Indirect Inhibitory Effects on Angiogenesis

The possibility that MDA-7/IL24, like many other antiangiogenic agents can inhibit the expression of proangiogenic growth factors (IL-8, bFGF, VEGF) produced by the tumor cells existed (67-70). Thus, inhibition of these tumor-derived growth factors by MDA-7/IL-24 will result in failure to support tumor vascularization thereby inhibiting angiogenesis. Preliminary cDNA array analysis demonstrated downregulation of VEGF, TGF-P, and IL-8 in human tumor cells treated with Ad-mda7 compared with tumor cells treated with Ad-luc (unpublished data). Subsequent in vitro studies demonstrated down-regulation of VEGF protein expression in Ad-mda7 treated human prostate cancer (LNCaP) cells compared with Ad-luc treated control cells (see Fig. 7A). Correlating with our in vitro findings is the recent report by Nishikawa et al. (68) who showed Ad-mda7 inhibited VEGF, bFGF, and IL-8 expression in human lung tumor xenografts. In the same study, combining radiation therapy with Ad-mda7 demonstrated enhanced radiosensitiza-tion of lung tumor xenografts that was associated with a significant inhibition of VEGF,

Fig. 7. MDA-7/IL-24 inhibits angiogenesis and tumor cell migration. (A) LNCap prostate tumor cells were treated with PBS, Ad-luc or Ad-mda7 (2000 vp/cell) and analyzed for MDA-7 and VEGF protein by western blot analysis. Ad-mda7 blocks VEGF expression. (B) H1299 NSCLC tumor cells were treated with 2000 vp/cell of Ad-luc or Ad-mda7 for 16 h and then replated. Cell migration was analyzed 24 h later. Control Ad-luc treated cells migrate whereas Ad-mda7 treated cells do not migrate.

Fig. 7. MDA-7/IL-24 inhibits angiogenesis and tumor cell migration. (A) LNCap prostate tumor cells were treated with PBS, Ad-luc or Ad-mda7 (2000 vp/cell) and analyzed for MDA-7 and VEGF protein by western blot analysis. Ad-mda7 blocks VEGF expression. (B) H1299 NSCLC tumor cells were treated with 2000 vp/cell of Ad-luc or Ad-mda7 for 16 h and then replated. Cell migration was analyzed 24 h later. Control Ad-luc treated cells migrate whereas Ad-mda7 treated cells do not migrate.

bFGF, and IL-8 and tumor neovascularization. In fact, combination therapy showed higher inhibition of proangiogenic factors compared with radiation or Ad-mda7 treatment alone. These studies demonstrated that intracellularly expressed MDA-7 (Ad-mda7), albeit at supraphysiological levels, can inhibit tumor vascularization indirectly by down-regulating proangiogenic growth factor expression.

Based on our results it is clear that both extracellular and intracellular MDA-7 protein can inhibit angiogenesis. Thus we speculate that the antiangiogenic activity observed in vivo is the result of interplay of the direct and indirect antiangiogenic effects mediated by the intracellular and secreted form of the MDA-7/IL-24 protein. We are currently examining the expression of these proangiogenic (VEGF, IL-8, bFGF, CD31) markers in Ad-mda7 treated tumor specimens obtained from the recently concluded phase I clinical trial (31,32).

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