Non-small Cell Lung Cancer Tumor Xenografts Research Articles

Abstract 3271: Inhibition of angiogenesis and tumor growth by itraconazole in primary xenograft models of human non-small cell lung cancer

Abstract Introduction: Lung cancer is the most common cause of cancer-related death in the US. Approximately 85 percent of cases are non-small cell lung cancer (NSCLC). Angiogenesis is a critical component to tumor growth. Bevacizumab is the only anti-angiogenic agent currently approved for NSCLC. Despite aggressive multimodality therapy, 5-year survival in patients with advanced stage NSCLC is exceedingly poor. Novel therapeutic strategies for NSCLC are critically needed. The azole anti-fungal agent, itraconazole (ITRA) has been identified as a potential anti-angiogenic agent. In this study we further characterize the activity of ITRA in human umbilical vascular endothelial cell (HUVEC) based assays. Preclinical efficacy of ITRA is also examined as a single-agent and in combination with cisplatin in xenograft models of NSCLC generated with primary tumor tissue. Methods: HUVECs were cultured in EGM-2 media (Lonza) and assayed in EGM-2 or EBM-2 media supplemented with recombinant growth factors, vascular endothelial growth factor (VEGF; 10 ng/ml), and basic fibroblast growth factor (bFGF; 12 ng/ml). Effects of ITRA on proliferation of HUVECs and three NSCLC cell lines, including H358 (bronchioalveolar carcinoma), H596 (adenosquamous carcinoma) and H1838 (adenocarcinoma) were evaluated by AqueousOne® (Promega). Additionally, HUVEC migration assays were carried out using an 8-micron pore Boyden chamber apparatus and endothelial tube formation assays were conducted using Geltrex® (Invitrogen) coated culture plates. To determine in vivo efficacy, mice bearing established xenografts derived from primary tumor tissue were treated orally with ITRA BID or QD at doses ranging from 75 to 100 mg/kg. In combination experiments, cisplatin (4 mg/kg) administered IP once weekly was given with ITRA. Mice were exposed to intravenous Hoechst 33342 prior to collection of tumors. Vascular area was determined by Hoechst 33342 and anti-CD31 staining. Tumor lysates were analyzed by immunoblot for Hif1α induction. Results: ITRA demonstrates potent dose-dependent inhibition of HUVEC proliferation (IC50 <0.7 μM). ITRA had no effect on proliferation in the panel of NSCLC cell lines tested (IC50 <100 μM). ITRA inhibits HUVEC migration and tube formation with similar potency. In vivo, ITRA inhibits NSCLC tumor xenograft growth with maximum single-agent activity seen with 100 mg/kg BID treatment. Addition of ITRA to a cisplatin regimen significantly enhanced anti-tumor effects compared to either single-agent alone (p <0.01). ITRA therapy resulted in increased tumor expression of HIF1α and significantly decreased tumor vascular area (p <0.01). Conclusion: The anti-angiogenic activity of ITRA and efficacy demonstrated in preclinical models of NSCLC supports development of this agent for the management of NSCLC in combination with standard of care chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3271. doi:10.1158/1538-7445.AM2011-3271

Phase II trial evaluating addition of fulvestrant to erlotinib in patients with stage IIIb/IV non-small cell lung cancer (NSCLC) who are stable on erlotinib and exhibit immunohistochemical (IHC) or PCR positivity for estrogen or progesterone receptor (PR).

