Manganese Porphyrin and Radiotherapy Improves Local Tumor Response and Overall Survival in Orthotopic Murine Mammary Carcinoma Models.

Novel synthetic compounds, known as manganese porphyrins (MnPs), have been designed to shift the redox status of both normal cells and cancer cells. When MnPs are coupled with cancer therapies, such as radiation, they have been shown to sensitize tumor cells to treatment and protect normal tissues from damage through the modulation of the redox status of various tissue types. Until now, our preclinical studies have focused on local effects of MnPs and radiation; however, we recognize that successful outcomes for cancer patients involve control of tumor cells throughout the body. In this study, using murine orthotopic mammary tumor models, we investigated how MnPs and radiation influence the development of distant metastasis. We hypothesized that the combination of MnP (MnP/RT), such as MnTnBuOE-2-PyP5+ and radiation treatment (RT) would increase local tumor control via a shift in the intratumoral redox environment, leading to subsequent downregulation of HIF-1 in the primary tumor. Secondarily, we hypothesized that these primary tumor treatment effects would result in a reduction in pulmonary metastatic burden. Balb/c mice with orthotopic 4T1 mammary carcinomas were treated with saline, MnP, RT or MnP/RT. We found MnP/RT did extend local tumor growth delay and overall survival compared to controls and was associated with increased intratumoral oxidative stress. However, the primary tumor growth delay observed with MnP/RT was not associated with a reduced pulmonary metastatic burden. Future directions to investigate the effects of MnP/RT on the development of distant metastasis may include modifications to the radiation dose, the experimental timeline or using a murine mammary carcinoma cell line with a less aggressive metastatic behavior. Clinical trials are underway to investigate the clinical utility of MnTnBuOE-2-PyP5+ for patients undergoing radiotherapy for various tumor types. The promising preclinical data from this study, as well as others, provides support that MnP/RT has the potential to improve local tumor control for these patients.

MnTE-2-PyP, a manganese porphyrin, reduces cytotoxicity caused by irradiation in a diabetic environment through the induction of endogenous antioxidant defenses.

Radiation is a common anticancer therapy for many cancer patients, including prostate cancer. Diabetic prostate cancer patients suffer from increased lymph node metastasis, tumor recurrence and decreased survival as compared to non-diabetic prostate cancer patients. These patients are also at increased risk for enhanced radiation-induced normal tissue damage such as proctitis. Diabetics are oxidatively stressed and radiation causes additional oxidative damage. We and others have reported that, MnTE-2-PyP, a manganese porphyrin, protects normal prostate tissue from radiation damage. We have also reported that, in an in vivo mouse model of prostate cancer, MnTE-2-PyP decreases tumor volume and increases survival of the mice. In addition, MnTE-2-PyP has also been shown to reduce blood glucose and inhibits pro-fibrotic signaling in a diabetic model. Therefore, to investigate the role of MnTE-2-PyP in normal tissue protection in an irradiated diabetic environment, we have treated human prostate fibroblast cells with MnTE-2-PyP in an irradiated hyperglycemic environment. This study revealed that hyperglycemia causes increased cell death after radiation as compared to normo-glycemia. MnTE-2-PyP protects against hyperglycemia-induced cell death after radiation. MnTE-2-PyP decreases expression of NOX4 and α-SMA, one of the major oxidative enzymes and pro-fibrotic molecules respectively. MnTE-2-PyP obstructs NF-κB activity by decreasing DNA binding of the p50-p50 homodimer in the irradiated hyperglycemic environment. MnTE-2-PyP increases NRF2 mediated cytoprotection by increasing NRF2 protein expression and DNA binding. Therefore, we are proposing that, MnTE-2-PyP protects fibroblasts from irradiation and hyperglycemia damage by enhancing the NRF2- mediated pathway in diabetic prostate cancer patients, undergoing radiotherapy.

Manganese porphyrin, MnTE-2-PyP, treatment protects the prostate from radiation-induced fibrosis (RIF) by activating the NRF2 signaling pathway and enhancing SOD2 and sirtuin activity.

