Osteoblast-Induced Prostate Cancer Cell Migration and Invasion Is Mediated Through TGF-β1/SMAD2 Signal Pathway and Blocked by 17α-Estradiol

Prostate cancer (PCa) is curable if it is diagnosed and treated in localized and regional stage. However, PCa outcome is poor once it has distant metastasis. Approximately 70% to 100% of PCa deaths have bone metastasis, which may be associated with a specific bone microenvironment. In this study, we investigated the effect and molecular mechanism of osteoblast cells on stimulation of PCa cell migration and invasion and examined the effectiveness of 17α-estradiol on blocking osteoblast-induced PCa cell migration and invasion using in vitro cell analysis. PCa cells (PC3, LNCaP and DU145), osteoblast hFOB, kidney CV-1, breast tumor MCF-7 and liver cancer Huh-7 cells (ATCC) were cultured in RMPI-1640 or DMEM media supplemented with or without fetal bovine serum (FBS) at 37 oC in a 5% CO2-humidified incubator. hFOB condition media (HCM) without FBS were collected at different times of hFOB cell culture. Transwell and wound-healing experiments were used to determine PCa cell migration and invasion. Cell migration and invasion in PC3, DU-145 and LNCaP PCa cells were markedly promoted by co-culturing hFOB osteoblast cells or HCM, but not by cells or condition media originated from kidney (CV-1), liver (Huh-7) and breast (MCF-7). Compared to other non-osteoblast cell conditioned media, HCM had much higher levels of several cytokines and chemokines including tumor growth factor (TGF) β1. Both HCM and TGF-β1 produced a dose- and time-dependent induction of PCa cell migration and invasion as well as SMAD2 phosphorylation without altering cell proliferation. These HCM and TGF-β1 effects were inhibited by a specific TGFβ receptor inhibitor, LY2157299, as well as by 17α-estradiol in a dose-dependent manner. Most intriguing, 17α-estradiol significantly inhibited the HCM and TGF-β1-induced PCa cell migration and invasion at very low nanomolar concentrations, presumably mediated through estrogen receptor β. These findings suggest that TGF-β1 is a major factor in mediating hFOB cell stimulation of PCa cell migration and invasion, and 17α-estradiol is a potential agent to block PCa cell bone metastasis, probably through inhibition of TGF-β1/SMAD2 signal pathway.

Interleukin 6 and Interleukin 17a Enhance Proliferation and Differentiation of Murine Osteoblast and Human Foetal Osteoblast Cell Lines

Introduction: Cytokines have been gaining great focus due to their role in enhancing osseointegration as well as their potential in bone reconstruction. Osseointegration often faces complications in its compatibility with the implant due to rejection by the recipients own immune system. Therefore, extensive studies are being carried out to enhance osteoblast development to minimize such complication. The aim of this study was to determine the effect of different concentrations of Interleukin 6 (IL-6) and Interleukin 17a (IL-17A) in the proliferation and differentiation of murine and human osteoblasts. Methods: Various concentrations (5, 10, 25 and 50 ng/ml) of rIL-6 and rIL-17A were tested on both murine osteoblast (MC3T3-E1) and human feotal osteoblast (hFOB) cell lines using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) and alkaline phosphatise (ALP) assays. MTS was carried out at 24, 48, 72, 96 and 120 hours while ALP assay was done on day 1, 3, 7, 10 and 14. Results: MC3T3-E1 cells showed steadier proliferation and differentiation compared to hFOB. Both cell lines expressed responses in dose-dependent manner. The concentration of 10ng for IL-6 and IL-17A in the case of MC3T3-E1 cell line was found to be the most suitable for further studies. Conclusion: IL-6 and IL-17A enhance proliferation and ALP activity of both MC3T3-E1 and hFOB cell lines.

NF-kappa B Plays a Key Role in the Hematopoietic Niche Function of Osteoblast after Ionizing Radiation.

Recent studies demonstrated that osteoblasts in the endosteal region of bone are a key cellular component of the hematopoietic niche that directly regulates hematopoietic stem cell (HSC) survival, proliferation and differentiation. In experiments with the human fetal osteoblast cell line hFOB1.19, we found survival of gamma-irradiated primary human hematopoietic CD34+ cells was significantly enhanced by co-culture with hFOB cells or by conditioned medium (CM) from hFOB cells, as shown by increased colony efficiency and colony size. In clonogenic assays of CD34+ cells starting with 5 × 103 cells/dish, co-culture with hFOB cells increased colony number from 428±53 (control) to 988±65 (co-culture) after 2 Gy (p<0.01), 301±44 to 739±56 after 4 Gy (p<0.01), and 45±15 to 72±10 after 6 Gy (p<0.05). Surprisingly, although radiation induces osteoblast cell damage, CM from 6 Gy-irradiated hFOB cells had a greater effect than CM from unirradiated hFOB cells in supporting CD34+ clonogenicity and resulted in colony number increase from 430±40 (CM from unirradiated hFOB cells) to 773±48 (CM from 6 Gy-irradiated hFOB cells) after 2 Gy IR (p<0.01). Using a cytokine array for 120 cytokines and chemokines, we detected 11 factors released from hFOB cells, and 4 of them (IL-6, G-CSF, GRO, and IL-8) were enhanced by ionizing radiation (IR). IR (2–8 Gy) caused hFOB cell cycle arrest in G2 phase after 24 h, and irreversible cell cycle block and apoptotic cell death were observed 72 h after IR in a radiation dose-dependent manner. IR induced NF-kappaB (NFkB) p65 phosphorylation and NFkB activity in hFOB cells. Inhibition of NFkB expression with siRNA blocked the ability of hFOB CM to support clonogenic survival of CD34+ cells, and abrogated the effect of IR on release of IL-6, G-CSF, GRO, and IL-8. Radiation upregulated p53 (ser15) phosphorylation within 4 h, and p21 expression after 24 h in hFOB cells. However, NFkB gene silencing accelerated p21 expression and induced a massive apoptosis of hFOB cells. We propose that IR induces NFkB and p53 activation simultaneously in hFOB cells. The fate of osteoblasts after irradiation depends on the amount and severity of injury, and on repair processes regulated by NFkB and p53. Silencing the NFkB gene disrupted this balance and resulted in hFOB cell apoptosis.