312 - The Mechanism of MnTE-2-PyP Functioning Differently in Cancer Cells and Normal Cells

Abstract

Rationale: Radiotherapy reduces prostate cancer growth but patients suffer from side effects caused by the accumulation of reactive oxygen species. MnTE-2-PyP5+ reduces radiation induced side effects, while simultaneously inhibiting cancer progression. We hypothesize that MnTE-2-PyP increases H2O2 levels in cancer cells but not normal cells due to the difference in redox capacities between the two cell types. Methods Three cell lines, PC3, LNCaP, primary mouse fibroblasts(PMF) were treated with PBS or 30µM MnTE-2-PyP. A Ros-Glo assay was performed to measure H2O2 levels in MnTE-2- PyP treated or untreated cells. In order to measure protein oxidation in PC3 cells treated with MnTE-2-PyP, biotinylated iodoacetamide (BIAM) probe was used which binds only to reduced thiol groups. A western for streptavidin was performed to identify proteins containing reduced thiols. Mass Spec was performed to determine protein contents difference in both drug treated or untreated cells. An in gel catalase activity assay was performed for cells treated with or without MnTE-2-PyP. The EPR assay was conducted to detect the basal levels of O2– in 3 cell lines. Results MnTE-2-PyP significantly increased H2O2 level in PC3 cells and LNCaP cells as compared to PBS treated cells but no in normal primary fibroblasts. Accordingly, we found that the increased H2O2 in the cancer cells caused global oxidation of thiols in many different proteins. MnTE-2-PyP treatment increased the amount of oxidized proteins in PC3 cells but not in PMF. Mass spectrometry was performed to identify oxidized proteins in the presence of MnTE-2-PyP. Preliminary results have identified two proteins that are oxidized with MnTE-2-PyP treatment: serine/threonine-protein phosphatase PP1-beta catalytic subunit and Pre-mRNA-processing factor 19. One explanation for why more H2O2 is produced in cancer cells vs. normal cells is that normal cells may have more active H2O2 removing enzymes than cancer cells, like catalase. However, we found that LNCaP cells have the highest catalase activity than PMF and PC3. But MnTE-2-PyP did not change the catalase activity in any kind of cell line. We also measured basal levels of superoxide production in cancer vs. normal cells, since MnTE-2-PyP scavenges superoxide to produce H2O2. We found that PMF have higher basal superoxide radicals than PC3 cells and LNCaP cells. Conclusion MnTE-2-PyP increases H2O2 levels in cancer cells but not normal cells, which leads to the oxidation of proteins in PC3 cells. We hypothesized that this difference in H2O2 production could be due to the fact that cancer cells have a reduced ability to remove H2O2; however, our preliminary data do not support this hypothesis. There are other important H2O2 removing enzymes that we still need to measure to obtain the over antioxidant capacity of the cells.