ER拮抗剂对Ishikawa细胞中p57~(kip2)、Cyclin D1表达及形态变化的影响研究
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摘要
目的:研究雌激素受体(ER)拮抗剂对人子宫内膜样癌(EC) Ishikawa细胞(高分化)形态变化及细胞中p57~(kip2)、Cyclin D1、CDK4表达情况的影响,探讨女性激素调控、细胞周期调控和EC发生、发展的影响。方法:分别用含雌二醇(E2)、他莫昔芬(TAM)、氟维司群(Faslodex,ICI182780)、E2+TAM、E2+ICI182780的培养基培养Ishikawa细胞,用不含药物的培养基作为对照。培养24、48、72、96h后,MTT法观察细胞增殖情况,培养24、48、72h后,通过光镜和电镜观察细胞的形态变化,Western blot法检测p57~(kip2)、Cyclin D1、CDK4蛋白表达。结果:MTT结果显示,与对照组比较,E2组、ICI182780组、E2+ICI182780组Ishikawa细胞增殖活性降低,以E2+ICI182780组更甚(P<0.05);与E2组比较,E2+ICI182780组的细胞增殖活性降低(P<0.05);实验组及对照组细胞的增殖活性均随着药物作用时间延长而增强(P<0.05)。光镜下:与对照组比较,E2组、ICI182780组、E2+ICI182780组细胞密度降低,以E2+ICI182780组更甚,TAM组则稍增加;与E2组比较,E2+ICI182780组细胞密度降低,E2+TAM组则增加;各组细胞形态变化均不明显。电镜下:与对照组比较,E2组部分细胞可见内质网、线粒体明显肿胀,其余各组内质网呈不同程度肿胀,可见凋亡现象;与E2组比较,TAM组、ICI182780组内质网肿胀较轻,E2+TAM、E2+ICI182780组肿胀明显。Western blot结果示:与对照组比较,随着药物作用时间延长,p57~(kip2)蛋白在E2组、ICI182780组、E2+ICI182780组表达逐渐增加,其中E2+ICI182780组表达最高(P <0. 05); Cyclin D1、CDK4蛋白则在E2组、ICI182780组、E2+ICI182780组表达逐渐降低,其中E2+ICI182780组表达最低(P <0. 05);与E2组比较,p57~(kip2)蛋白在E2+ICI182780组表达增加,在E2+TAM组表达降低(P<0.05); Cyclin D1、CDK4蛋白在E2+ICI182780组表达降低,在E2+TAM组表达增加(P<0.05)。结论:ER拮抗剂ICI182780单独或联合使用雌激素,可能通过诱导p57~(kip2)蛋白表达,下调Cyclin D1、CDK4蛋白表达,达到阻碍细胞周期进程,抑制Ishikawa细胞增殖的作用,且对细胞损伤较重; TAM单独或联合应用雌激素,可能通过上调Cyclin D1、CDK4蛋白表达,下调p57~(kip2)蛋白表达,对Ishikawa细胞产生较弱的促增殖作用,从而发挥TAM的弱雌激素样作用。ICI182780可能比TAM更适合用于EC患者的内分泌治疗,这为EC内分泌治疗药物的选择提供了一定的实验依据。
Objective: To study the effects of estrogen receptor antagonist on the expressions of p57~(kip2),Cyclin D1 and CDK4 protein and the morphological changes in human endometrioid carcinoma cells( EC) named Ishikawa( highly differentiated) in vitro,which to explore hormonal regulation,cell cycle regulation on the occurrence and development of EC.Methods: The Ishikawa cells cultured in vitro were treated with estradiol( E2),Tamoxifen( TAM),fulvestrant( ICI182780),E2+TAM,E2+ ICI182780 and another group was cultured without any drugs as control group.After 24,48,72,96 h,the cells proliferation and morphological changes were measured by MTT. After 24,48,72 h,the cells morphological changes were measured by light and electron microscopic.Western blot was used to detect the expressions of p57~(kip2),Cyclin D1 and CDK4 protein in cells.Results: The results of MTT showed that compared with the control group,the E2 group,ICI182780 group and E2 +ICI182780 group restrained the proliferation of Ishikawa cells,and E2+ICI182780 group was more obvious( P<0.05).Compared with the E2 group,E2 + ICI182780 group restrained the proliferation of Ishikawa cells( P < 0.05).The proliferative activity of the experimental groups and the control group increased with the prolongation of the time of action of the drug( P <0.05). Light microscope: Compared with the control group,the density of E2 group,ICI182780 group and E2 + ICI182780 group decreased in Ishikawa cell,the E2 +ICI182780 group was more obvious. The density of the TAM group increased slightly. compared with E2 control group,the density of E2 +ICI182780 group decreased in Ishikawa cell.The density of E2+TAM group increased.The morphological changes of cells in each group were not obvious.Electron microscope: compared with the control group,a part of E2 group cells of endoplasmic reticulum and mitochondria were swollen.The other groups dominated by vary degrees of endoplasmic reticulum swelling,and seen apoptotic phenomenon.Compared with E2 group,the TAM group