蝙蝠葛苏林碱调控Akt/mTOR信号通路对胶质瘤细胞增殖及自噬的影响

Effects of daurisoline on proliferation and autophagy of glioma cells through Akt/mTOR signaling pathway

摘要:

目的 探讨蝙蝠葛苏林碱（DAS）对胶质瘤U251和U87细胞生长与增殖的影响及其分子机制。方法 用2.5、5.0和10.0 μmol/L DAS（DAS组）、10.0 μmol/L替莫唑胺（TMZ组）和空白对照（对照组）处理U251和U87细胞。采用CCK-8法检测DAS对细胞活力的影响，细胞集落形成和Edu实验检测DAS对细胞增殖的影响，流式细胞术检测DAS对细胞凋亡的影响，透射电镜观察自噬小体的形成，Western blotting检测DAS对胶质瘤细胞凋亡、自噬和Akt/mTOR信号通路相关蛋白表达的影响。结果 CCK-8结果显示，与对照组比较，不同浓度DAS组胶质瘤细胞活力逐渐降低（P <0.05），DAS组细胞活力低于TMZ组（P <0.05）。胶质瘤U251和U87细胞IC₅₀分别为5.11 μmol/L和6.35 μmol/L。Edu和克隆结果显示，DAS能抑制胶质瘤细胞增殖，且随着药物浓度升高细胞增殖逐渐减少（P <0.05）。流式细胞术结果显示，DAS能诱导胶质瘤细胞凋亡，且随着药物浓度升高细胞凋亡增加（P <0.05）。DAS能增加胶质瘤细胞Bax、LC3Ⅱ/Ⅰ、p62、p-AKT、p-mTOR蛋白相对表达量（P <0.05），降低抗凋亡Bcl-2蛋白相对表达量（P <0.05）。透射电镜结果显示，DAS能增加胶质瘤细胞中自噬囊泡（P <0.05）。结论 DAS可以抑制胶质瘤细胞活力与增殖，并促进胶质瘤细胞凋亡，这可能与DAS激活Akt/mTOR信号通路抑制自噬有关。

Abstract:

Objective To investigate the effects of daurisoline (DAS) on growth and proliferation of glioma U251 and U87 cells and its underlying mechanisms.Methods U251 and U87 cells were treated with different concentrations of daurisoline (2.5, 5.0, and 10.0 μmol/L, DAS groups), 10.0 μmol/L of temozolomide (TMZ group), or left untreated (control group). The CCK-8 assay was used to detect the influence of DAS on cell viability, colony formation assay and EdU assay were used to evaluate the impact of DAS on cell proliferation, and flow cytometry was used to analyze the effect of DAS on cell apoptosis. The formation of autophagosomes was observed under the transmission electron microscopy. Western blotting was performed to detect the expressions of proteins associated with apoptosis, autophagy and Akt/mTOR signaling pathway in glioma cells.Results The CCK-8 assay showed that cell viability of glioma cells treated with DAS was decreased in a concentration-dependent manner compared with that in the control group (P < 0.05). In addition, the cell viability in the DAS group was lower than that in the TMZ group (P < 0.05). The IC50 of glioma U251 and U87 cells was 5.11 μmol/L and 6.35 μmol/L, respectively. The Edu assay and colony formation assay demonstrated that DAS suppressed the proliferation of glioma cells, and that the cell proliferation was successively inhibited with the increase in the drug concentration (P < 0.05). Flow cytometry exhibited that DAS induced apoptosis of glioma cells, and apoptosis aggravated with the increase in the drug concentration (P < 0.05). Western blotting revealed that DAS increased the relative protein expressions of Bax, LC3Ⅱ/Ⅰ, p62, p-AKT, and p-mTOR (P < 0.05) but decreased the relative expression of the anti-apoptotic Bcl-2 protein in glioma cells (P < 0.05). Transmission electron microscopy displayed that DAS promoted the formation of autophagosomes in glioma cells (P < 0.05).Conclusions DAS inhibits the viability and proliferation and promotes apoptosis of glioma cells, which may be associated with the suppression of autophagy by activating the AKT/mTOR signaling pathway.