白藜芦醇苷对人类乳腺癌MCF-7细胞增殖的影响及其机制
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摘要
目的:研究白藜芦醇苷(polydatin)在体外对乳腺癌细胞MCF-7增殖的作用,进一步探索其潜在的机制。方法:设定空白组及白藜芦醇苷(0.2,0.4,1.2,2.0,2.8 mg·L~(-1))组,5-氟尿嘧啶组,采用噻唑蓝(MTT)比色法测定不同质量浓度白藜芦醇苷对MCF-7细胞的生长抑制率;应用细胞克隆集落形成实验验证白藜芦醇苷对MCF-7细胞增殖的抑制作用;通过实时荧光定量PCR(Real-time PCR)检测5-氟尿嘧啶(1.0μg·L~(-1))和白藜芦醇苷(0.2,0.4,1.2 mg·L~(-1))对DNA聚合酶δ催化亚基P125(P125),P125编码基因(POLD1),p53基因(p53),细胞周期依赖性蛋白激酶抑制因子p21 mRNA表达水平的影响;应用蛋白质免疫印迹法(Western blot)检测白藜芦醇苷(0.2,0.4,1.2 mg·L~(-1))对P125,p53,p21蛋白表达水平的影响。结果:与空白组比较,白藜芦醇苷(0.2,0.4,1.2,2.0,2.8 mg·L~(-1))在体外对MCF-7细胞具有明显的抑制作用(P<0.05,P<0.01),并且这种抑制作用呈浓度依赖性。与空白组比较,白藜芦醇苷(0.2,0.4,1.2 mg·L~(-1))均能明显抑制细胞集落的形成(P<0.05,P<0.01)。与空白组比较,5-氟尿嘧啶组和白藜芦醇苷(0.4,1.2 mg·L~(-1))能够显著抑制POLD1 mRNA表达水平,同时显著抑制P125 mRNA和蛋白表达水平,明显升高p53,p21 mRNA和蛋白表达水平(P<0.05,P<0.01)。与5-氟尿嘧啶组比较,白藜芦醇苷(1.2 mg·L~(-1))对POLD1,P125,p53,p21 mRNA和蛋白表达水平影响均无显著差异。结论:白藜芦醇苷在体外能够显著抑制MCF-7细胞的增殖,其潜在机制可能与提高p53表达水平,抑制POLD1表达,促进p21表达有关。
Objective: To study the effect of polydatin on the proliferation of MCF-7 cells,in order to further explore its underlying mechanism.Method: MCF-7 cells were treated with polydatin at different doses(0,0.2,0.4,1.2,2.0,2.8 mg·L~(-1)).The inhibitory rate of the cells was detected by methylthiazolydiphenyltetrazolium bromide(MTT) assay after treatment for 48 h.To further verify the inhibitory effect of polydatinon on the proliferation of MCF-7 cells,the cell colonies were detected by the colony formation assay after treatment with polydatin(0.2,0.4,1.2 mg·L~(-1)) for 10 days.After treatment with polydatin for 48 h,the effect of polydatin(0.2,0.4,1.2 mg·L~(-1)) on mRNA expressions of DNA polymerase δ catalytic subunit gene POLD1,P125,p53 and p21 were detected by Real-time PCR.Protein expressions of P125,p53 and p21 were detected by Western blot.Result: Polydatin at different doses(0.2,0.4,1.2,2.0,2.8 mg·L~(-1)) showed an inhibitory effect on the proliferation of MCF-7 cells in a concentration-dependent manner(P < 0.05,P < 0.01).After administration for10 days,polydatin(0.2,0.4,1.2 mg·L~(-1)) significantly inhibited the formation of cell colonies,compared with control group(P < 0.05,P < 0.01).After treatment for 48 h,mRNA expressions of POLD1 and P125 were significantly lower in polydatin(0.4,1.2 mg·L~(-1)) groups and 5-FU group than control group,while mRNA expressions of p53 and p21 were increased significantly(P < 0.05,P < 0.01).After treatment for 48 h,protein expression of P125 was obviously lower in polydatin(0.4,1.2 mg·L~(-1)) groups and 5-FU group than control group,while protein expressions of p53 and p21 were increased significantly.The effect of polydatin on mRNA and protein expressions of PLOD1,P125,p53 and p21 in polydatin(1.2 mg·L~(-1)) group had no difference with 5-FU group.Conclusion: Polydatin shows a significantly inhibitory effect on the proliferation of MCF-7 cells.Its underlying mechanisms may be associated with the up-regulation of p53,the inhibition of expression of POLD1 and the promotion of p21.