Radiation therapy is a frequently used treatment for prostate cancer patients. Manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP or T2E or BMX-010) and other similar manganese porphyrin compounds that scavenge superoxide molecules have been demonstrated to be effective radioprotectors and prevent the development of radiation-induced fibrosis (RIF). However, understanding the molecular pathway changes associated with these compounds remains limited for radioprotection. Recent RNA-sequencing data from our laboratory revealed that MnTE-2-PyP treatment activated the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. Therefore, we hypothesize that MnTE-2-PyP protects the prostate from RIF by activating the NRF2 signaling pathway. We identified that MnTE-2-PyP is a post-translational activator of NRF2 signaling in prostate fibroblast cells, which plays a major role in fibroblast activation and myofibroblast differentiation. The mechanism of NRF2 activation involves an increase in hydrogen peroxide and a corresponding decrease in kelch-like ECH-associated protein 1 (KEAP1) levels. Activation of NRF2 signaling leads to an increase in expression of NAD(P)H dehydrogenase [quinone] 1 (NQO1), nicotinamide adenine dinucleotide (NAD+) levels, sirtuin activity (nuclear and mitochondrial), and superoxide dismutase 2 (SOD2) expression/activity. Increase in mitochondrial sirtuin activity correlates with a decrease in SOD2 (K122) acetylation. This decrease in SOD2 K122 acetylation correlates with an increase in SOD2 activity and mitochondrial superoxide scavenging capacity. Further, in human primary prostate fibroblast cells, the NRF2 pathway plays a major role in the fibroblast to myofibroblast transformation, which is responsible for the fibrotic phenotype. In the context of radiation protection, MnTE-2-PyP fails to prevent fibroblast to myofibroblast transformation in the absence of NRF2 signaling. Collectively, our results indicate that the activation of the NRF2 signaling pathway by MnTE-2-PyP is at least a partial mechanism of radioprotection in prostate fibroblast cells.

p66Shc protein through a redox mechanism enhances the progression of prostate cancer cells towards castration-resistance.

Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in U.S. men due to the development of the castration-resistant (CR) PCa phenotype. A useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies toward CR PCa. In this study, we established a PCa cell progressive model in three separate cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells. Those AI PCa cells obtain the biochemical properties of the clinical CR phenotype, including AR and PSA expression as well as enhanced proliferation and tumorigenicity under androgen-deprived conditions. Thus, those AI cells recapitulate CR PCa and exhibit increased oxidant species levels as well as enhanced signaling of proliferation and survival pathways. H2O2 treatment directly enhanced AS cell growth and migration, which was counteracted by antioxidant N-acetyl cysteine (NAC). We further identified p66Shc protein enhances the production of oxidant species which contributes to phenotypic and cell signaling alterations from AS to AI PCa cells. H2O2-treated LNCaP-AS cells had a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Conversely, the oxidant species-driven alterations of LNCaP-AI and p66Shc subclone cell signaling is mitigated by p66Shc knockdown. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules, correlating with p66Shc protein levels. Together, the data shows that p66Shc enhances oxidant species production that plays a role in promoting PCa progression to the CR stage.

Reactive Oxygen Species Drive Epigenetic Changes in Radiation-Induced Fibrosis.

Radiation-induced fibrosis (RIF) develops months to years after initial radiation exposure. RIF occurs when normal fibroblasts differentiate into myofibroblasts and lay down aberrant amounts of extracellular matrix proteins. One of the main drivers for developing RIF is reactive oxygen species (ROS) generated immediately after radiation exposure. Generation of ROS is known to induce epigenetic changes and cause differentiation of fibroblasts to myofibroblasts. Several antioxidant compounds have been shown to prevent radiation-induced epigenetic changes and the development of RIF. Therefore, reviewing the ROS-linked epigenetic changes in irradiated fibroblast cells is essential to understand the development and prevention of RIF.

The SOD Mimic, MnTE-2-PyP, Protects from Chronic Fibrosis and Inflammation in Irradiated Normal Pelvic Tissues.

Pelvic radiation for cancer therapy can damage a variety of normal tissues. In this study, we demonstrate that radiation causes acute changes to pelvic fibroblasts such as the transformation to myofibroblasts and the induction of senescence, which persist months after radiation. The addition of the manganese porphyrin, MnTE-2-PyP, resulted in protection of these acute changes in fibroblasts and this protection persisted months following radiation exposure. Specifically, at two months post-radiation, MnTE-2-PyP inhibited the number of α-smooth muscle actin positive fibroblasts induced by radiation and at six months post-radiation, MnTE-2-PyP significantly reduced collagen deposition (fibrosis) in the skin and bladder tissues of irradiated mice. Radiation also resulted in changes to T cells. At two months post-radiation, there was a reduction of Th1-producing splenocytes, which resulted in reduced Th1:Th2 ratios. MnTE-2-PyP maintained Th1:Th2 ratios similar to unirradiated mice. At six months post-radiation, increased T cells were observed in the adipose tissues. MnTE-2-PyP treatment inhibited this increase. Thus, MnTE-2-PyP treatment maintains normal fibroblast function and T cell immunity months after radiation exposure. We believe that one of the reasons MnTE-2-PyP is a potent radioprotector is due to its protection of multiple cell types from radiation damage.