Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity.

Interleukin (IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity.Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of IL-24-wild-type (IL-24wt) or IL-24 with all five phosphorylation sites replaced (IL-24mt) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24wt-expressing cells but not in IL-24mt-expressing cells. Secretion of IL-24mt protein was greatly reduced compared to IL-24wt protein. Further, IL-24wt and IL-24mt proteins markedly differed in their subcellular organelle localization. IL-24wt but not IL-24mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24wt but not IL-24mt mediated its anti-cancer activity by inhibiting the AKT signaling pathway.Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities.

Mechanisms of injury to normal tissue after radiotherapy: a review.

BACKGROUND: The benefits of radiotherapy for cancer have been well documented for many years, but many patients treated with radiation develop adverse effects. This study analyzed the current research into the biological basis of radiotherapy-induced normal tissue damage. METHODS: Using the PubMed and EMBASE databases, articles on adverse effects of radiotherapy on normal tissue published from January of 2005 through May of 2012 were identified. Their abstracts were reviewed for information relevant to radiotherapy-induced DNA damage and DNA repair. Articles in the reference lists that seemed relevant were reviewed with no limitations on publication date. RESULTS: Of 1751 publications, 1729 were eliminated because they did not address fundamental biology or were duplicates. The 22 included articles revealed that many adverse effects are driven by chronic oxidative stress affecting the nuclear function of DNA repair mechanisms. Among normal cells undergoing replication, cells in S phase are most radioresistant because of overexpression of DNA repair enzymes, while cells in M phase are especially radiosensitive. Cancer cells exhibit increased radiosensitivity, leading to accumulation of irreparable DNA lesions and cell death. Irradiated cells have an indirect effect on the cell cycle and survival of cocultured nonirradiated cells. Method of irradiation and linear energy transfer to cancer cells versus bystander cells are shown to have an effect on cell survival. CONCLUSIONS: Radiotherapy-induced increases in reactive oxygen species in irradiated cells may signal healthy cells by increasing metabolic stress and creating DNA lesions. The side effects of radiotherapy and bystander cell signaling may have a larger impact than previously acknowledged.

Tumor suppressor gene-based nanotherapy: from test tube to the clinic.

Cancer is a major health problem in the world. Advances made in cancer therapy have improved the survival of patients in certain types of cancer. However, the overall five-year survival has not significantly improved in the majority of cancer types. Major challenges encountered in having effective cancer therapy are development of drug resistance by the tumor cells, nonspecific cytotoxicity, and inability to affect metastatic tumors by the chemodrugs. Overcoming these challenges requires development and testing of novel therapies. One attractive cancer therapeutic approach is cancer gene therapy. Several laboratories including the authors' laboratory have been investigating nonviral formulations for delivering therapeutic genes as a mode for effective cancer therapy. In this paper the authors will summarize their experience in the development and testing of a cationic lipid-based nanocarrier formulation and the results from their preclinical studies leading to a Phase I clinical trial for nonsmall cell lung cancer. Their nanocarrier formulation containing therapeutic genes such as tumor suppressor genes when administered intravenously effectively controls metastatic tumor growth. Additional Phase I clinical trials based on the results of their nanocarrier formulation have been initiated or proposed for treatment of cancer of the breast, ovary, pancreas, and metastatic melanoma, and will be discussed.

Inhibition of nuclear factor-kappaB augments antitumor activity of adenovirus-mediated melanoma differentiation-associated gene-7 against lung cancer cells via mitogen-activated protein kinase kinase kinase 1 activation.

