碱性成纤维细胞生长因子影响卵巢癌CAOV3细胞生长的信号转导机制
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摘要
目的
碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)是对多种细胞的增殖、分化、存活以及功能具有强烈调节作用的多肽,通过和成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)结合实现其生物学活性。bFGF/FGFR信号传递与多种疾病有关,且与肿瘤发生、发展的关系极为密切。卵巢癌是女性生殖系统常见肿瘤,其死亡率高居妇科恶性肿瘤之首,对其早期诊断和治疗的研究仍面临巨大挑战。研究表明卵巢癌细胞中存在bFGF及其受体,可能通过自分泌或旁分泌机制在其发生、发展中起重要作用。
bFGF与受体结合后通过多条信号转导通路(如PLC/PKC、Ras-Raf-ERK、P13 K/PKB、JAK/STAT等)将信号传入细胞内。Ras-Raf-ERK信号转导级联是细胞内重要的信号转导通路,可被生长因子、激素、神经递质等激活,通过TPK→Grb2→SOS→Ras→Raf→MEKK→MEK→ERK→转录因子→相关基因表达,调节细胞分裂、增殖、存活等许多细胞过程。P13K/PKB是近年来发现的一个新的生长因子信号转导通路,有广泛的生物学效应,是许多细胞过程,如生长、增殖、分化、存活和恶性转化的调节者。
细胞周期调控在正常细胞的增殖、分化及肿瘤的演化过程中均起到重要的作用。细胞周期的超常快速进行或检查点的破坏将导致失控的细胞生长引起肿瘤的发生、发展,因而肿瘤形成的过程被认为是细胞周期机制失调的结果。细胞周期调控是在各期的控制点上进行的,G_1/S控制点是影响细胞周期的关键。细胞周期蛋白E(Cyclin E)、细胞周期蛋白依赖激酶2(cyclin dependent kinase 2,Cdk2)在细胞周期G_1/S转变过程中发挥重要作用。p27~(Kipl)通过与Cdk或Cyclin-Cdk复合物结合,以一种化学剂量关系抑制Cdk的活性,从而对细胞周期进行负调控。CyclinE-Cdk2是p27~(Kipl)的最适
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 G_1/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^ through Ras - Raf - ERK and PI3K/PKB pathway.
碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)是对多种细胞的增殖、分化、存活以及功能具有强烈调节作用的多肽,通过和成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)结合实现其生物学活性。bFGF/FGFR信号传递与多种疾病有关,且与肿瘤发生、发展的关系极为密切。卵巢癌是女性生殖系统常见肿瘤,其死亡率高居妇科恶性肿瘤之首,对其早期诊断和治疗的研究仍面临巨大挑战。研究表明卵巢癌细胞中存在bFGF及其受体,可能通过自分泌或旁分泌机制在其发生、发展中起重要作用。
bFGF与受体结合后通过多条信号转导通路(如PLC/PKC、Ras-Raf-ERK、P13 K/PKB、JAK/STAT等)将信号传入细胞内。Ras-Raf-ERK信号转导级联是细胞内重要的信号转导通路,可被生长因子、激素、神经递质等激活,通过TPK→Grb2→SOS→Ras→Raf→MEKK→MEK→ERK→转录因子→相关基因表达,调节细胞分裂、增殖、存活等许多细胞过程。P13K/PKB是近年来发现的一个新的生长因子信号转导通路,有广泛的生物学效应,是许多细胞过程,如生长、增殖、分化、存活和恶性转化的调节者。
细胞周期调控在正常细胞的增殖、分化及肿瘤的演化过程中均起到重要的作用。细胞周期的超常快速进行或检查点的破坏将导致失控的细胞生长引起肿瘤的发生、发展,因而肿瘤形成的过程被认为是细胞周期机制失调的结果。细胞周期调控是在各期的控制点上进行的,G_1/S控制点是影响细胞周期的关键。细胞周期蛋白E(Cyclin E)、细胞周期蛋白依赖激酶2(cyclin dependent kinase 2,Cdk2)在细胞周期G_1/S转变过程中发挥重要作用。p27~(Kipl)通过与Cdk或Cyclin-Cdk复合物结合,以一种化学剂量关系抑制Cdk的活性,从而对细胞周期进行负调控。CyclinE-Cdk2是p27~(Kipl)的最适
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 G_1/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^
引文
1. Tsuboi Y, Ichida T,Sugitani S, et al. Overexpression of extracellular signal - regulated protein kinase and its correlation with proliferation in human hepatocellular carcinoma [J]. Liver Int, 2004,24 ( 5 ) :432 - 436
2. Aoki K,Ogawa T,Ito Y,et al. Cisplatin activates survival signals in UM -SCC - 23 squamous cell carcinoma and these signal pathways are amplified in cisplatin - resistant squamous cell carcinoma [J]. Oncol Rep, 2004,11 (2):375-379
3. Oka N, Nakahara S,Takenaka Y,et al. Galectin -3 inhibits tumor necrosis factor - related apoptosis - inducing ligand - induced apoptosis by activating Akt in human bladder carcinoma cells[ J]. Cancer Res ,2005,65(17) :7546 -7553
