The Signal Mechanism Underlying Basic Fibroblast Growth Factor-mediated Cell Proliferation In Hepatic Cancer Bel-7402 Cells

ObjectiveBasic fibroblast growth factor(bFGF) is a potent mitogen that plays an important role in cell proliferation,differentiation and survival by phosphoinositide 3-kinase (PI3K)/Protein kinase B(PKB) after binding to fibroblast growth factor receptor (FGFR).Because the tumors are characterized of overgrowth and abnormal proliferation,the signal pathways underlying regualtion of cell cycle becomes the focus of medical research.Most of tumors result from dysregulated phase G1 and G2 in cell cycle.It is reported that bFGF and FGFR are overexpressed in hepatic cancer which may involve in carcinogenesis through activating PI3K/PKB signal pathway,promoting phase G1-S and strengthening the cell growth.FOXO1,a transcription factor,is an important downstream of PI3K/PKB pathway in the regulation of metabolism,cell cycle, proliferation and survival.As a tumor inhibitor,either low activity or dysfunction of FOXO1 will lead to the cell overgrowth and transformation of tumors.Differently,p27,the inhibitor of cyclin dependent kinase(CDK) and Cyclin/CDK, supresses the cell cycle and disrupt G1-S.In the tumor,S-phase kinase-associated protein 2(Skp2)-mediating ubiquitin-proteasome system(UPS) is the important pathway to degrade p27 and results in overgrowth.Most of human cancers overexpress Skp2,downexpress p27 and exhibit the abnormal proliferation.It indicates that degradation of p27 depedenting on Skp2 may paly an critical role in the carcinogenesis and development of tumors.However,the relationship between bFGF-mediating PI3K/PKB signal pathway and its downstream FOXO1,p27 and Skp2 has not been well defined.Hepatic cancer is one of the popular tumors and has the highest mortality among malignant tumors associated to the unbalance of proliferation and apoptosis.To evaluate the impact of bFGF-mediated activation on the cell proliferation in the hepatic cancer,we investigate the effects of bFGF on proliferation,activity of PKB,FOXO1,p27 and Skp2 expression in hepatic cancer Bel-7402 cells,and explore the relationship between these effects and PI3K/PKB signal pathway.Methods1.Bel-7402 cells were cultured in serum-free DMEM with bFGF which blocked by Wortmannin(PI3K inhibitor).2.The effects of bFGF and Wortmannin on the proliferation in starvated Bel-7402 cells were estimated by MTT and FCM analysis.3.The changes of the activity of PKB and FOXO1 induced by bFGF and Wortmannin were accessed by western blotting.p-FOXO1 distribution induced by bFGF was detected by immunofluorescence technique.4.The changes of the activity of the protein expression of p27 and Skp2 induced by bFGF,Wortmannin and MG132 were accessed by western blotting.5.The mRNA expression of p27 and Skp2 induced by bFGF and Wortmannin were determined by reverse transcription PCR(RT-PCR).Results1.As compared to starvation group,bFGF treatment activated PKB and phosphorylated FOXO1 dose-and time-dependently which were prevented by Wortmannin effectively P＜0.01).The phosphoralation of FOXO1 induced by bFGF occurred in cytoplasm.2.MTT and FCM analysis display that Bel-7402 cells were still viable after bFGF treatment,had increased proliferation rate and cell distribution from phase G1 to phase S,dose-dependently.Wortmannin would prevent the roles of bFGF all above effectively(P＜0.01).3.As compared to starvation group,bFGF treatment restraind the expression of p27,but accelerated the mRNA and protein expression of Skp2 time-dependently which peak at 6 h and 8 h after initiation of bFGF.Wortmannin would inhibit this process effectively(P＜0.01).bFGF treatment has no effect on the mRNA expression of p27.Proteasome inhibitor(MG132) could enhance the protein expression of p27. Conclusion1.bFGF could phosphorylate PKB and FOXO1.2.The activation of PKB and FOXO1 mediated by bFGF resulted from phosphorylating the kinases.3.bFGF could promote the proliferation of Bel-7402 cells by PI3K/PKB /FOXO1 signal pathway.4.bFGF could downregulate p27 protein expression and increase Skp2 mRNA and protein expression by PI3K signal transduction pathway that maybe contributes to the proliferation in Bel-7402 cells.