TPS294 Background: In NSCLCestrogen receptors (ER) α and β are expressed independent of sex and histology. Estrogen regulates gene expression and promotes growth of NSCLC cell lines and tumor xenografts. Epidermal growth factor receptor (EGFR) and ER pathways share and interact through downstream signaling molecules. Activation of EGFR results in phosphorylation of ERα via MAPK and ERK. In cell lines and xenographic models, the combination of an ER antagonist fulvestrant and an EGFR inhibitor gefitinib is additive in decreasing growth. This trial will determine a progression-free survival (PFS) rate of ER or PR positive NSCLC patients who are stable after 2 months of erlotinib monotherapy, and receiving fulvestrant + erlotinib. Methods: This is a single center, open-label, single-arm study. Eligible patients have measurable, stable disease as described, and ER a, ERb, and/or PR-positive tumors determined by IHC and PCR on paraffin-embedded tissue. Fulvestrant is added to daily eroltinib at 500 mg, based o...

Abstract 2334: A novel αV integrin antibody, intetumumab (CNTO 95), enhances anti-tumor activity against human non-small cell lung cancer through disruption of the FAK/ERK/AKT pathway and regulation of p27KIP1

Abstract Integrins are cell-surface adhesion proteins that interact with various components of the extracellular matrix and play an important role in intracellular signaling, cell adhesion, migration, and proliferation. The expression of αV integrins has been associated with poor clinical outcome in patients with non-small cell lung cancer (NSCLC). Intetumumab (CNTO 95) is a novel, fully human monoclonal antibody against all members of the αV integrin family including αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8. Intetumumab inhibits cell adhesion, migration, proliferation, and induces apoptosis in tumor and endothelial cells in vitro. Intetumumab inhibits tumor growth, metastasis, and angiogenesis in vivo in rat xenograft models. Here, we describe experiments to understand the mechanism of action of intetumumab in NSCLC models in vitro and in vivo. The focal adhesion complex and αV integrins play crucial roles in adhesion, motility, and migration in both normal and cancer cells. NSCLC cells were cultured on a vitronectin matrix to stimulate signaling through the αV integrin pathway and then treated with intetumumab (10 µg/ml). Intetumumab treatment resulted in the reduction of phosphorylated FAK (Y397) and Paxillin (Y31) compared to controls up through 48 hours. FAK has been shown to participate in downstream processes such as cell cycle progression by modulating ERK1/2, AKT and the cyclin-dependent kinase inhibitor p27KIP1. Intetumumab treatment of NSCLC cell lines in vitro resulted in the reduction of phosphorylated ERK1/2 and AKT and the subsequent upregulation of p27KIP1. Furthermore, intetumumab treatment activated the intrinsic pathway of apoptosis as evidenced by the cleavage of caspase 9 and PARP as well as increased Bim expression. In vivo administration of intetumumab (10 mg/kg, i.p., 3x weekly) alone or in combination with docetaxel (up to 20 mg/kg, i.p., weekly for 3 weeks) significantly inhibited the growth of established A549 human NSCLC tumor xenografts in nude rats as a monotherapy (p<0.030) or in combination with docetaxel (p<0.039). Moreover, in three separate experiments, combination treatment resulted in complete tumor regressions in 76% (25/33) of animals showing superior efficacy of the combination versus either agent alone (p=0.0138). In conclusion, the results presented here demonstrate the impact of intetumumab on the cellular and molecular mechanisms mediating cell survival through the αV integrin signaling pathway resulting in disruption of focal adhesion complexes, inhibition of cell cycle progression at the G1 phase and induction of the cell intrinsic pathway of apoptosis. Taken together these results demonstrate enhanced anti-tumor efficacy in an established NSCLC preclinical model and suggest the potential for clinical evaluation of intetumumab in NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2334.

