Effects Of Caveolin-1 On Proliferation And Metastasis Of Colorectal Cancer Cells By Regulating Epidermal Growth Factor Receptor Activation

Colorectal cancer is a common malignant tumor of the digestive system.In recent years,its incidence rate has been dramatically increased in China.Therefore,it is crucial to find out the valuable colorectal cancer biomarkers for early diagnosis and treatment so as to increase the survival rate.The pathogenesis of colorectal cancer is a complex series of heterogeneous diseases,with multiple stages,factors and multi-step processes,involving many biological changes including the disorders and missing expression of suppressor genes,activation of oncogenes,mutations and apoptosis regulation mechanism disorders.Moreover,it has been further confirmed that the development of colorectal cancer is the result of abnormal regulation of many tumors that are related with molecules and multiple signal transduction pathways.Signal transduction pathway is closely related to the differentiation,proliferation and apoptosis of cancer cells.Caveola,a flask-shaped invagination of a cell’s plasma membrane,is a specific microdomain about 50-100 nm in the cell membrane.It participates in many cell life activities,such as endocytosis,cholesterol transport,membrane assembly,signal transduction and tumorigenesis.Caveolin-1(Cav-1)is an important protein of Caveolae with 21-24 kDa.It interacts with many signal molecules through phosphorylation/ dephosphorylation pathway.It has been shown that Cav-1 binds directly to epidermal growth factor receptor(EGFR)and inhibits the activation of EGFR.Up to now,the effects of Cav-1 on the activation of EGFR and the signal pathways of MAPK/ERK,PI3K/Akt in colorectal cancer have not been reported.EGFR is a key molecule that participates in cell division,proliferation,migration and survival.After binding with ligands,homologous or hetero-dimerization of EGFR results in self-phosphorylation of its intracellular receptor tyrosine kinase and activation of many downstream signal transduction pathways including MAPK/ERK,PI3K/Akt,which consequently might lead to a variety of diseases,including tumors.PI3k/Akt is an important signal transduction pathway regulating cell survival,and related to the biological characteristics of many tumors.If PI3k/Akt is over-activated,it will inhibit cell apoptosis,promote cell invasion and metastasis,induce cell proliferation,tumor angiogenesis,hemotherapeutic resistance,radiotherapy resistance.After the activation of MEK-ERK signal transduction pathway,the activity of transcription factors will be regulated,which may result in cell proliferation and differentiation.In this study,we selected colorectal cancer SW480 cells as the research object with high expression of EGFR and low expression of Cav-1.The expression of Cav-1 was upregulated by plasmid transfection technique.And the transfection effects were confirmed by western blot.The effects of Cav-1 on the proliferation,migration and invasion of SW480 cells were observed by means of MTS test,wound healing and transwell cell invasion assays,respectively.Moreover the effect of Cav-1 expression on the phosphorylation of EGFR and its downstream signal was detected by western blot.The relationship between the expression of Cav-1 and clinical features of human colorectal cancer was analyzed by immunohistochemical method.The aim of the study was to provide the theoretical basis for Cav-1 as a new target for clinical diagnosis and gene therapy of colorectal cancer.This study consisted of the following three parts: Part One Effects of Caveolin-1 on proliferation,migration and invasion of SW480 cellsObjective: To investigate the effects of Cav-1 on proliferation,migration and invasion of SW480 cells.Methods:1.Cells were resuscitated and cultured.The colorectal cancer cells were removed from liquid nitrogen and melted in the water bath at 37 ℃.The cells were cultured in a 1640 culture medium with 10% fetal bovine serum,penicillin(100IU / ml),streptomycin(100IU/ml)and incubated in 37℃,5%CO2 incubator.2.Protein expression in different colorectal cancer cellsThe expression levels of Cav-1 in different colorectal cancer cells cell lines(HCT116,T84,HT29,SW837,SW620,SW480)were detected by western blot.3.Construction of stably transfected cell linesSW480 cells were stably transfected with the pcDNA3.1/Cav-1 plasmid and pcDNA3.1plasmid and selected with G418,resulting in SW480/Cav-1 and SW480/pcDNA3.1 cell lines.The expression efficiency of Cav-1 was measured by Western blot.4.Proliferation assaySW480/Cav-1 and SW480/pcDNA3.1 cells were seeded in 96-well plates,and serum-starved for 4h.After stimulating with different concentrations of EGF,MTS assay was used to measure the OD values at 490 nm and examine the proliferation ability of stably transfected cell lines.5.The wound-healing assay was used to measure the role of Cav-1 in colorectal cell migration in vitro.SW480/Cav-1 and SW480/pcDNA3.1 cells were seeded in 24-well plates,and serum-starved for 4 hours,Single cell layer formed after overnight