SN-38对K562细胞的增殖抑制、凋亡诱导及其与塞来昔布联合化疗的协同作用

Effect of SN-38 on Proliferation Inhibition and Apoptosis Induction in K562 Cells and Synergistic Effect of SN-38 and Celecoxib

目的观察SN-38对慢性粒细胞白血病(CML)急变细胞株K562增殖抑制及凋亡诱导作用,并探讨其作用机制及与塞来昔布联合化疗的协同作用。方法采用CCK-8法检测细胞增殖抑制率;流式细胞仪以AnnexinⅤ/PI双标记法检测细胞凋亡,PI单染法检测细胞周期;特异性荧光底物检测Caspase-3、Caspase-8、Caspase-9活性;Western blot法检测凋亡相关蛋白表达变化。以CalcuSyn药效学软件计算联合指数(CI),评价SN-38与塞来昔布对K562细胞的联合效应。结果 SN-38对K562细胞具有时间和浓度依赖性的增殖抑制作用,SN-38可以诱导K562细胞凋亡,伴随G2/M期细胞比例持续下降,Caspase-3、Caspase-8及Caspase-9活性以时间依赖方式升高,并显著下调K562细胞的Survivin蛋白表达。SN-38与塞来昔布联合化疗后,K562细胞增殖抑制率较两单药组明显增强;两药在低浓度时具有剂量依赖的协同效应(Fa<0.87)。结论 SN-38体外抑制K562细胞增殖并诱导其凋亡,其机制可能通过下调Survivin表达,并激活Caspase有关。SN-38与塞来昔布联合应用对K562细胞具有剂量依赖的协同细胞毒效应。

Objective To investigate the effects of SN-38 on the proliferation and apoptosis of human chronic myelocytic leukemia cell line K562 in vitro ,and to evaluate the synergistic effect of SN-38 and Celecoxib. Methods Cell inhibition rate was measured by CCK-8 assay. Apoptosis-inducing effects were determined with Annexin V/PI staining,and cell cycle distribution with PI staining by flow cytometry. Caspase-3,-8 and-9 activity was determined by using the specific fluorogenic substrates. The expression of apoptosis related protein was detected by using Western blot. The combination index（CI）with SN-38 and Celecox ib was calculated using Pharmaconamies CalcuSyn software. Results SN-38 inhibited proliferation of K562 cells in a time and dose-dependent manner in vitro. SN 38 induced apoptosis of K562 cells, reduced the number of cells in G2/M phase, elevated caspase-3,-8 and-9 activity in a time-dependent manner, and down regulated the expression of survivin protein in K562 cells. There was a synergistic effect at lower concentration of SN-38 and Celecoxib（Fa〈0.87）. The cell proliferation inhibition rate in the combination group with SN-38 and Celecoxib was higher than SN-38 and Celecoxib used alone. Conclusion SN-38 can inhibit proliferation and induce apoptosis of K562 cells in vitro, probably by reducing survivin protein expression and caspase activation. SN-38 combined with Celecoxib synergistically enhances cytotoxicity in K562 cells in vitro in a dose-depend ent manner.