Proanthocyanidins Inhibit In vitro and In vivo Growth of Human Non–Small Cell Lung Cancer Cells by Inhibiting the Prostaglandin E2 and Prostaglandin E2 Receptors

Overexpression of cyclooxygenase-2 (COX-2) and prostaglandins (PG) is linked to a wide variety of human cancers. Here, we assessed whether the chemotherapeutic effect of grape seed proanthocyanidins (GSP) on non-small cell lung cancer (NSCLC) cells is mediated through the inhibition of COX-2 and PGE(2)/PGE(2) receptor expression. The effects of GSPs on human NSCLC cell lines in terms of proliferation, apoptosis, and expression of COX-2, PGE(2), and PGE(2) receptors were determined using Western blotting, fluorescence-activated cell sorting analysis, and reverse transcription-PCR. In vitro treatment of NSCLC cells (A549, H1299, H460, H226, and H157) with GSPs resulted in significant growth inhibition and induction of apoptosis, which were associated with the inhibitory effects of GSPs on the overexpression of COX-2, PGE(2), and PGE(2) receptors (EP1 and EP4) in these cells. Treatment of cells with indomethacin, a pan-COX inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell growth and induced cell death. The effects of a GSP-supplemented AIN76A control diet fed to nude mice bearing tumor xenografts on the expression of COX-2, PGE(2), and PGE(2) receptors in the xenografts were also evaluated. The growth-inhibitory effect of dietary GSPs (0.5%, w/w) on the NSCLC xenograft tumors was associated with the inhibition of COX-2, PGE(2), and PGE(2) receptors (EP1, EP3, and EP4) in tumors. This preclinical study provides evidence that the chemotherapeutic effect of GSPs on lung cancer cells in vitro and in vivo is mediated, at least in part, through the inhibition of COX-2 expression and subsequently the inhibition of PGE(2) and PGE(2) receptors.

Inhibition of Insulin-Like Growth Factor 1 Receptor by CP-751,871 Radiosensitizes Non–Small Cell Lung Cancer Cells

Therapeutic strategies that target the insulin-like growth factor I receptor (IGF-1R) hold promise for a wide variety of cancers. We have now investigated the effect of CP-751,871, a fully human monoclonal antibody specific for IGF-IR, on the sensitivity of human non-small cell lung cancer (NSCLC) cell lines to radiation. The radiosensitizing effect of CP-751,871 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced damage was evaluated by immunofluorescence analysis of the histone gamma-H2AX and Rad51. A clonogenic survival assay revealed that CP-751,871 increased the sensitivity of NSCLC cells to radiation in vitro. CP-751,871 inhibited radiation-induced IGF-IR signaling, and potentiated the radiation-induced increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of the histone gamma-H2AX and Rad51 also showed that CP-751,871 inhibited the repair of radiation-induced DNA double-strand breaks. Finally, combination therapy with CP-751,871 and radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. These results show that CP-751,871 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of CP-751,871 is likely attributable to the inhibition of DNA repair and enhancement of apoptosis that result from attenuation of IGF-IR signaling. Combined treatment with CP-751,871 and radiation thus warrants further investigation in clinical trials as a potential anticancer strategy.

Grape Seed Proanthocyanidins Inhibit the Growth of Human Non-Small Cell Lung Cancer Xenografts by Targeting Insulin-Like Growth Factor Binding Protein-3, Tumor Cell Proliferation, and Angiogenic Factors

Lung cancer is a leading cause of cancer-related deaths worldwide. Here, we assessed the chemotherapeutic effect of grape seed proanthocyanidins (GSPs) on human non-small cell lung cancer (NSCLC) cells in vitro and in vivo using a tumor xenograft model. The effects of GSPs on human NSCLC cell lines in terms of cellular proliferation were determined. The chemotherapeutic effects of a GSP- supplemented AIN76A control diet fed to nude mice bearing tumor xenografts (A549 and H1299) were evaluated in terms of biomarkers of cell proliferation and angiogenesis and on insulin-like growth factor binding protein-3 using immunohistochemical detection, ELISA, and Western blotting. In vitro treatment of NSCLC cells with GSPs resulted in inhibition of cellular proliferation. Administration of GSPs (0.1%, 0.2%, and 0.5%, w/w) as a supplement of an AIN76A control diet resulted in a dose-dependent inhibition of the growth of NSCLC (A549 and H1299) tumor xenografts in athymic nude mice (25-76%; P < 0.05-0.001). The growth-inhibitory effect of GSPs on the NSCLC xenograft tumors was associated with the enhancement of the levels of insulin-like growth factor binding protein-3 in the tumor microenvironment and plasma and antiproliferative, antiangiogenic, and proapoptotic effects. This preclinical study reveals for the first time that dietary GSPs have the ability to inhibit the growth of human NSCLC tumor xenografts grown in vivo in athymic nude mice. More studies are needed to develop GSPs as a pharmacologically safe agent for the prevention of lung cancer in humans.

