摘要
目的:研究环氧合酶-2(cycloxygenase 2,COX-2)抑制剂尼美舒利(nimesulide)和表皮生长因子受体(epidermal growthfactor receptor,EGFR)单克隆抗体西妥昔单抗(cetuximab,C225)联用对结肠癌HT-29细胞株细胞增殖和凋亡的影响及其作用机制。方法:MTT法检测细胞增殖状态,吖啶橙/溴化乙锭(acridine orange/ethidium bromide,AO/EB)染色法及流式细胞仪检测细胞凋亡情况,RT-PCR检测COX-2和非甾体类消炎药活化基因-1(nonsteroidal anti-inflammatory drug-activated gene,NAG-1)的mRNA表达水平,Western印迹法检测EGFR通路下游相关蛋白Akt的磷酸化活性。结果:尼美舒利与C225联合作用于HT-29细胞48 h后,该组细胞的生长抑制率明显高于尼美舒利组[(72.8±2.3)%vs(51.8±1.8)%,P<0.05]。AO/EB染色观察到尼美舒利与C225联用组HT-29细胞发生典型的凋亡形态学改变,且FCM检测显示48 h时联用组的细胞凋亡率[(57.67±0.86)%]显著高于尼美舒利[(33.27±1.47)%]和C225单用组[(6.27±0.55)%,P<0.05]。C225下调COX-2 mRNA的表达,各药物干预组NAG-1 mRNA的表达均上调,联用组对Akt磷酸化的抑制作用明显强于尼美舒利和C225单用组。结论:尼美舒利与C225联用可增强二者各自对细胞的促凋亡作用,其作用机制可能是EGFR信号通路参与了结肠癌细胞内COX-2基因的表达调控,最终通过其下游促凋亡基因NAG-1、pAkt的蛋白表达变化,影响结肠癌HT-29细胞的增殖与凋亡。
Objective：The study was to investigate the effects of epidermal growth factor receptor（EGFR） antibody（cetuximab,C225） combined with cyclooxygenase 2（COX-2）inhibitor（nimesulide） on the proliferation and apoptosis of colon cancer HT-29 cells,and explore the potential molecular mechanism.Methods：C225 and nimesulide alone or in combination were incubated with HT-29 cells.MTT assay was used to measure the cell proliferation.AO/EB staining and flow cytometry were used to measure the cell apoptosis.RT-PCR was used to detect the COX-2 and non steroidal anti-inflammatory drug-activated gene（NAG-1） mRNA expression levels.The expression of Akt and phosphor-Akt protein was examined by Western blotting.Results：The inhibitory effect of nimesulide combined with C225 was significantly stronger than that of nimesulide alone [（72.8±2.3）% vs（51.8±1.8）%,P0.05]at 48 h.Typical changes of apoptosis in HT-29 cells were observed in nimesulide combined with C225 treatment group.The apoptosis rate of combined group was significantly higher than that of single nimesulide group [（57.67±0.86）% vs（33.27±1.47）%]and single C225 group [（6.27±0.55）%,P0.05].C225 down-regulated the expression of COX-2 mRNA.The expression of NAG-1 mRNA was up-regulated in all treatment groups and the expression of phosphor-Akt was significantly down-regulated in combined treatment group than single nimesulide or C225 group.Conclusion：Nimesulide combined with C225 has obvious synergistic effects in inducing apoptosis.The potential mechanisms may be that EGFR signaling pathway is involved in the regulation of cycloxygenase-2 expression in HT-29 cells and finally influence the proliferation and apoptosis of HT-29 cells through the up-regulation of NAG-1 and down-regulation of phosphor-Akt expression.
出处
《肿瘤》
CAS
CSCD
北大核心
2010年第1期26-30,共5页
Tumor
