蟾毒灵通过激活内质网应激通路诱导HCT116细胞凋亡

Bufalin induces apoptosis of HCT116 cells by activating endoplasmic reticulum stress pathway

目的研究蟾毒灵对人结直肠癌细胞HCT116增殖及凋亡的影响,并探讨内质网应激(ERS)在此过程中的作用。方法采用CCK-8法测定蟾毒灵对HCT116细胞增殖活性的影响;不同浓度蟾毒灵作用HCT116细胞48 h后,采用Annexin V/PI法检测细胞凋亡情况,用Western blot法检测凋亡相关蛋白Bax和Bcl-2的表达情况,同时用Western blot法检测内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、磷酸化蛋白激酶R样内质网激酶(p-PERK)、真核翻译起始因子2α(eIF2α)、磷酸化真核翻译起始因子2α(p-eIF2α)和C/EBP同源蛋白(CHOP)的表达情况;将HCT116细胞分为对照组、蟾毒灵组和联合组(蟾毒灵+4-苯基丁酸),Western blot法检测凋亡相关蛋白Bax和Bcl-2的表达变化。结果CCK-8法检测结果显示蟾毒灵对HCT116细胞的增殖活性有抑制作用;细胞凋亡实验表明蟾毒灵能引起HCT116细胞的凋亡;Western blot实验结果显示,蟾毒灵能够上调促凋亡蛋白Bax表达,下调抗凋亡蛋白Bcl-2表达,同时也能诱导ERS并激活PERK/eIF2α/CHOP通路;当蟾毒灵联合4-苯基丁酸后,蟾毒灵的促凋亡作用得到了抑制。结论蟾毒灵能有效地抑制HCT116的增殖活性,并能诱发HCT116的凋亡,而该作用在一定程度上是通过激活ERS来实现的。

Objective To study the effect of bufalin on the proliferation and apoptosis of human colorectal cancer cell line HCT116,and to explore the role of endoplasmic reticulum stress(ERS)in this process.Methods The effect of bufalin on the proliferation of HCT116 cells was determined by CCK-8 assay.After HCT116 cells were treated with different concentrations of bufalin for 48 hours,cell apoptosis was detected by Annexin V/PI assay,and the expression of apoptosis-related proteins Bax and Bcl-2 was detected by Western blot.At the same time,the expression of ERS-related proteins glucose regulated protein 78(GRP78),phosphorylated protein kinase R like endoplasmic reticulum kinase(p-PERK),eukaryotic translation initiation factor 2α(eIF2α),phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α)and C/EBP homologous protein(CHOP)was detected by Western blot.HCT116 cells were divided into control group,bufalin group and combination group(bufalin+4-phenylbutyric acid),and the expression of apoptosis-related proteins Bax and Bcl-2 was observed by Western blot.Results CCK-8 assay showed that bufalin could inhibit the proliferation of HCT116 cells.Apoptosis assay showed that bufalin could induce apoptosis of HCT116 cells.The results of Western blot showed that bufalin could up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2.It could also induce ERS and activate PERK/eIF2α/CHOP pathway.When bufalin combined with 4-phenylbutyric acid,the apoptosis-promoting effect of bufalin was inhibited.Conclusion Bufalin can effectively inhibit the proliferative activity and induce apoptosis of HCT116,which is achieved to some extent by activating ERS.