Synergistic Effects of Doxorubicin and Quercetin on ROS-Associated Apoptosis and EGFR/FOXP3 Modulation in OVCAR3...

Background/Objectives: Combination strategies involving natural compounds are increasingly being evaluated to improve the efficacy and safety of conventional chemotherapeutic agents. Quercetin (Q), a bioactive flavonoid, has been reported to regulate oxidative stress and apoptosis-associated signaling pathways. This study investigated whether Q enhances doxorubicin (DOX)-mediated cytotoxicity in OVCAR3 ovarian cancer cells, with particular emphasis on apoptosis, oxidative stress, and EGFR/FOXP3 signaling, while also assessing relative toxicity in HaCaT non-tumoral keratinocytes. Methods: Cell viability was determined using the MTT assay, and drug interactions were assessed according to the Combination Index (CI) method. Apoptosis was evaluated by Annexin V/PI flow cytometry. Caspase-3 and caspase-9 activities were measured using colorimetric assays. Intracellular reactive oxygen species (ROS) production was analyzed using the DCFH-DA assay. EGFR and FOXP3 gene expression levels were quantified by qRT-PCR, whereas caspase-9 protein expression was assessed immunocytochemically. Results: The DOX+Q combination produced synergistic cytotoxic effects in OVCAR3 cells (CI < 1). Compared with OVCAR3 cells, HaCaT cells displayed higher IC₅₀ values following DOX treatment (7.03 µM vs. 1.42 µM) and Q treatment (183.92 µM vs. 35.94 µM), indicating relatively lower treatment sensitivity and suggesting a potentially favorable selectivity tendency; however, these findings should be regarded as preliminary. Flow cytometric findings demonstrated markedly increased proportions of both early and late apoptotic cells following combination treatment. Caspase-3 and caspase-9 activities were significantly elevated after combined exposure (p < 0.01). ROS production increased substantially in response to DOX+Q treatment, corresponding to an approximately 6.82-fold elevation relative to the control group. qRT-PCR analysis demonstrated reduced EGFR and FOXP3 mRNA expression levels in the combination-treated group. Immunocytochemical evaluation additionally revealed stronger caspase-9 staining intensity in treated OVCAR3 cells. Conclusions: These findings suggest that Q may potentiate DOX-induced cytotoxicity through mechanisms associated with enhanced oxidative stress, activation of apoptotic pathways, and modulation of proliferative signaling. The comparatively lower sensitivity observed in HaCaT cells may indicate a possible selectivity tendency; however, these observations remain preliminary and require further validation through in vivo and translational studies.