银杏双黄酮介导AMPK/COX-2通路对黑色素瘤细胞凋亡的影响

Effect of ginkgo biloba biflavoneson apoptosis of melanoma cells by mediating AMPK/COX-2 pathway

目的:基于单磷酸腺苷活化蛋白激酶/环氧合酶-2(AMPK/COX-2)通路探究银杏双黄酮(GBB)对黑色素瘤细胞发生凋亡的影响。方法:体外培养人黑色素瘤细胞株A875,用不同浓度(50μmol/L、100μmol/L、200μmol/L)GBB、10μmol/LAMPK抑制剂Compound C、200μmol/L GBB+10μmol/L Compound C作用于A875细胞24 h。采用倒置显微镜观察细胞形态,Annexin V-FITC/PI双染色法检测GBB对A875细胞凋亡的影响,Western blotting法检测磷酸化乙酰辅酶A羧化酶(p-ACC)、磷酸化AMPK(p-AMPK)及COX-2蛋白表达。结果:随着GBB浓度的增加,细胞凋亡率升高(P<0.05),细胞形态回缩变小、变圆、变亮,细胞间接触消失,部分细胞脱落裂解为碎片状,悬浮细胞增多,折光性减弱,呈现典型的细胞凋亡形态。GBB可提高p-ACC、p-AMPK蛋白表达,降低COX-2蛋白表达(均P<0.05);Compound C逆转了GBB对p-ACC、p-AMPK蛋白表达的促进作用及对COX-2蛋白表达的抑制作用(均P<0.05)。结论:GBB可诱导人黑色素瘤A875细胞凋亡,且呈剂量依赖性,此过程可能是通过介导AMPK/COX-2通路实现的。

Objective:To investigate the effect of ginkgo biloba biflavones(GBB)on the apoptosis of melanoma cells based on adenosine 5'-monophosphate activated protein kinase/cyclooxygenase-2(AMPK/COX-2)pathway.Methods:Human melanoma cell line A875 cells were cultured in vitro,and treated with 0μmol/L,50μmol/L,100μmol/L,200μmol/L GBB,10μmol/L Compound C(AMPK inhibitor),or 200μmol/L GBB+10μmol/L Compound C respectively for 24 h.An inverted microscope was used to observe the cell morphology.Annexin VFITC/PI double staining was used to detect the effect of GBB on apoptosis of A875 cells.Western blotting was used to detect the expressions of phosphorylated(p-)acetyl-CoA carboxylase(ACC),p-AMPK and COX-2 protein.Results:With the increase of GBB concentration,the apoptosis rate of A875 cells increased(P<0.05).The cell morphology shrank,became smaller,rounder and brighter,and the contact between cells disappeared.Some cells fell off and lysed into fragments,the suspended cells increased,and the refraction also weakened,showing a typical phenomenon of apoptosis.GBB could induce apoptosis of A875 cells,and with the increase of GBB concentration,the rate of apoptosis also increased(P<0.05).GBB increased the expression of p-ACC and p-AMPK while reduced the expression of COX-2 protein(P<0.05).Compound C reversedthe promotive effect of GBB on the expressions of p-ACC and p-AMPK and inhibitive effect on COX-2 protein(P<0.05).Conclusion:GBB can induce apoptosis of human melano ma A875 cells in a dose-dependent manner,which may be achieved by mediating AMPK/COX-2 pathway.