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目的:探讨人硒结合蛋白1(SBP1)在结直肠癌DLD-1细胞中的表达水平对超氧化物歧化酶(SOD)的影响及其引起的DLD-1细胞对奥沙利铂的敏感性变化,揭示SBP1的部分抗肿瘤作用机制。方法:MTS法检测不同浓度奥沙利铂处理SBP1siRNA或SOD1siRNA转染细胞后的细胞存活率,CuZn/Mn-SOD活性检测试剂盒检测SBP1siRNA转染对DLD-1细胞SOD酶活性的影响;实时荧光定量PCR检测下调SBP1后,SOD1和SOD2mRNA水平的变化;Western blot法检测下调SBP1或SOD1后DLD-1细胞内SBP1、SOD1及SOD2蛋白水平的变化;超氧化物阴离子荧光探针DHE染色法检测抗肿瘤药物引起的细胞内超氧化物阴离子的水平。结果:下调SBP1后,奥沙利铂对DLD-1细胞的毒性作用[半数毒性浓度IC50为(36.7±1.6)μmol/L]相比对照组[IC50为(17.5±0.8)μmol/L]明显减小(P<0.05);同时下调SBP1和SOD1后,奥沙利铂对DLD-1细胞的毒性作用[IC50为(16.8±1.0)μmol/L]相比对照组[IC50为(17.3±1.2)μmol/L]无明显变化(P>0.05);下调SBP1分别在转录水平、蛋白水平及酶活性水平上调了SOD,并减少了由奥沙利铂引起的DLD-1细胞内产生的超氧阴离子。结论:下调SBP1能上调SOD的水平,进而减小了奥沙利铂对DLD-1细胞的毒性作用,故SBP1能通过抑制SOD增强奥沙利铂对DLD-1细胞的毒性作用。
Objective: To investigate the effect of human selenium binding protein 1 (SBP1) on the expression of superoxide dismutase (SOD) and the sensitivity of DLD-1 cells to oxaliplatin in colorectal cancer DLD-1 cells Changes, reveal part of SBP1 antitumor mechanism. Methods: MTS method was used to detect the cell viability after oxaliplatin treated with SBP1 siRNA or SOD1 siRNA transfected cells. CuZn / Mn-SOD activity assay kit was used to detect the effect of SBP1 siRNA transfection on SOD activity in DLD-1 cells. Real-time fluorescence Quantitative PCR was used to detect the changes of SOD1 and SOD2 mRNA levels after the down-regulation of SBP1. The protein level of SBP1, SOD1 and SOD2 in DLD-1 cells were detected by Western blot, and the levels of SOD1 and SOD2 in DLD-1 cells were detected by Western blot. Tumor drug-induced intracellular superoxide anion levels. Results: The toxic effect of Oxaliplatin on DLD-1 cells after IC50 downregulation (IC50 was (36.7 ± 1.6) μmol / L for IC50 was (17.5 ± 0.8) μmol / L compared with control group) (IC50 was (16.8 ± 1.0) μmol / L] compared with control group [IC50 was (17.3 ± 1.2), P <0.05] ) μmol / L] did not change significantly (P> 0.05). The down-regulation of SBP1 upregulated SOD at the level of transcription, protein and enzyme activity and decreased the production of superoxide induced by oxaliplatin in DLD- Anion. Conclusion: Down-regulation of SBP1 can up-regulate the level of SOD and decrease the toxic effect of oxaliplatin on DLD-1 cells. Therefore, SBP1 can enhance the toxic effect of oxaliplatin on DLD-1 cells by inhibiting SOD.