摘要
目的探讨高迁移率族蛋白1(HMGB1)-核因子κB(NF-κB)信号通路对人肝癌细胞自噬和化疗敏感性的影响及可能机制。方法体外培养肝癌细胞BEL-7402,分为对照组(不作任何处理的BEL-7402细胞)、多柔比星组、重组人HMGB1+多柔比星组、抗HMGB1中和抗体+多柔比星组、吡咯烷二硫氨基甲酸酯+多柔比星组以及3-甲基腺嘌呤+多柔比星组。四甲基偶氮唑盐(MTT)法检测细胞增殖抑制率;蛋白质印迹法检测HMGB1及NF-κB亚单位p-p65蛋白表达,同时检测自噬相关蛋白Beclin-1、LC3Ⅰ、LC3Ⅱ和凋亡相关蛋白bcl-2蛋白的表达;酶标法检测Caspase-9、Caspase-3活性。结果对照组、多柔比星组、重组人HMGB1+多柔比星组、抗HMGB1中和抗体+多柔比星组、吡咯烷二硫氨基甲酸酯+多柔比星组及3-甲基腺嘌呤+多柔比星组细胞增殖抑制率分别为(1.31±0.16)%、(47.80±6.30)%、(31.60±5.68)%、(67.20±6.83)%、(66.60±6.27)%、(68.60±11.19)%,差异有统计学意义(F=75.91,P<0.01),提示多柔比星对BEL-7402细胞有增殖抑制作用,重组人HMGB1预处理后,多柔比星对BEL-7402细胞的增殖抑制作用出现减弱;HMGB1表达水平分别为1.17±0.11、1.37±0.15、1.43±0.15、0.70±0.09、1.27±0.12、1.29±0.18,差异有统计学意义(F=18.70,P<0.01),提示多柔比星作用BEL-7402细胞可使HMGB1高表达,抗HMGB1中和抗体能阻断或减弱这一作用;采用抗HMGB1中和抗体、吡咯烷二硫氨基甲酸酯及3-甲基腺嘌呤预处理细胞后,多柔比星对BEL-7402细胞的增殖抑制作用增强。与对照组相比,多柔比星组、重组人HMGB1+多柔比星组、3-甲基腺嘌呤+多柔比星组p-p65蛋白表达均增加(均P<0.05);重组人HMGB1+多柔比星组、抗HMGB1中和抗体+多柔比星组、吡咯烷二硫氨基甲酸酯+多柔比星组p-p65蛋白表达均低于多柔比星组(均P<0.05)。与对照组相比,多柔比星组、抗HMGB1中和抗体+多柔比星组、吡咯烷二硫氨基甲酸酯+多柔比星组及3-甲基腺嘌呤+多柔比星组bcl-2蛋白表达减少(均P<0.05),Caspase-9、Caspase-3活性增强(均P<0.05);加入重组人HMGB1预处理后,细胞中bcl-2蛋白表达较单用多柔比星作用时增加(P<0.05),Caspase-9、Caspase-3活性减弱(均P<0.05)。对照组、多柔比星组、重组人HMGB1+多柔比星组、抗HMGB1中和抗体+多柔比星组、吡咯烷二硫氨基甲酸酯+多柔比星组及3-甲基腺嘌呤+多柔比星组自噬相关蛋白Beclin-1的表达水平分别为0.77±0.12、0.92±0.07、1.29±0.10、0.51±0.03、0.49±0.06、0.42±0.05,差异有统计学意义(F=97.01,P<0.01)。LC3Ⅱ表达水平分别为0.24±0.04、0.39±0.04、0.49±0.07、0.23±0.05、0.20±0.06、0.20±0.05,差异有统计学意义(F=26.98,P<0.01)。结论HMGB1-NF-κB信号通路活化可降低肝癌BEL-7402细胞对多柔比星的化疗敏感性,其机制可能与调控自噬进而下调多柔比星对肝癌细胞的凋亡诱导作用有关。
Objective To investigate the effect of high mobility group box-1 protein(HMGB1)-nuclear factor-κB(NF-κB)signaling pathway on autophagy and chemosensitivity of human hepatocellular carcinoma cells and its possible mechanism.Methods Human hepatocellular carcinoma BEL-7402 cells were cultured in vitro and divided into control group(BEL-7402 cells without any treatment),doxorubicin group,recombinant human HMGB1+doxorubicin group,anti-HMGB1 neutralizing antibody+doxorubicin group,pyrrolidine dithiocarbamate+doxorubicin group and 3-methyladenine+doxorubicin group.The methyl thiazolyl tetrazolium(MTT)method was used to detect cell proliferation inhibition rate.Western blot method was used to detect the expressions of HMGB1 and NF-κB subunit p-p65 protein,the autophagy-related proteins Beclin-1,LC3Ⅰ,LC3Ⅱand apoptosis-related protein bcl-2.Enzyme labeling method was used to detect Caspase-9 and Caspase-3 activity.Results The cell proliferation inhibition rates in the control group,doxorubicin group,recombinant human HMGB1+doxorubicin group,anti-HMGB1 neutralizing antibody+doxorubicin group,pyrrolidine dithiocarbamate+doxorubicin group and 3-methyladenine+doxorubicin group were(1.31±0.16)%,(47.80±6.30)%,(31.60±5.68)%,(67.20±6.83)%,(66.60±6.27)%,and(68.60±11.19)%,respectively,and the difference was statistically significant(F=75.91,P<0.01),suggesting that doxorubicin had a proliferation inhibitory effect on BEL-7402 cells;the expression levels of HMGB1 were 