桂枝茯苓丸抑制人乳腺癌细胞MCF-7增殖机制
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摘要
目的:通过观察桂枝茯苓丸对人乳腺癌细胞MCF-7增殖的影响,并检测细胞周期的变化和蛋白表达的改变,从而探讨桂枝茯苓丸对乳腺癌细胞产生抑制的作用机制。方法:体外培养人乳腺癌细胞MCF-7,以5-氟尿嘧啶(5-Fu,25 mg·L~(-1))作为阳性药,噻唑蓝(MTT)比色法检测不同剂量桂枝茯苓丸培养液和在不同作用时间对细胞的抑制作用;选用1.8,2.7 g·L~(-1)的桂枝茯苓丸培养液作用48 h,流式细胞技术检测5-Fu组和桂枝茯苓丸培养液组的周期,实时荧光定量聚合酶链式反应(Real-time PCR)检测表皮生长因子(EGFR),周期素A2(Cyclin A2)及周期素依赖性激酶2(Cdk2)mRNA表达,蛋白免疫印迹法(Western blot)检测其EGFR,Cyclin A2,Cdk2蛋白表达。结果:桂枝茯苓丸对人乳腺癌细胞MCF-7细胞有抑制作用(P<0.05),且具有时间-剂量依赖性。与空白组比较,1.8,2.7 g·L~(-1)桂枝茯苓丸作用MCF-7细胞48 h后S期细胞明显增加(P<0.05),EGFR,Cdk2,Cyclin A2mRNA表达明显降低(P<0.01);桂枝茯苓丸各质量浓度组中EGFR,Cdk2蛋白表达均下降(P<0.05),但Cyclin A2蛋白表达仅2.7 g·L~(-1)组有明显降低(P<0.05)。结论:桂枝茯苓丸在S期阻滞MCF-7细胞增殖,这可能与下调Cyclin A2,Cdk2,EGFR mRNA和其蛋白的表达有关。
Objective: To investigate the mechanism of Guizhi Fuling Wan in inhibiting the human breast cancer MCF-7 cell by observing its effect on MCF-7 cells and detecting the changes of cell cycle and protein expressions. Method: The human breast cancer MCF-7 cells were cultured in vitro,and 5-Fluorouracil(5-Fu,25 mg·L~(-1)) was used as a positive control medicine to detect the effect of Guizhi Fuling Wan culture on inhibiting the proliferation of the cells under different duration and different doses with the method of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT). Guizhi Fuling Wan cultures(1. 8,2. 7 g·L~(-1)) were used for treatment for 48 h. Then the cell cycles was measured by flow cytometry(FCM) in 5-fu group and Guizhi Fuling Wan culture groups; mRNA expression levels of epidermal growth factor receptor(EGFR),Cyclin A2 and cyclin-dependent kinase(Cdk2) were detected by Real-time quantitative polymerase chain reaction(Real-time PCR),and their protein expression levels were detected by Western blot. Result: Guizhi Fuling Wan can inhibit the proliferation of the human breast cancer MCF-7 cell in a time-dose dependent manner(P < 0. 05). As compared with blank control group,the number of S phase cells was increased significantly after treatment by 1. 8,2. 7 g·L~(-1) Guizhi Fuling Wan for 48 h(P < 0. 05); the mRNA expression levels of EGFR,Cdk2 and Cyclin A2 were significantly reduced(P < 0. 01); and the protein expression levels of EGFR and Cdk2 were also decreased(P < 0. 05); however,the expression of Cyclin A2 was only deceased in 2. 7 g·L~(-1) group(P < 0. 05).Conclusion: Guizhi Fuling Wan can inhibit the proliferation of MCF-7 cell in S phase which possiblly related to the expression of Cyclin A2,Cdk2 and EGFR mRNA and their proteins.
Objective: To investigate the mechanism of Guizhi Fuling Wan in inhibiting the human breast cancer MCF-7 cell by observing its effect on MCF-7 cells and detecting the changes of cell cycle and protein expressions. Method: The human breast cancer MCF-7 cells were cultured in vitro,and 5-Fluorouracil(5-Fu,25 mg·L~(-1)) was used as a positive control medicine to detect the effect of Guizhi Fuling Wan culture on inhibiting the proliferation of the cells under different duration and different doses with the method of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT). Guizhi Fuling Wan cultures(1. 8,2. 7 g·L~(-1)) were used for treatment for 48 h. Then the cell cycles was measured by flow cytometry(FCM) in 5-fu group and Guizhi Fuling Wan culture groups; mRNA expression levels of epidermal growth factor receptor(EGFR),Cyclin A2 and cyclin-dependent kinase(Cdk2) were detected by Real-time quantitative polymerase chain reaction(Real-time PCR),and their protein expression levels were detected by Western blot. Result: Guizhi Fuling Wan can inhibit the proliferation of the human breast cancer MCF-7 cell in a time-dose dependent manner(P < 0. 05). As compared with blank control group,the number of S phase cells was increased significantly after treatment by 1. 8,2. 7 g·L~(-1) Guizhi Fuling Wan for 48 h(P < 0. 05); the mRNA expression levels of EGFR,Cdk2 and Cyclin A2 were significantly reduced(P < 0. 01); and the protein expression levels of EGFR and Cdk2 were also decreased(P < 0. 05); however,the expression of Cyclin A2 was only deceased in 2. 7 g·L~(-1) group(P < 0. 05).Conclusion: Guizhi Fuling Wan can inhibit the proliferation of MCF-7 cell in S phase which possiblly related to the expression of Cyclin A2,Cdk2 and EGFR mRNA and their proteins.
