[Effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity].
OBJECTIVE
To explore the effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-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 beta-catenin protein expression and intracellular location were detected by cell immunofluorescence method. Intracellular p120 protein expression was detected by Western blot. beta-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 beta-catenin stain located in the cell membrane decreased in transfection group. Western blot showed that the total intracellular p120 protein decreased in transfection group and PI-3K inhibition group. Immunoprecipitation data showed that beta-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.


CONCLUSIONS
K-ras gene mutation stimulates the migration of colon cancer cell Caco-2, which may be achieved by decreasing the E-cadherin/beta-catenin/p120 protein complex formation via MAPK pathway and increasing the RhoA protein activity.
Department of Gastroenterology, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
Jing-nan
JN
Li
Xiao
X
Li
Jia-ming
JM
Qian
Xin-qing
XQ
Lu
Hong
H
Yang
D018938:Caco-2 Cells; D015820:Cadherins / Q000378:metabolism; D051177:Catenins / Q000378:metabolism*; D002465:Cell Movement; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D009362:Neoplasm Metastasis; D014162:Transfection; D051176:beta Catenin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D000095002:Delta Catenin
D015820:Cadherins; D051177:Catenins; D046912:Multiprotein Complexes; D051176:beta Catenin; D020742:rhoA GTP-Binding Protein; D000095002:Delta Catenin