Physiologically relevant oxygen tensions reshape anticancer responses under oxidative stress-permissive culture conditions.

Tumor hypoxia is a hallmark of solid malignancies and an important determinant of therapeutic response; however, most in vitro studies are still performed under atmospheric oxygen conditions that poorly reflect physiological tissue oxygenation. Conventional culture systems also rarely account for iron availability and polyunsaturated fatty acids (PUFAs), two important modulators of oxidative stress-associated cytotoxicity. This study investigated how physiologically relevant oxygen tension influences anticancer responses to oxidative stress-modulating compounds under iron- and PUFA-enriched conditions. Human colorectal, lung, and pancreatic cancer cell lines, together with non-tumorigenic HaCaT cells, were cultured under physioxic (10% O2) and hypoxic (1% O2) conditions in the presence of transferrin-bound iron and linoleic acid and exposed to artemisinin (ART), dihydroartemisinin (DHA), honokiol (HNK), and doxycycline (DOXY). The anticancer responses were strongly oxygen- and cell line-dependent. DHA exerted the strongest antiproliferative activity, inducing marked G0/G1 arrest and pronounced apoptosis, particularly under hypoxia. ART and HNK also displayed oxygen-dependent apoptotic and oxidative stress-associated effects, whereas DOXY primarily induced caspase activation accompanied by comparatively weak apoptosis. All compounds increased mitochondrial reactive oxygen species generation, while DHA and ART most consistently enhanced lipid peroxidation. In contrast, GPX4 protein expression remained largely unchanged in most cell lines, suggesting functional rather than expression-level modulation of ferroptosis-associated pathways. Collectively, these findings demonstrate that physiologically relevant oxygen tension profoundly reshapes anticancer responses to redox-active compounds and highlight the importance of incorporating physiologically relevant oxygen conditions into experimental cancer models.