Investigation Into The Effects And Mechanisms Of Macrophages On The Prostate Cancer Cells PC-3

Objective: In this study, we aim to investigate the effect of four subtypes of macrophages on the proliferation, apoptosis, migration, invasion and angiogenesis of prostate cancer using PC-3 cell lines, and to dress the mechanism underlying these effects.Methods: The human monocytes were obtained from healthy donor by gradient centrifugation using Ficoll, and purified by magnetic activated cell sorting.The monocytes were incubated for 7 days in RPMI1640 supplemented with 10% FBS and 50ng/ml M-CSF in order to generate macrophages. M0 cells were generated by treating the cells with serum-free medium without additional cytokines for 48 h. M1 macrophages were polarized by stimulating the cells with LPS(100ng/ml) and IFN-γ(100ng/ml) overnight. M2 macrophages were polarized by stimulating the cells with IL-4(20ng/ml) overnight. TAM were generated by culturing monocytes for 7 days in RPMI 1640 10% FBS with 50% of CM from PC-3. Characterization of subtypes of macrophages with anti CD-14 FITC and anti CD-163 PE was detected by High Throughput Connotation of Imaging System. The expression levels of IL-1β, IL-6, IL-10, IL-12, IL-23, CXCL9, CCL13 and micro RNA let-7 m RNA in four subtypes of macrophages were detected by real-time PCR. The expression levels of TGF-β, TNG-α, IL-10 and IL-12 in the supernate from cultures of four subtypes of macrophages were determined by ELISA. We then carried out MTT, Annexin V-FITC, wound healing, transwell assays to detect the effect of four subtypes of macrophages on the proliferation, apoptosis, migration and invasion ability of PC-3 respectively, we also conducted matrigel assay with HUVEC cell lines to detect the ability of four subtypes of macrophages on the angiogenesis of PC-3.Results: The expression levels of IL-1β, IL-6, IL-12, IL-23, CXCL9 m RNA in M1 cells were detected to be a significant increase as compared with M0, M2 and TAM, while the expression levels of IL-10 m RNA in M2 and TAM showed a significant increase as compared with M0 and M1. The expression levels of CCL13 m RNA in M2 cells demonstrated the highest level among four subtypes. The results of ELISA about IL-10 and IL-12 were in accordance with that of real-time PCR, the expression levels of TGF-β significantly increased in the supernate of TAM. In contrast, the expression levels of TNF-α was remarkably increased in M1. Additionally, we also detected the expression levels of micro RNA let-7 m RNA in four subtypes of macrophages. The relative expression levels of let-7b and let-7c in TAM were significantly up-regulated as compared with other subtypes. Moreover the relative expression levels of let-7g-5bp in M2 was significantly increased among the four subtypes. M0 and M1 cells could inhibit the proliferation of PC-3 cells, on the contrary, M2 and TAM could promote the proliferation significantly. However, four subtypes did not have obvious effect on the apoptosis of PC-3 cells. Furthermore, M2 and TAM could promote the migration, invasion and the angiogenesis of PC-3 cells.Conclusions: M1 cells can inhibit the proliferation, while M2 and TAM can promote the proliferation, migration, invasion and angiogenesis of PC-3. The possible mechanisms might be four subtypes of macrophages secreting different cytokines exerted critical roles in the biological functions on prostate cells, while epigenetically modulated by micro RNA let-7b and let-7g-5bp.