The Effects And Its Mechanism Of Endothelin-1 In Prostate Cancer Bone Metastasis

Part I The influence and its mechanism of endothelin-1 on proliferation, apoptosis, migration and invasion abilities of hormone refractory prostate cancer cell line PC3OBJECTIVE: To observe the influence of endothelin-1(ET-1) on proliferation, apoptosis, invasionand migration abilities of human hormone refractory prostate cancer cell line PC3, and to study themechanisms.METHODS: To culture PC3 cell in vitro. Experiments included control group and ET-1 group.Firstly, to detect the influence of ET-1 on tumor cell double time, distribution of cell cycle and thepercentage of apoptotic cells by cell growth curves and flow cytometry.Secondly, to detect the influence of ET-1 on migration and invasion abilities of PC3 cell by erasion trace test and Boyden-chamber chemotaxis and chemoinvasion assay. To further detect the mRNA expression of MMP-2 and MMP-9 by RT-PCR, and to detect protein synthesis and activation of MMP-2 and MMP-9 by gelatin zymography.At last, at 0, 5, 15, 30, 60min after ET-1 was added, we detected the level of FAK and phosphorylated FAK of PC3 cell by immunoprecipitation and western blot RESULTS: Compared with control group, ET-1 group showed a shortened tumor cell double time, a decreased G0-G1(%) and increased G2-M(%) in cell cycle and a decreased percentage of apoptotic cells. All results had significant difference, P<0.05.Compared with control group, the abilities of migration and invasion of PC3 cell in ET-1 group were statistically higher, the synthesis and activation of MMP-2 and MMP-9 of ET-1 group were statistically higher, P < 0.05.At every time-point after ET-1 was added, the expression of FAK had no significant change, but the level of phosphorylated FAK had a significant increase and had a trend of increasing at first and decreasing subsequently, with an intial peak at 15 min that fell sequently and approached basal level at 60min.CONCLUSIONS: (1) ET-1 could stimulate PC3 cell to enter S phase and G2-M phase from G0-G1 phase, promote mitogenesis of tumor cells, and inhibit apoptosis of PC3 cell. (2)Endothelin-1 promoted migration and invasion abilities of prostate cancer cell, one of the mechanisms is to promote the synthesis and activation of MMP-2 and MMP-9. (3)FAK might be one of the early signaling proteinmolecules ET-1 acts oa ET-1 triggers FAK signaling pathways through promoting phosphorylation of FAK and the signaling pathways is one of signaling pathways that ET-1 influences proliferation, apoptosis, invasion and migration abilities of prostate cancer cell.Part II The effects and its related links of endothelin-1 in the microenvironment of co-cultivation of prostate cancer cell and osteoblast cellOBJECTIVE: To investigate the interactions between prostate cancer cell and osteoblast cell in the microenvironment of co-cultivation, and the effects and its related links of ET-1 and its receptors in the interactions.METHODS: Regarding PC3 prostate cell or SaOS2 osteoblast cell that cultured alone as control, to observe the interaction on proliferation between PC3 cell and SaOS2 cell in the coculture microenvironment with an in vitro coculture experimental system. To observe whether blocking ET-1 recepters with selective ETAR antagonist BQ-123 or ETbR antagonist BQ-788 could impair the interaction between PC3 cell and SaOS2 cell in the coculture microenvironment To detect the concentration of ET-1 in culture media by ELISA and detect the mRNA expression of ET-1 and its ETaR of PC3 cells or SaOS2 cells by RT-PCR in the microenvironment of culturing alone and coculturing, and to compare the expression differences of those between two kinds of culture microenvironmentRESULTS: (DThe coculture microenvironment not only promoted proliferation of PC3 cell, but also promoted proliferation of SaOS2 cell. ?Selective ETbR antagonist BQ-788 could not block the effects of coculture microenvironment, but selective ETAR antagonist BQ-123 could completely block the growth-promotion