Radiosensitizing Effect of YM155, a Novel Small-Molecule Survivin Suppressant, in Non–Small Cell Lung Cancer Cell Lines

Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non-small cell lung cancer (NSCLC) cell lines to gamma-radiation. The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to gamma-radiation in vitro. The combination of YM155 and gamma-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone gamma-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and gamma-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.

Response of Non–Small Cell Lung Cancer Cells to the Inhibitors of Phosphatidylinositol 3-Kinase/Akt- and MAPK Kinase 4/c-Jun NH2-Terminal Kinase Pathways: An Effective Therapeutic Strategy for Lung Cancer

We previously showed that phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways cooperate to promote non-small cell lung cancer (NSCLC) cell proliferation in vitro. This study was designed to explore whether inhibition of these pathways effectively inhibits NSCLC tumor growth in vivo. The effects of PI3K/Akt inhibitors {LY294002, adenoviruses expressing dominant-negative mutant of the p85alpha adaptor subunit of PI3K (Ad-dnp85alpha), dominant-negative Akt [Ad-HA-Akt(KM)], or PTEN (Ad-PTEN)}, MKK4/c-jun NH2-terminal kinase (JNK) inhibitor [SP600215, adenovirus expressing dominant-negative MKK4, Ad-MKK4(KR)], and their combinations on proliferation and apoptosis in NSCLC cells were tested in vitro and in vivo using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a flow cytometry-based terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, Western blot and immunohistochemical analyses, and an NSCLC xenograft tumor model. Ad-dnp85alpha significantly inhibited proliferation of a subset of NSCLC cell lines used in our study. Intratumoral injection of Ad-dnp85alpha induced a significant decrease in the growth of H1299 NSCLC xenograft tumors. Concurrent inhibition of the PI3K/Akt and MKK4/JNK pathways showed enhanced antiproliferative effects on H1299 cells in vitro and in vivo by increasing apoptosis. PI3K/Akt and MKK4/JNK pathways cooperate to stimulate NSCLC cell proliferation by maintaining cell survival, suggesting that simultaneously targeting these two pathways might be an effective therapeutic strategy against NSCLC.

The retinoid X receptor agonist bexarotene (Targretin) synergistically enhances the growth inhibitory activity of cytotoxic drugs in non-small cell lung cancer cells

This study was designed to evaluate, using preclinical models of non-small cell lung cancer (NSCLC), the growth inhibitory effects of the retinoid X receptor (RXR) agonist bexarotene (LGD1069, Targretin) in combination with cytotoxic agents currently used as standard first-line therapy in advanced disease. Although single-agent bexarotene had modest growth inhibitory effects in several cell lines, efficacy was observed only in the micromolar range (>1muM), which approximates the plasma C(max) measured in pharmacokinetic studies in patients. However, when combined with paclitaxel or vinorelbine, bexarotene produced a concentration-dependent enhancement of the growth inhibitory activities of paclitaxel and vinorelbine. Formal synergy analysis using the Calu3 cell line demonstrated that the combination of bexarotene with either cytotoxic agent produced synergistic activity (combination index, CI<1). The in vitro observations were confirmed in vivo in a NSCLC xenograft tumor model (Calu3), where both bexarotene/paclitaxel and bexarotene/vinorelbine combinations produced significantly greater antitumor effects than the single agents. These results demonstrate that bexarotene can cooperate with widely used cytotoxic agents to decrease the growth of NSCLC tumor cells both in vitro and in vivo, and suggest the potential benefit of adding a RXR-selective agonist in combination with chemotherapy for NSCLC treatment. Furthermore, the data support the clinical observation from phase I/IIa trials suggesting that bexarotene has beneficial effects on survival when used in combination with cytotoxic agents in advanced NSCLC.

