Direct Inhibitory Effects on Angiogenesis

To test the direct inhibitory effects of MDA-7/IL-24 on angiogenesis, experiments were carried out using affinity purified human MDA-7/IL-24 protein (16). In vitro, MDA-7/IL-24 protein selectively inhibited ECD in a dose-dependent manner with complete inhibition occurring at concentrations above 10 ng/mL (see Fig. 5). MDA-7/IL-24 had no effect on endothelial cell proliferation. Note that at the concentrations of MDA-7/IL-24 protein used, no significant cytotoxicity against lung tumor cells was observed, indicating selective activity against endothelial cells (61). The specificity of the inhibitory effect of MDA-7/IL-24 on ECD was demonstrated by immunodepletion studies. Because MDA-7 belongs to the IL-10 family of cytokines and IL-10 has previously been reported to exhibit antiangiogenic activity studies, comparing the inhibitory effects of MDA-7 protein with those of recombinant IL-10 on ECD were also conducted. MDA-7, but not IL-10, inhibited ECD at the concentrations (5-50 ng/mL)

Fig. 5. Ad-mda7 and MDA-7/IL-24 inhibit endothelial cell differentiation. (A) HUVEC cells were treated with control media, Ad-luc or Ad-mda7 (5000 vp/cell), plated on matrigel and analyzed 24 h later for tube formation (in vitro angiogenesis assay). Ad-mda7 inhibits endothelial differentiation (tube formation) ut does not kill endothelial cells. (B) HUVEC cells were treated with control media, MDA-7 or IL-10, plated on matrigel and analyzed 24 h later for tube formation (in vitro angiogenesis assay). MDA-7 inhibits endothelial differentiation. (C) MDA-7 inhibits endothelial differentiation in a dose and ligand-dependent manner. Primary HUVEC and HMVEC endothelial cells were evaluated with PBS or increasing doses of MDA-7 (1; 5; 10; 50 ng/mL) or immunodepleted material.

Fig. 5. Ad-mda7 and MDA-7/IL-24 inhibit endothelial cell differentiation. (A) HUVEC cells were treated with control media, Ad-luc or Ad-mda7 (5000 vp/cell), plated on matrigel and analyzed 24 h later for tube formation (in vitro angiogenesis assay). Ad-mda7 inhibits endothelial differentiation (tube formation) ut does not kill endothelial cells. (B) HUVEC cells were treated with control media, MDA-7 or IL-10, plated on matrigel and analyzed 24 h later for tube formation (in vitro angiogenesis assay). MDA-7 inhibits endothelial differentiation. (C) MDA-7 inhibits endothelial differentiation in a dose and ligand-dependent manner. Primary HUVEC and HMVEC endothelial cells were evaluated with PBS or increasing doses of MDA-7 (1; 5; 10; 50 ng/mL) or immunodepleted material.

tested (see Fig. 5). Similarly, comparison between the inhibitory effects of MDA-7/IL24 protein on ECD and equimolar concentrations of recombinant endostatin, IFN-y, and IP10 agents that had previously shown to exhibit antiangiogenic activity (65) demonstrated MDA-7/IL-24 was 10 to 50x more potent than endostatin, IFN-y, and IP10 in vitro (61). The ability of MDA-7/IL-24 to inhibit ECD is similar to that seen with

IFN-y (66). Furthermore, IFN-y production in MDA-7/IL-24 treated PBMCs has been reported (16), raising the possibility that IFN-y or IP-10 produced by mda-7/IL-24 treated endothelial cells could be responsible for the observed inhibitory effects. However, the possibility of MDA-7-mediated inhibitory effect resulting from IFN-y or IP-10 was excluded by conducting antibody-blocking studies. Additionally, equimolar concentrations of recombinant IFN-y or IP-10, when added to endothelial cells, showed less inhibition of ECD compared with MDA-7/IL-24 protein. These results showed that the in vitro antiangiogenic activity of MDA-7/IL-24 was more potent than IFN-y or IP-10 and occurred via a novel mechanism.

Further evidence for MDA-7/IL-24-mediated antiangiogenic activity is its ability to potently inhibit VEGF-induced endothelial cell migration in a dose-dependent manner (60). Inhibition of cell migration by MDA-7/IL-24 was also obtained when bFGF was used as an inducer (unpublished data). These results demonstrate the direct and specific antiangiogenic activity of MDA-7/IL-24 in vitro.

Evidence for direct effect of MDA-7-mediated antiangiogenic activity in vivo was next examined. Subcutaneous implantation of MDA-7 producing 293 cells (293-mda7) mixed with A549 lung tumor cells (1:1 ratio) in nude mice resulted in significant suppression of tumor growth compared with tumor growth in mice implanted with a mixture of parental 293 cells and tumor cells (see Fig. 6). That the tumor growth inhibitory effects resulted from exogenous MDA-7 was demonstrated by detecting MDA-7 protein in the tumors. Note that A549 tumor cells do not express detectable endogenous MDA-7 protein. Tumor growth inhibition was demonstrated to occur via apoptotic death of tumor endothelial cells. Associated with tumor growth inhibition was a marked reduction in tumor vascular-ization as demonstrated by the reduced hemoglobin content, and less CD31+ endothelial cells (61). These results demonstrated the direct antiangiogenic activity for MDA-7/IL-24 in vivo. Although these experiments established the "proof of concept" it is to be realized that most cancers such as cancer of the lung, breast, colon, and melanoma are often not localized but disseminated to distant sites in the body. Therefore for MDA-7/IL-24 to be an effective antiangiogenic agent it has to inhibit tumor growth at a distant site systemi-cally. To test whether MDA-7 protein could exert its antiangiogenic effects systemically, two sets of experiments were performed. In the first set of experiment a mixture of 293 or 293-mda7 cells mixed with A549 tumor cells were implanted on the right lower flank of nude mice. On the contra lateral lower left flank of each mouse, tumor cells equivalent to that on the right flank were implanted. Animals were monitored daily for tumor growth on both the flanks. A significant delay in tumor growth was observed on both flanks of mice that were implanted with a mixture of tumor cells and 293-mda7 cells compared with animals that were implanted with a mixture of tumor cells and 293 cells (see Fig. 6). In the second set of experiments, lung tumor xenografts were established subcutaneously in the lower right flank of nude mice. Subsequently, when the tumors had grown to a size of 50 to 100 mm3, matrigel containing parental 293 cells or matrigel containing 293-mda7 cells were implanted subcutaneously into the upper right flank of tumor-bearing animals and the effects of mda-7 on tumor growth were measured. A significant growth inhibition with 40 to 50% reduction in tumor size was observed in mice that were implanted with matrigel containing 293-mda7 cells compared with mice that were implanted with matrigel containing 293 cells (see Fig. 6B). That the observed tumor growth inhibition resulted from systemic inhibitory effects of MDA-7 protein on tumor angiogenesis was demonstrated by detecting the protein in the blood and a reduction in CD31+ blood vessels (61). Importantly, no gross pathological changes were observed in the animals

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