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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 observe the effect of magnesium cantharidate on mitogen-activated protein kinase (MAPK) signaling pathway in SMMC-7721 hepatoma cells and to explore the anti-cancer mechanism of cantharidin magnesium. Methods The effect of magnesium cantharidate and okadaic acid (OA) on the activity of PP2A was assayed by the protein phosphatase 2A (PP2A) activity assay kit. The expression of extracellular signal-regulated kinase 1/2 (ERK1 / 2), p38MAPK and c-Jun N-terminal kinase 1/2 (JNK1 / 2) mRNA in SMMC-7721 human hepatocellular carcinoma cells were detected by real- The effect of magnesium cantharidate and OA on the expression of ERK1 / 2, p38MAPK, JNK and protein phosphorylation in SMMC-7721 cells was detected by Western blot. Results Magnesium cantharidate at 0.283μmol / L had no significant inhibitory effect on PP2A activity. At 0.567μmol / L magnesium cantharidate, the activity of PP2A was significantly inhibited. With the increase of drug concentration, the inhibitory effect became obvious. At the same time, 0.059 nmol / L OA on PP2A activity also significantly inhibited. Compared with the blank control group, the expression of ERK1 and ERK2 mRNA did not change significantly in the 0.283μmol / L magnesium cantharidate group, and the expression of ERK1 and ERK2 mRNA decreased significantly at the concentration of 0.567μmol / L, and decreased with the increase of the drug concentration More obvious; while 0.059 nmol / L OA group ERK1, ERK2 mRNA expression was significantly increased. The expressions of p38MAPK, JNK1 and JNK2 mRNA in 0.059 nmol / L OA and magnesium cantharidate groups were significantly increased. Compared with the blank control group, the level of ERK1 / 2 phosphorylation did not change significantly in 0.283μmol / L magnesium cantharidate magnesium group, but higher than 0.567μmol / L magnesium cantharidate significantly down-regulated phosphorylation level of ERK1 / 2, Dependent effect. However, ERK1 / 2 phosphorylation was significantly up-regulated in 0.059 nmol / L OA group. 0.059 nmol / L OA and different concentration of magnesium cantharidate magnesium group p38MAPK, JNK phosphorylation were significantly increased. Conclusion Magnesium cantharidin may inhibit the proliferation of SMMC-7721 hepatoma cells by inhibiting the activity of PP2A and inhibiting the ERK1 / 2 pathway.