The angiogenesis inhibitor NM-3 is active against human NSCLC xenografts alone and in combination with docetaxel

The novel isocoumarin 2-(8-hydroxy-6-methoxy-1-oxo-1 H-2-benzopyran-3-yl) propionic acid (NM-3) has completed phase I clinical evaluation as an orally bioavailable angiogenesis inhibitor. NM-3 directly kills both endothelial and tumor cells in vitro at low mM concentrations and is effective in the treatment of diverse human tumor xenografts in mice. The present work has assessed the activity of NM-3 against human non-small-cell lung cancer (NSCLC) cells when used alone and in combination with docetaxel. The results demonstrate that NM-3 decreases clonogenic survival of NSCLC cells at clinically achievable concentrations. The results also demonstrate that NM-3 is effective in the treatment of NSCLC (A549, NCI-H460) tumor xenografts in mice. Moreover, NM-3 potentiated the antitumor activity of docetaxel against NSCLC xenografts without increasing toxicity. Our findings indicate that NM-3 may be effective alone or in combination with docetaxel in the treatment of patients with NSCLC.

Effects of Insulin-like Growth Factor Binding Protein-3 and Farnesyltransferase Inhibitor SCH66336 on Akt Expression and Apoptosis in Non-Small-Cell Lung Cancer Cells

Overexpression of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in non-small-cell lung cancer (NSCLC) cells in vitro and in vivo. However, Ras-mediated signaling pathways could develop resistance to apoptotic activities of IGFBP-3 in NSCLC cells. We thus evaluated the therapeutic potential of the combination of IGFBP-3 and SCH66336, a farnesyltransferase inhibitor that blocks Ras activation, in NSCLC cell lines. The effects of the combination of adenoviral IGFBP-3 (Ad-IGFBP3) and SCH66336 on proliferation and apoptosis of NSCLC cell lines (H1299, H596, A549, H460, H358, H322, and H226B) were assessed in vitro and in vivo by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometry-based terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, western blot analyses, and an NSCLC xenograft tumor model. The specific effects of Ad-IGFBP 3 and SCH66336 on mitogen-activated protein kinase and Akt were assessed by using adenoviral vectors that express constitutively active MEK1 or constitutively active Akt. Synergy was assessed by median effect analysis. The combination of Ad-IGFBP3 and SCH66336 had synergistic antiproliferative effects in five cell lines (H1299, H596, A549, H460, and H322). Antiproliferative effects were accompanied by increased apoptosis in H460 cells in vitro. Overexpression of a constitutively active Akt but not a constitutively active MEK-1 rescued H460 cells from apoptosis induced by single or combined treatment of Ad-IGFBP3 and SCH66336. In H1299 tumor xenografts, Ad-IGFBP3 and SCH66336 was associated with decreased tumor volume, increased apoptosis, and decreased Akt levels. The combination of Ad-IGFBP3 and SCH66336 decreased Akt expression and increased apoptosis in NSCLC cells in vitro and in vivo. Simultaneous treatment with IGFBP-3 and SCH66336 may have the potential to be an effective therapeutic strategy in NSCLC.

