氧化苦参碱对人胃癌细胞杀伤作用的机制

Killing effect of oxymatrine on human gastric cancer cell line MKN45 and its mechanism

目的:探讨氧化苦参碱(oxymatrine,OM)对人胃癌细胞株MKN45的杀伤作用及其抗肿瘤作用的机制.方法:培养人胃癌MKN45细胞,以0.5,1,2,4,6 g/L OM处理.采用四唑蓝(MTT)比色法、流式细胞仪、聚合酶链反应(PCR),酶联免疫吸附测定(ELISA)和RT-PCR法检测OM对MKN45细胞的杀伤作用、细胞周期分布、端粒酶活性和端粒酶逆转录酶(hTERT)及其上游调控基因(c-myc,p53,mad1)表达的影响.结果:OM对MKN45细胞具有剂量依赖性杀伤作用,半数抑制浓度(IC_(50))78 g/L.OM作用48 h后,与对照组相比,在2 g/L剂量组,MKN45 G0/G1期细胞增加(62.2%±1.3% vs 56.7%±4.0%,P<0.05)、G2/M期细胞减少(5.4%±1.1% vs 10.0%±2.8%,P<0.05);在4 g/L剂量组,S期细胞减少(30.5%±1.3% vs 33.4%±1.2%,P<0.05).OM可抑制MKN45细胞的端粒酶活性,其作用呈剂量依赖性.OM可引起MKN45细胞hTERT基因表达下调,p53和mad1基因的表达升高,对c-myc基因的表达无影响.结论:OM对人胃癌细胞株MKN45具有杀伤作用,可能通过影响hTERT及其上游调控基因的表达来抑制肿瘤细胞端粒酶活性,发挥其抗肿瘤作用.

AIM： To explore the killing effects of oxymatrine （OM） on human gastric cancer cell line MKN45 and its anti-neoplastic mechanism. METHODS： Human gastric cancer cell line MKN45 was cultured and then treated with 0.5, 1, 2, 4 and 6 g/L OM. Methylthiazolyl tetrazolium analysis （MTT） was used to observe the killing effect of OM on MKN45 cells. Cell cycle distribution was measured by flow cytometry. The activity of telomerase was detected by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay （ELISA）. Reverse transcription （RT）-PCR was employed to examine the expression of hTERT, c-myc, p53 and rnadl genes in MKN45 cells. RESULTS： OM exhibited dose-dependent killing effects on MKN45 cells and its IC50 was 2.78 g/L After administration for 48 hours, OM induced an increase of G1/G0-phase cells （62.2% ± 1.3% vs 56.7% ± 4.0%, P 〈 0.05） and decrease of G2/ M-phase cells （5.4% ± 1.1% vs 10.0% ± 2.8%, P 〈 0.05） at a dose of 2 g/L, and a decrease of S-phass cells （30.5% ± 1.3% vs 33.4% ± 1.2%, P 〈 0.05） at a dose of 4 g/L. OM inhibited the activity of telomerase in MKN45 cells in a dose-dependent manner. The expression of hTERT gene in MKN45 cells was decreased, but the expression of p53 and mad1 genes were increased. However, and c-myc gene expression had no apparent changes. CONCLUSION： OM has dose-dependent killing effects on MKN45 cells, and it can inhibit the telomerase activity through hTERT gene and the up-stream regulation genes.