积雪草酸调控JAK2/STAT3信号通路对前列腺癌细胞凋亡的影响

Effect of asiatic acid on apoptosis of prostate cancer cells via regulation of JAK2/STAT3 signaling pathway

目的研究积雪草酸(AA)对前列腺癌PC-3细胞作用及分子机制。方法采用CCK8法检测积雪草酸(5、10、20、40、80、160μmol·L^(-1))对PC-3细胞活力的影响;采用Hochest法和Annexin V/PI法检测积雪草酸(20、30、40μmol·L^(-1))对PC-3细胞凋亡的影响;采用JC-1法检测积雪草酸对PC-3细胞线粒体膜电位的影响;采用Western blotting法检测积雪草酸对PC-3细胞线粒体凋亡途径和Janus激酶2(JAK2)/信号转导与转录活化因子3(STAT3)信号通路相关蛋白表达水平的影响。采用Annexin V/PI法、JC-1法、Western blotting法检测JAK2激动剂coumermycin A1(C-A1)对积雪草酸诱导PC-3细胞线粒体凋亡及相关蛋白表达的影响。结果与对照组比较,积雪草酸可浓度及时间相关性地抑制PC-3细胞增殖,20~160μmol·L^(-1)组差异显著(P<0.01),作用24、48、72 h后的半数抑制浓度(IC_(50))值分别为29.98、23.04、13.81μmol·L^(-1);20、30、40μmol·L^(-1)积雪草酸显著促进PC-3细胞凋亡(P<0.05、0.01);可明显导致PC-3细胞线粒体膜电位下降;显著上调PC-3细胞线粒体凋亡相关Bax、cleaved Caspase-3的蛋白表达(P<0.05、0.01),显著下调Bcl-2的蛋白表达(P<0.05、0.01),显著抑制JAK2/STAT3信号通路JAK2和STAT3的磷酸化水平(P<0.05、0.01)。与积雪草酸组比较,积雪草酸+C-A1显著抑制PC-3细胞的凋亡水平(P<0.01),明显抑制PC-3细胞线粒体膜电位下降,显著下调Bax、cleaved Caspase-3蛋白表达(P<0.01),显著上调Bcl-2蛋白表达(P<0.01),显著提升JAK2和STAT3的磷酸化水平(P<0.01)。结论积雪草酸可通过抑制JAK2/STAT3信号通路诱导前列腺癌细胞发生线粒体凋亡,进而发挥抗前列腺癌作用。

Objective To study the effects and molecular mechanism of asiatic acid(AA)on prostate cancer PC-3 cells.Method CCK8 assay was used to detect the effect of AA(5,10,20,40,80,and 160μmol·L^(-1))on PC-3 cell proliferation.Hochest assay and Annexin V/PI assay were used to detect the effect of AA(20,30,and 40μmol·L^(-1))on PC-3 cell apoptosis.JC-1 method was used to detect the effect of AA on mitochondrial membrane potential of PC-3 cells.Western blotting was used to detect the effect of AA on the expression level of mitochondrial apoptosis pathway and JAK2/STAT3 signaling pathway related proteins in PC-3 cells.Annexin V/PI method,JC-1 method and Western blotting method were used to detect the effects of JAK2 agonist Coumermycin A1(C-A1)on mitochondrial apoptosis and expression of related proteins in PC-3 cells induced by AA.Results Compared with control group,AA significantly inhibited the proliferation of PC-3 cells in a concentration-dependent and time-dependent manner,the difference of20—160μmol·L^(-1)group was significant(P<0.01).and the IC_(50)value was 29.98,23.04 and 13.81μmol·L^(-1)after 24,48 and 72 h.AA significantly induced the apoptosis of PC-3 cells compared with control group(P<0.05 and 0.01).AA also changed the MMP in PC-3 cells.AA significantly up-regulated mitochondrial apoptosis-related Bax and cleaved Caspase 3 expression compared with control group(P<0.05 and 0.01),and significantly down-regulated mitochondrial apoptosis-related Bcl-2 expression(P<0.05 and0.01).The phosphorylation levels of JAK2 and STAT3 in JAK2/STAT3 signaling pathway were also significantly inhibited by AA(P<0.05 and 0.01)compared with control group.Compared with AA group,AA+C-A1 significantly inhibited the apoptosis of PC-3cells(P<0.01),inhibited the MMP in PC-3 cells significantly,significantly down-regulated Bax and cleaved Caspase 3 expression in PC-3 cells(P<0.01),and significantly up-regulated Bcl-2 expression(P<0.01),as well as significantly increased the phosphorylation levels of JAK2 and STAT3(P<0.01).Conclusion AA could induce mitochondrial apoptosis in prostate cancer cells by inhibiting JAK2/STAT3 signaling pathway,and finally play an anti-prostate cancer role.