香加皮杠柳苷对MCF-7细胞周期及p21^(WAF1/CIP1)表达的影响

Effects of Periplocin from Cortex Periplocae on Cell Cycle of MCF-7 Cells and Expression of p21^(WAF1/CIP1)

目的研究香加皮杠柳苷(CPP)对人乳腺癌MCF-7细胞周期及p21WAF1/CIP1表达的影响,探讨其抗肿瘤作用及作用机制。方法采用MTT法检测不同浓度CPP(1.25、2.50、5.00、10.00、20.00ng/ml)作用不同时间(24、48、72h)对MCF-7细胞的增殖抑制作用;应用流式细胞术分析不同浓度CPP(2.50、5.00、10.00ng/ml)分别作用于MCF-7细胞6、12、24、48、72h对肿瘤细胞周期的影响;并采用RT-PCR和免疫细胞化学技术检测细胞周期相关因子p21WAF1/CIP1的表达。结果 CPP能明显抑制MCF-7细胞的增殖,并呈浓度及时间依赖性,作用于MCF-7细胞48h的IC50为(4.88±0.16)ng/ml。流式细胞术结果显示,CPP作用MCF-7细胞24h时,G0/G1期细胞明显增多,而S期和G2/M期细胞显著减少,与对照组相比差异有统计学意义(P<0.05),其中5.00ng/ml组G0/G1期细胞由对照组的(49.33±3.25)%升高至(79.47±2.40)%,S期和G2/M期细胞由对照组的(28.47±1.59)%和(22.20±2.09)%分别下降至(10.13±3.26)%和(10.40±1.41)%。经CPP处理的MCF-7细胞中p21WAF1/CIP1mRNA的表达明显增强,p21WAF1/CIP1/β-actin光吸度比值与对照组相比明显增高(P<0.05)。免疫细胞化学结果显示,CPP组MCF-7细胞中p21WAF1/CIP1蛋白的表达随作用浓度的增加而增强,其中10.00ng/ml组肿瘤细胞p21WAF1/CIP1的表达呈强阳性。结论香加皮杠柳苷(CPP)具有显著的体外抗肿瘤作用,且有效剂量很小,其IC50仅为(4.88±0.16)ng/ml。CPP可使MCF-7细胞发生G0/G1期阻滞,并可使细胞周期相关基因p21WAF1/CIP1的mR-NA及蛋白表达增强,提示阻滞细胞周期可能是CPP体外抗肿瘤的作用机制之一。

Objective To study the effect of Periplocin from Cortex Periplocae（CPP）on cell cycle of MCF-7 cells and expression of p21WAF1/CIP1,and demonstrate the possible mechanism of anti-tumor.Methods Inhibitory effects of CPP in different concentrations（1.25,2.50,5.00,10.00,20.00ng/ ml）and different time（24,48,72 h）on proliferation of MCF-7 were detected by MTT.The changes of cell cycle of tumor cells treated with CPP under different concentrations（2.50,5.00,10.00ng/ ml）and various time（6,12,24,48,72 h）were detected using flow cytometry,respectively.Expression of cell cycle associated gene p21WAF1/CIP1 was assessed by semi-quantitative RT-PCR and SP immunocytochemistry method.Results A dose and time-dependent proliferation inhibition of CPP was demonstrated in MCF-7 with IC50value（for 48h）of（4.88±0.16）ng/ml.Compared to control group,the number of G0/G1phase cells increased markedly,but which of S and G2/M phase cells decreased,after treatment with CPP for 24 hours,the difference was significant（P0.05）;in 5.00 ng/ml group,the proportion of tumor cells in the G0/G1phase was increased from（49.33±3.25）% to（79.47±2.40）%,the proportion of S and G2/M phase cells was decreased from（28.47±1.59）% and（22.20±2.09）%to（10.13±3.26）% and（10.40±1.41）%,respectively.p21WAF1/CIP1 mRNA level increased obviously in MCF-7 cells exposed to CPP,the ratio of p21WAF1/CIP1 to β-actin was much higher,compared with that in control group（P0.05）.The result of immunocytochemistry indicated that p21WAF1/CIP1 protein expression increased obviously in MCF-7 cells exposed to CPP in concentration-dependent manner.p21WAF1/CIP1 protein showed stronger positive staining in cells with 10.00 ng/ml CPP treatment.Conclusion CPP has marked anti-tumor effect in vitro,its curative dose is small,the IC50was（4.88±0.16）ng/ml.CPP could hinder the cell cycle of MCF-7 cells at G0/G1 phase.CPP elevated mRNA and protein expression of cell cycle associated gene p21WAF1/CIP1.The result suggested that blocking the cell cycle may be one of its anti-tumor mechanisms in vitro.