Elizabeth A. Kosmacek

GM-CSF induces neuroprotective and anti-inflammatory responses in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine intoxicated mice

Innate and adaptive immune responses can speed nigrostriatal neurodegeneration in Parkinsons disease (PD). We posit that GM-CSF can attenuate such responses. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice, GM-CSF given prior to MPTP protected nigral dopaminergic neurons coincident with altered microglial morphologies and regulatory T cell (Treg) induction. Adoptive transfer of GM-CSF-induced Treg to MPTP mice protected nigral neurons. Gene expression analyses revealed novel immune-based neuronal protection pathways linked to the upregulation of IL-27. The results provide evidence that GM-CSF modulation of immunity could be of clinical benefit for PD.

MnTE-2-PyP reduces prostate cancer growth and metastasis by suppressing p300 activity and p300/HIF-1/CREB binding to the promoter region of the PAI-1 gene.

To improve radiation therapy-induced quality of life impairments for prostate cancer patients, the development of radio-protectors is needed. Our previous work has demonstrated that MnTE-2-PyP significantly protects urogenital tissues from radiation-induced damage. So, in order for MnTE-2-PyP to be used clinically as a radio-protector, it is fully necessary to explore the effect of MnTE-2-PyP on human prostate cancer progression. MnTE-2-PyP inhibited prostate cancer growth in the presence and absence of radiation and also inhibited prostate cancer migration and invasion. MnTE-2-PyP altered p300 DNA binding, which resulted in the inhibition of HIF-1β and CREB signaling pathways. Accordingly, we also found that MnTE-2-PyP reduced the expression of three genes regulated by HIF-1β and/or CREB: TGF-β2, FGF-1 and PAI-1. Specifically, MnTE-2-PyP decreased p300 complex binding to a specific HRE motif within the PAI-1 gene promoter region, suppressed H3K9 acetylation, and consequently, repressed PAI-1 expression. Mechanistically, less p300 transcriptional complex binding is not due to the reduction of binding between p300 and HIF-1/CREB transcription factors, but through inhibiting the binding of HIF-1/CREB transcription factors to DNA. Our data provide an in depth mechanism by which MnTE-2-PyP reduces prostate cancer growth and metastasis, which validates the clinical use of MnTE-2-PyP as a radio-protector to enhance treatment outcomes in prostate cancer radiotherapy.

MnTE-2-PyP modulates thiol oxidation in a hydrogen peroxide-mediated manner in a human prostate cancer cell.

To improve the treatment of advanced prostate cancer, the development of effective and innovative antitumor agents is needed. Our previous work demonstrated that the ROS (reactive oxygen species) scavenger, MnTE-2-PyP, inhibited human prostate cancer growth and also inhibited prostate cancer migration and invasion. We showed that MnTE-2-PyP treatment altered the affinity of the histone acetyltransferase enzyme, p300, to bind to DNA. We speculate that this may be one mechanism by which MnTE-2-PyP inhibits prostate cancer progression. Specifically, MnTE-2-PyP decreased p300/HIF-1/CREB complex (p300/hypoxia-inducible factor-1/cAMP response element-binding protein) binding to a specific hypoxia-response element (HRE) motif within the plasminogen activator inhibitor-1 (PAI-1) gene promoter region, and consequently, repressed PAI-1 expression. However, it remains unclear how MnTE-2-PyP reduces p300 complex binding affinity to the promoter region of specific genes. In this study, we found that overexpression of Cu/ZnSOD (superoxide dismutase 1, SOD1) significantly suppressed PAI-1 gene expression and p300 complex binding to the promoter region of PAI-1 gene, just as was observed in cells treated with MnTE-2-PyP. Furthermore, catalase (CAT) overexpression rescued the inhibition of PAI-1 expression and p300 binding by MnTE-2-PyP. Taken together, the above findings suggest that hydrogen peroxide (H2O2) is likely the mediator through which MnTE-2-PyP inhibits the PAI-1 expression and p300 complex binding in PC3 cells. To confirm this, we measured the production of H2O2 following overexpression of SOD1 or catalase with MnTE-2-PyP treatment in the presence or absence of radiation. We found that MnTE-2-PyP increased the intracellular steady-state levels of H2O2 and increased nuclear H2O2 levels. As expected, catalase overexpression significantly decreased the levels of intracellular H2O2 induced by MnTE-2-PyP. We then determined if this increased H2O2 production could result in oxidized protein thiol groups. In the presence of MnTE-2-PyP, there was a significant increase in oxidized thiols in PC3 cell lysates and this was reversed with catalase overexpression. Specifically, we showed that p300 was oxidized after MnTE-2-PyP treatment, indicating that MnTE-2-PyP is creating a more oxidizing environment and this is altering the oxidation state of p300 thiol residues. Our data provide an in depth mechanism by which MnTE-2-PyP regulates gene transcription through induced H2O2 mediated oxidation of particular proteins, supporting an important role for MnTE-2-PyP as an effective and innovative antitumor agent to enhance treatment outcomes in prostate cancer radiotherapy.

