The Role of Nitric Oxide Dysregulation in Tumor Maintenance

The inflammatory nature of the tumor microenvironment provides a cytokine and chemokine rich proliferative environment. Much of the responsibility of this environment is due to the production of Reactive Oxygen Species (ROS). These studies examined the proliferative rich tumor environment from a new perspective of Nitric Oxide Synthase (NOS) dysregulation. NOS’s have the ability to become uncoupled and generate superoxide in lieu of nitric oxide (NO). A requirement of NOS for the production of NO is the cofactor tetrahydrobiopterin (BH4) and when it is missing NOS becomes uncoupled and turns into a peroxynitrite synthase. Here I demonstrate that NOS is uncoupled in tumor cells due to depleted BH4 levels. This uncoupling leads to decreased NO signaling and increased pro-inflammatory, pro-survival, signaling as a result of the increased generation of ROS/RNS from uncoupled NOS activity. I was able to recouple NOS through exogenous BH4 both in vitro and in vivo, reducing ROS/RNS and reestablishing NO signaling through cGMP protein associated kinase. Reduction of ROS/RNS resulted in the reduced activity of two major constitutively active transcription factors in breast cancer cells, NFκB and STAT3. In MCF-7 and MDA231 cells I found that increased NO-dependent PKG signaling led to tumor cell toxicity mediated by downregulation of β-catenin. Downregulation of β-catenin led to increased protein levels of p21 in MCF-7 and p27 in MDA 231cells, ultimately resulting in cell death. These results suggest that there is potential for BH4 as a therapeutic agent since exogenous dietary BH4 ameliorates chemically induced colitis, and reduced azoxymethane (AOM) induced colon and spontaneously developing mammary carcinogenesis

Molecular Imaging Investigations of Polymer-Coated Cerium Oxide Nanoparticles as a Radioprotective Therapeutic Candidate

Cerium oxide nanoparticles (CONPs) have a unique surface redox chemistry that appears to selectively protect normal tissues from radiation induced damage. Our prior research exploring the biocompatibility of polymer-coated CONPs found further study of poly-acrylic acid (PAA)-coated CONPs was warranted due to improved systemic biodistribution and rapid renal clearance. This work further explores PAA-CONPs’ radioprotective efficacy and mechanism of action related to tumor microenvironment pH. An ex vivo TUNEL assay was used to measure PAA-CONPs’ protection of the irradiated mouse colon in comparison to the established radioprotector amifostine. [18F]FDG PET imaging of spontaneous colon tumors was utilized to determine the effects of PAA-CONPs on tumor radiation response. In vivo MRI and an ex vivo clonogenic assay were used to determine pH effects on PAA-CONPs’ radioprotection in irradiated tumor-bearing mice. PAA-CONPs showed excellent radioprotective efficacy in the normal colon that was equivalent to uncoated CONPs and amifostine. [18F]FDG PET imaging showed PAA-CONPs do not affect tumor response to radiation. Normalization of tumor pH allowed some radioprotection of tumors by PAA-CONPs, which may explain their lack of tumor radioprotection in the acidic tumor microenvironment. Overall, PAA-CONPs meet the criteria for clinical application as a radioprotective therapeutic agent and are an excellent candidate for further study

Uncoupled nitric oxide synthase activity promotes colorectal cancer progression

Increased levels of reactive oxygen/nitrogen species are one hallmark of chronic inflammation contributing to the activation of pro-inflammatory/proliferative pathways. In the cancers analyzed, the tetrahydrobiopterin:dihydrobiopterin ratio is lower than that of the corresponding normal tissue, leading to an uncoupled nitric oxide synthase activity and increased generation of reactive oxygen/nitrogen species. Previously, we demonstrated that prophylactic treatment with sepiapterin, a salvage pathway precursor of tetrahydrobiopterin, prevents dextran sodium sulfate–induced colitis in mice and associated azoxymethane-induced colorectal cancer. Herein, we report that increasing the tetrahydrobiopterin:dihydrobiopterin ratio and recoupling nitric oxide synthase with sepiapterin in the colon cancer cell lines, HCT116 and HT29, inhibit their proliferation and enhance cell death, in part, by Akt/GSK-3β–mediated downregulation of β-catenin. Therapeutic oral gavage with sepiapterin of mice bearing azoxymethane/dextran sodium sulfate–induced colorectal cancer decreased metabolic uptake of [18F]-fluorodeoxyglucose and enhanced apoptosis nine-fold in these tumors. Immunohistochemical analysis of both mouse and human tissues indicated downregulated expression of key enzymes in tetrahydrobiopterin biosynthesis in the colorectal cancer tumors. Human stage 1 colon tumors exhibited a significant decrease in the expression of quinoid dihydropteridine reductase, a key enzyme involved in recycling tetrahydrobiopterin suggesting a potential mechanism for the reduced tetrahydrobiopterin:dihydrobiopterin ratio in these tumors. In summary, sepiapterin treatment of colorectal cancer cells increases the tetrahydrobiopterin:dihydrobiopterin ratio, recouples nitric oxide synthase, and reduces tumor growth. We conclude that nitric oxide synthase coupling may provide a useful therapeutic target for treating patients with colorectal cancer

Gene therapy with RALA/iNOS composite nanoparticles significantly enhances survival in a model of metastatic prostate cancer

Recent approvals of gene therapies by the FDA and the EMA for treatment of inherited disorders have further opened the door for assessment of nucleic acid pharmaceuticals for clinical usage. Arising from presence of damaged or inappropriate DNA, cancer is a condition particularly suitable for genetic intervention. The RALA peptide has been shown to be a potent non-viral delivery platform for nucleic acids. This study reports that complexation of RALA with a plasmid encoding inducible nitric oxide synthase (iNOS) DNA produces functional cationic nanoparticles with gene expression in PC-3 prostate cancer cells. Furthermore, repeated administrations of RALA/DNA nanoparticles to immunocompetent mice did not produce an immunological response, be that neutralization of the vector or release of inflammatory mediators. RALA/CMV-iNOS reduced the clonogenicity of PC-3 cells in vitro, and in an in vivo model of prostate cancer metastasis, systemically-delivered RALA/CMV-iNOS significantly improved the survival of mice. These results further validate RALA as a genetic cargo delivery vehicle and iNOS as a potent therapy for the treatment of cancer