Mechanism of Cantharidin-induced Morphological Changes and Dissociation of HCT116 Cells

Xin ZHANG; Hui ZHAO; Jie WEI; Jun GUO; Gang HU; Ya-jun LIN

doi:10.13422/j.cnki.syfjx.20191024

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Mechanism of Cantharidin-induced Morphological Changes and Dissociation of HCT116 Cells

Pharmacology | 更新时间：2021-02-09

- Mechanism of Cantharidin-induced Morphological Changes and Dissociation of HCT116 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 10, Pages: 20-25(2019)
- 作者机构：
  
  北京医院　国家老年医学中心　卫生部老年医学重点实验室，北京　 100730
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191024
  CLC： R22; R242; R2-031; R285.5
- Received：03 December 2018，
  
  Published Online：12 February 2019，
  
  Published：20 May 2019
- 稿件说明：
移动端阅览
Xin ZHANG, Hui ZHAO, Jie WEI, et al. Mechanism of Cantharidin-induced Morphological Changes and Dissociation of HCT116 Cells[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(10): 20-25.
DOI：

Xin ZHANG, Hui ZHAO, Jie WEI, et al. Mechanism of Cantharidin-induced Morphological Changes and Dissociation of HCT116 Cells[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(10): 20-25. DOI： 10.13422/j.cnki.syfjx.20191024.

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 (0，2.5，5，10，15，20 μmol·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10 μmol·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10 μmol·L

－1

CTD作用前后Rho家族RhoA，RhoB，RhoC，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10 μmol·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(

0.01)，CTD通过降低RhoA含量，逆转这一过表达效应(

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。

Abstract

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

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

RhoC

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 (

0.01). However

cantharidin can act on RhoA overexpressed HCT116 cells

reverse its overexpression(

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.

关键词

Keywords

references

国家药典委员会．中华人民共和国药典．一部 [M]. 北京：中国医药科技出版社， 2010 ： 311 - 312 .

Maroufi Y , Ghaffarifar F , Dalimi A , et al . Interferon-gamma and interlukin-4 patterns in BALB/c mice suffering from cutaneous leishmaniasis treated with cantharidin [J]. Jundishapur J Microbiol , 2014 , 7 ( 6 ): 10907 .

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 .

封艳艳，马齐襄，隋彤彤，等．斑蝥素对肝癌HepG2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 .

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 .

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 .

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 .

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 .

Aznar S , Lacal J C . Rho signals to cell growth and apoptosis [J]. Cancer Lett , 2001 , 165 ( 1 ): 1 - 10 .

Oxford G , Theodorescu D . Ras superfamily monomeric G proteins in carcinoma cell motility [J]. Cancer Lett , 2003 , 189 ( 2 ): 117 - 128 .

Hoffman G R , Nassar N , Cerione R A . Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI [J]. Cell , 2000 , 100 ( 3 ): 345 - 356 .

De Pascalis C , Etienne-Manneville S . Single and collective cell migration: the mechanics of adhesions [J]. Mol Biol Cell , 2017 , 28 ( 14 ): 1833 - 1846 .

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 .

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 .

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 .

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 .

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 .

Kang Y , Pantel K . Tumor cell dissemination: emerging biological insights from animal models and cancer patients [J]. Cancer Cell , 2013 , 23 ( 5 ): 573 - 581 .

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 .

Thompson E W , Newgreen D F , Tarin D . Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition？ [J]. Cancer Res , 2005 , 65 ( 14 ): 5991 - 5995 .

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 .

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 .

CHEN X , QIU J , YANG D , et al . Cantharidin promotes invasion and metastasis in invasive ductal breast carcinoma by inducing matrix metalloproteinase-9 [J]. PLoS One , 2013 , 8 ( 11 ): e78794 .

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