Objective: To study the effect of polydatin on the proliferation of MCF-7 cells,in order to further explore its underlying mechanism.Method: MCF-7 cells were treated with polydatin at different doses(0,0.2,0.4,1.2,2.0,2.8 mg·L~(-1)).The inhibitory rate of the cells was detected by methylthiazolydiphenyltetrazolium bromide(MTT) assay after treatment for 48 h.To further verify the inhibitory effect of polydatinon on the proliferation of MCF-7 cells,the cell colonies were detected by the colony formation assay after treatment with polydatin(0.2,0.4,1.2 mg·L~(-1)) for 10 days.After treatment with polydatin for 48 h,the effect of polydatin(0.2,0.4,1.2 mg·L~(-1)) on mRNA expressions of DNA polymerase δ catalytic subunit gene POLD1,P125,p53 and p21 were detected by Real-time PCR.Protein expressions of P125,p53 and p21 were detected by Western blot.Result: Polydatin at different doses(0.2,0.4,1.2,2.0,2.8 mg·L~(-1)) showed an inhibitory effect on the proliferation of MCF-7 cells in a concentration-dependent manner(P < 0.05,P < 0.01).After administration for10 days,polydatin(0.2,0.4,1.2 mg·L~(-1)) significantly inhibited the formation of cell colonies,compared with control group(P < 0.05,P < 0.01).After treatment for 48 h,mRNA expressions of POLD1 and P125 were significantly lower in polydatin(0.4,1.2 mg·L~(-1)) groups and 5-FU group than control group,while mRNA expressions of p53 and p21 were increased significantly(P < 0.05,P < 0.01).After treatment for 48 h,protein expression of P125 was obviously lower in polydatin(0.4,1.2 mg·L~(-1)) groups and 5-FU group than control group,while protein expressions of p53 and p21 were increased significantly.The effect of polydatin on mRNA and protein expressions of PLOD1,P125,p53 and p21 in polydatin(1.2 mg·L~(-1)) group had no difference with 5-FU group.Conclusion: Polydatin shows a significantly inhibitory effect on the proliferation of MCF-7 cells.Its underlying mechanisms may be associated with the up-regulation of p53,the inhibition of expression of POLD1 and the promotion of p21.
引文
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[10]孙海榕,王璐瑜,王忠鑫.白藜芦醇苷通过抑制长链非编码RNA HULC表达抑制肝癌SMMC-7721和Hep G2细胞的增殖和侵袭[J].中国生物化学与分子生物学报,2016,32(5):561-568.
[11]孙海榕,王璐瑜,王忠鑫.白藜芦醇苷通过抑制microRNA-21表达抑制肝癌细胞的增殖和侵袭[J].中国生物化学与分子生物学报,2015,31(11):1220-1226.
[12]张玉松,庄志祥,焦旸,等.白藜芦醇苷对乳腺癌细胞转移潜能影响观察[J].中华肿瘤防治杂志,2014,21(22):1788-1793.
[13]熊莎,高建蓉,胡祖良,等.鳖甲提取物对抑制TGF-β诱导的大鼠肝星状细胞活化的影响[J].中国实验方剂学杂志,2017,23(19):189-193.
[14]廖柳凤,欧贤红,刘华钢,等.POLD1基因在原发性肝癌中的表达及其意义[J].世界华人消化杂志,2011,19(2):151-155.
[15]吴琼,黄文涛,黄怡,等.RNA干扰技术抑制人肝癌细胞POLD1基因表达的研究[J].广西医学,2014,36(5):549-551.
[16]LI B,Lee M Y.Transcriptional regulation of the human DNA polymerase delta catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1[J].J Biol Chem,2001,276(32):29729-29739.
[17]石磊,徐恒,朱晓宇,等.p21对DNA聚合酶δ表达的抑制及其对MCF7细胞增殖和恶性表型的影响[J].自然科学进展,2006,16(6):672-679.
