Metformin Delays The Development Of Castration Resistance In Prostate Cancer By Inhibiting COX-2-mediated Inflammatory Cell Infiltration

Objective Inflammatory infiltration plays an important role in the development and metastasis of prostate cancer.Using the transgenic adenocarcinoma of the mouse prostate(TRAMP)mouse model and tumor specimens of prostate cancer patients,in vivo and in vitro experiments to investigate whether the use of metformin in the treatment of castration can inhibit COX-2 mediated inflammation Cell infiltration,at the same time,can delay the occurrence of prostate cancer castration resistance,thus providing a theoretical basis and research basis for the study of metformin combined with castration for prostate cancer.Methods(1)The effects of different concentrations of metformin on the proliferation of prostate cancer PC-3 and DU145 cells were detected by MTT assay.The proliferation activity of PC-3 and DU145 cells in the control group and metformin(20 mM)treatment group at different time points were detected by MTT assay.Flow cytometry was used to detect changes in cell cycle and apoptosis.(2)Immunohistochemical technique was used to detect the infiltration and density of CD68,CD163 and CD204 positive macrophages in prostate cancer tissues.The effects of metformin on inflammatory cell infiltration at various time points during prostate cancer were analyzed.(3)Immunohistochemical technique was used to detect the infiltration and density of CD68,CD163 and CD204 positive macrophages in prostate cancer tissues of control group,castration group,castration + metformin group,and analysis of the effect of metformin on inflammatory cell infiltration induced by castration.(4)Western Blot was used to detect the expression of COX-2 protein in prostate cancer PC-3 and DU145 cells treated with different concentrations of metformin.The effect of metformin on COX-2 protein expression was analyzed to explore the molecular mechanism of metformin inhibiting inflammatory infiltration.Results(1)MTT assay showed that different concentrations of metformin significantly inhibited the proliferation of prostate cancer PC-3 and DU145 cells in a dose-dependent manner.According to MTT assay,the proliferation of PC-3 and DU145 cells treated with 20 mM metformin decreased compared with the control group,and the effect of inhibition on PC-3 cell proliferation was more obvious with the prolongation of treatment time.The results of flow cytometry showed that metformin significantly inhibited the proliferation of prostate cancer PC-3 and DU145 cells.And ultimately leads to apoptosis;metformin blocks the S phase of PC-3 and DU145 cell cycle.(2)Immunohistochemical staining of macrophage markers(CD68,CD163 and CD204)in prostate cancer tissues of Tramp mice at 12 th week,25 th week,and 37 th week showed that at each time point,the staining intensity of metformin treatment group was weaker than that of the control group,and the number of staining positive cells was also less than that of the control group.(3)Immunohistochemical staining of macrophage markers(CD68,CD163 and CD204)in prostate cancer tissues and prostate cancer tissues of Tramp mice was performed.The results showed that in the prostate cancer tissues of Tramp mice,more macrophage infiltration was observed in the castration group than in the control group,while the number of staining positive cells and staining intensity were significantly lower in the castration+metformin group than in the castration group;Similarly,in the tissues of patients with prostate cancer,more inflammatory cell infiltration was observed in the bicalutamide treatment group than in the control group,while the number of staining positive cells was significantly lower in the bicalutamide+metformin group compared with the bicalutamide group.(4)After treatment of prostate cancer PC-3 and DU145 cells with different concentrations of metformin,the expression of COX-2 protein was detected by Western Blot.The results showed that the expression of COX-2 protein was decreased after metformin treatment,and it showed a dose-dependent manner.Conclusions 1.Metformin can inhibit the proliferation of prostate cancer cells and promote the apoptosis of prostate cancer cells;2.Metformin can inhibit the infiltration of inflammatory cells during the development of prostate cancer and castration;3.Metformin dose-dependently inhibits the expression of COX-2 in prostate cancer cells.In summary,metformin delays the development of castration resistance in prostate cancer by inhibiting COX-2 mediated inflammatory cell infiltration.