The Signal Mechanism Underlying Basic Fibroblast Growth Factor-mediated Antiapoptosis In Ovarian Cancer CAOV3 Cells

The signal mechanism underlying basic fibroblast growth factor-mediated anti-apoptosis in ovarian cancer CAOV3 cellsObjectiveAs a potent mitogen, basic fibroblast growth factor(bFGF), plays an important role in cell proliferation, differentiation, angiogenesis and survival through several signal pathways including Mitogen-activated protein kinase kinase (MEK)/Extracellullar signal regulated protein kinase (ERK), Phosphoinositide 3-kinase (PI3K)/Protein kinase B (PKB),Protein kinase C (PKC) by binding to fibroblast growth factor receptor (FGFR). However, its downstream mechanism has not been well defined.Ovarian cancer is one of the popular gynecology tumors and has the highest mortality among malignant gynecology tumors associated to the unbalance of proliferation and apoptosis. It is reported that bFGF and FGFR are overexpressed in ovarian cancer which may involve in carcinogenesis by autocrine or paracine mechanism.The tumor is characterized by increasing proliferation and decreasing apoptosis. Apoptosis involves a subset of effector caspase proteases .Activation of these caspases occurs via mitochondria-independent and -dependent mechanisms. It is well known that mitochondria-dependent apoptotic signal can be effectively blocked by anti-apoptotic members of Bcl-2 family such as Bcl-2 or Bcl-x1 and promoted by pro-apoptotic members such as Bax or Bad. Although Bcl-2 may have a direct action on the mitochondria membrance, it also resides and functions on the endoplasmic recticulum(ER),and there is increasing evidence for a role of the ER in apoptosis regulation as well. ER is an organelle responsible for the protein synthesis and homeostasis. Stress can bring about alterations of ER homeostasis which cause ER stress. Unfolded Protein Reaction(UPR) is an important genomic response to ER stress by synthesis ER chaperones proteins such as glucose regulated proteins 78(Grp78), which plays critical roles in cell survival as part of UPR.When the ER function is severely impaired,the organelle elicits apoptosis signal activated by growth arrest and DNA damage inducible gene (GADD153) and caspase-12. The apoptotic crosstalk between ER and mitochondria is also controlled by Bcl-2 family.To evaluate the impact of bFGF-mediated activation on the anti-apoptosis and the relationship between mitochondria and ER in ovarian cancer, by means of free-serum starvation to induce ER stress and apoptosis in vitro,we investigate the effects of bFGF on survival, activity of ERK,PKB,Bad and CREB,mRNA or protein expression of Bcl-2,Bcl-xl,Bax, Grp78 and GADD153 in ovarian cancer CAOV3 cells, and explore the relationship between these effects and MEK/ERK, PI3K/PKB signal pathway.Methods1. CAOV3 cells were cultured in serum-free DMEM with or without bFGF which blocked by PD98059 (MEK1 inhibitor) or Wortmarmin (PI3K inhibitor).2. The effects of bFGF, PD98059 and Wortmannin on the apoptosis in starvated CAOV3 cells were estimated by DNA ladder gelelectrophoresis, FCM analysis and staining with Hematoxylin-Eosin, Coomassie brilliant blue and Annexin-EGFP/PI, respectively.3. The changes of the activity of ERK, PKB and CREB, and the protein expression of Bcl-2,Bax,Bad,Grp78,GADD153 induced by starvation, bFGF,PD98059 and Wortmannin were accessed by western blotting.4. The mRNA expression of Bcl-2,Bcl-x1 and Grp78 induced by starvetion, bFGF, PD98059 and Wortmannin were determined by reverse transcription PCR(RT-PCR).Results1. Starvated CAOV3 cells displayed typical signs of apoptosis such as nuclear condensation or DNA fragments.As compared to starvation group, the cells after bFGF treatment were still viable and had increased activation of ERK and PKB, which was prevented by PD98059 and Wortmannin effectively(P＜0.01).Total ERK and PKB did not change under the same experimental conditions (P＞0.05).2. bFGF could activate CREB^ser133 and upregulate mRNA and protein expression of Bcl-2 time-dependently as compared to starvation group, which peaked at 45 min, 4 h and 8 h after initiation of bFGF treatment, respectively(P＜0.01).PD98059 could inhibit the effects of bFGF on Bcl-2 and CREB rather than Wortmannin. Total CREB remained unchanged under the same experimental conditions (P＞0.05).3. Both bFGF and starvation were ineffective on Bcl-x1 mRNA expression.4.Starvation induced Bax and GADD153 protein expression time-dependently, but bFGF restraind their expression which were blocked both by PD98059 and Wortmannin (P＜0.01).5.After bFGF exposure, the phosphorylated Badserf36 increased time-dependently as compared to starvation group (P＜0.01) and its peak was at 30 min which was prevented by Wortmannin but not PD98059. Total Bad protein levels did not change under the same experimental conditions (P＞0.05).6. bFGF could upregulate Grp78 mRNA and protein expression more effectively and persistently than starvation group.Grp78 mRNA and protein expression achieved their peaks at 4 h and 8 h after bFGF treatment,respectively (P＜0.01).Wortmannin could inhibit this process but PD98059 was ineffective on it.Conclusion1.Starvation could upregulate Bax and GADD153 that induce apoptosis in CAOV3 cells.bFGF could inhibit serum-free starvation-induced apoptosis through MEK/ERK, PI3K/PKB signal transduction pathway partly.3.bFGF activated MEK/ERK and PI3K/PKB by phosphorylating the kinases immediately rather than increasing their protein synthesis.4.bFGF upregulated Bcl-2 mRNA and protein expression through MEK/ERK/CREB^ser133 and increased Grp78 mRNA and protein expression through PI3K/PKB signal transduction pathway.5. bFGF induced Bad ^ser136 by PI3K/PKB signal transduction pathway and was ineffective on Bcl-x1 mRNA expression.6. bFGF blocked Bax protein expression through MEK/ERK and PI3K/PKB signal transduction pathway as well as GADD 153.