Nuclear factor-kappaB (NF-kappaB) activation promotes cell survival and growth. Reports show that chemotherapeutic agents and cytokines that are used for cancer therapy activate NF-kappaB expression in tumor cells and its suppression enhanced the antitumor activity. We hypothesized that adenovirus-mediated overexpression of melanoma differentiation-associated gene-7/interleukin-24 (Ad-mda7/IL-24) induces NF-kappaB expression and that inhibition of this expression results in enhanced tumor cell killing. Treatment of human lung tumor (H1299 and A549) cells with Ad-mda7 resulted in NF-kappaB activation in a dose- and time-dependent manner before activation of cell death pathways. To establish that inhibition of Ad-mda7-mediated NF-kappaB activation results in enhanced tumor cell killing, H1299 cells that overexpress the dominant-negative I kappa B alpha (dnI kappa B alpha) were treated with Ad-mda7 in vitro. An enhanced growth arrest and apoptosis was observed in Ad-mda7-treated H1299-dnI kappa B alpha compared with H1299-Neo cells. This Ad-mda7-mediated enhanced killing of H1299-dnI kappa B alpha cells involved cleavage of mitogen-activated protein kinase kinase kinase 1 (MEKK1) and caspase-3 in a feedback loop mechanism. The inhibition of MEKK1 or caspase-3 cleavage in H1299-dnI kappa B alpha cells resulted in reduced Ad-mda7-mediated cell killing. In vivo, the treatment of H1299-dnI kappa B alpha s.c. tumors with Ad-mda7 resulted in increased drug sensitivity and delayed the tumor growth rate compared with Ad-mda7-treated H1299-Neo tumors. Molecular analysis of Ad-mda7-treated H1299-dnI kappa B alpha tumors showed increased MEKK1 cleavage and activation of caspase-3 compared with Ad-mda7-treated H1299-Neo tumors. Our findings thus showed that the NF-kappaB activation induced by Ad-mda7 treatment of lung cancer cells is an intrinsic survival mechanism and that the inhibition of this NF-kappaB expression results in enhanced tumor cell killing.

mda-7 In combination with bevacizumab treatment produces a synergistic and complete inhibitory effect on lung tumor xenograft.

Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), has shown antitumor activity by inhibiting tumor angiogenesis in preclinical and clinical studies. However, bevacizumab monotherapy does not induce complete tumor regression. Therefore, additional treatments must be combined with bevacizumab to promote tumor regression. We previously showed that melanoma differentiation associated gene-7 (mda-7) protein exerts potent antitumor and antiangiogenic activity. Thus, in this study, we investigated the therapeutic effects of mda-7 in combination with bevacizumab using lung cancer as a model. In vitro, treatment of human umbilical vein endothelial cells with conditioned medium from Ad-mda7 plus bevacizumab-treated lung tumor cells showed reduced VEGF ligand-receptor binding, and decreased cell survival, resulting in growth arrest and apoptosis. In vivo, treatment of subcutaneous lung tumor xenografts with bevacizumab plus Ad-mda7 resulted in significant tumor growth inhibition and improved survival compared to tumor growth in control mice. Furthermore, tumors in all the Ad-mda7 plus bevacizumab-treated mice completely regressed, and these were tumor free through the study's end. Molecular analysis showed enhanced tumor cell apoptosis and reduced VEGF and CD31 expression in Ad-mda7 plus bevacizumab-treated tumors. Thus, Ad-mda7 and bevacizumab treatment produces a synergistic and complete therapeutic effect against human lung cancer.

MDA-7/IL-24-based cancer gene therapy: translation from the laboratory to the clinic.

Despite recent advances in treatment strategies, the overall 5-year survival rate for patients with common epithelial cancers is poor largely because of the difficulty in treating metastatic cancers. Therefore, therapeutic agents are urgently needed that can effectively inhibit both primary epithelial tumors and their metastases. One such agent that has shown promise in preclinical studies is the tumor suppressor/cytokine, melanoma differentiation associated gene-7 also known as interleukin-24 (mda-7/IL-24). Preclinical studies from our and other laboratories have shown that overexpression of MDA-7/IL-24 causes a strong tumor- suppressive effect in many human cancer cells but spares normal cells. This gene therapy also enhances the tumor-suppressive activity of radiotherapy and chemotherapy. Secreted MDA-7 protein that is glycosylated also has been shown to have potent antiangiogenic activity both in vitro and in vivo. Studies examining the immune properties of mda-7 have shown that MDA-7/IL-24 unlike the related IL-10, functions as a Th1 cytokine. Recently, an MDA-7 protein-mediated "bystander effect" on tumor cells has been documented. Building on these findings we successfully completed a Phase I clinical trial of adenovirus-based mda-7 cancer therapy that confirmed the safety of this gene therapy. Phase II trials evaluating the efficacy of mda-7-based gene therapy are warranted. The outcome of such ongoing mda-7-based gene therapy trials will allow us to better understand this therapy's clinical utility.

Inhibition of Src kinase activity by Ad-mda7 suppresses vascular endothelial growth factor expression in prostate carcinoma cells.

