MnTE-2-PyP protects the prostate from radiation-induced fibrosis (RIF) by activating Nrf2/Nqo1/SIRT signaling axis

Prostate cancer treated with radiation therapy is accompanied by damage to healthy tissues, resulting in radiation induced-fibrosis (RIF) and a reduction in quality of life. Radiation exposure increases reactive oxygen species (ROS) that leads to molecular remodeling of fibroblast cells to induce RIF. Our work demonstrates that MnTE-2-PyP, a scavenger of superoxide molecules, prevents acute and chronic changes to prostate fibroblast cells that are responsible for the development of RIF. Fibroblast cells exposed to radiation (≥ 3Gy) show a decrease in the redox-sensitive nuclear Nrf2 protein levels without any change in Nrf2 mRNA expression. This correlates with a decrease in mRNA and protein levels of a Nrf2 downstream target, Nqo1, which is a cytoprotective protein. Therefore, we hypothesize that MnTE-2-PyP can prevent ROS mediated molecular remodeling of fibroblast cells in RIF by increasing Nrf2 activity and Nqo1 expression. We have confirmed that MnTE-2-PyP increases nuclear Nrf2 levels and causes an upregulation of Nqo1 expression (2.23-fold ± 0.41). Upregulation of Nqo1 expression correlates with a decrease in Keap1 levels that control Nrf2 degradation and translocation to the nucleus (p=0.0161). Inhibition of Nqo1 activity using Dicoumarol (50μM) or siRNA (10nM) significantly decrease cell viability (40-50%). Interestingly, Nqo1 upregulation and its cytoprotective function correlate with an increase in NAD + levels (2.24-fold ± 0.60). NAD + is an essential cofactor for sirtuin (SIRT) proteins that can deacetylate other proteins involved epigenetic remodeling of fibroblast cells. We have observed a significant increase in sirtuin activity in irradiated cells treated with MnTE-2-PyP (1.88-fold) as compared to no treatment. Therefore, we believe that activating the Nrf2 signaling pathway that causes an increase in Nqo1 expression, followed by an increase in NAD + levels and sirtuin activity by MnTE-2-PyP treatment, is responsible for prevention of ROS mediated molecular alterations in RIF.