| Objective:1. To investigate the effect of human bone mesenchymal stem cells (hBMSCs) on proliferation、migration、invasion and Vasculogenic Mimicry in human prostate cancer cell line PC-3and22RV1. To investigate whether the SDF-1/CXCR4axis and PI3K/Akt pathway affected Vasculogenic Mimicry in human prostate cancer cell line and to reveal the related molecular mechanisms.2. To isolate and identify the prostate cancer stem-like cells (CSCs) by magnetic active cell sorting (MACS).3. To investigate the effect of Rosiglitazone (RSG),the ligand of Peroxisome proliferator-activated receptor y (PPARy),on Vasculogenic Mimicry in human prostate cancer cell line PC-3in vitro and to reveal the related molecular mechanisms.Methods:1. Primary hBMSCs were harvested from the bone marrow. The effect of BMSCs on prostate cancer proliferative ability was tested by Cell Counting Kit-8(CCK-8) assay. The invasion abilities were detected by Trans well experiment. Cell migration was evaluated using an in vitro wound healing assay. The Vasculogenic Mimicry abilities were detected by a well established in vitro3D model of Vasculogenic Mimicry formation. The expression of CXCR4was detected by realtime-PCR or immunofluorescent method. The expression of CXCR4, Akt and p-Akt were detected by Western blot.2. CD133+/CD44+cells were selected from PC-3human prostatic cancer cells lines by MACS, and they were identified as CSCs by immunohistochemistry, flow-cytometric analysis, CCK8assay, colony-forming assay and in vivo tumorigenicity assay.3. Human prostate cancer PC-3cell line was cultured in vitro. The effect of Rosiglitazone with different concentrations (0.1、1、10μmol/L) and different action times(1to6days)on the growth of prostate cancer PC-3cells was detected by CCK-8analysis. The invasion abilities were detected by Transwell experiment. Cell migration was evaluated using an in vitro wound healing assay. A3-dimensional cell culture system for human prostate cell line PC-3was established to observe vasculogenic mimicry formation. The change of vasculogenic mimicry formation under Rosiglitazone treated was observed. Realtime-quantitative polymerase chain reaction (RT-qPCR) was used to detect the effect of Rosiglitazone on the mRNA expression of VEGF and VE-cadherin. The expression of VEGF protein was analyzed by Elisa assay. The expression of Akt and p-Akt were detected by Western blot.Results:1. In addition to enhanced proliferation when in treatment with hMSCs conditioned medium, PC-3cells were found to have increased migration and invasion potential in vitro. MSC conditioned medium effectively promoted formation of capillary-like structures mediated by PC-3.Under in vivo conditions, tumor growth and VM formation was promoted by MSCs in nude mice. MSCs significantly enhanced CXCR4expression of prostate cancer cell lines in vitro. In addition, knocking down of CXCR4using RNA interference or inhibition of CXCR4function by an antagonist AMD3100blocked MSC-CM-induced VM formations of prostate cancer cell lines. Furthermore, PI3K inhibitor LY294002blocked MSC-CM-induced VM formations of Prostate cancer cell lines.2. There was a low proportion of CD133+/CD44+CSCs in the PC-3cells. The proliferation capacity of CSCs was significantly higher than PC-3cells. The proportion of these CD133+, CD44+and integinα2β1+cells could be increased in Sorted CSCs compared with PC-3cells. CD133+/CD44+cells had an enhanced colony-formation capability in vitro, and displayed greater tumorigenic properties in vivo when seeded at a low density. 3. Prostate cancer PC-3cells could form Patterned matrix VM or tubular VM in3D cultures in vitro and Rosiglitazone effectively inhibited the formation of VM structures in a dose and PPARy-dependent manner. In addition, Rosiglitazone significantly inhibited PC-3cells proliferation, migration and invasion. Rosiglitazone inhibited VEGF expression of PC-3cells in vitro. Furthermore, PI3K inhibitor LY294002blocked VM formations of Prostate cancer cell lines. VEGF and PI3K/AKT pathway exert a positive feedback regulation in the process of VM formation.Conclusion:1. This result establishes MSCs in the bone microenvironment might promote the VM formations and metastasis of prostate cancer. SDF-1/CXCR4axis and PI3K/Akt pathway played essential roles in the MSC-induced VM formations in prostate cancer.2. MACS was a simple and effective method to gain prostate CSCs from PC3cell lines.3. PPAR-y agonists have inhibitory effects on the proliferation, migration, invasion and VM formations of prostate cancer cell. The inhibitory effect of RSG on VM formation could be at least partially explained by a RSG-driven downregulation of VEGF and phosphorylation of Akt. |
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