Background/objectives: Hypoxia in the tumor microenvironment is linked to aggressiveness, epithelial–mesenchymal transition, metastasis, and therapy resistance. Targeting hypoxia to enhance antitumor immunity is crucial for overcoming therapeutic resistance. Here, we investigated the ability of Evofosfamide, a prodrug that gets activated under hypoxic conditions, to sensitize breast cancer cells to cell death. Evofosfamide is converted into bromo-isophosphoramide mustard, a potent DNA cross-linking agent that is expected to enhance the killing of cancer cells under hypoxic conditions, where these cells typically exhibit resistance. Methods: Representative breast cancer cell lines, MCF-7 and MDA-MB-231, were treated with Evofosfamide under normoxia and hypoxia. Changes in cell viability and the mechanism of cell death were measured using neutral red dye uptake, Annexin-FITC/propidium iodide staining, and Western blot analysis of markers—PARP1 and caspase 3/7. We tested Evofosfamide’s ability to counteract hypoxic suppression of type I Interferon signaling genes using quantitative PCR (qPCR), as well as its capacity to trigger natural killer (NK)-cell-mediated cytotoxicity. Results: Evofosfamide enhanced cell killing in both MCF-7 and MDA-MB-231 cells under hypoxic conditions compared to normoxic conditions. Cell killing was accompanied by increased cellular reactive oxygen species (ROS), diminished mitochondrial membrane potential, and induction of apoptosis, as demonstrated by the fragmentation or laddering of genomic DNA, the activation of caspase 3/7, and the cleavage of PARP. qPCR analysis revealed that Evofosfamide was capable of restoring type I interferon signaling in hypoxic breast cancer cells, leading to the subsequent cytolytic activity of NK cells against the tumor cells. Conclusions: Thus, conditioning the breast cancer cells with Evofosfamide resulted in enhanced cell killing under hypoxia, further underscoring its potential as a sensitizer to target hypoxia-driven tumors.

Waterpipe smoking has emerged as a major global health concern, with incompletely characterized effects on bronchial epithelial cells. In this study, we investigated the cytotoxic and adaptive responses of non-tumorigenic human bronchial epithelial BEAS-2B cells exposed to waterpipe smoke condensate (WPSC). Increasing concentrations of WPSC reduced cell viability in a dose-dependent manner; however, exposure to 0.1% WPSC caused only a modest, transient reduction in viability that was no longer significant by 72 hrs and did not significantly induce apoptosis over 72 hrs, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Annexin V/Propidium Iodide (PI) apoptosis assays. To elucidate the adaptive mechanisms underlying this resistance, we examined autophagic flux and found that WPSC induced time-dependent accumulation of LC3-II and p62, which was enhanced by bafilomycin A1 cotreatment, indicating the activation of autophagy. This was accompanied by increased phosphorylation of AMPK and decreased phosphorylation of mTORC1, consistent with an autophagy-mediated stress response. Functionally, the inhibition of autophagy with bafilomycin A1 increased apoptosis and necrosis, suggesting that autophagy could play a protective role. Moreover, WPSC exposure induced DNA damage, as evidenced by increased γH2AX and 53BP1 nuclear foci, and elevated proinflammatory cytokine expression (IL-1β and CCL2). Finally, WPSC treatment upregulated the expression of the cancer stem cell-associated markers CD44 and CD133. Collectively, our findings demonstrate that subcytotoxic exposure to waterpipe smoke activates autophagy in non-tumorigenic lung epithelial cells. This adaptive mechanism may represent an early cellular event linking waterpipe smoke exposure to lung carcinogenesis.

Ferroptosis has emerged as a promising strategy to overcome resistance to conventional cancer therapies. Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia, therapy resistance, and an immunosuppressive microenvironment. Although hypoxia is likely to influence ferroptosis susceptibility and the associated inflammatory pathways that regulate antitumor immunity, their impact on ferroptosis sensitivity and innate immune responses remains poorly understood. In this study, we investigated the effects of hypoxia on the induction of ferroptosis and immune-related signaling in PDAC cell lines. We examined how hypoxia affects the responses of Panc-1, BxPC3, and Capan-1 cells to the ferroptosis inducers RAS-selective lethal 3 (RSL3)/Imidazole ketone erastin (IKE) under normoxic and hypoxic (0.1% O₂) conditions. Cell viability assays were used to assess ferroptosis sensitivity, and rescue experiments were performed using liproxstatin-1 (LIP). Gene expression analysis was conducted to evaluate changes in immune, interferon, inflammatory, and hypoxia-related genes following ferroptosis induction. Panc-1 cells were the most sensitive, whereas Capan-1 cells were resistant, particularly under hypoxia. Ferroptosis triggered cell line-specific responses involving interferon signaling, inflammation, and stress pathways. Panc-1 cells showed over-expression of RIG-I, MAVS, IRF3/7/9, STAT1/2, and CXCL10, particularly under hypoxia, indicating activation of Type I interferon (IFN)-associated transcriptional program. BxPC3 cells demonstrated broader cytokine induction, including IL-8, CCL2, CXCL2, GM-CSF, and IL-11, whereas Capan-1 cells were minimally responsive. Hypoxia also increased ANGPTL4 and GDF15 expression following ferroptosis induction. These findings show that hypoxia differentially affects ferroptosis sensitivity and immune responses in PDAC, revealing complex interactions among ferroptosis, innate immunity, and the tumor microenvironment.