HGF and M-CSF modulate adhesion of MDA-231 breast cancer cell by increasing osteopontin secretion.

Recent studies reported an increased expression of osteopontin (OPN) in metastatic breast cancer cells, but the mechanisms modulating OPN production and the interaction of the cells with the secreted protein are far from clear. In this work, we utilized as an experimental system the cell line MDA-231 and we showed that HGF and M-CSF significantly enhance their adhesion onto OPN. Furthermore, in the presence of HGF and M-CSF, MDA-231 cells can adhere when plated onto BSA via increased OPN secretion. Moreover HGF and M-CSF induce de novo synthesis of OPN. In conclusion, these data suggest that HGF and M-CSF stimulate OPN production by MDA-231 cells, and that OPN is subsequently used as a substrate for cell adhesion.

Rat hindlimb unloading by tail suspension reduces osteoblast differentiation, induces IL-6 secretion, and increases bone resorption in ex vivo cultures.

In this research we utilized tail-suspended rats as an in vivo model for bone loss studies in order to investigate the effects of the tail suspension on the structure of the suspended bones and in ex vivo cultures the activities of trabecular osteoblasts, marrow-derived osteogenic cells, and osteoclasts obtained from treated animals, compared with untreated controls. After a 5-day hind limb unloading, trabecular thinning was already evidenced in the tibial primary spongiosa. In the secondary spongiosa, the bone formation activity was reduced whereas osteoclastic parameters were not yet altered. Bone marrow-derived osteogenic cells and differentiated osteoblasts from enzymatic digestion of posterior limb trabecular bone were prepared from 5 day tail-suspended rats and from normally loaded rats as controls. Cell morphology, alkaline phosphatase (ALPH) activity, production of mineral matrix, osteocalcin, and IL-6 secretion were evaluated in both cell populations. Tail suspension reduced the osteogenic potential of stromal marrow cells and of already differentiated osteoblasts. In fact, ALP positive colonies were significantly reduced in number and were smaller in size compared with controls and bone nodules formed in permissive conditions were also significantly fewer and smaller, whereas in cultures of cells from control conditions, large mineralizing nodules were formed. Osteocalcin secretion was not affected by unloading. Finally, IL-6 concentration was increased in marrow-derived cells from treated rats compared with controls. Primary cultures of osteoclasts were obtained from the nonadherent fraction of the bone marrow of the same animals. The number of TRAP positive cells in culture from tail-suspended rats was significantly increased, as well as bone resorption activity, measured as resorbed surfaces of a suitable synthetic hydroxyapatite, compared with controls. These data clearly suggest that skeletal unloading not only reduces the osteogenic potential of osteoblastic cells but induces an increased osteoclastogenesis and osteoclast activity in ex vivo cultures. They also indicate for the first time that a possible mediator responsible for the increased osteoclastogenesis could be represented by the IL-6 whose secretion by bone marrow cells was significantly enhanced by unloading.

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts.

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.

Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces.

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40-45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as alphaVbeta3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.