| The dissertation is divided into three parts:Part â… Establishment of Anoikis-Resistance Model in Human Prostate Cancer Cells and Study on Their Biological CharacteristicsObjective:To establish of anoikis resistance model in PC3, DU145cells of human prostate cancer, and to observe changes in biological characteristics.Methods:The expression levels of TrkB and BDNF of LNCaP, PC3, PC3M, DU145cells were measure by Western Blot and qRT-PCR. Then, we chose PC3, DU145to establish anoikis-resistance model. To induce parental PC3, DU145cells into anoikis-resistant colonies, we Shifted PC3, DU145cells from adhesive to ULC dishes. After7days, cells were transferred back to regular, adhesive tissue culture dishes, to allow anoikis-resistant cells to expand. And then, these expanded cells were re-transferred to ULC plates. After7days, surviving cells were transferred to6-well adhesive plates, and allowed to expand. Transwell assay and soft agar cloning assay were applied to observe migration, invasion and anchorage-independent growth of PC3, DU145cells and anoikis-resistant colonies. The expression levels of TrkB and BDNF of parental cells and anoikis-resistant colonies were measured by Western Blot and qRT-PCR.Results:1. Western blotting and qRT-PCR revealed that TrkB were detected in all prostate cancer cells which mentioned above, but BDNF was detected only in PC3, PC3M, and DU145cells.2. Compared to parental cells, anoikis-resistant colonies showed higher capability of anchorage-independent growth, migration and invasion.3. The mRNA and protein expression levels of TrkB in anoikis-resistant colonies were significantly higher than those in parental cells, but no statistical difference of BDNF expression levels were observed between parental cells and anoikis-resistant colonies.Conclusion:Variable levels of TrkB and BDNF were expressed in prostate cancer cell lines. A method of suspension culture in ULC plates and subsequent re-adherent culture could be applied to establish a reliable anoikis-resistance model in prostate cells. Anoikis-resistant colonies showed higher capability of anchorage-independent growth, migration and invasion, and increased expression levels of TrkB. Part â…¡ Study on The Effects of BDNF/TrkB Pathway on Epithelial-Mesenchymal Transition, Migration, Invasion and Anoikis in Prostate Cancer Cells in VitroObjective:To investigate the effects of BDNF/TrkB pathway on epithelial-mesenchymal transition, migration, invasion and anoikis in human prostate cancer cells in vitro.Methods:Anoikis-resistant colonies were transiently transfected with siRNA constructs against TrkB, and flow cytometry and transwell assay were applied to observe migration, invasion, anoikis of anoikis-resistant colonies. Flow cytometry and transwell assay were applied to investigate the effects of rhBDNF and the Trk antagonist K252a on anoikis, migration and invasion of parental prostate cancer cells in vitro. TrkB was overexpressed in PC3cells by stable transfection. The properties of anoikis-resistance, anchorage-independent growth, migration and invasion in transfectants were assessed by flow cytometry, soft agar cloning assay, cck-8assay and transwell assay, and the morphological changes of the cells were observed with inverted phase contrast microscope..Results:1. The invasion and anoikis-resistant ability of anoikis-resistant colonies were reduced when TrkB was silenced by siRNA in vitro.2. rhBDNF coluld promote migration, invasion and inhibition of anoikis of prostate cancer cells, but these effects were generally inhibited by K252a.3. Stable overexpression of TrkB could promote anoikis-resistance, anchorage-independent growth, constitutive and BDNF-mediated migration and invasion in PC3cells, and resulted in epithelial-mesenchymal transition-like transformation in cell morphology.Conclusion:TrkB/BDNF pathway induces epithelial-mesenchymal Transition, anoikis resistance, and the invasive behavior in prostate cancer cells. These data suggest that TrkB/BDNF pathway may play an important role in the multi-step tumor progression of prostate cancer. Part â…¢ Study on The Mechanisms of BDNF/TrkB pathway-Induced Epithelial-Mesenchymal Transition, Migration, Invasion and Anoikis-Resistance in Prostate Cancer Cells Objective:To explore the mechanisms of BDNF/TrkB pathway on epithelial-mesenchymal transition, migration, invasion and anoikis in human prostate cancer cells.Methods:1.The protein levels of AKT, p-AKT, ERK and p-ERK in parental cells, anoikis-resistant cells, anoikis-resistant cells transfected with siRNA-NT and anoikis-resistant cells transfected with siRNA-TrkB were measured by western blot.2. The effects of rhBDNF and the Trk antagonist K252a on protein expression levels of AKT, p-AKT, ERK, p-ERK, MMP9and Cleaved caspase3in suspension/adherent cultured cells were measured by Western blot.3. The protein expression levels of AKT, p-AKT, ERK, p-ERK, E-cadherin, N-cadherin, Vimentin, Snail, Slug, Twist, MMP9and Cleaved caspase3in adherent/suspension cultured transfectants were measured via Western blot.4. We also observed the effect of AKT antagonist MK2206on migration, invasion, and anoikis in both parental and transfectants cells.Results:1. The AKT and ERK pathway were activated in anoikis-resistance prostate cancer cells. Transient suppression of TrkB resulted in downregulation of p-AKT and p-ERK expression and promotion of Cleaved caspase3production.2. Increased expression of MMP9, p-AKT, p-ERK and reduced expression of Cleaved caspase3were noted under BDNF stimulation. These effects were generally inhibited by K252a.3. Stable overexpression of TrkB in PC3cells could upregulate expression of p-AKT, p-ERK, N-cadherin, Vimentin, Snail, Twist, MMP9and downregulate expression of E-cadherin and inhibit production of Cleaved caspase3.4. MK2206could inhibit BDNF/TrkB-induced migration, invasion and anoikis-resistance in prostate cancer cells.Conclusion:The BDNF/TrkB pathway induces epithelial-mesenchymal Transition, anoikis-resistance, and the invasive behavior in prostate cancer cells through regulating a variety of proteins. AKT is critical for TrkB-induced migration, invasion and anoikis-resistance in prostate cancer cells. |
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