249 - Redox Proteomics of 4T1 Breast Cancer Cell After Treatment with MnTE-2-PyP5+/Ascorbate System

Abstract

We have recently demonstrated that cationic Mn(III) N-substituted pyridylporphyrins (MnPs) couple with ascorbate (Asc) thereby inducing cancer cell death. Mechanism of such anticancer effect has been attributed to the tumoricidal H 2 O 2 production as a result of MnP-catalyzed Asc oxidation. Among MnPs explored, that differ vastly with regards to their redox properties, charge, size/bulkiness and lipophilicity, MnTE-2-PyP 5+ (Mn1) was identified as the best catalyst of Asc oxidation and demonstrated the highest ability to promote cancer cell death. Herein we have analyzed oxidative modifications of proteins via mass spectrometry-based redox proteomics in 4T1 mouse breast cancer cells treated with Mn1/Asc system. Cells were treated for 4 hours in a growth medium with 5 µM Mn1 and 1 mM ascorbate - an optimized treatment scheme that had shown the antitumor effect in vivo. Such treatment affected 3605 peptidyl cysteins (Cys) in total, out of which 1577 were oxidized 1.3-fold or higher compared with control, untreated samples. Distribution analyses of Mn1/Asc-induced oxidation of peptidyl Cys show that >50% of oxidized peptidyl Cys bear 1.3-2.0 fold oxidation level. MnP/Asc system affected 1762 proteins in total, among which 942 proteins were associated with >1.3-fold oxidized peptidyl Cys. Analysis of potential functional pathways revealed that Mn1/Asc oxidized peptides are associated with regulation of cytoskeleton rearrangement, transcription – mRNA processing, translation, protein folding, cell cycle and adhesion. As expected, analyses of total thiol levels and GSSG/GSH and Cys/CySS ratios in treated vs control cells revealed significant oxidative burden imposed by Mn1/Asc system. Evaluation of biological networks affected by Mn1/Asc system using Analyze network algorithm revealed NF-κB-associated pathway as a key affected network. When tested in 4T1 flank mouse tumor model, Mn1/Asc treatment alone or even more so in combination with radiation, induced significant tumor growth delay Support: NIH 1R03-NS082704-01, 5-P30-CA14236-29, NIH R01 ES023485 and R21 ES 025632.