4. Fernandes DJ, Ravenhall CE, Harris T,et al. Contribution of the p38MAPK signaling pathway to proliferation in human cultured airway smooth muscle cells is mitogen - specific [J]. Br J Pharmacol, 2004,142 ( 7 ) : 1182 - 1190
5. The SH,Hill AK,Foley DA,et al. COX inhibitors modulate bFGF - induced cell survival in MCF -7 breast cancer cells[J]. J Cell Biochem,2004,91 (4):796-807
6. Gu Q,Wang D,Wang X,et al. Basic fibroblast growth factor inhibits radiation - induced apoptosis of HUVECs. I. The PI3K/AKT pathway and induction of phosphorylation of BAD[ J]. Radiat Res ,2004,161 (6) :692 -702
7. Gu Q, Wang D,Wang X,et al. Basic fibroblast growth factor inhibits radiation - induced apoptosis of HUVECs. Ⅱ. The RAS/MAPK pathway and phosphorylation of BAD at serine 112. [J]. Radiat Res,2004,161 (6) :703 -711
8. Aikawa T, Segre GV, Lee K. Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E -Cdk2[J]. J Biol Chem,2001,276(31) :29347 -29352
9. Messmer UK, Briner VA, Pfeilschifter J. Basic fibroblast growth factor selectively enhances TNF - alpha - induced apoptotic cell death in glomerular en- dothelial cells;effects on apoptotic signaling pathways[J]. J AM Soc Nephrol,2000,ll(12):2199-2211
10. Sturla LM,Westwood G,Selby PJ,et al. Induction of cell death by basic fi- broblast growth factor in Ewing's sarcoma [J]. Cancer Res,2000,60(21) : 6160-6170
11. Arbabi S, Maier RV. Mitogen - activated protein kinase[ J]. Crit Care Med, 2002,30(1 Supp):S74-S79
12. Hemmings B A. Akt signaling:linking membrane events to life and death decision [ J ]. Science, 1997,275 (5300): 628 - 630
13. Yao R, Cooper GM. Requirement for phosphatidylinositol 3 - kinase in the prevention of apoptosis by the nerve growth factor[J]. Science, 1995,267 (5206): 2003 -2006
14. Takeda A,Osaki M,Adachi K,et al. Role of the phosphatidylinositol 3 - kinase - Akt signal pathway in the proliferation of human pancreatic ductal carcinoma cell lines[J]. Pancreas,2004,28(3);353 -358
15. Ford MD, Cauchi J, Greferath U, et al. Expression of fibroblast growth factors and their receptors in rat glomeruli[ J]. Kidney Int, 1997,51 (6) : 1729 - 1738
16. Nguyen HB,Estacion M,Gargus JJ. Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF - induced calcium signals in human diploid fibroblasts[J]. Hum Mol Genet, 1997 ,6(5) :681 -688
17. Crickard K,Gross JL,Crickard U,et al. Basic fibroblast growth factor and receptor expression in human ovarian cancer[J]. Gynecol Oncol, 1994,55 (2):277-284
18. Di Blasio AM,Cremonesi L,Vigano P,et al. Basic fibroblast growth factor and its receptor messenger ribonucleic acids are expressed in human ovarian epithelial neoplasma[ J]. AM J Ohstel Gynecol,1993,69(6) :1517 - 1523
2. Aoki K,Ogawa T,Ito Y,et al. Cisplatin activates survival signals in UM -SCC - 23 squamous cell carcinoma and these signal pathways are amplified in cisplatin - resistant squamous cell carcinoma [J]. Oncol Rep, 2004,11 (2):375-379