and ICI182780 group endoplasmic reticulum swelling and lighter,but E2 + TAM group,E2 + ICI182780 group were obvious. Western blot results showed that compared with the control group,with prolonging the drug action time,the expressions of p57~(kip2) protein in the E2 group,ICI182780 group and E2+ ICI182780 group were gradually increased,with the highest expression in E2 +ICI182780 group( P < 0.05). However,the expressions of Cyclin D1 and CDK4 protein in the E2 group,ICI182780 group and E2 +ICI182780 group were decreased gradually,with the lowest expression in E2+ICI182780 group( P<0.05).Compared with E2 group,the expression of p57~(kip2) protein in E2 +ICI182780 group was increased( P<0.05),the expression in E2 +TAM group was decreased. The expressions of Cyclin D1 and CDK4 protein in E2+ ICI182780 group were decreased( P<0.05),the expressions in the E2 + TAM group was increased. Conclusion: The application of ER antagonist ICI182780 alone or in combination with estrogen could inhibit the proliferation of Ishikawa cells by inducing the expression of p57~(kip2),down-regulating the expressions of Cyclin D1 and CDK4,hinder the cell cycle progression,and have a severe effect on cell injury.The application of TAM alone or in combination with estrogen may play a weak estrogen-like role by up-regulating the expressions of Cyclin D1 and CDK4 protein,down-regulating the expression of p57~(kip2) protein,and weakening proliferation in Ishikawa cells. ICI182780 may be more suitable than TAM for endocrine therapy in patients with EC.This provides a certain experimental basis for EC endocrine therapy drug selection.
Objective: To study the effects of estrogen receptor antagonist on the expressions of p57~(kip2),Cyclin D1 and CDK4 protein and the morphological changes in human endometrioid carcinoma cells( EC) named Ishikawa( highly differentiated) in vitro,which to explore hormonal regulation,cell cycle regulation on the occurrence and development of EC.Methods: The Ishikawa cells cultured in vitro were treated with estradiol( E2),Tamoxifen( TAM),fulvestrant( ICI182780),E2+TAM,E2+ ICI182780 and another group was cultured without any drugs as control group.After 24,48,72,96 h,the cells proliferation and morphological changes were measured by MTT. After 24,48,72 h,the cells morphological changes were measured by light and electron microscopic.Western blot was used to detect the expressions of p57~(kip2),Cyclin D1 and CDK4 protein in cells.Results: The results of MTT showed that compared with the control group,the E2 group,ICI182780 group and E2 +ICI182780 group restrained the proliferation of Ishikawa cells,and E2+ICI182780 group was more obvious( P<0.05).Compared with the E2 group,E2 + ICI182780 group restrained the proliferation of Ishikawa cells( P < 0.05).The proliferative activity of the experimental groups and the control group increased with the prolongation of the time of action of the drug( P <0.05). Light microscope: Compared with the control group,the density of E2 group,ICI182780 group and E2 + ICI182780 group decreased in Ishikawa cell,the E2 +ICI182780 group was more obvious. The density of the TAM group increased slightly. compared with E2 control group,the density of E2 +ICI182780 group decreased in Ishikawa cell.The density of E2+TAM group increased.The morphological changes of cells in each group were not obvious.Electron microscope: compared with the