唑来膦酸抑制 U937细胞增殖及诱导凋亡

摘要

目的：研究唑来膦酸（ ZOL）对人类急性髓系白血病细胞U937的增殖抑制及促凋亡作用。方法：CCK－8法检测不同时间ZOL对U937细胞的生长抑制率；流式细胞术检测ZOL对 U937细胞周期的影响；Annexin V－PI法及Hoechst 33342法检测ZOL作用前后细胞凋亡情况变化， JC－1检测ZOL对U937细胞线粒体膜电位变化的影响；克隆形成实验检测U937细胞克隆形成能力；Western blot法检测ZOL对U937细胞周期和凋亡相关蛋白的变化。结果：CCK－8结果显示ZOL可以抑制U937细胞的活力，并呈时间－剂量依赖性；Annexin V－PI 及Hoechst 33342结果显示ZOL可以促进U937细胞凋亡，且呈时间－剂量依赖性；JC－1结果显示ZOL可以明显降低U937细胞线粒体膜电位；PI法证实ZOL将U937细胞周期阻滞在S期，克隆形成实验证实0．2 mmol／L ZOL可以完全抑制U937细胞的克隆形成能力；Western blot结果显示ZOL作用于U937细胞48 h后细胞周期相关蛋白p21表达显著增强，促凋亡蛋白Bax表达增强，抑凋亡蛋白Bcl－2表达明显减弱。结论： ZOL抑制U937细胞的增殖和克隆形成主要是由于抑制了细胞周期相关蛋白表达，同时ZOL可以促进U937细胞凋亡，这种作用主要是通过调节线粒体凋亡途径相关蛋白来实现的。%[ ABSTRACT] AIM:To study the antiproliferation and proapoptotic effects of zoledronic acid ( ZOL) on human a-cute myeloid leukemia cell line U937.METHODS:The viability of U937 cells was detected by CCK-8 assay.The cell cy-cle of the U937 cells was analyzed by flow cytometry with PI staining.Apoptotic rate was assessed by flow cytometry with Annexin V-PI and Hoechst 33342 staining.Mitochondrial membrane potential was detected by JC-1 assay.Methylcellulose was used to assess colony formation.The protein levels of p21, Bcl-2 and Bax were determined by Western blot.RE-SULTS:ZOL inhibited the viability of U937 cells.ZOL induced S-phase cell cycle arrest in the U937 cells.The results of flow cytometry analysis with Annexin V-PI and Hoechst 33342 staining showed that ZOL also induced apoptosis in a dose-and time-dependent manner.Mitochondrial membrane potential assay was also used to verify the apoptosis.The apoptotic rate was consistent with the reduction of mitochondrial membrane potential.Colony formation assay showed that ZOL signifi-cantly inhibited the colony formation capacity of the U937 cells.This was achieved by the induction of S-phase cell cycle arrest, and up-regulation of Bax and p21, and down-regulation of Bcl-2.CONCLUSION:ZOL inhibits cell proliferation by regulating the expression of cell cycle-related protein, and induces apoptosis via the mitochondrial apoptotic pathway.