3. Oka N, Nakahara S,Takenaka Y,et al. Galectin -3 inhibits tumor necrosis factor - related apoptosis - inducing ligand - induced apoptosis by activating Akt in human bladder carcinoma cells[ J]. Cancer Res ,2005,65(17) :7546 -7553
4. Fernandes DJ, Ravenhall CE, Harris T,et al. Contribution of the p38MAPK signaling pathway to proliferation in human cultured airway smooth muscle cells is mitogen - specific [J]. Br J Pharmacol, 2004,142 ( 7 ) : 1182 - 1190
5. The SH,Hill AK,Foley DA,et al. COX inhibitors modulate bFGF - induced cell survival in MCF -7 breast cancer cells[J]. J Cell Biochem,2004,91 (4):796-807
6. Gu Q,Wang D,Wang X,et al. Basic fibroblast growth factor inhibits radiation - induced apoptosis of HUVECs. I. The PI3K/AKT pathway and induction of phosphorylation of BAD[ J]. Radiat Res ,2004,161 (6) :692 -702
7. Gu Q, Wang D,Wang X,et al. Basic fibroblast growth factor inhibits radiation - induced apoptosis of HUVECs. Ⅱ. The RAS/MAPK pathway and phosphorylation of BAD at serine 112. [J]. Radiat Res,2004,161 (6) :703 -711
8. Aikawa T, Segre GV, Lee K. Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E -Cdk2[J]. J Biol Chem,2001,276(31) :29347 -29352
9. Messmer UK, Briner VA, Pfeilschifter J. Basic fibroblast growth factor selectively enhances TNF - alpha - induced apoptotic cell death in glomerular en- dothelial cells;effects on apoptotic signaling pathways[J]. J AM Soc Nephrol,2000,ll(12):2199-2211
10. Sturla LM,Westwood G,Selby PJ,et al. Induction of cell death by basic fi- broblast growth factor in Ewing's sarcoma [J]. Cancer Res,2000,60(21) : 6160-6170
11. Arbabi S, Maier RV. Mitogen - activated protein kinase[ J]. Crit Care Med, 2002,30(1 Supp):S74-S79
12. Hemmings B A. Akt signaling:linking membrane events to life and death decision [ J ]. Science, 1997,275 (5300): 628 - 630
13. Yao R, Cooper GM. Requirement for phosphatidylinositol 3 - kinase in the prevention of apoptosis by the nerve growth factor[J]. Science, 1995,267 (5206): 2003 -2006
14. Takeda A,Osaki M,Adachi K,et al. Role of the phosphatidylinositol 3 - kinase - Akt signal pathway in the proliferation of human pancreatic ductal carcinoma cell lines[J]. Pancreas,2004,28(3);353 -358
15. Ford MD, Cauchi J, Greferath U, et al. Expression of fibroblast growth factors and their receptors in rat glomeruli[ J]. Kidney Int, 1997,51 (6) : 1729 - 1738
16. Nguyen HB,Estacion M,Gargus JJ. Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF - induced calcium signals in human diploid fibroblasts[J]. Hum Mol Genet, 1997 ,6(5) :681 -688
17. Crickard K,Gross JL,Crickard U,et al. Basic fibroblast growth factor and receptor expression in human ovarian cancer[J]. Gynecol Oncol, 1994,55 (2):277-284
18. Di Blasio AM,Cremonesi L,Vigano P,et al. Basic fibroblast growth factor and its receptor messenger ribonucleic acids are expressed in human ovarian epithelial neoplasma[ J]. AM J Ohstel Gynecol,1993,69(6) :1517 - 1523