1,4-二(2-苄硒乙氧基)蒽诱导人喉癌Hep-2细胞凋亡的研究

The research on apoptosis of human laryngeal cell line Hep-2 induced by 1,4-bis[2-(benzylselanyl)ethoxy] anthracene

背景与目的:硒是人类生命活动中必不可少的微量元素之一,对人类健康的巨大作用是其他物质无法替代的。硒的小分子化合物1,4-二(2-苄硒乙氧基)蒽{1,4-bis[2-(benzylselanyl)ethoxy]anthracene,BSEA}具有杀菌消炎的作用,但BSEA的抗癌作用尚未见报道。该研究旨在探讨BSEA诱导人喉癌Hep-2细胞凋亡及其作用机制。方法:不同浓度的BSEA处理人喉癌Hep-2细胞24 h后,采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测细胞生长抑制率;在荧光显微镜下观察细胞形态;AnnexinⅤ-FITC法检测细胞凋亡;JC-1检测线粒体膜电位;酶标分析仪检测caspase-3和caspase-8活性;实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)分别检测Bax、XIAP mRNA和蛋白表达水平。结果:BSEA处理人喉癌Hep-2细胞24 h后,BSEA对其生长抑制呈剂量依赖性,IC_(50)为35.74μmol/L;80μmol/L BSEA处理的Hep-2细胞在荧光显微镜下可观察到明显的凋亡小体;磷脂酰丝氨酸外翻加剧;线粒体膜电位开始下降;caspase-3活性的影响呈剂量依赖趋势,而caspase-8活性未见显著变化;Bax的mRNA和蛋白表达水平上升,XIAP的mRNA和蛋白表达水平下降。结论:BSEA对人喉癌Hep-2细胞生长有明显的抑制作用,并能通过线粒体途径诱导细胞凋亡。

Background and purpose： Selenium is one of the essential trace elements for human activities, and plays an incomparable role in maintaining human health. It was reported that selenium compound 1,4-bis [ 2-（benzylse- lanyl）ethoxy ] （BSEA） anthracene has antiseptic and antiphlogistic effects. However, the mechanisms underlying anti- cancer effects of BSEA are rarely reported. BSEA-induced apoptosis in human laryngeal carcinoma Hep-2 cells and its mechanisms were studied. Methods： Methyl thiazolyl tetrazolium （MTF） assay was used to determine inhibition ratio of Hep-2 cells 94 hours after Hep-2 cells were treated with different concentrations of BSEA. Fluorescence microscope was used to observe the morphology change of apoptosis in Hep-2 cells. The apoptosis was detected by Annexin V-FITC. Mi- tochondrial membrane potential was assayed by JC-1. Mieroplate reader detected the activity of caspase-3 and caspase-8. The mRNA and protein levels of Bax and XIAP were measured by real-time fluorescent quantitative polymerase chain reaction （RTFQ-PCR） and Western blot. Results： The results showed that BSEA caused a dose-dependent inhibition of the growth of human laryngeal carcinoma cell line Hep-2 in vitro, and ICso was 35.74 μmol/L. The apoptotic bodies were distinctly observed at a concentration of 80 μmol/L of BSEA by AO fluorescence staining. This study found that the eversion of pbosphatidyl serine intensified, and mitochondrial membrane potential also began to decline. The activity of caspase-3 appeared the tendency of dependence on dosage, while the activity of caspase-8 did not change significantly. The mRNA and protein expression level of Bax increased, whereas the mRNA and protein expression level of XIAP decreased. Conclusion： Therefore, BSEA could obviously inhibit human laryngeal carcinoma Hep-2 cells proliferation and induce apoptosis via the mitochondrial pathway.