Abstract
BackgroundHuman melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown.MethodsHerein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied.ResultsMice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNFα and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion- and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFα induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1.ConclusionsWe demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion- and proliferation-stimulating effects of TNFα, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing melanoma cells. These data suggest COX-2 neutralization as a potential anti-metastatic therapy in melanoma patients at high risk of systemic and bone dissemination due to intercurrent infectious and inflammatory diseases.
Highlights
A significant proportion of cancer patients with no clinical evidence of systemic dissemination will develop recurrent disease after primary tumor therapy because they already had a subclinical systemic spread of the disease [1]
Inhibition of Melanoma Bone Marrow Metastasis by Celecoxib Mice developed a mean number of 35 ± 6 macroscopic metastases by day 15 after LCV injection of B16 melanoma (B16M) cells
The histological examination of bones by day 10 after cancer cell injection prior to macroscopic development of metastases, revealed subclinical micrometastases limited to the hematopoietic tissue of red bone marrow (BM), which indicates that bone-infiltrating B16M cells colonized extravascular compartments of BM (Figure 1A and 1B)
Summary
A significant proportion of cancer patients with no clinical evidence of systemic dissemination will develop recurrent disease after primary tumor therapy because they already had a subclinical systemic spread of the disease [1]. The understanding of complex interactions between cancer and bone cells/bone marrow stromal cells leading to these prometastatic events is critical for the design of an organ-specific therapy of bone metastasis. A non-epithelial tumor characterized by a marked inflammatory stromal response and osteolytic metastases, overexpresses COX-2 gene [22], which may be correlated with the development and progression of disease [23]. As shown by immunohistochemistry, COX-2 expression in primary melanomas is restricted to melanoma cells and significant correlation between immunohistochemical staining, tumor thickness and disease-specific survival has been reported [24], suggesting that COX-2 is a prognostic marker and a potential therapeutic target, its role in the complex pathogenic process of bone metastasis is unclear [3]. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma its pathogenic role in bone marrow melanoma metastasis is unknown
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