尖吻蝮蛇毒抑瘤组分I通过增强子结合蛋白同源蛋白/半胱氨酸天冬氨酸蛋白酶-12途径诱导人舌鳞癌Tca8113细胞凋亡的研究  

Apoptosis of Tca8113 cell induced by antitumor component-I from Agkistrodon acutus venom through CHOP/Caspase-12 pathway

作　　者：柴琳 王振杰 徐冉 CHAI Lin;WANG Zhenjie;XU Ran(School of Stomatology, Wannan Medical College, Wuhu 241001, Anhui, China;Department of Pathophysiology, Wannan Medical College, Wuhu 241001, Anhui, China)

机构地区：[1]皖南医学院口腔医学院,安徽芜湖241001 [2]皖南医学院病理生理学教研室,安徽芜湖241001

出　　处：《中国临床药理学与治疗学》2020年第3期271-277,共7页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：安徽省高校自然科学研究重点项目(KJ2017A255);皖南医学院重点科研培育基金(WK2015Z08);芜湖市科技计划项目(2015CXY11);皖南医学院大学生科研资助金项目(WK2019S24)。

摘　　要：目的:探讨内质网应激途径在尖吻蝮蛇毒抑瘤组分I(antitumor component-I from Agkistrodon acutus venom,AAVC-I)诱导人舌鳞癌Tca8113细胞凋亡中的作用。方法:本实验选择人舌鳞癌Tca8113细胞作为研究对象,体外条件下传代培养取对数生长期细胞进行实验。根据实验目的设为正常对照组、二硫苏糖醇(DL-dithiothreitol,DTT)阳性对照组和AAVC-I实验浓度组。MTT法检测不同浓度的DTT和AAVC-I处理Tca8113细胞24 h后的增殖抑制作用并筛选合适的DTT阳性对照实验浓度和AAVC-I实验浓度组,采用HE染色、Annexin V-FITC/PI双荧光染色法观察细胞凋亡情况,Western blot检测内质网应激葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、增强子结合蛋白同源蛋白(enhance-binding protein-homologous protein,CHOP)、半胱氨酸天冬氨酸蛋白酶-12(Caspase-12)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达水平。结果:随着AAVC-I实验浓度的增加,其对Tca8113细胞的增殖抑制作用增强(P<0.05),并观察到细胞皱缩变小间隙增大,胞核浓缩,细胞破碎,出现凋亡小体,凋亡率增高(P<0.05),蛋白GRP78、CHOP、Caspase-12、Caspase-9、Caspase-3表达水平上调(P<0.05)。结论:在AAVC-I诱导人舌鳞癌Tca8113细胞凋亡中,内质网应激CHOP/Caspase-12途径发挥着正向调节作用。AIM:To investigate the effect of endoplasmic reticulum stress pathway on the apoptosis of tongue squamous cancer Tca8113 cells induced by antitumor component-I from Agkistrodon acutus venom(AAVC-I).METHODS:The in vitro experiments were performed on subculture tongue squamous cancer Tca8113 cells in their growth period.A normal control group,a DL-dithiothreitol(DTT)positive control group and different AAVC-I concentrations were set according to the experiment objective.MTT assay was used to detect the proliferation inhibition of Tca8113 cells after been treated with different concentrations of DTT and AAVC-I for 24 h.The results were used to choose appropriate concentrations of DTT and AAVC-I in DTT positive control group and AAVC-I treated group,respectively.HE staining and Annexin V-FITC/PI double fluorescence staining were used to monitor the apoptosis of Tca8113 cells.Western blot was used to identify the expression levels of apoptosis-related proteins including endoplasmic reticulum stress glucose-regulatory protein 78(GRP78),enhance-binding protein-homologousprotein(CHOP),cysteine-containing aspartate specific protease-12(Caspase-12),cysteine-containing aspartate specific protease-9(Caspase-9)and cysteine-containing aspartate specific protease-3(Caspase-3).RESULTS:The proliferation inhibition of Tca8113 cells increased with an increased concentration of AAVC-I concentration(P<0.05),causing cell shrinkage,increased cell gaps,cytonuclear condensation,cell fragmentation,the appearance of apoptotic bodies,and increased rate of apoptosis(P<0.05).In addition,the expression level of GRP78 protein,CHOP protein,proteins of Caspase-12,Caspase-9 and Caspase-3 were increased(P<0.05).CONCLUSION:Endoplasmic reticulum stress CHOP/Caspase-12 pathway plays an important role in AAVC-I induced Tca8113 cells apoptosis.

关 键 词：内质网应激 人舌鳞癌Tca8113细胞 蛇毒 

分 类 号：R965.2[医药卫生—药理学]