| Background and objectivesColorectal cancer(CRC) is one of the major malignancies in the world.The prognosis of CRCs is poor,due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually,amounting to492 000 related deaths.With the many changes having taken place in people's diet and lifestyle,CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing.The overall incidence is identical in men and women,with the risk beginning at age 40 and increasing with age.Thus colorectal cancer ranks as the frequent cause of cancer deaths among China.Despite the rate of improvements in surgery,radiotherapy and chemotherapy,unfortunately,the prognosis of CRCs has not been gained progress over the past decades,with an overall five-year survival rate of around 40%-50%.Therefore,novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium.In recent years,molecular biology has applied to the study of colonic carcinoma,both in the human and in the experimental animal. The data obtained have enriched our understanding of colonic carcinogenesis and are of potential interest for CRCs diagnosis and prevention.Phosphatidylinositol-3-kinase(PI3K) is a lipid kinase and is responsible for the phosphorylation of 3 position of the inositol ring of PI(4,5)P2,to generate PI(3,4,5) P3,a potent second-messenger required for fundamental cellular functions such as transcription,translation,proliferation,growth,and survival.There are three members in PI3K family.Signaling pathway composed of Class-IA PI3K and serine/threonine kinase Akt/PKB has close relationship with tumor progression.This pathway regulates proliferation and survival of cancer cells.The disturbed activation of PI3K signaling leads to not only neoplastic transformation of normal cells,but also correlation with tumor cell migration,adhesion,tumor angiogenesis,as well as the degradation of extracellular matrix.PTEN is a tumor-suppressor gene located on human chromosome 10q23.3.PTEN is both a lipid phosphatase and a protein phosphatase.As a lipid phosphatase,PTEN classically converts phosphatidylinositio-3, 4,5-trisphosphate(PIP3) in thecytoplasm to phosphatidylinositol-4,5-bisphosphate (PIP2),thereby directly antagonizing the activity of PI3 kinase(PI3K).Its inactivation results in constitutive activation of the PI3K/AKT pathway and in subsequent increases in protein synthesis,proliferation,migration and survival.It has been reporetd that deletion and mutation of PTENcould activate PI3K/Akt signaling pathway.Most studies focused on the mutation and the mutation form of PTEN in cancer.Reports about the amplification of PTEN in tumor and the biological changes result from the amplification are rare.FoxO transcription factors belong to the Forkhead family of proteins,a family characterized by a conserved DNA binding domain termed the Forkhead box(Fox). The family is primarily regulated by PI3K pathway.Direct phosphorylation by PKB/Akt inhibits transcriptional activation by FoxO factors,causing their displacement from the nucleus into the cytoplasm.Deregulation of cell cycle and cell apoptosis are colosely related with tumor progression.FoxO transcription factors may be involved and play an important role in regulating cell cycle and cell apoptosis:①Inactivation of FoxO transcription factors leads to cell cycle progression which contributes to the development of tumor.②Inactivation of FoxO transcription factors leads to impaired ability to repair DNA damage which results in genomic instability.③Depletion of FoxO transcription proteins leads to inhibition of apoptosis which may contributes to tumor progression.