control group,a part of E2 group cells of endoplasmic reticulum and mitochondria were swollen.The other groups dominated by vary degrees of endoplasmic reticulum swelling,and seen apoptotic phenomenon.Compared with E2 group,the TAM group and ICI182780 group endoplasmic reticulum swelling and lighter,but E2 + TAM group,E2 + ICI182780 group were obvious. Western blot results showed that compared with the control group,with prolonging the drug action time,the expressions of p57~(kip2) protein in the E2 group,ICI182780 group and E2+ ICI182780 group were gradually increased,with the highest expression in E2 +ICI182780 group( P < 0.05). However,the expressions of Cyclin D1 and CDK4 protein in the E2 group,ICI182780 group and E2 +ICI182780 group were decreased gradually,with the lowest expression in E2+ICI182780 group( P<0.05).Compared with E2 group,the expression of p57~(kip2) protein in E2 +ICI182780 group was increased( P<0.05),the expression in E2 +TAM group was decreased. The expressions of Cyclin D1 and CDK4 protein in E2+ ICI182780 group were decreased( P<0.05),the expressions in the E2 + TAM group was increased. Conclusion: The application of ER antagonist ICI182780 alone or in combination with estrogen could inhibit the proliferation of Ishikawa cells by inducing the expression of p57~(kip2),down-regulating the expressions of Cyclin D1 and CDK4,hinder the cell cycle progression,and have a severe effect on cell injury.The application of TAM alone or in combination with estrogen may play a weak estrogen-like role by up-regulating the expressions of Cyclin D1 and CDK4 protein,down-regulating the expression of p57~(kip2) protein,and weakening proliferation in Ishikawa cells. ICI182780 may be more suitable than TAM for endocrine therapy in patients with EC.This provides a certain experimental basis for EC endocrine therapy drug selection.
引文
[1]Felix AS,Yang HP,Bell DW,et al.Epidemiology of endometrial carcinoma:etiologic importance of hormonal and metabolic influences[J].Adv Exp Med Biol,2017,943:3-46
[2]Brinton LA,Felix AS,McMeekin DS,et al.Etiologic heterogeneity in endometrial cancer:evidence from a gynecologic oncology group trial[J].Gynecol Oncol,2013,129(2):277-284
[3]Setiawan VW,Yang HP,Pike MC,et al.Type I and II endometrial cancers:have they different risk factors?[J].JClin Oncol,2013,31(20):2607-2618
[4]Hammes SR,Davis PJ.Overlapping nongenomic and genomic actions of thyroid hormone and steroids[J].Best Pract Res Clin Endocrinol Metab,2015,29(4):581-593
[5]郭瑞霞,王建六,赵丹,等.子宫内膜癌细胞系Ishikawa和HEC-1A细胞雌激素受体表达[J].中国妇产科临床杂志,2005,6(4):272-274+304-323
[6]Tokino T,Urano T,Furuhata T,et al.Characterization of the human p57kip2gene:alternative splicing,insertion/deletion polymorphisms in VNTR sequences in the coding region,and mutational analysis[J].Hum Genet,1996,97(5):625-631
[7]Siegel RL,Miller KD,Jemal A.Cancer statistics,2015[J].CA Cancer J Clin,2015,65(1):5-29
[8]Dedes KJ,Wetterskog D,Ashworth A,et al.Emerging therapeutic targets in endometrial cancer[J].Nat Rev Clin Oncol,2011,8(5):261-271
[9]Hill EK,Dizon DS.Medical therapy of endometrial cancer:current status and promising novel treatments[J].Drugs,2012,72(5):705-713
[10]Nathan MR,Schmid P.A review of fulvestrant in breast cancer[J].Oncol Ther,2017,5(1):17-29
[11]An KC.Selective estrogen receptor modulators[J].Asian Spine J,2016,10(4):787-791
