55 - MnTE-2-PyP and NOX4 Deletion Protects Radiation Induced Damage in Mouse Primary Prostate Fibroblasts by Inhibiting TGFβ1 Signaling Pathway

Abstract

Patients with prostate cancer, receiving ionizing radiation, frequently suffer from radiation induce damage in normal tissues adjacent to the tumor. These irradiated normal cells lead to tissue fibrosis and senescence which ultimately leads to post-irradiation related chronic complexities like damage in prostate tissue, urinary urgency and frequency, erectile dysfunction, urethral stricture, and incontinence etc. Radiation-induced reactive oxygen species (ROS) was reported as the most potent causative factor for radiation damage to normal tissue. MnTE-2-PyP, a ROS scavenger, can protect the normal cell by radiation-induced damage whereas it can inhibit the growth of the cancer cell after irradiation, which is necessary for any radio protector. However, the mechanism by which MnTE-2-PyP protects from radiation-induced fibrosis is unclear. Our current study reveals the underlined 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 presence or absence of MnTE-2-PyP and exposed to 2 Gy of X- rays. Results of this study indicate that, MnTE-2-PyP can protect primary prostate fibroblasts from irradiation mediated activation as measured by contraction of collagen disc and radiation-induced senescence as detected by β-galactosidase staining. We observed that MnTE-2-PyP inhibits TGFβ mediated fibroblast activation pathway by reducing the expression of the TGF beta receptor II, which in turn reduces the activation and/or expression of SMAD2, SMAD3 and SMAD4. As a result, SMAD2/3 mediated transcription of pro-fibrotic markers is reduced by MnTE-2-PyP. Due to the inhibition of the TGFβ pathway, fibroblasts treated with MnTE-2-PyP can resist radiation induced activation and senescence of the prostate fibroblast. NADPH oxidase 4 (NOX4) produces ROS and its expression is upregulated after irradiation. 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 suggests that, reduction of ROS levels by MnTE-2-PyP or reducing NOX4 activity significantly protects normal prostate tissues from radiation induced tissue damage, but work on different components of the TGF-β signaling pathway. This study proposes a crucial insight for molecular mechanism executed by MnTE-2-PyP during its action as a radio protector. Understanding how this molecule works as a radio protector will lead to better and controlled mode of treatment during post irradiation complexities in prostate cancer patients.