[18]朱晓宇,徐恒,李莉,等.p53亚细胞定位变化对POLD1基因启动子活性的影响[J].自然科学进展,2006,16(4):555-562.
[19]赵剑平.5-氟尿嘧啶对人乳腺癌MCF-7细胞增殖的影响及其机制探讨[J].山东医药,2013,53(40):26-28.
[2]Hirschey M D,De Berardinis R J,Diehl A M,et al.Dysregulated metabolism contributes to oncogenesis[J].Semin Cancer Biol,2015,35(Suppl):S129-150.
[3]Gérard C,Goldbeter A.Dynamics of the mammalian cell cycle in physiological and pathological conditions[J].Wiley Interdiscip Rev Syst Biol Med,2016,8(2):140-156.
[4]林晓燕,张叔人,徐恒,等.真核生物DNA聚合酶δ的研究现状[J].自然科学进展,2005,15(1):1-7.
[5]Lee M Y,ZHANG S,LIN S H,et al.Regulation of human DNA polymerase delta in the cellular responses to DNA damage[J].Environ Mol Mutagen,2012,53(9):683-698.
[5]李锦源.乳腺癌组织POLD1基因的表达研究[D].南宁:广西医科大学,2014.
[6]廖柳凤,欧贤红,刘华钢,等.氯化两面针碱对MCF-7细胞增殖的抑制及对P125表达的影响[J].中国实验方剂学杂志,2016,22(19):85-89.
[7]Dalva M,Correia A T,Jatene N F,et al.New contribution tothe study of ventricular remodeling and valve rings in dilatedcardiomyopathy:anatomical and histological evaluation[J].Rev Bras Cir Cardiovasc,2014,29(4):478-486.
[8]YE X,JIANG F,LI Y,et al.Glabridin attenuates the migratoryand invasive capacity of breast cancer cells by activating microRNA-200c[J].Cancer Sci,2015,105(7):875-882.
[9]ZHANG Q,TAN Y,ZHANG N,et al.Polydatin prevents angiotensin II-induced cardiac hypertrophy and myocardial superoxide generation[J].Exp Biol Med(Maywood),2015,240(10):1352-1361.
[10]孙海榕,王璐瑜,王忠鑫.白藜芦醇苷通过抑制长链非编码RNA HULC表达抑制肝癌SMMC-7721和Hep G2细胞的增殖和侵袭[J].中国生物化学与分子生物学报,2016,32(5):561-568.
[11]孙海榕,王璐瑜,王忠鑫.白藜芦醇苷通过抑制microRNA-21表达抑制肝癌细胞的增殖和侵袭[J].中国生物化学与分子生物学报,2015,31(11):1220-1226.
[12]张玉松,庄志祥,焦旸,等.白藜芦醇苷对乳腺癌细胞转移潜能影响观察[J].中华肿瘤防治杂志,2014,21(22):1788-1793.
[13]熊莎,高建蓉,胡祖良,等.鳖甲提取物对抑制TGF-β诱导的大鼠肝星状细胞活化的影响[J].中国实验方剂学杂志,2017,23(19):189-193.
[14]廖柳凤,欧贤红,刘华钢,等.POLD1基因在原发性肝癌中的表达及其意义[J].世界华人消化杂志,2011,19(2):151-155.
[15]吴琼,黄文涛,黄怡,等.RNA干扰技术抑制人肝癌细胞POLD1基因表达的研究[J].广西医学,2014,36(5):549-551.
[16]LI B,Lee M Y.Transcriptional regulation of the human DNA polymerase delta catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1[J].J Biol Chem,2001,276(32):29729-29739.
[17]石磊,徐恒,朱晓宇,等.p21对DNA聚合酶δ表达的抑制及其对MCF7细胞增殖和恶性表型的影响[J].自然科学进展,2006,16(6):672-679.
[18]朱晓宇,徐恒,李莉,等.p53亚细胞定位变化对POLD1基因启动子活性的影响[J].自然科学进展,2006,16(4):555-562.
[19]赵剑平.5-氟尿嘧啶对人乳腺癌MCF-7细胞增殖的影响及其机制探讨[J].山东医药,2013,53(40):26-28.