Inhibitory Effects of Zoledronic Acid on Proliferation and Apoptosis in Human Nasopharyngeal Carcinoma Cell Line HNE-1 in vitro
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摘要: 目的 观察唑来膦酸对鼻咽癌细胞系HNE-1的增殖抑制及凋亡诱导作用并探索其相关机制。方法 MTT法检测体外细胞增殖能力；流式细胞仪分析细胞凋亡和细胞周期变化；原位末端标记法 (TUNEL) 检测DNA片段化以标记凋亡细胞；Real-time PCR和Western blot检测凋亡相关基因Bcl-2、Bad、Bax及Caspase-9 mRNA和蛋白表达。结果 MTT显示不同浓度唑来膦酸处理组(2.5、5、10、20、40 μmol/L)作用24 h，对 HNE-1细胞增殖无抑制作用(P>0.05)；作用48和72 h均能明显抑制 HNE-1 细胞增殖(P<0.05)；但抑制增殖能力与药物浓度和作用时间未呈时效和量效关系(P>0.05)。10~40 μmol/L唑来膦酸处理后，48和72 h 的早期凋亡率明显高于对照组；S期细胞比例较对照组升高 (P＜0.01)；TUNEL 法染色显示，作用72 h后凋亡细胞明显增多(P<0.05)；Real-time PCR和Western blot显示，唑来膦酸下调Bcl-2 mRNA和蛋白的表达，同时上调Bad、Bax及Caspase-9 mRNA和蛋白表达。结论 唑来膦酸可以抑制HNE-1细胞增殖，诱导HNE-1细胞凋亡，可能与促进Bad、Bax及Caspase-9的表达并降低Bcl-2的表达有关。Abstract: Objective To demonstrate the effects of apoptosis-inducing and proliferation-inhibiting of zoledronic acid (ZOL) in human nasopharyngeal carcinoma cell HNE-1 and explore its potential mechanism. Methods The ability of cell proliferation was detected by MTT assay. Cell apoptosis and cycle were analyzed by fl ow cytometry. DNA fragmentation of apoptotic cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) assay. The expression of mRNA and protein of apoptosis-related genes, such as Bcl-2, Bad, Bax and Caspase-9, were investigated by Real-time PCR and Western blot. Results Compared with control group, different concentrations of ZOL(2.5, 5, 10, 20, 40 μmol/L) did not show an inhibition effect on the proliferation of HNE-1 cells at the time point of 24 h by MTT assay. While the anti-proliferative effect was found obviously over a period of incubation of 48 h as well as 72 h(P<0.05), but not shown in a doseor time-dependent manner(P>0.05). The rate of early apoptosis was signifi cantly higher than that of control group over an incubation time of 48 and 72 h. After incubated with ZOL for 48 h, HNE-1 cells showed a cell cycle arrest in S phases. TUNEL method showed more apoptotic cells in the experiment group (P<0.05). Realtime PCR and Western blot revealed that the proapoptotic genes, Bad, Bax, and Caspase-9, were up-regulated in ZOL-treated HNE-1 cells, whereas the antiapoptotic gene Bcl-2 was down-regulated both in mRNA and protein levels. Conclusion ZOL could inhibit cell proliferation and induce cell apoptosis in HNE-1 cells, which might be related with down-regulating protein expressions of antiapoptotic Bcl-2 gene and upregulating proapoptotic genes, Bax, Bad and Caspase-9.
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