芒柄花黄素对鼻咽癌细胞增殖和周期的影响及其可能机制

Effect of formononetin on proliferation and cycle of nasopharyngeal cancerous cells and its possible mechanism

目的 探讨芒柄花黄素对鼻咽癌5-8F细胞增殖和周期的影响及其可能的作用机制。方法 将5-8F细胞分为溶剂对照组(加入完全培养液)、不同浓度芒柄花黄素组(加入终浓度为10μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L的芒柄花黄素)、5-氟尿嘧啶(5-Fu)组(加入5-Fu)。分别干预24 h、48 h后,采用MTT法检测各组5-8F细胞增殖率。干预24 h后采用PI染色检测溶剂对照组、20μmol/L芒柄花黄素组、40μmol/L芒柄花黄素组、80μmol/L芒柄花黄素组、5-Fu组的5-8F细胞周期。干预24 h后采用Western blot检测溶剂对照组、20μmol/L芒柄花黄素组、40μmol/L芒柄花黄素组、5-Fu组的5-8F细胞增殖细胞核抗原(PCNA)和细胞周期蛋白依赖性激酶1(CDK1)的蛋白表达水平。结果 (1)干预24 h和48 h后,20μmol/L、40μmol/L、80μmol/L、160μmol/L芒柄花黄素组及5-Fu组5-8F细胞增殖率低于溶剂对照组(P<0.05)。干预24 h后,10μmol/L、20μmol/L、40μmol/L芒柄花黄素组5-8F细胞增殖率高于5-Fu组;干预48 h后,各浓度芒柄花黄素组5-8F细胞增殖率高于5-Fu组(P<0.05)。(2)与溶剂对照组相比,20μmol/L、40μmol/L、80μmol/L芒柄花黄素组和5-Fu组的G0/G1期细胞比例降低、G2/M期细胞比例增加(P<0.05);20μmol/L、40μmol/L、80μmol/L芒柄花黄素组的G0/G1期细胞比例高于5-Fu组,G2/M期细胞比例低于5-Fu组(P<0.05)。(3)与溶剂对照组相比,20μmol/L、40μmol/L芒柄花黄素组和5-Fu组的CDK1和PCNA蛋白表达水平下降(P<0.05);5-Fu组的CDK1和PCNA蛋白表达水平高于40μmol/L芒柄花黄素组,PCNA蛋白表达水平低于20μmol/L芒柄花黄素组(P<0.05)。结论 芒柄花黄素能够抑制鼻咽癌5-8F细胞增殖,并阻滞细胞周期于G2/M期,其作用机制可能与下调PCNA和CDK1的表达水平有关。但药物的抗肿瘤作用机制复杂,芒柄花黄素的抗鼻咽癌效果仍有待优化,其具体作用机制亦仍有待深入探究。

Objective To investigate the effect of formononetin on proliferation and cycle of nasopharyngeal carcinoma 5-8F cells and its possible mechanism.Methods Cells of 5-8F were assigned to solution control group(adding complete culture solution),formononetin in different concentrations groups(adding formononetin with final concentration of 10μmol/L,20μmol/L,40μmol/L,80μmol/L,and 160μmol/L),or 5-fluorouracil(5-Fu)group(adding 5-Fu).After 24 and 48 hours of respective intervention,the MTT method was used to detect the proliferation rate of 5-8F cells in various groups.After 24 hours of intervention,the PI staining was employed to detect 5-8F cells cycle in the solution control group,20μmol/L formononetin group,40μmol/L formononetin group,80μmol/L formononetin group,and 5-Fu group.After 24 hours of intervention,the Western blot was used to detect protein expressions of 5-8F cells proliferating cell nuclear antigen(PCNA)and cyclin dependent kinase 1(CDK1)in the solution control group,20μmol/L formononetin group,40μmol/L formononetin group,and 5-Fu group.Results(1)After 24 and 48 hours of intervention,the 20μmol/L,40μmol/L,80μmol/L,and 160μmol/L formononetin groups and 5-Fu group exhibited lower proliferation rates of 5-8F cells as compared with the solution control group(P<0.05).After 24 hours of intervention,the proliferation rates of 5-8F cells in the 10μmol/L,20μmol/L,40μmol/L formononetin groups were higher than those in the 5-Fu group.After 48 hours of intervention,the proliferation rates of 5-8F cells in the formononetin with various concentrations groups were higher than those in the 5-Fu group(P<0.05).(2)Compared with the solution control group,the proportion of cells in G 0/G 1 phase was decreased,while proportion of cells in G 2/M phase was increased in the 20μmol/L,40μmol/L,and 80μmol/L formononetin groups and the 5-Fu group(P<0.05).The proportion of cells in G 0/G 1 phase in the 20μmol/L,40μmol/L,and 80μmol/L formononetin groups was higher than that in the 5-Fu group,whereas the proportion of cells in G 2/M phase was lower than that in the 5-Fu group(P<0.05).(3)Compared with the solution control group,the protein expressions of CDK1 and PCNA were decreased in the 20μmol/L and 40μmol/L formononetin groups and the 5-Fu group(P<0.05);moreover,the 5-Fu group exhibited higher protein expressions of CDK1 and PCNA as compared with the 40μmol/L formononetin group,and a lower PCNA protein expression as compared with the 20μmol/L formononetin group(P<0.05).Conclusion Formononetin is able to inhibit proliferation of nasopharyngeal carcinoma 5-8F cells,and the cell cycle is blocked in G 2/M phase.Its possible mechanism may be related to the down-regulated expressions of PCNA and CDK1;however,the anti-tumor mechanism of drug is complex,the anti-nasopharyngeal carcinoma effect of formononetin remains to be optimized,and its specific mechanism remains to be further explored.