The ability of an adenoviral vector expressing the melanoma differentiation-associated gene-7 (Ad-mda7) to mediate inhibition of vascular endothelial growth factor (VEGF) has recently been reported. However, the molecular mechanism by which Ad-mda7 inhibits VEGF is unknown. In an attempt to elucidate this mechanism, we studied the effects of Ad-mda7 on VEGF expression using human prostate cancer cells as a model. We found that Ad-mda7 treatment of prostate cancer cells (LNCaP and DU145) in vitro resulted in a significant (P < 0.05) inhibition of VEGF expression. Analysis of the VEGF signaling pathway showed that Ad-mda7 inhibited c-Src kinase activity and abrogated STAT-3 binding to the VEGF promoter. Correlating with these observations were reductions in VEGF mRNA and protein levels in Ad-mda7-treated cells. Furthermore, Ad-mda7 inhibited VEGF in Src(+/+) but not in Src(-/-) mouse embryo fibroblasts. These results showed that Ad-mda7 inhibited VEGF by inhibiting the Src signaling pathway. Finally, conditioned medium from Ad-mda7-treated tumor cells containing reduced VEGF inhibited VEGF receptor signaling, resulting in reduced endothelial cell proliferation and apoptosis. Our results provide evidence for the mechanism by which Ad-mda7 inhibits VEGF in tumor cells and of the effects of this VEGF inhibition on endothelial cell proliferation, a requirement for angiogenesis. Our findings demonstrate that MDA-7 protein, in addition to inhibiting tumor angiogenesis directly, inhibits angiogenesis indirectly by inhibiting VEGF production by tumor cells.

Sulindac enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human lung cancer.

Several studies have shown antitumor activities of the melanoma differentiation-associated gene 7 (mda-7) and the nonsteroidal anti-inflammatory drug sulindac when used as a monotherapies against a wide variety of human cancers. However, the combined effects of mda-7 and sulindac have not previously been tested. Therefore, we tested the antitumor activity of an adenoviral vector expressing mda-7 (Ad-mda7) in combination with sulindac against non-small cell lung cancer cells in vitro and in vivo. When treated with Ad-mda7 in combination with sulindac, human lung cancer cells (A549 and H1299) underwent growth suppression resulting in apoptosis. The growth inhibition induced by Ad-mda7 in combination with sulindac was significantly greater than that observed with Ad-mda7 or sulindac alone. Furthermore, the degree of growth inhibition induced using this combination was dose-dependent for sulindac. Treatment with Ad-mda7 in combination with sulindac had no growth inhibitory effects on human normal lung (CCD-16) fibroblasts. We then investigated the mechanism by which sulindac enhances Ad-mda7-mediated apoptosis. Sulindac increased expression of ectopic MDA-7 protein in tumor cells, thereby increasing the expression of downstream effectors RNA-dependent protein kinase, p38MAPK, caspase-9, and caspase-3 and enhancing apoptosis of non-small cell lung cancer cells. Pulse-chase experiments showed that the increased expression of MDA-7 protein in sulindac-treated cells was due to increased half-life of the MDA-7 protein. Finally, treatment of human lung tumor xenografts in nude mice with Ad-mda7 plus sulindac significantly suppressed growth (P = 0.001) compared with Ad-mda7 or sulindac alone. Our results show for the first time that combined treatment with Ad-mda7 plus sulindac enhances growth inhibition and apoptosis of human lung cancer cells. The increased antitumor activity observed with the combination treatment is a result of increased half-life of MDA-7 protein. Regulation of protein turnover is a heretofore-unrecognized mechanism of this nonsteroidal anti-inflammatory drug.

Selective induction of cell cycle arrest and apoptosis in human prostate cancer cells through adenoviral transfer of the melanoma differentiation-associated -7 (mda-7)/interleukin-24 (IL-24) gene.

We have previously reported that overexpression of the melanoma differentiation-associated gene -7 (mda-7) using a replication-defective adenovirus (Ad-mda7), results in tumor-specific growth suppression and induction of apoptosis in wide variety of cancer cells. In the present study, we investigated the antitumor activity of Ad-mda7 and the underlying mechanism in human prostate cancer cells and normal prostate epithelial cells. Overexpression of MDA-7 induced significant (P=.001) suppression of cell growth and apoptosis in prostate cancer cells (DU 145, LNCaP, and PC-3). In normal prostate epithelial cells (PrEC) some degree of growth inhibition but not apoptosis was observed. However, the inhibitory effects in normal cells were less compared to tumor cells. Growth inhibitory effects were mediated by the intracellular and not by extracellular MDA-7 protein. Molecular effectors that are involved in Ad-mda7-mediated tumor killing included activation of the caspase cascade, and the induction of G2 phase cell cycle arrest through the inhibition of Cdc25C pathway. These results demonstrate the mechanisms by which Ad-mda7 exerts its antitumor activity in human prostate cancer cells. The antitumor activity combined with previously reported antiangiogenic and proimmune properties of Ad-mda7 can serve as a potential therapeutic agent for treatment of primary and disseminated prostate cancer.