[12]Jameera Begam A,Jubie S,Nanjan MJ.Estrogen receptor agonists/antagonists in breast cancer therapy:A critical review[J].Bioorg Chem,2017,71:257-274
[13]Kümler I,Knoop AS,Jessing CA,et al.Review of hormone-based treatments in postmenopausal patients with advanced breast cancer focusing on aromatase inhibitors and fulvestrant[J].ESMO Open,2016,1(4):e000062
[14]Zhang L,Li Y,Lan L,et al.Tamoxifen has a proliferative effect in endometrial carcinoma mediated via the GPER/EGFR/ERK/cyclin D1 pathway:A retrospective study and an in vitro study[J].Mol Cell Endocrinol,2016,437:51-61
[15]Emons G,Günthert A,Thiel FC,et al.Phase II study of fulvestrant 250 mg/month in patients with recurrent or metastatic endometrial cancer:a study of the Arbeitsgemeinschaft Gynakologische Onkologie[J].Gynecol Oncol,2013,129(3):495-499
[16]郭瑞霞,魏丽惠,赵丹等.雌激素拮抗剂ICI182780(Faslodex)对17β-雌二醇作用下子宫内膜癌细胞增殖和凋亡的影响[J].北京大学学报(医学版),2006,38(5):470-474
[17]Borriello A,Caldarelli I,Bencivenga D,et al.p57(Kip2)and cancer:time for a critical appraisal[J].Mol Cancer Res,2011,9(10):1269-1284
[18]Zhao Y,Wang Y,Yang Y,et al.MicroRNA-222 Controls Human Pancreatic Cancer Cell Line Capan-2 Proliferation by P57 Targeting[J].J Cancer,2015,6(12):1230-1235
[19]Lee JY,Shin JY,Kim HS,et al.Effect of combined treatment with progesterone and tamoxifen on the growth and apoptosis of human ovarian cancer cells[J].Oncol Rep,2012,27(1):87-93
[20]Li L,Li X,Han X,et al.An ovarian cancer model with positive ER:Reversion of ER antagonist resistance by Src blockade[J].Oncol Rep,2014,32(3):943-950
[21]格桑志玛,周正平,罗祖强,等.人子宫内膜癌中p57kip2、Cyclin D1的表达及意义[J].临床与实验病理学杂志,2014,30(9):975-978
[22]Motokura T,Bloom T,Kim HG,et al.Anovel cyclin encoded by a bcl-1 linked candidate oncogene[J].Nature,1991,350(6318):512-515
[23]Thangavel C,Dean JL,Ertel A,et al.Therapeutically activating RB:reestablishing cell cycle control in endocrine therapy-resistant breast cancer[J].Endocr Relat Cancer,2011,18(3):333-345
[24]Fribbens C,O'Leary B,Kilburn L,et al.Plasma ESR1mutations and the treatment of estrogen receptor-positive advanced breast cancer[J].J Clin Oncol,2016,34(25):2961-2968
[25]Kim CW,Go RE,Choi KC.Treatment of BG-1 ovarian cancer cells expressing estrogen receptors with lambdacyhalothrin and cypermethrin caused a partial estrogenicity via an estrogen receptor-dependent pathway[J].Toxicol Res,2015,31(4):331-337
[26]In SJ,Kim SH,Go RE,et al.Benzophenone-1 and nonylphenol stimulated MCF-7 breast cancer growth by regulating cell cycle and metastasis-related genes via an estrogen receptorα-dependent pathway[J].J Toxicol Environ Health A,2015,78(8):492-505
[27]Ichikawa A,Ando J,Suda K.G1 arrest and expression of cyclin-dependent kinase inhibitors in tamoxifen-treated MCF-7 human breast cancer cells[J].Hum Cell,2008,21(2):28-37
[2]Brinton LA,Felix AS,McMeekin DS,et al.Etiologic heterogeneity in endometrial cancer:evidence from a gynecologic oncology group trial[J].Gynecol Oncol,2013,129(2):277-284
[3]Setiawan VW,Yang HP,Pike MC,et al.Type I and II endometrial cancers:have they different risk factors?[J].JClin Oncol,2013,31(20):2607-2618
[4]Hammes SR,Davis PJ.Overlapping nongenomic and genomic actions of thyroid hormone and steroids[J].Best Pract Res Clin Endocrinol Metab,2015,29(4):581-593
[5]郭瑞霞,王建六,赵丹,等.子宫内膜癌细胞系Ishikawa和HEC-1A细胞雌激素受体表达[J].中国妇产科临床杂志,2005,6(4):272-274+304-323
[6]Tokino T,Urano T,Furuhata T,et al.Characterization of the human p57kip2gene:alternative splicing,insertion/deletion polymorphisms in VNTR sequences in the coding region,and mutational analysis[J].Hum Genet,1996,97(5):625-631
