Effects And Mechanisms Of Over-expression Of Angiomotin By Lentivirus On Bone-metastatic Prostate Cancer Cells

Objective: To determine effects and mechanisms of Amot on bone-metastatic prostate cancer cells.Methods: Stable PC3mm2-Amot cells overexpressing Amot and negative control PC3mm2-CDH were established by lentivirus packaging technology. Moreover, the effects of up-regulation of Amot on migration of PC3mm2-Amot cells were detected by wound scratch assay and transwell migration assay. Then m RNA levels of some genes were detected the by quantitative RT-PCR, which are chemokines related to tumor cells homing to bone and osteolytic factors. Finally, competition between amot and β-catenin to bind Cad11 CBS domain was identified by immunoprecipitation using PC3 cells overexpressing Amot or β-catenin, and the interaction of Amot and Patj was investigated by immunoprecipitation.Results: Stable PC3mm2-Amot cells overexpressing Amot was established. Wound scratch assay showed that significant increased cell migration at 5h and 10 h was observed in PC3mm2-Amot cells compared with PC3mm2-CDH cells. Transwell migration assay showed that compared with PC3mm2-CDH cells, there was a significant increased in cell migration when the low chamber contained 10% FBS medium,and significant enhanced cell migration was observed when the low chamber contained osteoblast conditioned culture medium.After co-culturing with MC3T3-E1, there was a significant increased in cell migration in PC3mm2-Amot cells compared with PC3mm2-CDH cells.The m RNA levels of some genes were detected by quantitative RT-PCR, which are chemokines related to tumor cells homing to bone and osteolytic factors. Compared with control cells, there is no significant difference in the expression of CXCR4、MCP-1、IL-6 and PTHr P in the PC3mm2-Amot cells, while sharply higher expression of COX-2 and C3. Immunoprecipitation showed that transfection of p IRES2-Amot led to translocation of β-catenin to nucleus. The increased protein expression of Amot bound to Cad11 was observed, while the protein expression of β-catenin bound to Cad11 decreased. Furthermore, enhanced expression of β-catenin in PC3 cells led to a decreased protein expression of Amot and increased protein expression of β-catenin. Finally the interaction of Amot andPatj was investigated by immunoprecipitation and there is no expected band appeared in western blotting.Conclusion: Up-regulation of Amot promote migration of bone-metastatic prostate cancer cells PC3-mm2. Amot and β-catenin competed for binding to Cad11 leading to translocation of β-catenin to the nucleus, resulting in increased migratory activity in these cells. Higher expression of COX-2 and C3 is also involved. The Patj/Syx/Rho signaling is not an essential pathway by which Amot controls metastasis of Prostate cancer to bone.