引文
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[18]李琰,张娟.MCM7、CDK2及Ki-67蛋白在甲状腺癌中表达的临床意义[J].实用癌症杂志,2015,30(3):359-361.
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[20]刘思远,李志鹏,郑心.肺抑瘤合剂联合AP化疗方案治疗非小细胞肺癌临床疗效观察[J].中国实验方剂学杂志,2018,24(3):185-190.
[21]林炜明,罗茂春,陈彤,等.CDK2在非小细胞肺癌组织中的表达[J].龙岩学院学报,2014,32(2):76-79.
[2]石建伟,唐智柳,蔡美玉,等.2008-2012年我国女性乳腺癌流行状况的系统性综述[J].中国妇幼保健,2014,29(10):1622-1626.
[3]张保宁.乳腺肿瘤学[M].北京:人民卫生出版社,2013:198.
[4]高国璇,辛灵,刘倩,等.St Gallen国际乳腺癌会议专家共识10年历程回顾[J].中国实用外科杂志,2014,34(1):70-72,76.
[5]韩晓雪.乳腺癌病因病机及证治的文献研究[D].北京:北京中医药大学,2012.
[6]王英,高洪泉.桂枝茯苓丸诱导卵巢癌HO8910细胞凋亡的研究[J].牡丹江医学院学报,2003,24(6):1-4.
[7]石建伟,唐智柳,蔡美玉,等.2008-2012年我国女性乳腺癌流行状况的系统性综述[J].中国妇幼保健,2014,29(10):1622-1626.
[8]刘丽云.乳腺癌证治的中医文献研究[D].北京:北京中医药大学,2008.
[9]于晓红,郑瑞茂,杨宝华,等.桂枝茯苓丸中单味药实验免疫学研究进展[J].中医药信息,2000,17(5):31-32.
[10]秦泗涟,马利华,贾晓斌,等.桂枝茯苓方物质基础及其制剂质量控制模式研究进展[J].中国实验方剂学杂志,2011,17(13):270-273.
[11]Takahashi N,Iwasa S,Taniguchi H,et al.Prognostic role of ERBB2,MET and VEGFA expression in metastatic colorectal cancer patients treated with antiEGFR antibodies[J].Br J Cancer,2016,doi:10.1038/bjc.2016,74.
[12]王学军,刘国希,赵艳玲,等.乳腺癌治疗的新进展[J].中国医疗前沿,2012,7(13):16-18.
[13]王春艳.EGFR和VEGF联合检测对评估乳腺癌淋巴结转移的意义[J].中国实验诊断学,2015,19(3):440-442.
[14]Evan G I,Vouaden K H.Proliferation,cell cycle and apoptosis in cancer[J].Nature,2001,411(6835):342-348.
[15]Diederichs S,Banmer N,JI P,et al.Identification of interaction Partners and Substrates of the Cyclin A1-CDK2 complex[J].Biol Chem,2004,279(32):33727-33741.
[16]Pagano M,Pepperkok R,Verde F,et al.Cyclin A is required at two points in the human cell cycle[J].EMBO J,1992,11(3):961-971.
[17]杨丽萍,罗庆丰,吴毓东,等.周期蛋白依赖激酶-2表达对乳腺癌新辅助化疗的疗效判断价值[J].实用癌症杂志,2014,29(11):1374-1376.
[18]李琰,张娟.MCM7、CDK2及Ki-67蛋白在甲状腺癌中表达的临床意义[J].实用癌症杂志,2015,30(3):359-361.
[19]刘雷,陈国昌,宋振云,等.胃癌中Cdk2和p21的表达及其与临床病理和预后的关系[J].江苏医药,2014,40(17):2045-2047,1980.
[20]刘思远,李志鹏,郑心.肺抑瘤合剂联合AP化疗方案治疗非小细胞肺癌临床疗效观察[J].中国实验方剂学杂志,2018,24(3):185-190.
[21]林炜明,罗茂春,陈彤,等.CDK2在非小细胞肺癌组织中的表达[J].龙岩学院学报,2014,32(2):76-79.