The Effect Of PI3K P85α Depletion On Survival Of Colorectal Cancer Cells And The Signaling Pathway Involved In

Colorectal cancer(CRC) is one of the major malignancies which has high attack rate and death rate in the world.The morbidity is higher in North America and Oceania than in Europe and Asia.In China,the morbidity is higher in South than in North.In recent years,the morbidity of CRC has increased year by year. Conventional chemotherapy regiments for the treatment of colorectal cancer have limited efficacy and are associated with significant toxicity.Therefore,a great deal of effort and resources have been put into better understanding the mechanism of colorectal cancer development and developing novel targeted approaches for the management of this disease.Unlimited proliferation and resistance to apoptosis are major biological characters of CRC and are major causes lead to poor prognosis.With the rapid development of molecular biology recently,signaling pathways that involved in tumor progression have been payed more and more attention.Truly,an increasing knowledge about the genetic control of cellular proliferation and the modulation of the signaling pathways that are aberrant in colorectal cancer has the potential to provide an effective and better approach for its management.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.Class-IA PI3K are heterodimers composed of a catalytic subunit(p110) and an adapter/regulatory subunit(p85).p85 regulatory subunit is essential for stabilization and collection of p110 catalytic subunit and for activation of Class-IA PI3K.p85αis the most abundant regulatory subunit of PI3K family and is encoded by PIK3R1 gene.It has been reporetd that mutation of PI3K p85αcould activate PI3K/Akt signaling pathway.Evidences have showed that PI3K p85αis a kind of oncogene.Most studies focused on the mutation and the mutation form of PI3K p85αin cancer.Reports about the amplification of PI3K p85αin 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:â‘ Inactivation of FoxO transcription factors leads to cell cycle progression which contributes to the development of tumor.â‘¡Inactivation of FoxO transcription factors leads to impaired ability to repair DNA damage which results in genomic instability.â‘¢Depletion of FoxO transcription proteins leads to inhibition of apoptosis which may contributes to tumor progression.â‘£FoxO transcription proteins are found in human translocation mutational tumors.At present,the relationship between biological changes resulted from the changes of PI3K p85αexpression levels and the changes of FoxO transcription factors is not reported.In this study,we investigate the expression and significance of PI3K p85αin the progression of colorectal cancer,including normal colorectal tissue,colorectal adenoma and primary colorectal carcinoma.Then we construct shRNA vector to transfect Lovo and SW480 cells.After stable transfection,we observed the effect of PI3K p85αdepletion 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 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 PI3K p85αin progression of colorectal cancerImmunohistochemical staining was used to detect the expression and significance of PI3K p85αin the progression of colorectal cancer,including normal colorectal tissue,colorectal polyp,colorectal adenoma and primary colorectal carcinoma.The relationship between the expression of PI3K p85αprotein and clinicopathological factors was also analyzed.The effect of PI3K p85αdepletion on proliferation,cell cycle and apoptosis in colorectal cancer cells1.Construction of human PI3K p85αshRNA vectors The oligonucleotides encoding four 21-mer hairpin sequences were designed of PI3K p85αmRNA (Genebank:NM₁81504).A scramble siRNA with the same nucleotide composition as the siRNA but which lacks significant sequence homology to the genome was also designed.The oligonucleotides are synthesized,annealed,and ligated into the linearized pGPU6/Neo siRNA Expression Vector by GnenPharma company.2.Transfection and selection of stable transfected cell clones The shRNA vectors 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 antibiotic-resistant cells were passaged in medium containing 500μg/ml G418 as needed.3.PI3K p85αexpression detecting The interference effect was evaluated by Western-blot analysis.Selected stable cell lines transfected with the shRNA that had the highest imerference effect for the following experiments.4.Cell proliferation assays Cells were cultured for 96 h 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 shRNA 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 survival signaling pathway PI3K p85αinvolved in coloreetal cancer cellsWestern-blot was used to analyzed expression changes of signaling pathway proteins after depletion of PI3K p85α.The cell proliferation and apoptosis signaling pathway proteins include Akt,p-Akt,cytoplasmic proteins p-FoxO1(FKHR,Ser²⁵⁶),p-FoxO3a(FKHRL1,8er²⁵³),p-FoxO4(AFX,Ser¹⁹³),nuclear proteins Foxo1 (FKHR),FoxO3a(FKHRL1),FoxO4(AFX),and cell cycle associated proteins cyclinD1,cdk4,cdk6 and p27/Kip1.Statistical AnalysisAll experiments results were from at least three separate experiments.For immunohistochemistry results,Kruskal-Wallis test was used.Spearman correlations test was used for analyzing the relationship between PI3K p85αexpression and colorectal tissue type and Dukes stage.For other results,one-way analysis of variance (ANOVA) and Student's t test were used in group comparison.Dates are expressed as the(?)