[7]Siegel RL,Miller KD,Jemal A.Cancer statistics,2015[J].CA Cancer J Clin,2015,65(1):5-29
[8]Dedes KJ,Wetterskog D,Ashworth A,et al.Emerging therapeutic targets in endometrial cancer[J].Nat Rev Clin Oncol,2011,8(5):261-271
[9]Hill EK,Dizon DS.Medical therapy of endometrial cancer:current status and promising novel treatments[J].Drugs,2012,72(5):705-713
[10]Nathan MR,Schmid P.A review of fulvestrant in breast cancer[J].Oncol Ther,2017,5(1):17-29
[11]An KC.Selective estrogen receptor modulators[J].Asian Spine J,2016,10(4):787-791
[12]Jameera Begam A,Jubie S,Nanjan MJ.Estrogen receptor agonists/antagonists in breast cancer therapy:A critical review[J].Bioorg Chem,2017,71:257-274
[13]Kümler I,Knoop AS,Jessing CA,et al.Review of hormone-based treatments in postmenopausal patients with advanced breast cancer focusing on aromatase inhibitors and fulvestrant[J].ESMO Open,2016,1(4):e000062
[14]Zhang L,Li Y,Lan L,et al.Tamoxifen has a proliferative effect in endometrial carcinoma mediated via the GPER/EGFR/ERK/cyclin D1 pathway:A retrospective study and an in vitro study[J].Mol Cell Endocrinol,2016,437:51-61
[15]Emons G,Günthert A,Thiel FC,et al.Phase II study of fulvestrant 250 mg/month in patients with recurrent or metastatic endometrial cancer:a study of the Arbeitsgemeinschaft Gynakologische Onkologie[J].Gynecol Oncol,2013,129(3):495-499
[16]郭瑞霞,魏丽惠,赵丹等.雌激素拮抗剂ICI182780(Faslodex)对17β-雌二醇作用下子宫内膜癌细胞增殖和凋亡的影响[J].北京大学学报(医学版),2006,38(5):470-474
[17]Borriello A,Caldarelli I,Bencivenga D,et al.p57(Kip2)and cancer:time for a critical appraisal[J].Mol Cancer Res,2011,9(10):1269-1284
[18]Zhao Y,Wang Y,Yang Y,et al.MicroRNA-222 Controls Human Pancreatic Cancer Cell Line Capan-2 Proliferation by P57 Targeting[J].J Cancer,2015,6(12):1230-1235
[19]Lee JY,Shin JY,Kim HS,et al.Effect of combined treatment with progesterone and tamoxifen on the growth and apoptosis of human ovarian cancer cells[J].Oncol Rep,2012,27(1):87-93
[20]Li L,Li X,Han X,et al.An ovarian cancer model with positive ER:Reversion of ER antagonist resistance by Src blockade[J].Oncol Rep,2014,32(3):943-950
[21]格桑志玛,周正平,罗祖强,等.人子宫内膜癌中p57kip2、Cyclin D1的表达及意义[J].临床与实验病理学杂志,2014,30(9):975-978
[22]Motokura T,Bloom T,Kim HG,et al.Anovel cyclin encoded by a bcl-1 linked candidate oncogene[J].Nature,1991,350(6318):512-515
[23]Thangavel C,Dean JL,Ertel A,et al.Therapeutically activating RB:reestablishing cell cycle control in endocrine therapy-resistant breast cancer[J].Endocr Relat Cancer,2011,18(3):333-345
[24]Fribbens C,O'Leary B,Kilburn L,et al.Plasma ESR1mutations and the treatment of estrogen receptor-positive advanced breast cancer[J].J Clin Oncol,2016,34(25):2961-2968
[25]Kim CW,Go RE,Choi KC.Treatment of BG-1 ovarian cancer cells expressing estrogen receptors with lambdacyhalothrin and cypermethrin caused a partial estrogenicity via an estrogen receptor-dependent pathway[J].Toxicol Res,2015,31(4):331-337
[26]In SJ,Kim SH,Go RE,et al.Benzophenone-1 and nonylphenol stimulated MCF-7 breast cancer growth by regulating cell cycle and metastasis-related genes via an estrogen receptorα-dependent pathway[J].J Toxicol Environ Health A,2015,78(8):492-505
[27]Ichikawa A,Ando J,Suda K.G1 arrest and expression of cyclin-dependent kinase inhibitors in tamoxifen-treated MCF-7 human breast cancer cells[J].Hum Cell,2008,21(2):28-37