奥沙利铂通过内质网应激PERK-eIF2α-ATF4通路诱导口腔鳞癌HSC-3细胞凋亡发生的研究

Oxaliplatin induced apoptosis of oral squamous cell carcinoma HSC-3 cells through PERK-eIF2α-ATF4 pathway of endoplasmic reticulum stress

目的研究奥沙利铂对口腔鳞癌HSC-3细胞的杀伤作用及ERS中PERK-eIF2α-ATF4通路的影响。方法CCK8检测不同浓度奥沙利铂对HSC-3细胞增殖的影响;DAPI染色和Annexin V/PI双染法检测奥沙利铂对HSC-3细胞凋亡诱导情况;RT-qPCR和Western blotting分别检测奥沙利铂激活HSC-3细胞中内质网应激及PERK-eIF2α-ATF4通路相关基因和蛋白的表达。结果CCK-8结果显示,不同浓度的(10、20、30、40、50 mg/L)奥沙利铂对HSC-3细胞生长增殖具有抑制作用,且呈一定的剂量依赖性,其中半数有效浓度为40 mg/L。DAPI染色和Annexin V/PI双染法结果证明不同浓度(20、40、80 mg/L)奥沙利铂对HSC-3细胞具有促进凋亡的作用;与对照组相比,奥沙利铂组细胞核染色质固缩加重,形成更多的核碎裂片段和凋亡小体,表明凋亡进一步加重。RT-qPCR及Western blot结果显示奥沙利铂促进内质网应激标志性分子GRP78、CHOP及信号通路PERK-eIF2α-ATF4的标志性分子PERK、eIF2α、ATF4表达的上调,同时促进PERK磷酸化水平和Caspase 3发生剪切激活,最终诱导细胞凋亡的发生。结论本研究明确了奥沙利铂在体外具有杀伤口腔鳞癌HSC-3细胞的作用,其分子机制之一可能是通过内质网应激途径及其PERK-eIF2α-ATF4信号通路的激活实现药物对细胞的抑制作用,此研究结果为临床上治疗口腔鳞癌提供了一定的理论基础和依据。

Objective This study examined the role of PERK-eIF2α-ATF4 signaling pathway,the main pathway of endoplasmic reticulum stress(ERS),in the oxaliplatin inhibition of oral squamous cell carcinoma HSC-3 cell proliferation.Methods CCK8 detected the effects of 10,20,30,40 and 50 mg/L oxaliplatin on HSC-3 cell proliferation.Apoptosis induced by oxaliplatin on HSC-3 cells was detected by DAPI staining and Annexin V/PI staining.The expression of genes and proteins related to ERS and PERK-eIF2α-ATF4 pathways in the HSC-3 cells were detected by RT-qPCR and Western blot respectively.Results The results of CCK-8 showed that different concentrations of oxaliplatin(10,20,30,40 and 50 mg/L)could inhibit the growth and proliferation of HSC-3 cells in a dose-dependent manner,and its half maximal inhibitory concentration was 40 mg/L.The results of DAPI and Annexin V/PI staining proved that different concentrations of oxaliplatin(20,40,and 80 mg/L)could promote the apoptosis of HSC-3 cells.Compared to the control group,the pyknosis of nuclear chromatin in oxaliplatin group was aggravated,and more nuclear fragmentation and apoptotic bodies were formed,indicating the further aggravation of apoptosis.The results of RT-qPCR and Western blot showed that oxaliplatin promoted the up-regulation of ERS marker GRP78,CHOP and PERK-eIF2α-ATF4 signaling pathway marker PERK,eIF2α,ATF4,the phosphorylation level of PERK and the shear activation of Caspase 3,which finally contributed to cell apoptosis.Conclusion It was suggested that ERS pathway and its PERK-eIF2α-ATF4 signaling pathway may be one of the molecular mechanisms of oxaliplatin-induced inhibition of oral squamous cell carcinoma HSC-3 cell proliferation,which could provide a theoretical basis for clinical treatment of oral squamous cell carcinoma.