基于YAP与Akt/mTOR通路交叉对话探究没药甾酮抑制脑胶质瘤细胞增殖的作用机制

The effect and mechanism of Z-guggulsterone inhibit the proliferation of glioblastoma cells based on the crosstalk between YAP and Akt/mTOR pathway

关键词:  没药甾酮  YAP  Akt/mTOR通路  人脑胶质瘤  T98G细胞

Key words:  guggulsterone  YAP  Akt/mTOR signaling  glioma  T98G cells

摘要:

目的 探究没药甾酮(Z-guggulsterone，Z-GS)通过YAP与Akt/mTOR通路交叉对话调控脑胶质瘤细胞增殖的作用机制。方法 免疫组织化学染色法测定脑胶质瘤临床标本中Mst1、Lats1、YAP、p-YAP、Akt、p-AktThr308、p-AktSer473、mTOR、p-mTORSer2448和p-mTORSer2481蛋白表达；Western Blot法筛选蛋白表达量较高的人脑胶质瘤细胞株。人脑胶质瘤细胞T98G经Z-GS处理后，CCK-8法检测细胞活力确定给药浓度和时间；平板克隆实验检测T98G细胞增殖情况；Western Blot法检测T98G细胞中Mst1、p-Mst1、Lats1、p-Lats1、YAP、p-YAP、PTEN、Akt、p-AktThr308、p-AktSer473、mTOR、p-mTORSer2448和p-mTORSer2481蛋白表达。结果 与正常人脑组织相比，Ⅳ级人脑胶质瘤组织中YAP、p-YAP、Akt、p-AktThr308、mTOR、p-mTORSer2448阳性表达更强，p-YAP、Akt、p-mTORSer2448的阳性表达与脑胶质瘤组织有显著性关联。四种人脑胶质瘤细胞中T98G细胞上述蛋白表达较高。Z-GS(12.5，25，50 μmol·L-1)给药24 h后对人脑胶质瘤T98G细胞无细胞毒性作用且能抑制细胞增殖能力。与对照组相比，Z-GS显著下调T98G细胞中YAP、p-YAP蛋白表达和p-mTORSer2448水平，显著上调p-Mst1和p-Lats1水平。结论 Z-GS可能通过恢复Hippo通路活性，下调mTORC1活性，抑制人脑胶质瘤细胞增殖。

Abstract:

OBJECTIVE To explore the effect and mechanism of Z-guggulsterone regulating glioblastoma cells proliferation through YAP and Akt/mTOR pathway crosstalk. METHODS Determine the expression of expression of YAP, p-YAP, Akt, p-AktThr308, p-AktSer473, mTOR, p-mTORSer2448 and p-mTORSer2481 proteins in glioma clinical specimens by immunohistochemical staining. Screen the glioblastoma cell lines with high expression of above proteins by western blotting. Assess the concentration and time of administration of Z-guggulsterone in glioblastoma cells by CCK-8. After Z-guggulsterone treatment of human glioma cells, measure the cell proliferation by plate cloning experiment; Determine the expression of Mst1, p-Mst1, Lats1, p-Lats1, YAP, p-YAP, PTEN, Akt, p-AktThr308, p-AktSer473, mTOR, p-mTORSer2448 and p-mTORSer2481 proteins in glioblastoma cells treated with Z-guggulsterone by western blotting. RESULTS Compared with normal brain tissue, the positive expression of YAP, p-YAP, Akt, p-AktThr308, mTOR and p-mTORSer2448proteins in grade IV glioma tissue is stronger. The positive expression of p-YAP, Akt, and p-mTORSer2448 is significantly correlated with the glioblastoma tissue. The expression level of above proteins in T98G cells is higher among the four glioblastoma cell lines. After T98G cells treatment with Z-guggulsterone (12.5, 25, 50 μmol·L-1) for 24 hours, there was no cytotoxic effects and it could inhibit the cell proliferation. Meanwhile, Z-guggulsterone significantly reduced the proteins expression of p-YAP, YAP and the level of p-mTORSer2448, and significantly increased the level of p-Mst1 and p-Lats1 in T98G cells. CONCLUSION Z-guggulsterone can restored the activity of Hippo pathway, and down-regulated the activity of mTORC1 to inhibit the proliferation of glioma cells.