斑蝥素诱导HCT116细胞形态改变及脱黏附机制
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摘要
目的:探讨斑蝥素(cantharidin,CTD)引起人结肠癌HCT116细胞形态改变及脱黏附作用的分子机制与抑制肿瘤转移的相关性,为CTD的临床使用提供新依据。方法:将HCT116细胞接种于6孔板后,每孔加入不同浓度CTD (0,2. 5,5,10,15,20μmol·L~(-1))培养7～10 d,使用结晶紫染色液染色,对形成的克隆进行计数及统计分析,确定CTD对HCT116细胞的抑制效果;使用鬼笔环肽(phalloidin)特异性染色检测10μmol·L~(-1)CTD对HCT116细胞聚合态微丝骨架(F-actin)的影响,间接免疫荧光染色法观察CTD作用后整合素蛋白含量的改变;蛋白免疫印迹法分析10μmol·L~(-1)CTD作用前后Rho家族RhoA,Rho B,Rho C,Cdc42,Rac1/2/3蛋白表达量变化,根据实验结果确定RhoA为研究目标蛋白;使用质粒瞬时转染方法构建过表达RhoA的HCT116结肠癌细胞,观察10μmol·L~(-1)CTD对过表达细胞骨架形态及黏附的影响。结果:CTD可引起HCT116结肠癌细胞微丝骨架重构及整合素含量减少; RhoA蛋白是CTD作用后含量变化最大的Rho酶家族成员,构建的RhoA过表达细胞中带绿色荧光的转染细胞比例约为60%,与野生型细胞相比,目标蛋白含量显著增高(P <0. 01),CTD通过降低RhoA含量,逆转这一过表达效应(P <0. 01),对细胞的形态及黏附均产生影响。结论:CTD可通过抑制RhoA蛋白的表达,引起HCT116结肠癌细胞形态改变及与基底的黏附减弱,游离的肿瘤细胞无法锚定于继发部位,从而抑制细胞迁移。
Objective: To explore the molecular mechanism of cantharidin( CTD) in inducing morphological changes and dissociation in human colon cancer HCT116 cells,in order to study the correlation with tumor metastasis and provide a new basis for clinical use of cantharidin. Method: Different concentrations of CTD( 0,2. 5,5,10,15,20 μmol·L~(-1)) were added to each hole to culture for 7 to 10 days,so as to determine the inhibitory effect of CTD on HCT116 cells; and changes of F-actin cytoskeleton and integrin in HCT116 cells were detected by immunofluorescence staining. Western blot analysis of protein expressions of RhoA,RhoB,Rho C,Cdc42 and Rac1/2/3 of Rho family were performed before and after cantharidin treatment. overexpression of RhoA was constructed by plasmid transient transfection,and effect of 10 μmol·L~(-1) cantharidin on the morphology and adhesion of overexpressing cells was also observed. Result: Cantharidin induced cytoskeletal remodeling and decreased integrin content in HCT116 colon cancer cells. RhoA protein was a member of Rho enzyme family with the greatest variation after cantharidin action. The proportion of transfected RhoA cells with green fluorescence was about 60%,the expression of RhoA protein in constructed RhoA overexpression cells was significantly increased,compared with wild-type HCT116 cells( P < 0. 01). However,cantharidin can act on RhoA overexpressed HCT116 cells,reverse its overexpression( P < 0. 01),and affect its morphology. Conclusion: Cantharidin can inhibit the expression of RhoA protein,induce the morphological changes of HCT116 cells and weaken the adhesion to the basement,thereby inhibiting cell migration.
Objective: To explore the molecular mechanism of cantharidin( CTD) in inducing morphological changes and dissociation in human colon cancer HCT116 cells,in order to study the correlation with tumor metastasis and provide a new basis for clinical use of cantharidin. Method: Different concentrations of CTD( 0,2. 5,5,10,15,20 μmol·L~(-1)) were added to each hole to culture for 7 to 10 days,so as to determine the inhibitory effect of CTD on HCT116 cells; and changes of F-actin cytoskeleton and integrin in HCT116 cells were detected by immunofluorescence staining. Western blot analysis of protein expressions of RhoA,RhoB,Rho C,Cdc42 and Rac1/2/3 of Rho family were performed before and after cantharidin treatment. overexpression of RhoA was constructed by plasmid transient transfection,and effect of 10 μmol·L~(-1) cantharidin on the morphology and adhesion of overexpressing cells was also observed. Result: Cantharidin induced cytoskeletal remodeling and decreased integrin content in HCT116 colon cancer cells. RhoA protein was a member of Rho enzyme family with the greatest variation after cantharidin action. The proportion of transfected RhoA cells with green fluorescence was about 60%,the expression of RhoA protein in constructed RhoA overexpression cells was significantly increased,compared with wild-type HCT116 cells( P < 0. 01). However,cantharidin can act on RhoA overexpressed HCT116 cells,reverse its overexpression( P < 0. 01),and affect its morphology. Conclusion: Cantharidin can inhibit the expression of RhoA protein,induce the morphological changes of HCT116 cells and weaken the adhesion to the basement,thereby inhibiting cell migration.
