Selective Tumor Cytotoxicity via Singlet Oxygen: Investigating Eosinophil Peroxidase and Myeloperoxidase in Cancer Therapy.

INTRODUCTION: Eosinophil peroxidase (EPO) and myeloperoxidase (MPO) are large, cationic enzymes secreted by granulocytes that bind preferentially to negatively charged cancer cell membranes generated by Warburg metabolism. In the presence of halide cofactors and hydrogen peroxide (H2O2), and under acidic conditions that potentiate catalysis, they generate singlet oxygen (1O2*), a metastable oxygen state with a microsecond lifetime and hundred nanometer-scale diffusion radius. This confines cytotoxicity to enzyme-bound surfaces, producing a spatially restricted therapeutic effect. METHODS: Human bladder cancer cell lines (5637, T24) and normal urothelial cells (SV-HUC1) were treated with porcine EPO or porcine MPO aggregate formulations in acidic medium (pH 5.3). Activation occurred when 10 millimolar (mM) H2O2 was added immediately prior to cell contact. Viability was assessed by MTS assay, and IC50 values were determined by nonlinear regression. Mixed cultures of GFP+ SV-HUC1 and mCherry+ malignant cells were analyzed by fluorescence microscopy and flow cytometry. MPO binding was assessed by immunofluorescence, and DNA damage was evaluated by Western blotting for γH2AX and phospho-ATM. Independent toxicity of individual components was also tested. RESULTS: Aggregate formulations selectively eliminated bladder cancer cells while sparing SV-HUC1. IC50 values were in the nanomolar haloperoxidase range for malignant cells, with SV-HUC1 remaining viable at concentrations up to 200 nM. In mixed cultures, malignant cells were preferentially eliminated, while GFP+ SV-HUC1 remained intact. Immunofluorescence confirmed MPO binding to malignant membranes, and DNA damage markers were induced only in cancer cells. Component testing showed no cytotoxicity from enzymes, cofactors, or 10 mM H2O2 alone; only higher peroxide concentrations produced injury. CONCLUSION: Selective cytotoxicity arises from concurrent enzyme binding and 1O2* generation in mild acidic conditions rather than from direct peroxide toxicity. Haloperoxidase therapy thus offers a precise, contact-driven approach for post-bulk tumor treatment in non-muscle invasive bladder cancer.