The combination of a Retinoid X Receptor (RXR) agonist LGD1069 (bexarotene) and vinorelbine produce synergistic growth inhibitory activity in non-small-cell lung cancer (NSCLC) cell lines

7358 Background: NSCLC is one of the leading causes of cancer-related deaths world wide. Targretin® (bexarotene) is a selective RXR agonist approved for the treatment of CTCL. Bexarotene has also shown phase I/IIa activity by enhancing survival and delaying progression in NSCLC patients (Clin Cancer Res 5:1658, 1999; J Clin Oncol 19:2626, 2001). Methods: We evaluated the anti-proliferative effects in vitro of LGD1069 as a single agent or in combination with the cytotoxic agents vinorelbine or paclitaxel in 10 NSCLC cell lines of different histologies. Cells were treated with LGD1069 alone or in combination with cytotoxic agents, growth and viability were examined. The combinations were carried out either concomitantly or sequentially. Results: LGD1069 as a single agent had modest growth inhibitory effects (at > 1 μM) in a subset of the NSCLC cell lines. LGD1069 combined with vinorelbine produced enhanced growth inhibitory activity (a left-ward shift of 3 –10 fold in the IC50) when compared to vinorelbine alone. Analysis of synergy between LGD1069 and vinorelbine showed synergistic (CI<1) growth inhibition in several NSCLC cell lines. The extent of synergy of the LGD1069/vinorelbine combo was greater at a modest drug effect (≤IC50). Moreover, when several NSCLC cell lines were pretreated with vinorelbine and then allowed to recover LGD1069 strongly inhibited re-growth as much as 75% when compared to the absence of LGD1069. We observed similar results when LGD1069/paclitaxel combos were evaluated. We also found enhanced anti-tumor efficacy (compared to single agents) in vivo with a LGD1069/paclitaxel combination in two different NSCLC xenograft tumor models. Conclusions: These results suggest that LGD1069 can cooperate with cytotoxic agents to decrease proliferation of NSCLC cells both in vitro and in vivo. These data support the use of Targretin/chemotherapy combinations in NSCLC. Targretin is currently in pivotal phase III trials in advanced NSCLC in combination regimens with cytotoxic chemotherapeutic agents (cisplatin/vinorelbine or carboplatin/paclitaxel). Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand Pharmaceuticals Ligand Pharmaceuticals

Adenovirus-mediated mda-7 (IL24) gene therapy suppresses angiogenesis and sensitizes NSCLC xenograft tumors to radiation

Melanoma differentiation-associated gene-7 (mda-7), recently classified as interleukin-24 (approved gene symbol IL24), is thought to be a tumor suppressor gene based on the loss of its expression in many different types of cancer. Gene therapy by adenovirus-mediated mda-7 (Ad-mda7) gene transfer has been shown to inhibit the growth of several different tumor cell lines, in vitro and in vivo. We previously demonstrated that Ad-mda7 radiosensitized non-small-cell lung cancer (NSCLC) cell lines by enhancing an apoptosis pathway through the activation of JNK and c-Jun. In the present study, we investigated the efficacy of intratumoral administration of Ad-mda7 combined with ionizing radiation for treating A549 xenograft tumors in nude mice. Substantial and long-lasting inhibition of tumor growth was evident following the combined treatment. Histological examination revealed marked reduction of angiogenic factors (bFGF, VEGF) and microvessel density and enhanced apoptosis in the tumors treated with the combination therapy compared to those treated with Ad-mda7 alone or radiation alone. To confirm the radiosensitizing effect of secreted MDA-7 protein, we performed clonogenic survival assays using human umbilical vein endothelial cells (HUVECs), A549 cells, and normal human lung fibroblasts, CCD16 cells, pretreated with the conditioned medium from 293 cells that had been stably transfected with mda-7 or a control vector. The results showed that MDA-7 protein sensitized HUVECs to ionizing radiation but not A549 cells or CCD16 cells. Our results suggest that Ad-mda7 in combination with radiation enhances apoptosis in the tumors and that secreted MDA-7 protein inhibits angiogenesis by sensitizing endothelial cells to ionizing radiation without affecting other normal cells. We conclude that the combination of mda-7 gene therapy and radiotherapy may be a feasible and effective strategy for treatment of NSCLC.