PKB-dependent Pathway In Colorectal Cancer Induction By BFGF

ObjectiveTo study the mechanism of occurrence,development,infiltration and metastasis in colorectal cancer is the focus viewpoint in ontology.In recent years, fibroblast growth factor has been found as a kind of polypeptide growth factor which is widely spreading in vivo,its biology activity is close correlation with neoplasm growth.Growth factor receptor binding activates multiple intracellular pathways,the appropriate integration of these pathways in proliferation,differentiation,and growth arrest are crucial for the function of a cell.The final outcome of growth factor signaling is the result of complicated interplay between activated pathways and feedback loops,and it is also dependent on the amplitude and duration of the signals as well as type of cell involved.The fibroblast growth factors(FGFs) are a family of angiogenic factors,that are potent mitogens for fibroblast and endothelial cells,and overexpression of the growth factor and its receptors have been implicated in transformation and malignant progression.Mitogenic signals are transmitted from the activated tyrosine kinase receptors through several known pathways.Triggering these pathways depends on direct protein- protein interactions,posttranslational modifications,and the induction of second messenger molecules.The synthesis of one class of such molecules involves the phosphorylation of phosphatidylinositol and its D -4 - and D -5 -phosphorylated derivates in the D -3 position,in a reaction catalyzed by the phosphatidylinisitol 3 - kinase.More and more studies have shown that the PI3 - kinase exhibits both lipid and protein kinase activities,and in many cell types, plays a critical role in mitogenic or other signaling.PKB is activated in response to activation by many different GFs,including IGF 1,epidermal GF,basic fibroblast GF,insulin,interlukin 3,interlukin 6, and macrophage stimulating factor.PKB is the cellular homologue of the product of the v akt oncogene and has three isoforms:PKBα,-β,and -γ.PKBβand PKBγare overexpressed in ovarian,pancreatic,and breast cancer cells. Activation of three isoforms is similar in that phosphorylation of two sites,one in the activation domain and the other in the GOOH - terminal hydrophobic motif, are necessary for full activity.For PKBα,phosphorylation of T308 in the activation domain by PDK1 is dependent on the products of PI3 - K,PIP₂ and PIP₃. PIP₂ and PIP₃ bind to the pleckstrin homology domains of PKB and PDK1,which relieves steric hindrance,fully activates PDK1,and translocates PKB to the plasma membrane.The mechanism of 5473 phophorylation is less clear.Kinase potentially responsible for S473 phosphorylation include PDK1,integrin - linked kinase,or an integrin -linked kinase -associated kinase,PKB itself or an as yet uncharacterized PDK2.PKB activation may also be achieved through PI3 -K independent means,either through phosphorylation of PKB by kinases such as PKA or CAMKK,or under conditions of cellular stress.Cell proliferation is ultimately controlled at the level of cell cycle.Under appropriate stimuli,cells exit quiescens and enter the cell cycle at G1.Progression through the cell - cycle is dependent onthe balance of positive and negative regulatory cell -cycle proteins.During each phase of the cell -cycle there is an increase in expression for specific cyclins,which bind to their catalytic partners, the cyclin - dependent kinases.This results in the formation of active cyclin -CDK complexes,which are responsible for the phosphorylation of the retinoblastoma protein as well as other substrates,an event required for G1/S transition and DNA synthesis,The D -cyclins bind CDK4 in G1,whereas cyclin E and CDK2 complex at G₁/S transition.Cychn A is complexed with CDK2 during S phase.Cyelin -kinase inhibitors negatively regulate the cell -cycle by inhibiting cyelin- CDK complexes.In this study,we use the PKB specific inhibitor. genistein and observe the inhibition of the proliferation in colorectal cancer cell line LoVo which has been induced by bFGF and observe the cell apoptosis phenomenon, and then to,explore the signal transduction in tumor cells and provide the basis of inhibiting the tumor cells proliferation by inhibiting the signal transduction pathway.To elucidate the mechanism for PKB -mediated growth control, therefore,we investigate whether bFGF regulates cyclin A expression via PI3K/PKB pathway. Materials and Methods1.The expression of bFGF PKB and cyclin A in colorectal cancer tissue was evaluated by RT- PCR technique.2.MTT assay was used to determine the suppressive effect of LY294002 on human colorectal cancer cell line LoVo induced by bFGF.3.Assay[γ-32^p]ATP incorporation assay to detect the PKB kinase activity.4.The expression of cyclin A - mRNA was assessed by RT- PCR.5.The expression of PKB and cyclin A protein was detected by Western blot.Analysis.Results1.Expreesion of bFGFmRNA PKBmRNA and Cyclin AmRNA in colorectal cancer.Thepositive rates bFGF PKB and Cyclin A in colorectal cancer tissue were 50%,72%and 57%,respectively the positive rates of bFGF PKB and Cyclin A in the peritumoral tissue were 4%,35%and 5%,respectively.There was no positive cases in the control group.The expression levels of bFGFmRNA PKBmRNA and Cyclin AmRNA in Dukes stage C and D were significantly higher than those in Dukes stage A and B(P＜0.05).There were no difference in the differentiated grade(P＞0.05).2.The effect of bFGF and LY294002 on the proliferation of LoVo cell. bFGF could improve the proliferation of LoVo significantly.The proliferation of LoVo cell decreased significantly with LY294002,especially in the cells treated with bFGF(P＜0.05).3.We found that both membrane and cytosol activity of PKB in LoVo cell (75ng/ml)reached to the peak at 10min(3.65 - fold and 3.39 - fold induction, P＜0.05).Both membrane and cytosol activity of PKB in LoVo cell treated with bFGF(25ng/ml)reached to the peak at 10min(2.53 -fold and 2.78 -fold induction,P＜0.05).Inhibition of phosphatidylinositol 3 -kinase by LY294002 inhibits bFGF- stimulated PKB activity(P＜0.05).4.Cyclin A mRNA level was up- regulated in LoVo cell.LY294002 efficiently inhibited the level of cyclin A mRNA.Conclusion1.There was overexpression of bFGF PKB and Cyclin A in colorectal cancer. The expression levels were related to Dukes stage.2.bFGF modulated cyclin A expression via PI3K/PKB pathway,which stimulated the proliferation of LoVo cell.