紫草素通过抑制PKM2/PHD3/HIF-1α通路诱导肝癌细胞凋亡  

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

作　　者：张换换 陈卓 赵想弟 霍强 程秀 ZHANG Huanhuan;CHEN Zhuo;ZHAO Xiangdi;HUO Qiang;CHENG Xiu(Anhui Provincial Engineering Technology Research Center of Biochemical Drugs,Bengbu 233030,China)

机构地区：[1]安徽省生化药物工程技术研究中心,安徽蚌埠233030

出　　处：《南方医科大学学报》2023年第1期92-98,共7页Journal of Southern Medical University

基　　金：安徽省自然科学基金(1908085MH292);安徽省高校优秀青年人才基金(gxyq2019038);蚌埠医学院科技发展基金(BYKF1721)。

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、16μmol/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8.041μmol/L与31.75μmol/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。Objective To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721cells.Methods Cultured SMMC-7721 cells and normal hepatocytes(L-02 cells)were treated with 4,8,or 16μmol/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 PHD3and HIF-1αexpressions in SMMC-7721 cells were detected using Western blotting.Results The IC50of shikonin against SMMC-7721 and L-02 cells was 8.041μmol/L and 31.75μmol/L,respectively.Treatment with shikonin significantly inhibited the protein expressions of PKM2,HIF-1αand PHD3 and nuclear translocation of PKM2 and HIF-1αin SMMC-7721 cells.Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1αcomplex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells(P<0.05).The expressions of PHD3 and HIF-1αdecreased 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-1αsignaling pathway to cause energy supply dysfunction in the cells.

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 

分 类 号：R285[医药卫生—中药学]