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.

Viral-mediated gene transfer for cancer treatment.

Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses that retain the capability of the virus for efficient gene delivery but are safer than the native virus due to modifications that eliminate or alter one or more essential viral functions. The field of viral-based gene transfer vectors for the treatment of cancer has now entered the final stage of clinical testing prior to possible product approvals. Three viral vectors are currently undergoing this Phase III or Phase II/III clinical testing for cancer treatment. All three of these vectors are based on adenovirus, a common human virus that in its native state can cause cold or flu-like symptoms. In two of these vectors, genes essential for viral replication have been replaced with the wild-type p53 tumor suppressor gene, a gene that is deleted or mutated in over 50% of human cancers and which, when transferred into tumor cells, can induce tumor cell death. The third vector retains more of the natural adenoviral functions and relies on replication in tumor cells to induce cell killing. These three vectors represent two of the approaches now being taken to develop viral-based gene transfer vectors for cancer treatment. Additional approaches include the transfer of genes capable of converting non-toxic prodrugs into toxic forms, using anti-angiogenic gene transfer to block the formation of tumor blood vessels, inhibiting the activity of oncogenes through blocks to transcription or translation, stimulating the body's own immune system with immunomodulatory genes, and "cancer vaccination" with genes for tumor antigens. The data derived to date from clinical trials with viral-based vector systems are promising. The vectors have been generally well-tolerated without the severe toxicities common to standard cancer treatments. Many of these vectors have been demonstrated to have anti-tumor activity in a clinical setting and to lead to tumor regressions or to reductions in the rate of tumor growth. Furthermore, the safety profile of these vector systems has allowed for their clinical testing in combination with conventional cancer treatments to determine their benefit in multi-modality therapy. As part of their clinical development, all three of the vectors in Phase III and Phase II/III clinical trials are being tested for their benefit in combination with chemotherapy in randomized trials. The field is also looking to future product opportunities with improvements that will further increase potency, safety, and/or ease of administration. These developments may expand the number of cells that are susceptible to infection or may target the vector to particular tumor types following an intravenous administration. Some of these approaches are already in clinical testing. Additional opportunities may utilize multigene strategies to target multiple pathways in cancer cells or expand the use of cytotoxic or cytostatic gene transfer in combination with immunotherapy.

Inhibition of radiation-induced nuclear factor-kappaB activation by an anti-Ras single-chain antibody fragment: lack of involvement in radiosensitization.

We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-kappaB (NF-kappaB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-kappaB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IkappaB kinase activity and IkappaBalpha phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-kappaB. Whereas AV1Y28 inhibited NF-kappaB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-alpha-induced NF-kappaB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-kappaB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-kappaB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-kappaB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-kappaB activation. However, inhibition of NF-kappaB activation via the expression of a dominant-negative form of IkappaBalpha in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-kappaB activation, this process does not appear to play a direct role in its radiosensitizing actions.

The effects of exogenous p53 overexpression on HPV-immortalized and carcinogen transformed oral keratinocytes.

BACKGROUND: Overexpression of p53 in head and neck carcinoma cells has demonstrated tumor growth suppression using in vitro and in vivo models. The effects of exogenous overexpression of wild-type p53 on human papilloma virus (HPV)-immortalized and carcinogen transformed oral keratinocytes were determined. METHODS: The p53 gene was overexpressed in IHGK (immortalized human gingival keratinocyte), IHGKN [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)]-carcinogen transformed keratinocytes, and two head and neck squamous carcinoma (HNSCC) cell lines, HN30 and HN12. The transfection efficiency, growth suppression, and inhibition of the cell cycle along with the induction of apoptosis were measured. RESULTS: Transfections with adenoviruses were more efficient for IHGK cells than for IHGKN, HN12, and HN30 cells. Inhibition of proliferation in all cell lines was proportional to the viral particle to cell (VPC) ratios. IHGK cells were more sensitive to p53 than IHGKN cells. HN12 cells were more suppressed than HN30 cells. HN12 were the most suppressed at 72 hours whereas HN30 cells were most suppressed at 24 hours. Expression of exogenous p53-induced G1 cell cycle arrest and p21 expression as VPC ratios increased in IHGK and IHGKN cell lines. Apoptosis also was induced in these cells by p53 as VPC increased. IHGK cells were more sensitive to p53-induced growth inhibition, cell cycle regulation, p21 expression and apoptosis than IHGKN cells. HN12 (mutated p53) cells were more sensitive to p53 overexpression than HN30 (wild-type p53) cells. Gene transfer and expression of exogenous p53 by using Ad-p53 demonstrates suppressive effects on HPV immortalized and carcinogen transformed oral keratinocytes. CONCLUSIONS: Cell cycle regulation by gene transfer is feasible in immortalized oral keratinocytes. Carcinogen transformed cells are less susceptible to the effects of p53 overexpression. Expression of exogenous p53 through p53 gene transfer can suppress HPV immortalization and carcinogen transformation in oral keratinocytes. The sensitivity of HNSCC cell lines to p53-induced cell cycle regulation and apoptosis is variable and dependent on the cell line and duration of exposure. In vitro results using p53 gene transfer must be validated in clinical studies with patients at risk for HNSCC.