臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

Apoptosis-inducing effect of ailanthone on human melanoma A375 cells and its active mechanism

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。

To investigate the effect of ailanthone (AIL) on the apoptosis of human melanoma A375 cells and its mechanism,the effect of AIL on proliferation of A375 cells was studied by MTT assay.The morphological change of A375 cells treated by AIL was observed by inverted phase contrast microscope.Hoechst33258 staining was used to observe nuclear change of A375 cell treated by AIL.The apoptosis of A375 cells induced by AIL was analyzed by flow cytometry.Caspase-3 and caspase-9 activity was detected using commercial kits.The expression level of p-PI3K β(Ser1070),PI3K β,p-Akt (Ser473) and Akt was tested by Western blot.LY294002 was invited to prove the effect of AIL on PI3K/Akt signaling pathway.The result showed that AIL significantly inhibited the proliferation of A375 cells.Decrease of cell number and shrinkage of nuclear could be observed in A375 cells treated by AIL.The apoptosis rate of A375 cells was increased by AIL.The activity of caspase-3 and caspase-9 was elevated by AIL in A375 cells.Protein phosphorylation of PI3K and Akt was inhibited by AIL in A375 cells.PI3K and Akt phosphorylation level deceased and apoptosis rate increased more significantly by AIL co-treated with LY294002.This further noted that AIL induced A375 cells apoptosis via PI3K/Akt signaling pathway.