| ObjectiveBasic fibroblast growth factor ( bFGF) , being a polypeptide, has powerful effects on proliferation, differentiation, survival and function of many kinds of cells through binding to fibroblast growth factor receptor ( FGFR). The signal transduction of bFGF/FGFR is related to a lot of diseases and carcinogenesis. Ovarian cancer is one of the popular tumors of gynecology and its mortality is the highest among gynecology malignant tumors. There are great challenge in the re-seach about diagnosis and treatment of ovarian cancer. bFGF and its receptor in ovarian cancer have been reported which may play important roles in carcinogenesis through autocrine or paracrine mechanism.Mitogenic signals are transmitted from the activated tyrosine kinase receptors through several known pathways ( such as PLC/PKC, Ras - Raf - ERK, PDK/PKB, JAK/STAT). Ras - Raf - ERK cascade which is the important signal transduction pathway can be activated by growth factor, hormone etc. and regulate cell division, proliferation, survival etc. through TPK→Grb2→SOS→ Ras→Raf→MEKK→MEK→ERK→transcription factor→gene expression. The recently discovered PDK/PKB pathway has comprehensive functions and can regulate growth, proliferation , differentiation, survival, transformation and so on.Cell cycle mechanism has important roles in proliferation, differentiation and carcinogenesis. Rapid progress of cell cycle or destruction of checkpoint may make proliferation out of control then carcinogenesis. Thus carcinogenesis is believed to be the result of turbulence of cell cycle. Control of cell cycle undergoes at different restriction point and the G1/S restriction point is very impor-tant. Cyclin E and Cdk2 funtion at Gj/S transition. p27K!pl binding to Cdks and Cyclin - Cdk complexes, dose dependently inhibits activity of Cdks and negatively regulates cell cycle. CyclinE - Cdk2 is the most optimum substrate of p27K>pl. Mdm2, the production of oncogene, can regulate proliferation and survival and play important roles in carcinogenesis.To elucidate the effects of bFGF in ovarian cancer, we investigate the effects of bFGF on growth, ERK activity, expression of Cyclin E, Cdk2, p27Kipl, Mdm2 in ovarian cancer cell CA0V3 and the ralationship between these effects and Ras -Raf - ERK, PI3K/PKB pathways.Methods1. The cell cycle distribution of ovarian cancer cell CA0V3 after treated with bFGF and the roles of Ras - Raf - ERK,PI3K/PKB pathways in cell cycle progression mediated by bFGF were determined by FACScan flow cytometer (FCM).2. Effects of bFGF,PD98059 - the inhibitor of MEK1 and wortmannin - the inhibitor of PDK on proliferation of ovarian cancer cell CA0V3 were detected by MTT assay.3. Effects of bFGF on resistance to apoptosis induced by starvation in ovarian cancer cell CAOV3 and the roles of Ras - Raf - ERK,PI3K/PKB pathways in these process were determined by FCM.4. Change of ERK activity, protein expression of Cyclin E, Cdk2, p27 ipl, Mdm2 induced by bFGF in ovarian cancer cell CA0V3 and the roles of Ras -Raf-ERK,PI3K/PKB pathways in these process were determined by Western blot.5. mRNA expression of Cyclin E, Mdm2 induced by bFGF in ovarian cancer cell CA0V3 and the roles of Ras - Raf - ERK, PDK/PKB pathways in these process were determined by RT - PCR.Resultsl.-FCM analysis showed that;In CAOV3 cells treated with bFGF for 24h, the proportion of cells in Gl phajse decreased (60. 64 ± 1. 75% —?41. 51 ± 1.26% ) and the proportion of cells in S phase increased (35. 40 ± 1. 75% —? 58.02 ± 1. 25% ) compared with control group. bFGF induced S - phase entry( P <0. 05) , which was inhibited by PD98059 ( G, phase :54. 78 ± 1. 11%;S phase;37.26 ± 1.27% ) and wortmannin ( G! phase:53. 85 ± 2. 23%;S phase: 40. 83 ±3.15% ) respectively( P <0. 05 ).2. MTT assay showed that: Treated CAOV3 cells with bFGF, the cell pro-liferative rate increased dose dependently. When the concentration of bFGF was at 75 ng/ml, the cell proliferative rate was at the peak as 140%. The difference between bFGF group and control group has statistics significance ( P < 0. 05 ). The cell proliferative rate decreased by PD98059 and wortmannin respectively, which has statistics significance ( P <0.05).3. Apoptosis peak, appeared in front of Gj phase peak in ovarian cancer cell CAOV3 induced by starvation for 72h. bFGF decreased the apoptosis rate, which was inhibited by PD98059 and wortmannin respectively (P <0.05).4. Western blot showed that;(1) bFGF up - regulated ERK activity in ovarian cancer cell CAOV3, which could be inhibited by PD98059, the inhibitor of MEK1. Treated cells with 75ng/ml bFGF for 5,10,30,60min respectively, activity of ERK in CAOV3 cell began to increase at 5 min and reached the peak at lOmin. The difference between bFGF group and control group has statistics significance ( P <0. 05). Pre - treated with PD98059, ERK activity decreased compared with treated with bFGF alone, which has statistics significance(P <0.05).(2)bFGF induced Cyclin E protein expression time and dose dependently (P <0.05) , which was inhibited by PD98059 and wortmannin respectively(P < 0.05). Treated CAOV3 cells with 75ng/ml bFGF for 18h, Cyclin E protein expression reached the peak.(3)bFGF time and dose dependently induced Cdk2 protein expression( P <0. 05 ) , which was inhibited by PD98059 and wortmannin respectively ( P < 0.05). Treated CA0V3 cells with 75ng/ml bFGF for 12,16,24h , Cdk2 protein expression increased gradually.(4)bFGF time and dose dependently down -regulated p27Kipl protein ex-pression(P<0. 05) , which was inhibited by PD98059 and wortmannin respectively^ |
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