In the course of other studies on an experimental athymic mouse model, tumor regression was associated with extensive tissue necrosis associated with endothelial cell damage and intravascular thrombosis. This suggested that tissue ischemia might be central to tumor regression, and suggested that unbalanced angiogenesis might be responsible for tumor regression. Analysis of cytokine expression in the regressing tumor tissues showed the presence of a variety of inflammatory cytokines , including TNF-alpha and IL-6, and , in addition, the CXC chemokines IP-10 and Mig. Additional studies using cytokines, chemokines and neutralizing antibodies have demonstrated that complete tumor regressions could not be achieved through known mediators. In particlular, while tumor necrosis was consistently achieved, tumor tissue regrowth was also noted. We therefore looked for mediators that might inhibit endothelial cell proliferation rather than agents that would damage exsisting tumor vasculature. Using the EBV-immortalized VDS-O cell line, we have purified an endothelial cell inhibitor and have identified it calreticulin and an N-terminal calreticulin fragment. The purified recombinant calreticulin and N-terminal domain of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, vasostatin significantly inhibited the growth of human Burkitt lymphoma, colon carcinoma, lung adenocarcinoma and other malignant cell lines. Compared to other inhibitors of angiogenesis, calreticlulin and the N-terminal domain of calreticulin are soluble and stable molecules that are easy to produce and deliver. As angiogenesis inhibitors that specifically target endothelial cells, calreticulin and calreticulin N-domain have a unique potential as cancer therapeutics. Ongoing and future studies are focused on understanding the mechanism of action of these molecules and exploiting their potential as anticancer agents in experimental murine models.