④FoxO transcription proteins are found in human translocation mutational tumors.At present,the relationship between biological changes resulted from the changes of PTEN expression levels and the changes of FoxO transcription factors is not reported.In this study,we investigate the expression and significance of PTEN in the progression of colorectal cancer,including normal colorectal tissue,colorectal adenoma and primary colorectal carcinoma.Then we construct pc-DNA3.1-PTEN expressional vector to transfect LoVo and SW480 cells.After stable transfection,we observed the effect of PTEN expression on colorectal cancer cell proliferation,cell cycle and cell apoptosis.We also investigated the expression changes of PI3K signaling pathway proteins including Akt,FoxO transcription proteins and associated cell cycle proteins.We aim not only to investigate the molecular mechanism of tumor proliferation and apoptosis,but also to search new effective target for gene therapy of CRC.Materials and methodsThe expression and significance of PTEN in progression of colorectal cancerImmunohistochemical staining was used to detect the expression and significance of PTEN in the progression of colorectal cancer,including normal colorectal mucosa,colorectal proliferational plopy,colorectal adenoma and primary colorectal carcinoma.The relationship between the expression of PTEN protein and clinicopathological factors was also analyzed.The effect of PTEN expression on proliferation and apoptosis in colorectal cancer cells1.Transfection and selection of stable transfected cell clones The pcDNA3.1-PTEN expressional vector and control vector were transfected into Lovo and SW480 cells using Lipofectamine 2000(Invitrogen) according to the manufacturer's protocol and cultured for 24 h without antibiotic selection.Then the cells were cultured in medium containing 1000μg/ml G418 until all of the cells in the nontransfected control culture were killed and antibiotic-resistant clones were formed in the transfected cells.Visible clones were picked up and expanded for another 4 weeks. Then the antib(?)tic-resistant cells were passaged in medium containing 500μg/ml G418 as needed. 2.PTEN expression detecting The interference effect was evaluated by RT-PCR and Western blot analysis.Selected stable cell lines transfected with PTEN that had the highest interference effect for the following experiments.4.Cell proliferation assays Cells were cultured for 96h after stable transfection. Cell proliferation was determined using Cell Counting Kit-8(CCK-8) solution.5.Cell cycle analysis FACSC alibur Flow Cytometer was used to determine the cell cycle after stable transfection.6.Apoptosis Assays Lovo and SW480 cells that expressing pc-DNA3.1-PTEN expressional vector and the control cells were treated with 5-FU for 48 h at final concentration of 0.01μmol/ml and 0.03μmol/ml,respectively.Annexin V-FITC Kit was used to determine the apoptosis.Research of cell proliferation and apoptosis signaling pathway PTEN involved in colorectal cancer cellsWestern blot was used to analyzed expression changes of signaling pathway proteins after expression of PTEN.The cell proliferation and apoptosis signaling pathway proteins include Akt,p-Akt,cytoplasmic proteins p-FoxO1a(FKHR, Ser256),p-FoxO3a(FKHRL1,Ser253)),p-FoxO4(AFX,Ser193),nuclear proteins FoxO1a(FKHR),FoxO3a(FKHRL1),FoxO4(AFX),and cell cycle associated proteins cyclinD1,cdk4,cdk6 and p27/Kipl.Statistical AnalysisAll experiments results were from at least three separate experiments.For immunohistochemistry results,Kruskal-Wallis test was used.For other results, one-way analysis of variance(ANOVA) and Student's t test were used in group comparison,repeatedly measured data analysis of variance were cell proliferation, LSD analysis method was used in group comparison.Dates are expressed as the mean±SD.A value of P＜0.05 was considered statistically significant.ResultsThe expression and significance of PTEN in progression of colorectal cancerImmunohistochemistry was performed to examine PTEN expression levels in paraffin-embeded tissue from colorecta mucosa,benign polypi and adenomas to primary colorectal cancers.PTEN expression was highest in surface epithelium of paraffin-embeded tissue from colorecta mucosa.The diffierence for the expression of PTEN protein in normal colorectal tissue,colorectal proliferational plopy,colorectal adenoma and primary colorectal carcinoma was significant(P＜0.05).No obvious correlation was found between expression of PTEN and pathological diagnosis (P＞0.05).The effect of up-regulating PTEN expression on proliferation and apoptosis in colorectal cancer cells1.Constructed human pc-DNA3.1-PTEN expression vetors successfully,which were identified by restriction enzyme digestion analysis and DNA sequencing.2.After pc-DNA3.1-PTEN expression