Nanoparticle based systemic gene therapy for lung cancer: molecular mechanisms and strategies to suppress nanoparticle-mediated inflammatory response.

Cancer gene therapy for the treatment of lung cancer has shown promise in the laboratory and in Phase I/II clinical trials. However, it is currently limited to treating localized tumors due to host-immunity against the gene delivery vector and the transgene. Therefore, there is a tremendous effort to develop and test alternate gene delivery vectors that are efficient, non-immunogenic, and applicable for systemic therapy. One such gene delivery vehicle is the non-viral vector, DOTAP:cholesterol (DOTAP:Chol) nanoparticle. Preclinical studies from our laboratory has shown that DOTAP:Chol. nanoparticles are effective systemic gene delivery vectors that efficiently deliver tumor-suppressor genes to disseminated lung tumors. Based on our findings we have recently initiated a Phase-I trial for systemic treatment of lung cancer using a novel tumor suppressor gene, FUS1. Although DOTAP:Chol. nanoparticles complexed to DNA (DNA-nanoparticles) are efficient vectors for systemic therapy, induction of an inflammatory response in a dose-dependent fashion has also been observed thereby limiting its use. A better understanding of the underlying mechanism for DNA-nanoparticles-mediated inflammatory response will allow us to develop strategies to suppress inflammation and expand the therapeutic window in treating human cancer. In the present study we conducted experiments examining the mechanism of nanoparticle-mediated inflammatory response in vitro and in vivo. We demonstrate that systemic administration of DNA-nanoparticles induced multiple signaling molecules both in vitro and in vivo that are associated with inflammation. Use of small molecule inhibitors against the signaling molecules resulted in their suppression and thereby reduced inflammation without affecting transgene expression. Our results provide a rationale to use small molecule inhibitors to suppress nanoparticle-mediated inflammation when administered systemically. Further development and testing will allow us to incorporate this strategy into future clinical trials that is based on systemic non-viral vector gene therapy.

Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo.

Lung cancer is one of the leading causes of death in the world. The underlying cause for lung cancer has been attributed to various factors that include alteration and mutation in the tumor suppressor genes. Restoration of normal function of the tumor suppressor gene is a potential therapeutic strategy. Recent studies have identified a group of candidate tumor suppressor genes on human chromosome 3p21.3 that are frequently deleted in human lung and breast cancers. Among the various genes identified in the 3p21.3 region, we tested the antitumor activity of the FUS1 gene in two human non-small-cell lung cancer (NSCLC) xenografts in vivo. Intratumoral administration of FUS1 gene complexed to DOTAP:cholesterol (DOTAP:Chol) liposome into subcutaneous H1299 and A549 lung tumor xenograft resulted in significant (P = .02) inhibition of tumor growth. Furthermore, intravenous injections of DOTAP:Chol-FUS1 complex into mice bearing experimental A549 lung metastasis demonstrated significant (P = .001) decrease in the number of metastatic tumor nodules. Finally, lung tumor-bearing animals when treated with DOTAP:Chol-FUS1 complex demonstrate prolonged survival (median survival time: 80 days, P = .01) compared to control animals. This result demonstrates the potent tumor suppressive activity of the FUS1 gene and is a promising therapeutic agent for treatment of primary and disseminated human lung cancer.

Persistent transgene expression following intravenous administration of a liposomal complex: role of interleukin-10-mediated immune suppression.

Studies conducted in non-tumor-bearing, immunocompetent mice have shown that intravenous administration of liposome-DNA complex elicits an inflammatory response that results in a failure to sustain adequate transgene expression. In the present study, however, we investigated the effects of a cationic liposomal DOTAP:cholesterol (DOTAP:Chol)-DNA complex on cytokine production and transgene expression in both experimental lung tumor-bearing (TB) mice and non-tumor-bearing (NTB) syngeneic mice and nude mice. Intravenous injection of DOTAP:Chol-luciferase (luc) DNA complex resulted in tumor necrosis factor-alpha levels that were 50% lower and interleukin-10 levels that were 50-60% higher in TB mice than in NTB mice. Furthermore, a significant increase in luc expression (P = 0.001) that persisted for 7 days was observed in TB mice. In contrast, luc expression decreased significantly from day 1 to day 2 in NTB mice. Also, luc expression was two- to threefold higher in TB mice that were given multiple injections of DOTAP:Chol-luc complex than in mice who received a single injection. In contrast, luc expression was significantly suppressed following multiple injections in NTB mice (P = 0.01). Further analysis revealed IL-10 protein expression by the tumor cells in TB mice. Injection of anti-IL-10 antibody in TB mice resulted in a significant decrease in luc expression (P = 0.01) compared with that in mice injected with a control antibody. Based on these findings, we conclude that transgene expression persists in TB mice and is partly mediated by IL-10. Additionally, multiple injections of liposome-DNA complex can increase transgene expression in TB mice. These findings have clinical applications in the treatment of cancer.

