Antiangiogenic Activity Of Mda7il24

Angiogenesis, the formation of new blood vessels is important for the normal physiologic function of the body and is most commonly observed during the early stages of development (47). Angiogenesis can also occur in the later stages of life, however, most frequently as a pathology-associated angiogenesis, such as tumor-related angiogenesis. Tumor-related angiogenesis is a multistep process that involves many cell types and is a finely orchestrated series of events that include endothelial cell proliferation, new capillary formation, attraction of pericytes and macrophages, disruption of existing extracellular matrix and deposition of new matrix (48,49). A role for tumor-related angiogenesis is now well established, and recent studies support the concept that for tumors to grow and metastasize there is a need for blood supply that is provided by newly formed blood vessels adjacent to the tumor cells (50). Importantly, extensive data exists demonstrating that solid tumors express genes coding for angiogenic mediators (e.g., VEGF, basic fibroblast growth factor [bFGF], platelet derived growth factor [PDGF], IL-8) in the local tumor milieu resulting in the production of new blood vessels (51,52). Thus, metastasis from solid tumors is facilitated by angiogenesis of the primary tumor. Therefore inhibition of tumor angiogenesis is an effective means of inhibiting cancer growth and spread.

A number of angiogenesis inhibitors have evolved and can be used to block tumor growth (53). These inhibitors can be broadly classified into four categories: (1) endothelial cell inhibitors such as thalidomide, TNP-70, angiostatin and endostatin (54-56); (2) inhibitors of angiogenic factors, such as VEGF and VEGF receptor inhibitor (51,57,58); (3) inhibitors of endothelial and smooth muscle migration, such as MMP inhibitor and integrin inhibition (59,60); and (4) retinoids and cytokines such as IFN and IL-12 (61-63). Although these antiangiogenic molecules have been shown to inhibit angiogenesis in preclinical studies, very few of them have demonstrated a therapeutic effect in clinical trials (54). The failure to demonstrate activity and potency in clinical trials may result from the lack of understanding of the mechanism of angiogenic regulation by some of these inhibitors. Additionally, the optimal strategies for the use, monitoring, and validation of antiangiogenic agents in the clinic remain unclear. Angiogenesis is likely regulated on multiple levels; some inhibitors may function to block the formation of new blood vessels (antiangiogenic) whereas others may disrupt or modify the existing vessels (antivascular). A better understanding of the mechanism of action of vascular-targeted drugs will help in improving the treatment strategies that include combining with chemotherapy or radiotherapy.

Although antiangiogenic agents show great promise in preclinical models of cancer, their use may be limited in part by their delayed onset of inhibitory activity on tumor growth (e.g., tumors progressed as much as 400% in the first several days after initiation of antiangiogenic therapy) (64,65). Given that the doubling time of murine tumors is several-fold higher than is observed in the presentation of human cancer, this delay in onset of activity could translate to several months in patients. For patients with metastatic and/or locally advanced cancer, this delay in onset of activity may make the use of these agents impractical. Another potential limitation with these agents is the high dose and prolonged treatment course that are required. Although recent work suggests that the delivery of antiangiogenic agents via continuous infusion or sustained release may allow for a reduction of the bolus dose, the amount of protein needed for widespread use will still be challenging (56,65). Perhaps the most significant limit of current antiangiogenic therapy is the inability of these agents to completely eradicate the disease. Thus, there is an urgent to develop and test new and novel antiangiogenic agents that may overcome some of the limitations described above.

The concept to test the antiangiogenic properties of mda-7/IL-24 arises from several key observations made by us and several other investigators and are as follows: identification and demonstration of mda-7/IL-24 as a member of the IL-10 family with limited homology to IL-10 (4), demonstration of IL-10-mediated antiangiogenic activity in vivo (62,63), and reduced vascularization in tumors treated with an adenoviral vector expressing mda-7 (Ad-mda7) compared with tumors treated with control vectors (61).

Initial in vitro studies demonstrated Ad-mda7 inhibited endothelial differentiation (ECD) and cell migration, assays that are routinely used to test the antiangiogenic activity of an agent (23). Surprisingly, Ad-mda7 did not inhibit endothelial cell proliferation, an activity common to many antiangiogenic agents. The ability of Ad-mda7 to inhibit ECD and cell migration was similar to that observed with other antiangiogenic agents and suggested that mda-7/IL-24 may have antiangiogenic activity (61). However, realizing the potential caveat that in vitro results do not always correlate with in vivo studies, a dorsal air-sac chamber assay was utilized to test the antiangiogenic activity of mda-7/IL-24 in vivo. In these experiments, human A549 lung tumor cells were used as the angiogenesis inducers. Tumor cells were treated with Ad-mda7 or Ad-luc (vector control) and loaded into chambers that were implanted into the dorsal side of nude mice. Seven to ten days later, the chambers were removed and observed for angiogenesis or neovascularization. A significant reduction in neovascularization was observed in chambers that contained Ad-mda7 treated tumor cells compared with chambers that contained Ad-luc treated tumor cells (see Fig. 4). That inhibition of tumor-vascularization was the result of tumor cell death was excluded by performing a viability assay and nuclear staining of cells inside the chamber (unpublished data). These results indicated that MDA-7/IL-24 possesses antiangiogenic activity.

An additional line of evidence supporting the antiangiogenic activity of MDA-7/IL-24 comprises the findings from in vitro tumor-endothelial cell mixing experiments that

Fig. 4. Ad-mda7 inhibits angiogenesis in vivo. A549 tumor cells treated with Ad-luc or Ad-mda7 were plated on a semipermeable membrane (shown in circle) and implanted under the skin of a mouse. Five days later, the disc was isolated and neoangiogenesis evaluated by microscopy. Ad-luc treated cells demonstrate robust angiogenesis whereas Ad-mda7 treated cells shown significantly reduced vasculature.

Fig. 4. Ad-mda7 inhibits angiogenesis in vivo. A549 tumor cells treated with Ad-luc or Ad-mda7 were plated on a semipermeable membrane (shown in circle) and implanted under the skin of a mouse. Five days later, the disc was isolated and neoangiogenesis evaluated by microscopy. Ad-luc treated cells demonstrate robust angiogenesis whereas Ad-mda7 treated cells shown significantly reduced vasculature.

mimic the in vivo conditions. Ad-mda7 infected A549 lung tumor cells when mixed with human umbilical vein endothelial cells (HUVEC) showed marked inhibition of ECD. In contrast, inhibition of ECD was not observed when Ad-luc (vector control) infected tumor cells were mixed with endothelial cells. These results demonstrated MDA-7/IL-24 expressing tumor cells when in close proximity to endothelial cells inhibited ECD.

Although the above findings support MDA-7-mediated antiangiogenic activity, several questions related to MDA-7-mediated antiangiogenic activity remain unanswered and are as follows: (1) is the antiangiogenic activity mediated by the intracellular MDA-7 protein or by the secreted MDA-7 protein, (2) can MDA-7/IL-24 directly affect the tumor vasculature or indirectly via inhibiting proangiogenic factors, and (3) what is the underlying mechanism for mda-7-mediated antiangiogenic activity?

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