as Bcl2(B-cell lymphoma 2),Bax(Bcl-2-associated X protein),cleaved-caspase-3 and glycolysis related proteins such as Glut-1(Glucose transporter-1),Glut3,HK2(Hexokinase 2),PDK-1(Pyruvate dehydrogenase lipoamide kinase-1),PDK3 and HIF-1α(hypoxia-inducible factor-1α)in OVCAR-8 cells after astragaline treatments in 2 D and 3 D culture conditions.HK2 activity was detected in OVCAR-8 cells by Elisa.Results Astragaline at doses of 4~100μmol/L significantly inhibited the proliferation of OVCAR-8 cells in 2 D culture conditions,and showed a dose-and time-dependent manner(P<0.05).Astragalin significantly inhibited the proliferation of OVCAR-8 cells in 3 D culture conditions(P<0.05).Astragalin also significantly inhibited the migration ability of OVCAR-8 cells(P<0.05).In addition,astragaline significantly increased apoptosis rate,decreased the levels of Bcl2,Glut1,Glut3,HK2,PDK1 and PDK3 proteins and increased the levels of Bax and cleaved-caspase-3 proteins in OVCAR-8 cells both in 2 D and 3 D culture conditions(P<0.05).Astragaline significantly decreased the expression of HIF-1αin OVCAR-8 cells in 3 D culture conditions(P<0.05).In addition,astragalin decreased HK2 activity in OVCAR-8 cells under 2 D culture conditions in a dose-dependent manner(P<0.05).Conclusion Astragalin has an inhibitory effect on the proliferation of OVCAR-8 cells both in 2 D and 3 D culture conditions.Its mechanism may be related to inhibiting glycolytic and the mitochondrial apoptotic pathways induced by HIF-1α.