1.17±0.11,1.37±0.15,1.43±0.15,0.70±0.09,1.27±0.12,1.29±0.18,and the difference was statistically significant(F=18.70,P<0.01),suggesting that doxorubicin could increase the expression of HMGB1 in BEL-7402 cells,and the anti-HMGB1 neutralizing antibody could block or attenuate this effect;after pretreatment with recombinant human HMGB1,the proliferation inhibitory effect of doxorubicin on BEL-7402 cells was weakened;after pretreatment with anti-HMGB1 neutralizing antibody,pyrrolidine dithiocarbamate and 3-methyladenine,the proliferation inhibitory effect of doxorubicin on BEL-7402 cells was enhanced.Compared with the control group,the expression of p-p65 protein in the doxorubicin group,recombinant human HMGB1+doxorubicin group and 3-methyladenine+doxorubicin group increased(all P<0.05).The expression of p-p65 protein in the recombinant human HMGB1+doxorubicin group,anti-HMGB1 neutralizing antibody+doxorubicin group and pyrrolidine dithiocarbamate+doxorubicin group was lower than that in the doxorubicin group(all P<0.05).Compared with the control group,the expression of bcl-2 protein in doxorubicin group,anti-HMGB1 neutralizing antibody+doxorubicin group,pyrrolidine dithiocarbamate+doxorubicin group and 3-methyladenine+doxorubicin group decreased(all P<0.05),and the activity of Caspase-9 and Caspase-3 was enhanced(all P<0.05);after adding recombinant human HMGB1 pretreatment,the expression of bcl-2 protein in the cells increased compared with doxorubicin alone,and the activity of Caspase-9 and Caspase-3 was weakened(all P<0.05).The expression levels of autophagy-related protein Beclin-1 in the control group,doxorubicin group,recombinant human HMGB1+doxorubicin group,anti-HMGB1 neutralizing antibody+doxorubicin group,pyrrolidine dithiocarbamate+doxorubicin group,and 3-methyl adenine+doxorubicin group were 0.77±0.12,0.92±0.07,1.29±0.10,0.51±0.03,0.49±0.06,and 0.42±0.05,and the difference was statistically significant(F=97.01,P<0.01).The expression levels of LC3Ⅱ were 0.24±0.04,0.39±0.04,0.49±0.07,0.23±0.05,0.20±0.06,and 0.20±0.05,and the difference was statistically significant(F=26.98,P<0.01).Conclusion The activation of HMGB1-NF-κB signaling pathway can reduce the chemosensitivity of hepatocellular carcinoma cells to doxorubicin,and its mechanism may be related to the regulation of autophagy and down-regulation of doxorubicin inducing apoptosis of hepatocellular carcinoma cells.
作者
周薇
徐细明
高丹丹
李军华
Zhou Wei;Xu Ximing;Gao Dandan;Li Junhua(Department of Gastroenterology,the First People's Hospital of Jingmen,Jingmen 448000,China;Cancer Center,Renmin Hospital of Wuhan University,Wuhan 430060,China;Department of Infectious Diseases,the First People's Hospital of Jingmen,Jingmen 448000,China)
出处
《肿瘤研究与临床》
CAS
2020年第10期673-679,共7页
Cancer Research and Clinic
基金
国家自然科学基金(31971166)
湖北省荆门市重点科技项目(YFZD2017037)。
关键词
高迁移率族蛋白质类
NF-κB
癌
肝细胞
自噬
多柔比星
High mobility group proteins
NF-kappa B
Carcinoma,hepatocellular
Autophagy
Doxorubicin
