Abstract Many natural compounds have been shown to possess anticancer properties against breast cancer cells in vitro and/or in vivo, and there is increased interest in understanding the effects and mechanisms of action of such products as an alternative to traditional chemotherapy. Oleuropein is a polyphenolic compound derived from olive leaves that has demonstrated antioxidant and anticancer properties in breast cancer and other cancers. Punicic acid is a poly-5-unsaturated fatty acid found in pomegranate seeds that has also shown antioxidant and anticancer effects in various cancers. The MCF-7 cell line is an important model for estrogen receptor-positive (ER+) breast cancer which is useful for studying the effects and mechanisms of these natural products. We sought to compare oleuropein and punicic acid in this model and hypothesized that both would induce cytotoxicity in MCF-7 cells by increasing the oxidative stress and disrupting mitochondrial function. Cells were treated with 50 or 200 ug/ml oleuropein, 10 or 50 ug/ml punicic acid, or 70% ethanol as a control for 72 hours. Cytotoxicity was measured using an lactate dehydrogenase (LDH) release assay, and oxidative stress was assessed using the MitoSOX assay for mitochondrial ROS and malondialdehyde (MDA) quantification for lipid peroxidation. We found that oleuropein induced a significant increase in LDH release, and morphological changes consistent with apoptosis, but no significant increase in ROS. Punicic acid induced a dose-dependent increase in LDH release, and additionally led to an increase in mitochondrial ROS and lipid peroxidation. Since we previously reported significant induction of expression of the Peroxiredoxin antioxidant genes by both oleuropein and punicic acid in these cells, we also set out to examine the effects of Prdx suppression on susceptibility to these compounds. Using transient siRNA transfections targeting each of the six Prdx genes, we found that suppression of Prdx2, Prdx3, Prdx4 and Prdx5 significantly increased MCF-7 susceptibility to punicic acid, but not to oleuropein. Together, our data suggest that these compounds induce cytotoxicity through different mechanisms, with oxidative stress playing a major role in punicic acid-induced toxicity. Targeting peroxiredoxins may be an effective strategy for increasing breast cancer cell susceptibility to these compounds in future therapeutic applications. Citation Format: Samantha Diiorio, Braden Quitmeyer, Shelley A. Phelan. Differential mechanisms of MCF-7 cytotoxicity by oleuropein and punicic acid and the role of oxidative stress and peroxiredoxin antioxidants abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3646.
Diiorio et al. (Fri,) studied this question.