+s.A value of P＜0.05 was considered statistically significant.ResultsThe expression and significance of PI3K p85αin progression of colorectal cancerImmunohistochemistry was performed to examine PI3K p85αexpression levels in paraffin-embeded tissue from colorecta mucosa,benign polypi and adenomas to primary colorectal cancers.PI3K p85αexpression was highest in surface epithelium of colorectal mucosa,and expression in the stroma was limited to inflammatory cells, with a predominantly cytoplasmic distribution.Representative shows that PI3K p85αexpression is negative in normal colorectal mucosa and polyp,begins to increase in adenoma and over-expressed in primary colorectal adenocarcinoma(P＜0.001). Furthermore,there was a significant difference in the positive rates of the PI3K p85αexpression duiring different Dukes' stage(P＜0.050).No obvious correlation was found between expression of PI3 K p85αand pathological diagnosis(P＞0.050).The effect of PI3K p85αdepletion on proliferation,cell cycle and apoptosis in colorectal cancer cells1.Constructed human PI3K p85αshRNA expression vetors successfully,which were identified by restriction enzyme digestion analysis and DNA sequencing.2.After four shRNA vectors were transfected into Lovo and SW480 cells,stable colnes were formed after G418 selection.Then the cells were name as Lovo shRNA/89;Lovo shRNA/324;Lovo shRNA/1073;Lovo shRNA/1123;SW480 shRNA/89;SW480 shRNA/324;SW480 shRNA/1073;SW480 shRNA/1123.Cells transfected with contro shRNA vectors were named as Lovo shRNA/N and SW480 shRNA/N.3.After stable transfection,Western-blot analysis was used to investigate the expression changes of PI3K p85α.The expression levels were significantly inhibited in Lovo shRNA/324 and SW480 shRNA/324 cells compared to those of control cells. Thus,cells expressing shRNA/324 vectors were used for the following experiments.4.To determine the effect of PI3K p85αknockdown on proliferation of colorectal cancer cells,CCK-8 assay was performed at 0h,24h,48h,72h and 96h, respectively.Compared with the control cells,Lovo shRNA/324 cells grew much slowly at 24h,48h,72h and 96h(P＜0.005),SW480 shRNA/324 cells grew much slowly at 48h,72h and 96h(P＜0.050).The results indicate that knockdown of PI3K p85αcould inhibit coloredtal cell proliferation.5.To examine if RNA interference against PI3K p85αhas an impact on cell cycle of colorectal cancer cells,flow cytometry analyses were performed and the results showed that depletion of PI3K p85αcause a significant increase in the proportion of Lovo and SW480 cells at the G1 phase(P＜0.050).The observed increase in/G1 cell population in Lovo and SW480 cells was accompanied by a reduction of cells in the S phase(P＜0.050).The findings suggest that depletion of PI3K p85αcould obviously induce cell cycle arrest.6.In order to evaluate the effect of PI3K p85αknockdown on the induction of apoptosis,colorectal cancer cells expressing PI3K p85αshRNA and control shRNA 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 PI3K p85αknockdown Lovo and SW480 cells than in control cells(P＜0.050).The results indicate that depletion of PI3K p85αsensitized colorectal cancer cells to 5-FU induced apoptosis.PI3K p85αdepletion 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 silencing of PI3K p85α,we examined expression levels of AKT,phospho-Akt,FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm.The results showed that PI3K p85αdepletion led to substantial reduction in the levels of AKT and phospho-AKT(P＜0.050).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 PI3K p85α-knocked down cells(P＜0.050),the prominent accumulation of FoxO1 (FKHR),FoxO3a(FKHRL1) and FoxO4(AFX) in nucleus was simultaneously observed(P＜0.050).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 PI3K p85αdepletion cells(P＜0.050),while the expression level of cyclin kinase inhibitor p27/Kip1 was markedly induced(P＜0.050).Conclusions1.Immunohistochemical results show that the expression levels of PI3K p85αgradually increases from normal colorectal mucosa,adenoma to primary colorectal adenocarcinoma,indicating that PI3K p85αplays an important role in the progression of colorectal cancer.2.Depletion of PI3K p85αcould inhibit LoVo and SW480 cell proliferation.3.Depletion of PI3K p85αcould induce G1 phase cell cycle arrest and reduction of S phase cells.4.Depletion of PI3K p85αcould sensitize LoVo and SW480 cells to 5-FU induced apoptosis,indicating that combination of 5-FU and PI3K p85αdepletion may be an effective approach in treating colorectal cancer cells.5.Depletion of PI3K p85αcould 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 inhibited cell proliferation and cell cycle arrest of colorectal cancer cells result from PI3K p85αdepletion are closely related with activation of FoxO transcription factors,and the activation of FoxO transcription factors maybe involved in the enhanced sensibility to 5-FU induced apoptosis.