【摘要】 目的:探讨PI3K/mTOR信号通路抑制剂XL765(SAR245409, Voxtalisib)对人急性髓系白血病细胞株KG-1细胞的抑制效应及可能的作用机制。方法:应用CCK-8法检测XL765对KG-1细胞增殖的影响;平板集落形成实验检测XL765对KG-1细胞集落形成的抑制情况;Annexin V/PI双染色流式细胞术检测XL765对细胞凋亡的影响;实时荧光定量PCR检测细胞凋亡相关基因的表达;Western blot法检测凋亡相关蛋白的表达水平及信号通路分子磷酸化水平的变化。结果:XL765能有效抑制KG-1细胞的增殖,抑制率呈剂量依赖性增加;与DMSO处理组相比,加入XL765后,KG-1细胞的集落形成能力显著下降(P=0.0002);XL765能有效诱导细胞凋亡(P<0.001);XL765作用KG-1细胞48 h后,细胞的凋亡相关基因BCL-2表达下调,BAX及Caspase3表达上调,其差异均有统计学意义(P<0.05);与DMSO组对比,实验组BAX及Caspase3活化蛋白表达上调,同时BCL-2蛋白表达下调,信号蛋白PI3K、AKT及S6K磷酸化表达下调,其差异均有统计学意义(P值均<0.005)。结论:XL765能有效抑制KG-1细胞增殖及集落形成,并诱导细胞凋亡,其机制可能与调节凋亡蛋白BCL2、BAX及Caspase3水平及抑制PI3K、AKT及S6K磷酸化水平相关。更多还原

【Abstract】 Objective: To explore the effect and possible mechanism of PI3K/mTOR inhibitor XL765 on KG-1 cells in vitro. Methods: The effect of XL765 on cell proliferation was detected by CCK-8 assay. The colony formation test(200 cells were plated in a plate for 9 days) was used to detect the effect of XL765 on the colony forming ability of KG-1 cells. The apoptosis was assessed by flow cytometry with Annexin V-FITC/PI double staining. Quantitative real-time polymerase chain reaction(q-PCR) was used to detect the expression of cell apoptosis-related genes BCL-2,BAX and caspase-3, Western blot was performed to detect the expression levels of BCL-2, BAX, Caspase-3, and the phosphorylation change of p-PI3K, p-AKT and p-S6 K. Results: XL765 effectively inhibited the proliferation and the colony formation of KG-1 cells(P=0.0002). XL765(150 nmol/L) induced KG-1 cell apoptosis(31.87±1.376%), very statistically significant different from(3.533±0.4179%) in the control group(P<0.01). Treatment with 150 nmol/L XL765 could in a significantly increase the expression levels of BAX and active caspase-3, and decreases expression level of the BCL-2(P<0.01). In accordance with these results, the Western blot further confirmed the expression decrease of BCL-2 protein along with the increase BAX and cleaved caspase-3 activity. XL765 statistically significantly downregulated the phosphorylation levels of PI3K, AKT and S6 K. Conclusion: PI3K/mTOR inhibitor XL765 substantially suppresses KG-1 cell proliferation and induces apoptosis by inhibiting the activation of PI3K-AKT-mTOR signaling pathway, and regulating the apoptosis-related proteins.更多还原