Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica ›› 2010, Vol. 32 ›› Issue (1): 46-50.doi: 10.3881/j.issn.1000-503X.2010.01.012

• Original Articles • Previous Articles     Next Articles

Effects of K-ras Gene Mutation on Colon Cancer Cell Line Caco-2 Metastasis by Regulating E-cadherin/β-catenin/p120 Protein Complex Formation and RhoA Protein Activity

LI Jing-nan1;LI Xiao1,2;QIAN Jia-ming1;LU Xin-qing1;YANG Hong1   

  1. 1Department of Gastroenterology,PUMC Hospital,CAMS and PUMC,Beijing 100730,China
    2Emergency Department, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China
  • Received:2009-08-24 Revised:1900-01-01 Online:2010-02-28 Published:2010-02-28
  • Contact: QIAN Jia-ming

Abstract: ABSTRACT:Objective To explore the effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/β-catenin/p120 protein complex formation and RhoA protein activity. Methods K-ras wild-type colon cancer cell line Caco-2 was transiently transfected by phr-GFP vector (control group), transfected by mutant K-ras gene phr-K-ras (Val12) vector (transfection group), transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific MAPK pathway inhibitor PD98059 (MAPK inhibition group), or transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific PI-3K pathway inhibitor LY294002 (PI-3K inhibition group), respectively. Cell migration was tested by Transwell experiment. E-cadherin and β-catenin protein expression and intracellular location were detected by cell immunofluorescence method. Intracellular p120 protein expression was detected by Western blot. β-catenin protein level which combined with E-cadherin was detected by immunoprecipitation. RhoA activity was analyzed by Pull-down assay. Results The Caco-2 cell migration rate was (19.8±5.6)% in transfection group, which was significantly higher than that in control group [(14.0±4.2)%] (P=0.001) and in MAPK inhibition group [(15.8±1.2)%] (P =0.044), but was not significantly different from that in PI-3K inhibition group [(17.5±2.8)%] (P=0.095). Immunofluorescence method showed that the E-cadherin and β-catenin stain located in the cell membrane decreased in transfection group. Western blot showed that the total intracellular p120 preotein decreased in transfection group and PI-3K inhibition group. Immunoprecipitation data showed that β-catenin protein level combined with E-cadherin decreased in transfection group and PI-3K group. Pull-down test showed that RhoA protein activity was up-regulated in transfection group. Conclusion K-ras gene mutation stimulates the migration of colon cancer cell Caco-2, which may be achieved by decreasing the E-cadherin/β-catenin/p120 protein complex formation via MAPK pathway and increasing the RhoA protein activity.

Key words: K-ras cene, mutation, e-cadderin/β-catenin/p120 protein complex, rdoa protein, colon cancer, metastasis