The Role Of Ezrin In The Invasion And Metastasis Of Colorectum Cancer And Effect Of Baicalein On Its Expression

Objectives:Tumor metastasis is a multi-step process in which several molecules are involved.Micro tumor thrombosis is formated with homotype and iostype adhesion increasing after tumor cells entered blood vessels.Compared with single tumor cell, micro tumor embolus is convenient for tumor cells survival, invasion,and liver metastasis formation.In the process of cell adhesion and invasion,siginal conduction mediated by adhesion molecules induce cytoskeleton reorganization.Adhesion molecules on the cell surface redistribute dramatically from even condition to aggregation. This process is blocked when cytoskeleton reorganization is inhibited.So it is necessary for further investigation the relationship between adhesive molecules redistribution and cytoskeleton reorganization.Ezrin,a crosslink protein of cytoskeleton and transmemberane molecule,may play a key role in the control of cell morphology.It is over-expressed in many tumors such as pancreatic carcinoma,endometrium carcinoma,and esophagealcarcinoma.Several researches showed that ezrin was significantly overexpressd in tumors with highly metastatic potential,whereas inhibition of ezrin by stable expression of short hairpin RNA or antisense constructs directed at ezrin,or an established dominant negative ezrin mutant reduces the metastatic capability of tumor cell lines significantly.When cells adhesion,the actin based cytoskeleton reorganizes beneath the cytoplasma membrane to facillitate the adhesion molecules aggregation.When ezrin bind the side branches of F-actin,it can promote actin to assemble and F-actin enlongate.Cytochalasin D can inhibit actin polymerization and actin based cytoskeleton remoding.The purpose of our study is to investigate the expression of ezrin in human Colorectum Cancer cell line and human Colorectum Cancer,and the role of ezrin in tumor adhesion,motility,invasion and liver metastasis formation. Methods:This study was carried out in human colorectum cancer,9 specimens of benign Colorectum disease and 50 specimens of colorectum cancer.The expression of ezrin protein was investigated by immunohistochemistry.The immunohistochemistry method was also used to invastigate the ezrin distribution.Ezrin expression was inhibited by transfecting antisense oligonucleotide into cells with liposomes and tumor's adhesion,motility,invasion was inhibited meanwhile.Baicalein suppressed significanily sw620 cell growth by inhibiting its Proliferation in a time-and dose-dependent manner. Reverse transcriptase- polymerase chain reaction (RT-PCR)andWestern blotting results revealed that baicalein notably down-regulated Ezrin expression at both mRNA and protein levels,and inhabited its phosphorylation as well as.Results:To investinate the expression of Ezrin and its relationship with clinicopatholonical characteristics in Colorectum cancer. Research suggested that ezrin was overexpressed in Colorectum Cancer (P<0.05).Higher levels of ezrin expression were showed in colorectum cancer with low differentiation than in those with high differentiation(P?0.05).The ezrin expression in normal colorectal tissue was concentrated on cell membrane, and in colorectum cancer was stained in cell plasma in a scattered manner. Higher levers of ezrin expression were showed incolorectum cancer with liver metastases than in those without liver metastases (P?0.05).Inhibition of ezrin expression by antisense oligonucleotide Transfection of antisense oligonucleotide of ezrin into SW620 cell by liposomes dramatically inhibited ezrin expression.At the optimal concentration of antisense oligonucleotide and liposomes, more than 90% cells were transfected after 48 hours incubabtion.Ezrin mRNA decrease from 1.9258±0.0456 to 0.7963±0.0842, inhibition rate was 60%.At the same time,it is safe to SW620 cell.Inhibition of ezrin expression affect tumor cells motility,adhesion and invasion .As ezrin inhibited by asODN, SW620 cell adhesion was suppressed.The adhesion rate decreased from 75.69±4.35% to 42.3±4.65%(P<0.05),while random oligonucleotide has no significant effect(P>0.05).Suppression of ezrin expression inhibited tumor cells migration.After suppressing ezrin expression by antisense oligonucleotide,number of SW620 cells invasing membrane significantly decreased from 46.78±3.70/200 to 20.5±3.6/200(P<0.05).Transfection of random oligonucleuotid didn't had this effect, the number is 42.68±3.60/200,P>0.05.Suppression of ezrin expression can also inhibit cell invasion.After suppressing ezrin expression by antisense oligonucleotide,number of sw620 cells invased significantly decreased from 36.3±5.4/200 to 12.7±1.84/200 (P<0.05).Their expression levels were correlation with invasive potential. Transfecting antisense oligonuleotide of ezrin into SW620 cells by liposomes can dramatically suppress ezrin expression. This method is effective and safe. Suppressing ezrin expression in SW620 cells can significantly suppress cell adhesion, motility and invasive ability. Ezrin regulated cell surface adhesion receptors cluster by promoteing actin polymerization and actin based cytoskeleton reoganization.Baicalein may suppress colorectum cancer cell sw620 growth by inhibiting its proliferation in a time-and dose-dependent manner,and down-regulate the expression level of ezrin mRNA and ezrin Protein as well as its Phosphorylation,which is related with the effects of baicalein on colorectum cancer cell.These results suggest that baicalein is likely to be valuable for the treatment of human colorectum cancer .