The Effects Of Human Bone Marrow-Derived Mesenchymal Stem Cells(hBMSCs) In Prostate Cancer Bone Metastasis

Prostate carcinoma(PCa) is one of the most common malignances among the urological neoplasms and the incidence of PCa is getting higher and higher in China today with the aging and the diet habit changing.However,the mechanisms of PCa bone metastasis are not illuminated.Bone marrow-derived mesenchymal stem cells(MSCs) are multipotent adult progenitors capable of differentiating into bone,cartilage,muscle,fat, and marrow stroma.MSCs have important effects on growth of some tumors.The effects of MSCs on PCa bone metastasis are not clear.In this study,fast,human MSCs derived from human bone marrow(hBMSCs) were isolated and culture-expanded by Percoll density gradient centrifugation and their property of adherence to plastic flasks.The adherent cells displayed themselves as fibroblast-like in morphology.Flow cytometric analysis showed that the expression of CD14,CD34,CD45, and HLA-DR were negative and the expression of CD29,CD44,CD90,CD105,and HLA -ABC were positive,which meaned they were phenotypically homogenous devoid of hematopoietic and endothelial cells.The ALP,Von Kossa,and Oil Red O staining were positive,which meaned they had the ability of osteogenesis and adipogenesis in vitro. These results reveal that the cells gained by these methods meet the well-accepted criteria for MSC identification.Second,the effects ofhBMSCs on PC-3 ceils migration and invasion behaviors were explored with Boyden Chamber method in vitro.The number of PC-3 cells migration and invasion toward hBMSCs CM was more than that toward PC-3 medium(PC-3M)(325±21 vs 225.33±21.60 and 245.33±11.50 vs 110.67±11.68,respectly.P＜0.01).These results indicate that hBMSCs enhance PC-3 cells migration and invasion capability in vitro. The results of quantitative RT-PCR showed the RQ values of PC-3 cells matrix metalloproteinase-2,9(MMP-2,9) mRNA in MSCs CM group were more than those in control group(1.19±0.05 vs 0.96±0.04 and 25.40±1.32 vs 10.67±0.984,respectly.P＜0.01).The results of western blot showed the RGS values of PC-3 cells MMP-2 and MMP-9 protein in hBMSCs CM group were more those in control group(1.04±0.18 vs 0.50±0.12 and 0.46±0.07 vs 0.17±0.01,respectly.P＜0.01).These results illuminate that hBMSCs enhance the expression of MMP-2 and MMP-9 in PC-3 cells.Third,the interactions on growth between PC-3 cells and hBMSCs were investigated. In hBMSCs,the OD of control group from 1st day to 7th day were 0.41±0.02,0.53±0.04, 0.66±0.01,0.96±0.02,1.37±0.02,1.72±0.03,and 1.79±0.01,respectly;the OD of PC -3 CM group from 1st day to 7th day were 0.44±0.03,0.64±0.01,0.80±0.02,1.26±0.01,1.91±0.06,2.20±0.04,and 2.30±0.06,respectly.There were significant differences between the two groups(P＜0.01) except for 1st day(P＞0.01).The cytometry analysis showed that S phase+G2/M phase population/fraction of PC-3 CM group at 4th day were significantly increased compared to control group(49.38±0.54% vs 37.90±1.15%,P＜0.01).In PC-3 cells,the OD of control group from 1st day to 7th day were 0.116±0.01,0.208±0.004,0.405±0.04,0.845±0.01,1.404±0.01,1.957±0.01,and 2.08±0.05,respectly;the OD of hBMSCs conditioned medium(hBMSCs CM) group from 1st to 7th day were 0.120±0.02,0.285±0.01,0.617±0.01,1.111±0.03, 1.625±0.02,2.277±0.01,and 2.283±0.01,respectly.There were significant differences between the two groups(P＜0.01) except for 1st day(P＜0.01).The cytometry analysis showed that S+G2/M phase population/fraction of hBMSCs CM group at 4th day were significantly increased compared to control group(43.80±0.77%vs 31.46±1.20%,P＜0.01).There were lower ratio of the nucleus to cytoplasm and more organelles in CM groups compared to control groups under transmission electron microscope.These findings suggest that PC-3 cells and hBMSCs promote cell growth and proliferation each other.Finally,we assessed the role of prostate canoe PC-3 cells in the differentiation of MSCs to an osteoblastic lineage,hBMSCs were devided into four groups:①,control group;②,PC-3 conditioned medium(PC-3CM) group;③,osteoblast differentiation medium group;④,osteoblast differentiation medium group containing PC-3 CM.In 14th day,The ALP activity(U/mg protein) in the four groups were 0.29±0.03,1.30±0.03, 2.13±0.08,and 3.80±0.03,respeetly.There were significant differences among them(P＜0.0001).The intensity of ALP staining gradually increased.In 21th day,the calcium quantitation(μmol/well) in the four groups were 0.04±0.01,0.44±0.05,0.98±0.03, and 1.27±0.04,respectly.There were significant differences among them(P＜0.01).The intensity of Von Kossa staining gradually increased.These results demonstrate that PC-3 cells effectively predetermine hBMSCs to differentiate to osteoblasts and increase bone formation in vitro.To sum up,the findings here demonstrate that PC-3 cells effectively predetermine hBMSCs to differentiate to osteoblasts and increase bone formation in vitro.PC-3 cells and MSCs promote cells growth and proliferation each other,hBMSCs enhance the PC-3 cells migration and invasion capability,which are related to the expression and activity of MMP -2,9 in PC-3 cells.