Effects Of FGF8b On Epithelial-Mesenchymal Transition And Invasion And Metastasis In Prostate Cancer

Background and Objective:prostate cancer is one of the most common male urogenital carcinomas. In Europe and United states, especially in the latter, the incidence of prostate cancer is the leading cancer and higher than lung cancer. In China, the incidence has increased rapidly. Tumor invasion and metastasis was the maximal barrier influencing therapeutic effect and was the leading cause of death in patients with prostate cancer.It's unclear that how prostate cancer cells obtain the potential of invasion and metastasis.To explore molecular mechanism about invasion and metastasis of prostate cancer and try to find new therapeutic targets exhibited important theoretical significance and clinically practical value.Present study is firstly planning to evaluate expression of FGF8b, epithelial marker (E-cadherin), mesenchymal marker (Vimentin) in prostate cancer and benign prostatic hyperplasia (BPH) in surgical specimens and to assess correlation between FGF8b expression and clinicopathologic factors of prostate cancer and correlation between FGF8b and cell phenotype markers, to clarify the possible role of FGF8b expression in the process of invasion and metastasis of prostate cancer. Secondly, we select DU 145 prostate cancer cell line as target cells in vitro for following experimental study. Reverse transcription polymerase chain reaction(RT-PCR) and western blotting were performed to assess changes of epithelial marker E-cadherin and mesenchymal marker Vimentin after FGF8b transfection to investigate whether FGF8b could induce epithelial-mesenchymal transition (EMT) in prostate cancer cells,MTT method and Transwell chamber were used to examine adherent,migration and invasion ability of transfected cells.Lastly, ERK kinase MEK1 inhibitor PD98059 were added to investigate the signal transduction pathway of epithelial-mesenchymal transition(EMT)induced by FGF8b. Through this experimental research we will initially reveal the role of FGF8b and possible mechanisms in promoting invasion and metastasis of prostate cancer, and try to find the key factors in the process and provide a new and effective target for interferencing invasion and metastasis of prostate cancer.Part one Expression of FGF8b,E-cadherin and Vimentin in prostate cancer and BPHObjetive:To investigate expression of FGF8,E-cadherin and Vimentin in prostate cancer and BPH, and relationship between their expressions and clinical stage and pathological grade in prostate cancer.Materials and Methods:20 cases of BPH (55-78 years old, averagely 62.4 years old) and 49 cases of prostatic cancer (61-82 years old, averagely 67.3 years old) paraffin-embeded tissues were provided by urological department of XiangYa Hospital during 2002-2009. Prostatic cancer tissues were categorized according to Gleason scoring. No prostate cancer patients received endocrine therapy or other treatment. Consecutive resection specimens of 4μm were performed. Immunohistochemistry was carried out to detect FGF8b,E-cadherin and Vimentin expression. Count of positive cells in ten representive high power fields was performed. The relationship between FGF8b, E-cadherin and Vimentin expression and related clinicopathologic features was statistically analyzed.Results:FGF8b was expressed in 33 prostate cancer tissue specimen(33/49,67.35%), while no expression in BPH.22 prostate cancer specimen expressed no E-cadherin(22/49,44.90%) and 27 expressed E-cadherin(27/49,55.10%); 29 PC specimen expressed Vimentin (29/49,59.18%).It demonstrated by statistical analysis that in prostate cancer deletion expressing of epithelial marker E-cadherin and positive expressing of mesenchymal marker Vimentin correlated significantly with Clinical stage and pathological grade which showed that the attenuated expressing of epithelial marker and accentuated expressing of mesenchymal marker synergially contributed to invasion and migration of PC. The expressing of FGF8b correlated strikingly with clinical stage,pathological grade and deleted expressing of E-cadherin, positive expressing of Vimentin in prostate cancer.Conclusions:(1) FGF8b was expressed in prostate cancer and correlated strikingly with clinical stage and pathological grade. (2) Deletion expressing of epithelial marker E-cadherin and positive expressing of mesenchymal marker Vimentin correlated significantly with clinical stage and pathological grade in proatate cancer.Part two Effects of FGF8b on Epithelial-Mesenchymal Transition and invasion and metastasis in prostate cancer cell DU145Objective:Our investigation attempts to study the effect of transfected FGF8b gene on Epithelial-Mesenchymal Transition and invasion and metastasis in prostate cancer cellMethods:The DU145 cells were transfected with pcDNA3.1/FGF8b sense plasmid by Liposome-mediated transfection and gained the stable FGF8b/DU145 cell model with high expression of FGF8b protein. The expression of EMTmarker(E-cadherin, vimentin) were detected by western-blot analysis and polymerase chain reaction;Adherent ability of transfected cells was detected by MTT method; The ability of migration and invasion of transfected cells was examined by by a Transwell chamber.Results:The cell models (FGF8b/DU145) steadily expressing FGF8b protein were obtained; After FGF8b transfecting DU145 cells,we observed differences in the gross appearance of FGF8b/DU145 compared with untreated cells.The phenotypic changes observed included increased full-length form increased intercellular separation. RT-PCR,western blotting were performed to assess expressing of epithelial marker E-cadherin and mesenchymal marker Vimentin and the results showed that expressing of epithelial markers E-cadherin decreased,mesenchymal markers Vimentin increased.Results of cell migration and invasion test showed the cell adherenee, migration and invasion ability of FGF8b/DU145 cells were increased obviously(P<0.05)compared with the control.Conclusions:(1) Over expression of FGF8b could promote the cell adherence, migration, and invasion ability of DU145 cells. (2) FGF8b promote DU145 cells invading and migrating which is associated with epithelial-mesenchymal transitionPart three molecular mechanism of FGF8b regulating epithelial-mesenchymal transition in prostate cancerObjective:To investigate the molecular mechanism of FGF8b promoting epithelial-mesenchymal transition in prostate cancer DU145 cell.Methods:ERK1/2 pathway activity of cells were detected by western-blot and then the FGF8b-DU145 cells and DU145 cells were cultured with PD98059 (ERK kinase MEK1 inhibitor) for two days. Morphological changes was observed everyday with microscope;the expression of EMT markers(E-cadherin,vimentin) were detected by western-blot analysis and cell migration ability were detected by the Transwell chamber.Results:ERK1/2 pathway activity of FGF8b/DU145 group was significantly higher than the control; after a ERK kinase MEK1 inhibitor PD98059 was added to treat FGF8b/DU145 cell, the epithelial marker E-cadherin protein expression were significantly increased compared with the control group (P<0.05) and mesenchymal marker Vimentin protein expression significantly reduced compared with the control group (P<0.05); cell migration test results suggest that the cell migration were decreased obviously(P<0.05)compared with the control group.Conclusions:Epithelial mesenchymal transition in prostate cancer induced by FGF8b was mediated by ERK kinase pathway,in which MEK1 as a key factor could be an effective target for interferencing in invasion and migration of prostate cancer...