南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (1): 92-98.doi: 10.12122/j.issn.1673-4254.2023.01.12

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紫草素通过抑制PKM2/PHD3/HIF-1α通路诱导肝癌细胞凋亡

张换换，陈 卓，赵想弟，霍 强，程 秀   

  1. 安徽省生化药物工程技术研究中心，安徽 蚌埠 233030
  • 出版日期:2023-01-20 发布日期:2023-02-23

Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway

ZHANG Huanhuan, CHEN Zhuo, ZHAO Xiangdi, HUO Qiang, CHENG Xiu   

  1. Anhui Provincial Engineering Technology Research Center of Biochemical Drugs, Bengbu 233030, China
  • Online:2023-01-20 Published:2023-02-23

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摘要： 目的 探讨紫草素诱导人肝癌细胞SMMC-7721死亡的可能机制。方法 体外培养人肝癌细胞（SMMC-7721）和正常肝细胞（L-02），实验分为对照组和紫草素给药组（4、8、16 μmol/L）。 采用3（-4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐 3（-4,5-二甲基噻唑-2-yl）-2,5-二苯基四氮唑溴盐，MTT法检测细胞活力，试剂盒检测三磷酸腺苷（ATP）和乳酸水平，免疫共沉淀和免疫荧光双染实验明确M2型丙酮酸激酶（PKM2）、脯氨酰酸羟化酶3（PHD3）、缺氧诱导因子1α（HIF-1α）三者之间的作用关系及蛋白表达情况；Annexin V/PI检测细胞凋亡；Western blot观察PKM2、PHD3、HIF-1α 及凋亡蛋白Bax、cleaved caspase-3、Bcl-2表达水平；采用小干扰核糖核酸（siRNA）干扰法建立 PKM2低表达的人肝癌SMMC-7721细胞，Western blot检测 PKM2低表达对人肝癌SMMC-7721细胞中的 PHD3、HIF-1α 蛋白表达水平的影响。结果 紫草素对SMMC-7721和L-02细胞的半抑制浓度（IC50）分别是8.041 μmol/L与31.75 μmol/L，与对照组相比紫草素能抑制肝癌SMMC-7721 细胞中 PKM2、HIF-1α和PHD3蛋白表达及PKM2、HIF-1α入核（P<0.05）。免疫共沉淀和免疫荧光双染实验证实，紫草素可以抑制PKM2/PHD3/HIF-1α复合体形成（P<0.05），并且抑制有氧糖酵解产物乳酸和ATP含量（P<0.05）。与对照组相比，PKM2敲低后PHD3和HIF-1α表达明显降低（P<0.05）；与未处理组相比，紫草素处理后凋亡率明显升高且凋亡蛋白Bax和cleaved caspase-3表达升高，Bcl-2表达下降（P<0.05）。结论 紫草素靶向PKM2调控PHD3/HIF-1α复合物，从而抑制肝癌细胞的有氧糖酵解，破坏其供能途径，导致肝癌细胞凋亡。

关键词: 肝细胞癌；紫草素；M2型丙酮酸激酶；脯氨酰酸羟化酶3；缺氧诱导因子1α；凋亡

Abstract: Objective To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells. Methods Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting. Results The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC- 7721 cells. Co-immunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF- 1α complex and significantly reduced the contents of lactic acid and ATP in SMMC- 7721 cells (P<0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P<0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P<0.05). Conclusions Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.

Key words: hepatocellular carcinoma; shikonin; pyruvate kinase M2; prolyl hydroxylase 3; hypoxia-inducible factor-1α; apoptosis