鸦胆子苦醇抑制H1299非小细胞肺癌细胞增殖和促进其凋亡的机制

Research of proliferation inhibition and apoptosis promotion mechanism of brusatol on H1299 cells

目的研究鸦胆子苦醇(brusatol,BRU)对H1299非小细胞肺癌细胞增殖和凋亡的影响及其机制。方法使用CCK-8法与EdU实验检测BRU对细胞增殖的影响;克隆形成实验检测BRU对细胞克隆形成的影响;Hoechst33258染色实验与流式细胞仪观察细胞凋亡情况;Western blot检测Bax、Bcl-2、Bcl-xL、cleaved-caspase-3、caspase-3、Gadd45α、PI3K、p-PI3K、Akt、p-Akt、细胞核与胞质中NF-κB-p65的蛋白质水平。结果CCK-8实验发现,随着BRU浓度与作用时间的增加,H1299细胞受到的抑制作用逐渐增强;EdU实验显示,BRU处理组的EdU掺入率明显下降;克隆形成实验结果显示,BRU能抑制H1299细胞的克隆形成能力;Hoechst33258实验与流式细胞仪检测发现,BRU能使H1299细胞胞核呈现凋亡状态并增加其凋亡率;Western blot显示BRU可上调Bax、cleaved-caspase-3、Gadd45α,下调Bcl-xL、Bcl-2、caspase-3、p-PI3K、p-Akt与胞核中p65的蛋白质水平。结论BRU以浓度和时间依赖抑制H1299细胞的增殖并诱导其凋亡,其机制可能与抑制PI3K/Akt信号通路的激活并抑制NF-κB-p65的核转运有关。

Aim To preliminarily investigate the effect of brusatol(BRU),the monomer components from Chinese medicines on H1299 cells and its mechanisms.Methods CCK-8 and EdU staining experiment were used to detect the effect of BRU on cell prolifera-tion.Clone formation experiment was performed to measure the effect of drugs on cell clone formation.Hoechst33258 staining experiment and flow cytometry were employed to observe the cell apoptosis.Western blot assay was used to detect the protein expression levels of Bcl-xL,Bax,Bcl-2,cleaved-caspase-3,caspase-3,Gadd45α,PI3K,p-PI3K,Akt,p-Akt and NF-κB-p65.Results CCK-8 assay revealed that the inhibitory effect of H1299 cells gradually increased with the rising of BRU concentration and action time.Compared with control group,the EdU incorporation rate of the BRU treatment group decreased significantly.Treated with different concentrations of BRU for 24 h,the clone formation rate was significantly reduced in a concentration-dependent manner.Hoechst33258 experiment and flow cytometry showed that BRU could induce apoptosis in H1299 cell nucleus and increase its apoptotic rate.Western blot results revealed that BRU could significantly up-regulate the protein levels of Bax,cleaved-caspase-3,Gadd45α,and significantly down-regulate the expression of Bcl-xL,Bcl-2,caspase-3.In addition,BRU could significantly decrease the expression level of p-PI3K,p-Akt,NF-κB-p65 in cell nucleus.Conclusions BRU can inhibit the proliferation and induce apoptosis of H1299 cells in a concentration and time-dependent manner.The mechanism may be related to the inhibition of PI3K/Akt signaling pathway and the nuclear shuttle of NF-κB-p65.