In the present study, we analyzed the effects of rhBMP-2 in mesenchymal stem cells for use in the regenerative therapy of canine osteosarcoma utilizing OST cells as a model. The OST cells remained spindle-shaped during cell growth and the confluent stages.
Treatment of OST cells with rhBMP-2 compromised the osteoblastic phenotype. Osteoprogenitors are either pre-determined or inducible, depending on additional signals necessary to cause differentiation. This difference is important because it reflects the variation between cell compromise, which is when cell fate is programmed, and cell differentiation, which is when cells are compromised by permissive microenvironment signs [23
Although the exact function and interrelation of each type of BMP are not completely understood, evidence indicates that they are a part of a series of complex factors that regulate cell differentiation, specifically maturation intochondroblasts and osteoblasts. The structural and functional evolutionary conservation of genes encoding BMPs suggest that these genes have critical regulatory functions in the process of differentiation during development and neoplastic transformation. In human colorectal carcinoma, for example, rhBMP-2 acts as a tumor suppressor [24
]. Similarly, rhBMP-2 shows anti-proliferative and pro-apoptotic potential in gastric, prostate, and ovarian cancer cells. In breast cancer cell lines, rhBMP-2 treatment decreases cell proliferation. The effects of the BMPs varies with tumor progression. In other words, in the early stages of carcinogenesis, the TGF beta superfamily acts by suppressing tumor growth, and at later stages, the superfamily actually promotes tumor progression [25
In our findings, we observed that when mesenchymal stem cells were exposed to rhBMP-2, there was a significant reduction in the expression of the marker Nanog. We noted a significant decrease in expression of the cell proliferation marker Oct 3/4 in OST cells treated with rhBMP-2. The treatment of OST cells with rhBMP-2 after Transwell culture inhibited the proliferative response (Ki-67 expression) and promoted an increase in cell death mediated by the pro-apoptotic proteins (Bax and Bad), resulting in suppression of proliferation and an increase of phosphorylation of caspase-3.
The treatment of bone marrow cells with rhBMP-2 stimulates the production of growth and differentiation factors. Thus, rhBMP-2 treatment may be a relevant treatment for canine osteosarcoma cells. Eliseev et al. [26
], showed that rhBMP-2 increases the expression of Bax via Runx2, thus increasing the sensitivity of osteosarcoma cells to apoptosis. In our experiments, we also observed an increase in Bax expression when canine osteosarcoma cells were treated with rhBMP-2. These results suggest that rhBMP-2 treatment increases the susceptibility of OST cells to death by apoptosis.
Kawamura et al. [17
] observed that rhBMP-2 inhibits the growth of human multiple myeloma U266 cells by arresting the cells in the G1 phase of the cell cycle, leading to apoptosis. The combined treatment with rhBMP-2 induces cell cycle inhibitory proteins, such as p21 and p27, and induces other proteins associated with apoptosis, such as Bcl-xl, Bcl2, Bax, and Bad. Thus, rhBMP-2 may be an important tool for the treatment of multiple myeloma due to its anti-tumor and bone regeneration effects. Kawamura et al. [17
] investigated the antiproliferative effect of rhBMP-2 in myeloma cells and found that BMPs inactivate the STAT3 protein, which is a signal transducer activated by IL-6. BMPs were also found to increase the expression of cell cycle inhibitors leading to a cell replication blockage via pRb.
Based on the studies of Hsu et al. [27
], BMPs can function either as an oncogene or as a tumor suppressor, depending on the stage of disease. The effects of BMPs are cell type-specific and may vary among different tumors. BMPs are also reported as tumor suppressors and act on the cell cycle by inducing apoptosis of abnormal cells, such as tumors. Hardwick et al. [24
] used cell lines of colorectal cancer to evaluate the role of rhBMP-2. They observed that rhBMP-2 reduced cell growth and stimulated apoptosis due to high levels of phosphorylated caspase-3. In our results, we also observed a decrease in cell growth when OST cells were treated with rhBMP-2, and we observed an increase of caspase-3 levels.
Treatment with rhBMP-2 may be a new therapeutic option for canine osteosarcoma. We found that rhBMP-2 decreases the expression of embryonic stem cell markers Nanog and Oct 3/4 in different treatment regimens, and it is also associated with tumorigenesis of many types of cancer [28
]. Because rhBMP-2 has the potential to inhibit the expression of markers such as Nanog and Oct 3/4, it may also exhibit anti-tumor effects in animal models in vivo
. Oct 3/4 and Nanog are important factors in the regulation of self-renewal and the pluripotency of embryonic stem cells. There are studies showing a correlation of these cells with cases of tumorigenesis [29
]. Oct 3/4 is a marker for both adult stem cells and cancer stem cells. Inhibition of this factor can inhibit the expression of proteins associated with tumorigenesis.
Osteogenesis is defined by a series of events, which starts with a commitment to an osteogenic lineage by mesenchymal cells. Subsequently, these cells proliferate and demonstrate an upregulation of osteoblast-specific genes and mineralization. Multiple signaling pathways have been demonstrated to participate in the differentiation of an osteoblast progenitor to a committed osteoblast [30
An association between the expression of STRO-1 and the presence of cells with osteogenic potential has been demonstrated in precursor adult human bone marrow. STRO-1+
population of human bone marrow cells is capable of osteogenic differentiation. The expression STRO-1 is complicated by the fact that a considerable proportion of STRO-1+
cells are not of the osteogenic lineage and the exact stage of osteogenic differentiation at which STRO-1 is expressed remains unclear, especially when working with cultured cell populations and the coexpression of STRO-1 and a panel of antibodies recognizing cell surface determinants which may be regulated during osteogenic differentiation [32
BMP-2 alone does not induce osteogenesis in isolates of human bone marrow stromal cells as measured by stimulation of alkaline phosphatase expression. However, BMP-2 does induce other markers associated with differentiation of osteoblasts. This osteogenic capacity is seen in stromal cells isolated from mice, rats, rabbits, and humans; however, cell behavior and efficacy of inducers varies in a species-dependent manner [33
BMP-2 stimulates surrounding tissues; however, more robust data is needed to demonstrate that BMP-2 also augments the osteogenic potential of implanted MSCs cells. In the present study, probably the effects of MSCs and rBMP-2 treated model culture Transwell, controlled environmental niches and alterations in this microenvironment can dramatically modify their behavior and differentiation capacities.
Langenfeld et al. [34
] showed that cell culture conditions and the intra- and extra-cellular antagonist concentrations interfere with the biological activities of BMPs. Wang et al. [23
] observed that rhBMP-2 inhibits the tumorigenic potential of human osteosarcoma OS99-1 cells. The inhibition was due to a decrease in the expression of proteins associated with tumorigenesis and an increase of osteosarcoma cell differentiation in response to rhBMP-2. Thus, rhBMP-2 could be considered a novel tool for the treatment of human osteosarcoma. Our study clearly showed that the association of mesenchymal stem cells derived from canine fetal bone marrow, combined with rhBMP-2, decreases the tumorigenic potential of canine osteosarcoma cells in vitro