Angiocidin: A novel immunomodulatory molecule

In 1993 our laboratory discovered a novel thrombospondin-1-binding protein which we later termed "angiocidin". In vitro and in vivo experiments suggested that this protein might play a significant role in the negative regulation of tumor growth. Angiocidin protein was highly over-expressed in many different cancer types relative to normal tissue. The protein was also expressed in the blood of patients with advanced melanoma, as well as cancers of the colon, prostate, and breast. Angiocidin expressed and purified from bacteria, or recombinant angiocidin ("r-angiocidin"), was able to potently inhibit tumor and endothelial cell proliferation, as well as induce apoptosis in these cell types. Additionally, r-angiocidin inhibited invasion of tumor cells through collagen, and inhibited endothelial cell tube formation. The purified protein was also a potent inhibitor of tumor growth and metastatic spread in vivo.; The observation that a majority of tumors exist in an inflammatory microenvironment has raised the hypothesis that leukocyte infiltrates may be able to suppress tumor growth. Intratumoral leukocytes have been correlated with reduced metastasis and improved patient survival. Likewise, some pro-inflammatory cytokines have been demonstrated to inhibit tumor development and progression. As such, our laboratory has recently begun to investigate a novel immunomodulatory function for angiocidin.; We hypothesize that r-angiocidin's ability to elicit tumor cell death may be mediated in part by the molecules ability to induce inflammatory changes within the tumor microenvironment by acting as a secreted, cytokine-like molecule. We have shown that r-angiocidin-treated THP-1 cells, a monocytic cell line, experienced dramatic morphological changes resembling tissue macrophages. These cells became firmly attached and spread on tissue culture plastic. The phenotypic changes were accompanied by focal adhesion formation and cytoskeletal re-arrangements. Coincident with monocyte-to-macrophage differentiation, r-angiocidin-treated THP-1s also had reduced proliferative activity.; As with cytokines, endogenous angiocidin protein was inducibly secreted by both THP-1s and freshly isolated peripheral blood mononuclear cells. r-angiocidin itself was also able to induce the secretion of endogenously-synthesized angiocidin. We have also demonstrated that various cancer cell lines constitutively secrete basal levels of angiocidin protein into their conditioned media.; We have also begun to characterize the functional changes that occur in THP-1 cells upon treatment with r-angiocidin. These cells presented with increased phagocytic and antigen presentation activity, as well as cytokine and matrix metalloproteinase secretion. Generation of the observed cell adhesive effect was mediated in part by the cytokines present in r-angiocidin-treated conditioned media.; Additionally, we have begun to elucidate the signal transduction mechanisms whereby r-angiocidin is able to exert its pro-inflammatory effects on THP-1 cells. Several different signal transduction pathways became activated as a result of r-angiocidin treatment. The NF-kappaB p65/p50 heterodimer became phosphorylated and exhibited cytoplasmic to nuclear shuttling. In addition, r-angiocidin also increased phosphorylation of the MAPK molecules p44/42. Our data indicates that NF-kappaB may be functioning downstream of MAPK p44/42, as inhibition of p44/42 reduced r-angiocidin-induced NF-kappaB p65 phosphorylation. In addition, we have demonstrated that inhibition of MAPK p44/42 or NF-kappaB p65 completely abrogated r-angiocidin-induced cytokine and matrix metalloproteinase (MMP) secretion. Interestingly, inhibition of these two pathways had no effect on r-angiocidin-induced cellular adhesion, indicating that in our system, the processes of cell adhesion and cytokine secretion may be separate. Finally, we have shown that inhibition of PI3-kinase also completely abrogated r-angiocidin-induced cytokine a...