adhesion.Then scratched with a 10μl pipette tip.Cells were divided into EGF stimulated group and no EGF group.The images of cells in the same field were taken every 2h until the end of the scratch.6.Transwell assay was used to measure the role of Cav-1 in colorectal cell invasion in vitroSerum-starved cells were placed in the top chamber of transwell invasion chambers with serum-free 1640 medium.The lower chamber was filled with 1640 with 10% FBS.After 24-hour incubation,noninvasive cells were removed with a cotton swab from the upper surface of the transwell membrane,and invasive cells on the lower membrane surface were fixed with methanol,stained with H&E,photographed,and counted under microscope.Results:1.Expressions of Cav-1 in different colorectal cell linesCav-1and EGFR levels were detected by western blot in different kinds of colorectal cell lines.Moreover the expression levels of Cav-1were the lowest and the expression levels of EGFR were the highest in SW480 cell line.2.The expression of Cav-1 in SW480 cell lineThe relative levels of Cav-1 expression in SW480/Cav-1 cells were significantly higher than those of SW480/pc DNA3.1(P<0.01).3.Proliferation abilities of stably transfected SW480 cells.After stimulating by different concentrations of EGF(0nM,4nM,20 nM,100nM),the OD values detected by MTS assay in SW480/Cav-1 group were significantly lower than those in SW480/pcDNA3.1 group(P<0.01).4.Migration abilities of stably transfected SW480 cells.Wound healing test was used.The migration rates of SW480/Cav-1 cells at different time points(8h,16 h,24h,32h)were significantly lower than those in SW480/pcDNA3.1 group with or without EGF stimulating(P<0.01).5.Invasion abilities in stably transfected SW480 cells.In Transwell assays,the invasive potential of SW480/Cav-1 cells was lower by 56% than that in SW480/ pcDNA3.1 cells(P<0.05).Part Two Effects of Caveolin-1 on activation of EGFR signaling pathway in colorectal cancer cellsObjective: To detect the effects of Cav-1 on phosphorylation of EGFR and its downstream signal.Methods: SW480/Cav-1 and SW480/pcDNA3.1 cells were seeded,starved with serum-free 1640 medium for 4 hours.After stimulating by EGF in different time points,total proteins were extracted from cells.The expression levels of Cav-1,EGFR,p-EGFR,Akt,p-Akt,ERK,p-ERK and β-actin were detected by western blot.Results:The results of statistics demonstrated that the expression levels of Cav-1 in SW480/Cav-1 group were significantly higher than those in SW480/pcDNA3.1 group at every time points(P<0.01).The expression levels of p-EGFR in SW480/Cav-1 group after stimulating by EGF(20nM)at different time point were all significantly lower than those of SW480/ pcDNA3.1group(P<0.01).The levels of p-ERK and p-AKT expressions in SW480/Cav-1 group after stimulating by 20 nM EGF at different time points were significantly lower than those in SW480/ pc DNA3.1 group.Part Three The expression of Cav-1 in colorectal cancer tissue with EGFR positive expression and relationships with clinicopathological featuresObjective: To investigate the relationship between the expression of Cav-1 and clinical features in human colorectal cancer.Methods: PV Immunohistochemistry was applied to detect the expression levels of EGFR in 82 cases with colorectal cancer.Further analysis of the expression of Cav-1 and Ki-67 was performed in 76 patients with EGFR positive expression,using the same method.Also,the expression of Cav-1 and its correlation with clinicopathological features were analyzed.Results:1.In colorectal cancer tissues,Cav-1 and EGFR expressed in the cell membrane and cytoplasm with brownish yellow or brown granules.The positive rate of EGFR expression was 92.68%(76/82).The positive expression rate of Cav-1 was 56.58%(43/76).Ki-67 distributed in the nucleus with brownish yellow or brown granules.The positive rate of Ki-67 was 52.63%(40/76).2.The relationship between Cav-1/Ki-67 and clinical pathological features of colorectal cancerIn 76 cases of colorectal cancer tissue with EGFR positive expression,there was a close correlation between expression of Cav-1 and the lymph node metastasis as well as TNM stage,the differences were statistically significant(P<0.05).The expression of Ki-67 was related to stage,and the Ki-67 expression of III,IV was higher than that of I,II(P<0.05).3.The correlation between Cav-1 and Ki-67 in colorectal cancer tissue with EGFR positive expressionThe expression of Cav-1 and ki-67 were negatively correlated(r=-0.283,P=0.024).Conclusions:1.In vitro,overexpression of Cav-1 could inhibit the proliferation,migration and invasion of SW480 cells.2.Cav-1 may affect biological behavior of colorectal cancer cells through inhibiting the phosphorylation of EGFR and regulating it down steam signaling pathway factors to inhibit the growth of colorectal cancer cells.3.The expression levels of Cav-1 are negatively correlated with the TNM stage,lymph node and distant metastasis in colorectal cancer,which are negatively correlated with that of Ki-67,so as to inhibit the growth of colorectal carcer.