Cynthia D. Branch

Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro.

The melanoma differentiation-associated gene-7 (mda-7), cloned from a human melanoma cell line H0–1, is known to induce tumor cell-selective growth inhibition in breast cancer cells in vitro and loss of tumorigenicity ex vivo. Yet, the mechanisms underlying these effects are still unknown. Therefore, we investigated these mechanisms on the molecular level in human non-small cell lung carcinoma (NSCLC) cells in vitro. Overexpression of mda-7 protein by Ad-mda-7 significantly suppressed proliferation and induced G2/M cell cycle arrest in wild-type p53 (A549, H460), and p53-null (H1299) non-small cell lung cancer cell lines, but not in normal human lung fibroblast (NHLF) cells. p53, Bax, and Bak protein expression was up-regulated in wild-type p53 tumor cell lines, but not in p53-null cells, suggesting that an intact p53 pathway was required for Bax and Bak induction. However, in all three cancer cell lines tested, activation of the caspase cascade and cleavage of poly(ADP-ribose) polymerase (PARP) appeared to be independent of the p53 mutational status. Together, these results suggest that apoptosis may be induced via multiple pathways by Ad-mda-7 in lung cancer cells and that Ad-mda-7 has the potential to become a novel therapeutic for clinical cancer gene therapy.

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.

Comparison of cell adhesion molecule expression between glioblastoma multiforme and autologous normal brain tissue

We investigated glioblastoma multiforme (GBM) for a pattern of consistent alterations in cell adhesion molecules (CAM) expression that might distinguish tumor from normal autologous brain tissue. We used frozen section immunohistochemistry with anti-CAM and computerized image analysis to quantify staining intensity which we expressed as relative intensity units (RIU). Our results showed that normal brain tissue generally did not express alpha 1 beta 1, intercellular CAM-1 (ICAM-1), and sialylated Lewisx, slightly expressed alpha 2, alpha 4, alpha 5, alpha 6 beta 1, alpha v beta 3, lymphocyte function-associated antigen-3 (LFA-3), Lewisx, sialylated LewisLewisx, had a good expression of alpha 3 beta 1 and CD44, and strongly expressed neural CAM (NCAM). GBM expressed alpha 2, alpha 3, alpha 5, alpha 6 beta 1, alpha v beta 3, ICAM-1, LFA-3, CD44, Lewisx, sialylated Lewisx, and sialylated LewisLewisx significantly higher (2-11-fold RIU) than normal brain tissue. ICAM-1 and LFA-3 were the most distinctive markers of GBM. The small blood vessel endothelial cells of the normal brain and the GBM showed a few differences. The tumor endothelium expression of alpha 2 beta 1, alpha 4 beta 1, and LFA-3 RIU appeared twice higher than in normal endothelium and alpha 6 beta 1 showed an average of 40% RIU decrease in comparison to normal. These results show that the expression of several CAM is consistently altered in GBM and its microvasculature when compared with autologous normal brain tissue.

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.

5-aza-2′-deoxycytidine upregulates caspase-9 expression cooperating with p53-induced apoptosis in human lung cancer cells

Treating lung cancer cell lines using low-dose 5-aza-2′-deoxycytidine (DAC) caused an accumulation of procaspase-9 through mRNA upregulation, but the cells did not undergo apoptosis. However, when cells were treated with DAC and infected with a low dose of a recombinant wild-type p53 adenovirus vector (Ad-p53), a synergistic growth inhibitory effect was observed. Combination treatment induced Apaf-1 and procaspase-9 expression in which cytochrome c releases by Ad-p53 triggered the mitochondrial pathway of apoptosis. Selective blockage of caspase-9 activities by Z-LEHD-FMK completely attenuated DAC-induced enhancement of apoptosis mediated by Ad-p53 infection, and ectopic overexpression of procaspase-9 sensitized cells to Ad-p53-induced apoptosis in p53-null cells. In addition, DAC sensitized lung cancer cells to cisplatin and paclitaxel. Induction of the mitochondrial pathway of apoptosis using a slightly toxic dose of DAC may therefore be a strategy for treating lung cancer, and DAC treatment may have clinical implications when combined with chemotherapy or apoptosis-inducing gene therapy.

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.

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.

Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo.

The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers.

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.

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.