effects of microenvironment on PC3 cell, and partially block the growth-promotion effects of microenvironment on SaOS2 cell, which suggested that in coculture microenvironment ET-1 was the main cytokines that promoted proliferation of SaOS2 cell and besides ET-1 other cytokines from osteoblast also participated in promoting proliferatics of PC3 cell. @PC3 cells expressed ET-1 and ETAR, and SaOS2 cells only expressed ETaR- The coculture microenvironment could promote PC3 cell to synthesize and secrete ET-1. Comparing with control, the ETaR expression of PC3 cell or SaOS2 cell in coculture microenvironment showed no significant change, which implied the coculture microenvironment did not influence the ETaR expression of two kinds of cells. CONCLUSIONS: ?In the coculture microenvironment, prostate cancer cells and osteoblast cells mutually interact and promote growth through secreting cytokines. ET-1 secreted by prostate cancer cells promotes proliferation of tumor cells in an autocrine fashion, and promotes proliferation of osteoblast cells in a paracrine fashioa In turn, cytokines secreted by osteoblast cells stimulate prostate cancer cells to produce and secrete ET-1 in a paracrine fashion, which further promote proliferation oftumor cells. Taken together, these formed a positive feedback regulation system. ?ET-1 acts on prostate cancer cell and osteoblast cell both through ETA receptor and the ETAR antagonist could impair the influence of the coculture microenvironment on prostate cell and osteoblast cell.PartEQ The effects and its mechanisms of ETAR antagonist BQ123 in combination with paclitaxel on prostate cancer cell line PC3OBJECTIVE: To observe the influence of ET-1 on tolerability of PC3 cell to apoptosis-induced drug paclitaxel. To observe the effects of BQ123 combined with paclitaxel on hormone refractory prostate cancer cell line PC3. To further imitate the influence of bone microenvironment on tolerability of PC3 cell to chemotherapeutic agent through coculturing PC3 cell and osteoblast cell, and observe the effects of BQ123 combined with paclitaxel on PC3cell.METHODS: Before adding drugs, cells were starved for 24 h in serum-free medium to reach quiescence. Firstly, experiment included control group^ Paclitaxel groupN Paclitaxel+ET-1 group. To detect distribution of cell cycles and percentage of apoptotic cells by flow cytometry and detect the level of Bcl-2> Bax and Bax/Bcl-2 heterodimer by immunoprecipitation and western blotSecondly, experiment included control group> BQ123 group> Paclitaxel group> Paclitaxel+ BQ123 group. To detect distribution of cell cycles and percentage of apoptotic cells by flow cytometry and detect the level of Bcl-2, Bax and Bax/Bcl-2 heterodimer by immunoprecipitation and western blotAt last, before and after intervention of BQ123, to further observe the influence of coculture microenvironment on die therapeutic effects of paclitaxel.RESULTS: Compared with control group, paclitaxel group showed a decreased GO-G1(%) and increased G2-M(%) in cell cycle and a increased percentage of apoptotic cells, and showed a increased phosphorylation of Bcl-2 and decreased Bax/Bcl-2 heterodimer. Compared with paclitaxel group, all indexes of paclitaxel + ET-1 group were reversed partially. All results had significant difference, P<0.05.Compared with control group, BQ123 group showed a increased percentage of apoptotic cells, P <0.05, but cell cycle showed no significant difference. Compared with paclitaxel group, paclitaxel+BQ123 group showed a decreased GO-G1(%) and increased G2-M(%) in cell cycle and a increased percentage of apoptotic cells, and showed a increased phosphorylation of Bcl-2 and decreased Bax/Bcl-2 heterodimer. All results had significant difference, P<0.05.Compared with PC3 culture alone group, in coculture group paclitaxel-induced G2/M phase blockade were reversed partially and percentage of apoptotic cells of PC3 cell had an significant decrease, P<0.05. After BQ123 was added, the therapeutic effect of paclitaxel was enhanced...