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery.

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.

Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor.

The melanoma differentiation-associated gene 7 (mda-7), also called interleukin (IL)-24, suppresses the growth of some cancers in vitro and in vivo as a result of the ectopic expression of its protein. However, the function of the secreted form of the protein in cancer has not been previously studied. The purpose of this study was to determine the antiangiogenic function of a secreted form of the MDA-7/IL-24 protein (sMDA-7/IL-24). In vitro, sMDA-7/IL-24 inhibited both endothelial cell differentiation and migration of endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor. The sMDA-7/IL-24-mediated inhibitory effect was 10-50 times more potent than endostatin, IFN-gamma, and IFN-inducible protein 10 in vitro. Furthermore, the inhibitory effect was not mediated by IFN or IFN-inducible protein 10. IL-22 receptor mediated the antiangiogenic activity of sMDA-7/IL-24. Administration of a blocking antibody to IL-22 receptor in conjunction with sMDA-7/IL-24 led to abrogation of inhibition of endothelial differentiation. sMDA-7/IL-24 inhibited vascular endothelial growth factor-induced angiogenesis as evidenced by reduced vascularization and hemoglobin content in in vivo Matrigel plug assays. In vivo, the growth of human lung tumor cells was significantly inhibited, and vascularization was reduced when the cells were mixed with 293 cells stably expressing sMDA-7/IL-24. Systemic administration of sMDA-7/IL-24 inhibited lung tumor growth in a mouse xenograft model. Associated with tumor growth inhibition was decreased tumor microvessel density and hemoglobin content, indicating the presence of antiangiogenic activity. These data demonstrate that sMDA-7/IL-24 is a novel and potent antiangiogenic effector and support the development of MDA-7/IL-24-based therapeutics.

Increased uptake of liposomal-DNA complexes by lung metastases following intravenous administration.

We have investigated the effects of an improved liposomal formulation (extruded DOTAP:cholesterol (DOTAP:Chol)-DNA complex) on transgene expression in tumor cells and normal cells of murine and human origin both in vitro and in vivo. In vitro, transgene expression was significantly increased (P = 0.01) in human tumor cells compared to normal human cells. The increased transgene expression was due to increased uptake of the liposome-DNA complex by tumor cell phagocytosis. Furthermore, immunohistochemical analysis demonstrated a greater transgene expression in lung tumors than in surrounding normal tissues. Increased transgene expression due to enhanced uptake of the liposome-DNA complexes by tumor cells in vivo was also demonstrated using fluorescently labeled DOTAP:Chol liposomes. Finally, evaluation of lung tissue explants obtained from patients undergoing pulmonary resection demonstrated significantly higher (P = 0.001) transgene expression in tumor cells than in normal cells. Thus, we demonstrated that intravenous injection of DOTAP:Chol-DNA complex results in increased transgene expression in tumor and is due to increased phagocytosis of the complexes by tumor cells.

Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo.

Overexpression of the melanoma differentiation associated gene-7 (mda-7) in vitro results in suppression of lung cancer cell proliferation. However, the ability of MDA-7 to suppress lung cancer in vivo has not been previously demonstrated. In this study, we investigated the possibility of inducing overexpression of the mda-7 gene in human non-small cell lung carcinoma cells in vivo and its effects on tumor growth. Adenovirus-mediated overexpression of MDA-7 in p53-wild-type A549 and p53-null H1299 subcutaneous tumors resulted in significant tumor growth inhibition through induction of apoptosis. In addition, decreased CD31/PECAM expression and upregulation of APO2/TRAIL were observed in tumors expressing MDA-7. In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. These data demonstrate that Ad-mda7 functions as a multi-modality anti-cancer agent, possessing both, pro-apoptotic and anti-angiogenic properties. We demonstrate for the first time the potential therapeutic effects of Ad-mda7 in human lung cancer.