摘要
目的观察斑蝥素酸镁对SMMC-7721肝癌细胞丝裂原激活蛋白激酶(MAPK)信号通路的影响,探讨斑蝥素酸镁的抗癌机制。方法使用蛋白磷酸酶2A(PP2A)活性检测试剂盒分别检测斑蝥素酸镁和冈田酸(OA)对PP2A活性的影响。实时定量PCR检测斑蝥素酸镁、OA对SMMC-7721人肝癌细胞胞外信号调节激酶1/2(ERK1/2)、p38MAPK、c-Jun N末端激酶1/2(JNK1/2)mRNA表达水平的影响;Western blot法检测斑蝥素酸镁、OA对SMMC-7721细胞ERK1/2、p38MAPK、JNK蛋白表达以及蛋白磷酸化水平的影响。结果 0.283μmol/L斑蝥素酸镁对PP2A活性无明显抑制作用,0.567μmol/L斑蝥素酸镁能显著抑制PP2A活性,且随着药物浓度的增加,抑制作用愈趋明显;同时0.059 nmol/L OA对PP2A活性也有显著抑制作用。与空白对照组比较,0.283μmol/L斑蝥素酸镁组ERK1、ERK2 mRNA表达量无明显变化,当浓度为0.567μmol/L时,ERK1、ERK2mRNA表达量显著下降,且随着药物浓度的增加下降更明显;而0.059 nmol/L OA组ERK1、ERK2 mRNA表达量却显著升高。0.059 nmol/L OA和不同浓度斑蝥素酸镁组的p38MAPK、JNK1、JNK2 mRNA表达量均显著升高。与空白对照组比较,0.283μmol/L斑蝥素酸镁组ERK1/2磷酸化水平无明显变化,高于0.567μmol/L斑蝥素酸镁处理显著下调ERK1/2磷酸化水平,其下调程度具有浓度依赖效应;而0.059 nmol/L OA组ERK1/2磷酸化水平却显著上调。0.059 nmol/L OA和不同浓度斑蝥素酸镁组的p38MAPK、JNK磷酸化水平均显著上调。结论斑蝥素酸镁可能是通过抑制PP2A活性进而抑制ERK1/2通路来实现对SMMC-7721肝癌细胞增殖的抑制作用。
Objective To investigate the anticancer mechanism of magnesium cantharidate by observing its effect on the mitogen-activated protein kinase（MAPK） signaling pathway in human hepatoma SMMC-7721 cells. Methods The protein phosphatase 2A（PP2A） activity detection kit was used to detect the effects of magnesium cantharidate and okadaic acid（OA） on PP2 A activity. After the treatment of SMMC-7721 cells with magnesium cantharidate and/or OA,mRNA levels of extracellular signal-regulated kinase 1（ERK1）,ERK2,p38 MAPK,c-Jun N-terminal kinase 1（JNK1） and JNK2 were detected by real-time quantitative PCR,and the protein expression levels and protein phosphorylation of ERK1,ERK2,p38 MAPK and JNK were determined by Western blotting. Results The effect of magnesium cantharidate on the activity of PP2 A in SMMC-7721 cells was not evident at the concentration of 0. 283 μmol/L,but the activity of PP2 A was declined significantly at 0. 567 μmol/L or higher concencentrations in a concentration-dependent manner. Likewise,OA also displayed apparent inhibitory effect on the activity of PP2 A at 0. 059 nmol/L. Compared with the control group,mRNA levels of ERK1 and ERK2 were not changed by magnesium cantharidate at 0. 283 μmol/L,but they significantly declined at the concentrations greater than0. 567 μmol/L. In contrast,mRNA levels of ERK1 and ERK2 were significantly elevated by 0. 059 nmol/L OA. mRNA levels of p38 MAPK,JNK1 and JNK2 significantly increased after the treatment of 0. 059 nmol/L OA or magnesium cantharidate at varying concentrations. Compared with the control group,phosphorylation levels of ERK1 and ERK2 were not changed by0. 283 μmol/L magnesium cantharidate,but decreased significantly when the concentration was 0. 567 μmol/L or above. In contrast,the phosphorylation levels of ERK1 and ERK2 showed a significant increase in 0. 059 nmol/L OA treated group. The phosphorylation levels of p38 MAPK,JNK1 and JNK2 were also significantly increased by 0. 059 nmol/L OA or magnesium cantharidate in a concentration-dependent manner. Conclusion Magnesium cantharidate may inhibit the proliferation of SMMC-7721 cells by inhibiting the activity of PP2 A and ERK1/2 signaling pathway.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第3期347-351,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81560669
81260488)
贵州省科技合作计划项目(黔科合LH字[2015]7509号
黔科合LH字[2015]7516号)
贵州省科技厅中药现代化项目(黔科合ZY字[2013]3012)
贵州省卫计委科学基金项目(gzwjk2015-1-013)
医学昆虫资源开发利用创新团队(黔合教人才团队字[2014]39号)
贵州省教育厅特色重点实验室建设项目(黔教合KY字[2014]212)