MnTE-2-PyP Treatment, or NOX4 Inhibition, Protects against Radiation-Induced Damage in Mouse Primary Prostate Fibroblasts by Inhibiting the TGF-Beta 1 Signaling Pathway

Prostate cancer patients who undergo radiotherapy frequently suffer from side effects caused by radiation-induced damage to normal tissues adjacent to the tumor. Exposure of these normal cells during radiation treatment can result in tissue fibrosis and cellular senescence, which ultimately leads to postirradiation-related chronic complications including urinary urgency and frequency, erectile dysfunction, urethral stricture and incontinence. Radiation-induced reactive oxygen species (ROS) have been reported as the most potent causative factor for radiation damage to normal tissue. While MnTE-2-PyP, a ROS scavenger, protects normal cells from radiation-induced damage, it does not protect cancer cells during radiation treatment. However, the mechanism by which MnTE-2-PyP provides protection from radiation-induced fibrosis has been unclear. Our current study reveals the underlying molecular mechanism of radiation protection by MnTE-2-PyP in normal mouse prostate fibroblast cells. To investigate the role of MnTE-2-PyP in normal tissue protection after irradiation, primary prostate fibroblasts from C57BL/6 mice were cultured in the presence or absence of MnTE-2-PyP and exposed to 2 Gy of X rays. We found that MnTE-2-PyP could protect primary prostate fibroblasts from radiation-induced activation, as measured by the contraction of collagen discs, and senescence, detected by beta-galactosidase staining. We observed that MnTE-2-PyP inhibited the TGF-β-mediated fibroblast activation pathway by downregulating the expression of TGF-β receptor 2, which in turn reduced the activation and/or expression of SMAD2, SMAD3 and SMAD4. As a result, SMAD2/3-mediated transcription of profibrotic markers was reduced by MnTE-2-PyP. Due to the inhibition of the TGF-β pathway, fibroblasts treated with MnTE-2-PyP could resist radiation-induced activation and senescence. NADPH oxidase 4 (NOX4) expression is upregulated after irradiation and produces ROS. As was observed with MnTE-2-PyP treatment, NOX4–/– fibroblasts were protected from radiation-induced fibroblast activation and senescence. However, NOX4–/– fibroblasts had reduced levels of active TGF-β1, which resulted in decreased TGF-β signaling. Therefore, our data suggest that reduction of ROS levels, either by MnTE-2-PyP treatment or by eliminating NOX4 activity, significantly protects normal prostate tissues from radiation-induced tissue damage, but that these approaches work on different components of the TGF-β signaling pathway. This study proposes a crucial insight into the molecular mechanism executed by MnTE-2-PyP when utilized as a radioprotector. An understanding of how this molecule works as a radioprotector will lead to a better controlled mode of treatment for post therapy complications in prostate cancer patients.

The Addition of Manganese Porphyrins during Radiation Inhibits Prostate Cancer Growth and Simultaneously Protects Normal Prostate Tissue from Radiation Damage

Radiation therapy is commonly used for prostate cancer treatment; however, normal tissues can be damaged from the reactive oxygen species (ROS) produced by radiation. In separate reports, we and others have shown that manganese porphyrins (MnPs), ROS scavengers, protect normal cells from radiation-induced damage but inhibit prostate cancer cell growth. However, there have been no studies demonstrating that MnPs protect normal tissues, while inhibiting tumor growth in the same model. LNCaP or PC3 cells were orthotopically implanted into athymic mice and treated with radiation (2 Gy, for 5 consecutive days) in the presence or absence of MnPs. With radiation, MnPs enhanced overall life expectancy and significantly decreased the average tumor volume, as compared to the radiated alone group. MnPs enhanced lipid oxidation in tumor cells but reduced oxidative damage to normal prostate tissue adjacent to the prostate tumor in combination with radiation. Mechanistically, MnPs behave as pro-oxidants or antioxidants depending on the level of oxidative stress inside the treated cell. We found that MnPs act as pro-oxidants in prostate cancer cells, while in normal cells and tissues the MnPs act as antioxidants. For the first time, in the same in vivo model, this study reveals that MnPs enhance the tumoricidal effect of radiation and reduce oxidative damage to normal prostate tissue adjacent to the prostate tumor in the presence of radiation. This study suggests that MnPs are effective radio-protectors for radiation-mediated prostate cancer treatment.

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.