vector and pc-DNA3.1 vector were transfected into LoVo and SW480 cells,stable colnes were formed after G418 selection.Then the cells were name as LoVo pc-DNA3.1-PTEN;LoVo pc-DNA3.1; SW480 pc-DNA3.1-PTEN;SW480 pc-DNA3.1.3.After stable transfection,western blot analysis was used to investigate the expression changes of PTEN.The expression levels were significantly enhanced in LoVo pc-DNA3.1-PTEN and SW480 pc-DNA3.1-PTEN cells compared LoVo pc-DNA3.1 and SW480 pc-DNA3.1cells.4.To determine the effect of PTEN expression on proliferation of colorectal cancer cells,CCK-8 assay was performed at Oh,24h,48h,72h and 96h,respectively. Compared with the control cells,LoVo pc-DNA3.1-PTEN andSW480 pc-DNA3.1-PTEN cells grew much slowly at 24h,48h,72h and 96h(P＜0.05).The results indicate up-regulating PTEN expression could inhibit coloredtal cell proliferation.5.To examine if expression of PTEN has an impact on cell cycle of colorectal cancer cells,flow cytometry analyses were performed and the results showed that expression of PTEN cause a significant increase in the proportion of LoVo and SW480 cells at the G0/G1 phase(P＜0.05).The observed increase in G0/G1 cell population in Lovo and SW480 cells was accompanied by a reduction of cells in the S phase(P＜0.05).The findings suggest that expression of PTEN could obviously induce cell cycle arrest.6.In order to evaluate the effect of up-regulating PTEN expression on the induction of apoptosis,colorectal cancer cells expressing pc-DNA3.1-PTEN and pc-DNA3.1 were treated with 5-Fu at final concentration of 0.01μmol/ml(LoVo) or 0.03μmol/ml(SW480) for 48h,followed by assessing early apoptotic rate by means of flow cytometric analysis.Results showed that the proportion of positive cells for Annexin V was significantly higher in up-regulating PTEN expression LoVo and SW480 cells than in control cells(P＜0.05).The results indicate that expression of PTEN sensitized colorectal cancer cells to 5-Fu induced apoptosis.Up-regulating PTEN expression activated FoxO transcription factors associated with regulating cell cycle-associated protein expression in colorectal cancer cellsTo further explore the mechanism underlying the enhancement of cell cycle arrest and 5-FU-induced apoptosis by the expression of PTEN,we examined expression levels of Akt,phospho-Akt,FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm.The results showed that up-regulating PTEN expression led to substantial reduction in the levels of Akt and phospho-Akt(P＜0.05).Consistent with this reduction in the phospho-Akt level, Western blot analysis showed significantly decreased expression of phospho-FoxO1 (FKHR),phospho-FoxO3a(FKHRL1) and phospho-FoxO4(AFX) in cytoplasm of PTEN expresion cells(P＜0.05),the prominent accumulation of FoxO1(FKHR), FoxO3a(FKHRL1) and FoxO4(AFX) in nucleus was simultaneously observed (P＜0.05).As potential downstream targets of FoxO transcription factors,the expression levels of cell cycle-associated protein were also determined.Results showed that the expression levels of cyclin D1,cdk4 and cdk6 were significantly decreased in up-regulating PTEN expression cells(P＜0.05),while the expression level of cyclin kinase inhibitor p27/Kip1 was markedly induced(P＜0.05).Conclusions1.Immunohistochemical results show that the expression levels of PTEN gradually decreases from normal colorectal mucosa,colorectal proliferational plopy,adenoma to primary colorectal adenocarcinoma,indicating that PTEN plays an important role in the progression of colorectal cancer.2.Up-regulating PTEN expression could inhibit LoVo and SW480 cell proliferation.3.Up-regulating PTEN expression could induce G1 phase cell cycle arrest and reduction of S phase cells.4.Up-regulating PTEN expression could sensitize LoVo and SW480 cells to 5-Fu induced apoptosis.5.Up-regulating PTEN expression could inhibit the activity of Akt and activate FoxO transcription factors,which,in turn,activated transcription of target genes such as those involved in cell cycle regulation and apoptosis.Thus,the colorectal cancer cell cycle arrest and enhanced sensibility to 5-Fu induced apoptosis result from expression of PTEN are closely related with activation of FoxO transcription factors. |