引文
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[19] Lieto E,Galizia G,Orditura M,et al. CD26-positive/CD326-negative circulating cancer cells as prognostic markers for colorectal cancer recurrence[J]. Oncol Lett,2015,9(2):542-550.
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[21] Fujihara K, Takahashi K, Koyama K, et al.Triterpenoid saponins from Polaskia chichipe Backbg.and their inhibitory or promotional effects on the melanogenesis of B16 melanoma cells[J]. J Nat Med,2017,71(4):606-616.
[22]隋彤彤,田丽春,张鑫,等.斑蝥素与培美曲塞对结直肠癌HCT116细胞的联合作用[J].中国实验方剂学杂志,2018,24(16):43-48.
[23] CHEN X, QIU J, YANG D, et al. Cantharidin promotes invasion and metastasis in invasive ductal breast carcinoma by inducing matrix metalloproteinase-9[J]. PLo S One,2013,8(11):e78794.
[2] Maroufi Y,Ghaffarifar F,Dalimi A,et al. Interferongamma and interlukin-4 patterns in BALB/c mice suffering from cutaneous leishmaniasis treated with cantharidin[J]. Jundishapur J Microbiol,2014,7(6):10907.
[3] CHEN R T,HUA Z,YANG J L,et al. Studies on antitumor actions of cantharidin[J]. China Med J,1980,93(3):183-187.
[4]封艳艳,马齐襄,隋彤彤,等.斑蝥素对肝癌Hep G2细胞增殖、凋亡的影响及其可能机制[J].中国实验方剂学杂志,2017,23(15):112-117.
[5]范潇婷,于睿鹏,董瑞娟,等.斑蝥素及去甲斑蝥素对小鼠毒性靶器官的影响[J].中国实验方剂学杂志,2017,23(15):118-123.
[6]邵好珍,马齐襄,胡晓炜,等.斑蝥素引起小鼠急性中毒的器官损伤[J].中国实验方剂学杂志,2018,24(16):55-60.
[7]孙笑,盛鸿昊,胡晓炜,等.斑蝥素引起小鼠急性膀胱炎的作用及相关机制[J].中国实验方剂学杂志,2018,24(16):49-54.
[8]张鑫.斑蝥素通过影响微丝骨架抑制HCT116结肠癌细胞增殖和迁移[D].北京:北京中医药大学,2018.
[9] Aznar S,Lacal J C. Rho signals to cell growth and apoptosis[J]. Cancer Lett,2001,165(1):1-10.
[10] Oxford G,Theodorescu D. Ras superfamily monomeric G proteins in carcinoma cell motility[J]. Cancer Lett,2003,189(2):117-128.
[11] Hoffman G R,Nassar N,Cerione R A. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator Rho GDI[J]. Cell,2000,100(3):345-356.
[12] De Pascalis C, Etienne-Manneville S. Single and collective cell migration:the mechanics of adhesions[J]. Mol Biol Cell,2017,28(14):1833-1846.
[13] Huehn A,CAO W,Elam W A,et al. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments[J]. J Biol Chem,2018,293(15):5377-5383.
[14]宗洋,李伟,陈政,等.斑螯素抑制胰腺癌细胞株生长、侵袭及其机制[J].中华实验外科杂志,2011,28(2):177-179.
[15]张琼妍.斑蝥素抑制乳腺癌细胞与血小板粘附的机制研究[D].苏州:苏州大学,2013.
[16]郑乔丹,李伟,殷红.斑蝥素抑制舌癌细胞生长及转移能力[J].中山大学学报:医学科学版,2014,35(5):650-656.
[17] Amano M,Nakayama M,Kaibuchi K. Rho-Kinase/ROCK:a key regulator of the cytoskeleton and cell polarity[J]. Cytoskeleton,2010,67(9):545-554.
[18] Kang Y,Pantel K. Tumor cell dissemination:emerging biological insights from animal models and cancer patients[J]. Cancer Cell,2013,23(5):573-581.
[19] Lieto E,Galizia G,Orditura M,et al. CD26-positive/CD326-negative circulating cancer cells as prognostic markers for colorectal cancer recurrence[J]. Oncol Lett,2015,9(2):542-550.
[20] Thompson E W,Newgreen D F,Tarin D. Carcinoma invasion and metastasis:a role for epithelialmesenchymal transition?[J]. Cancer Res,2005,65(14):5991-5995.
[21] Fujihara K, Takahashi K, Koyama K, et al.Triterpenoid saponins from Polaskia chichipe Backbg.and their inhibitory or promotional effects on the melanogenesis of B16 melanoma cells[J]. J Nat Med,2017,71(4):606-616.
[22]隋彤彤,田丽春,张鑫,等.斑蝥素与培美曲塞对结直肠癌HCT116细胞的联合作用[J].中国实验方剂学杂志,2018,24(16):43-48.
[23] CHEN X, QIU J, YANG D, et al. Cantharidin promotes invasion and metastasis in invasive ductal breast carcinoma by inducing matrix metalloproteinase-9[J]. PLo S One,2013,8(11):e78794.
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