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.
背景/目的：肿瘤微环境中的缺氧状态与肿瘤侵袭性、上皮-间质转化、转移及治疗抵抗密切相关。靶向缺氧以增强抗肿瘤免疫对于克服治疗抵抗至关重要。本研究探讨了前药依沃福酰胺（一种在缺氧条件下被激活的药物）增强乳腺癌细胞对死亡敏感性的能力。依沃福酰胺在缺氧条件下转化为溴代异磷酰胺芥，这是一种强效DNA交联剂，有望在通常表现出抵抗性的缺氧癌细胞中增强其杀伤效果。 方法：在常氧和缺氧条件下，用依沃福酰胺处理代表性乳腺癌细胞系MCF-7和MDA-MB-231。采用中性红染料摄取法、Annexin-FITC/碘化丙啶染色法以及通过Western blot检测PARP1和caspase 3/7等标志物，测定细胞活力变化和细胞死亡机制。我们使用定量PCR（qPCR）检测了依沃福酰胺逆转缺氧对I型干扰素信号通路基因抑制的能力，以及其触发自然杀伤（NK）细胞介导的细胞毒性的能力。 结果：与常氧条件相比，依沃福酰胺在缺氧条件下增强了对MCF-7和MDA-MB-231细胞的杀伤作用。细胞杀伤伴随着细胞内活性氧（ROS）水平升高、线粒体膜电位降低以及细胞凋亡的诱导，这通过基因组DNA的片段化或梯状条带、caspase 3/7的激活以及PARP的裂解得到证实。qPCR分析显示，依沃福酰胺能够恢复缺氧乳腺癌细胞中的I型干扰素信号传导，从而引发NK细胞对肿瘤细胞后续的溶细胞活性。 结论：因此，用依沃福酰胺预处理乳腺癌细胞可在缺氧条件下增强细胞杀伤，进一步凸显了其作为靶向缺氧驱动肿瘤的增敏剂的潜力。
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