抗高迁移率族蛋白1中和抗体对肝癌细胞BEL-7402/ADM药物敏感性的影响  

Reversal effects of anti-high mobility group box-1 neutralizing antibody on drug sensitivity of human hepatocellular carcinoma cells BEL-7402/ADM

作　　者：周薇[1] 高丹丹 李军华[1] ZHOU Wei;GAO Dandan;LI Junhua(Basic and Clinical Medical Research Center,Department of Gastroenterology,the First People's Hospital of Jingmen,Hubei Jingmen 448000,China;Department of Infectious Diseases,the First People's Hospital of Jingmen,Hubei Jingmen 448000,China)

机构地区：[1]荆门市第一人民医院消化内科,临床医学实验室,湖北荆门448000 [2]荆门市第一人民医院感染科,湖北荆门448000

出　　处：《现代肿瘤医学》2021年第5期744-748,共5页Journal of Modern Oncology

基　　金：荆门市重点科技计划项目(编号:YFZD2017037)。

摘　　要：目的:探讨抗高迁移率族蛋白1(high mobility group box-1,HMGB1)中和抗体对肝癌细胞药物敏感性的影响及可能机制。方法:采用阿霉素(adriamycin,ADM)小剂量持续诱导法建立肝癌细胞耐药细胞株BEL-7402/ADM。实验分为BEL-7402/ADM组、ADM组及ADM+抗HMGB1中和抗体组,MTT法检测细胞对ADM的敏感性及抗HMGB1中和抗体对细胞的低毒性,Annexin V-FITC流式细胞法检测细胞凋亡情况,酶标法检测Caspase-9、Caspase-3活性,Western-blot方法检测核因子-κB(nuclear factor-κB,NF-κB)亚单位p-p65、Bcl-2蛋白的表达。结果:ADM对亲本株的半数抑制浓度(IC50)为0.23μg/mL,对肝癌细胞耐药株BEL-7402/ADM的IC50上升至4.07μg/mL,耐药指数为17.7。联合抗HMGB1中和抗体能明显提升ADM对BEL-7402/ADM细胞增殖抑制率,IC50下降至0.91μg/mL;加用逆转浓度(19 ng/mL)的抗HMGB1中和抗体组的BEL-7402/ADM细胞凋亡率较单用ADM组的凋亡率明显上升(P<0.01)。与BEL-7402/ADM组相比,ADM组细胞的Caspase-9、Caspase-3活性有一定程度上升(P<0.01),联合抗HMGB1中和抗体作用后,Caspase-9、Caspase-3活性增强则更加明显(P<0.01)。BEL-7402/ADM组及ADM组间p-p65、Bcl-2表达无显著差异(P> 0.05),加用抗HMGB1中和抗体处理后,细胞p-p65、Bcl-2表达明显下降(P<0.01)。结论:抗HMGB1中和抗体对BEL-7402/ADM细胞耐药性有逆转作用,其机制可能与下调NF-κB介导的Bcl-2表达,促进肝癌细胞凋亡有关。Objective:To investigate the reversal effect of anti-high mobility group box-1(HMGB1) neutralizing antibody on drug sensitivity of human hepatocellular carcinoma cells(HCC) BEL-7402/ADM in vitro.Methods:Human hepatocellular carcinoma cells BEL-7402 were exposed to continuous increasing concentration of adriamycin(ADM) to induce resistant HCC cell line BEL-7402/ADM.Three groups were studied,including BEL-7402/ADM group,ADM group and ADM+anti-HMGB1 group.Drug sensitivity of BEL-7402 and BEL-7402/ADM to ADM,and the toxicity of anti-HMGB1 neutralizing antibody to BEL-7402/ADM were examined by MTT assay.Half lethal concentration(IC50) of ADM to cells and resistance index(RI) of BEL-7402/ADM were calculated.Apoptosis of cells was detected by Annexin V-FITC flow cytometry.The expression of p-p65,Bcl-2 was determined by Western-bolt assessment and the activity of Caspase-9,Caspase-3 was measured with acolorimetric assay kit.Results:Compared to parent cells,the induced resistant cells showed less sensitivity to ADM,IC50 of ADM to BEL-7402/ADM was significantly increased.When combined with anti-HMGB1 neutralizing antibody,IC50 of ADM to BEL-7402/ADM was reduced,and the growth inhibition rate of BEL-7402/ADM was increased.Anti-HMGB1 neutralizing antibody could promote BEL-7402/ADM cell apoptosis(P<0.01). The activity of Caspase-9,Caspase-3 in ADM group cells was higher than those in BEL-7402/ADM cells(P<0.01),and increased significantly after treated with anti-HMGB1 neutralizing antibody(P<0.01).There were no differences in the expression of p-p65 and Bcl-2 between BEL-7402/ADM group and ADM group(P>0.05),p-p65 and Bcl-2 expression decreased significantly when combined with anti-HMGB1 neutralizing antibody(P<0.01).Conclusion:The anti-HMGB1 neutralizing antibody can reverse drug resistance of BEL-7402/ADM cells and the mechanism may be related to down-regulation of Bcl-2 mediated by NF-κB and potentiated apoptosis of BEL-7402/ADM.

关 键 词：高迁移率族蛋白1 肝癌细胞 药物敏感性 阿霉素 核因子-ΚB 

分 类 号：R735.7[医药卫生—肿瘤]