Abstract
<title>Abstract</title> <p>Hypoxia is a defining feature of the tumor microenvironment and is classically associated with chemoresistance through HIF-1α-driven adaptive signaling. However, whether mild intermittent oxygen fluctuation, rather than chronic severe hypoxia, can sensitize breast cancer cells to chemotherapy remains unclear. Here, we investigated the effects of mild intermittent hypoxia on tumor progression and chemosensitivity using complementary in vitro and in vivo models. In SKBR3 and MDA-MB-231 breast cancer cells, hypoxia was chemically induced with CoCl₂ (100–300 µM), while TNF-α was added to mimic the inflammatory component of the tumor microenvironment, thereby recreating hypoxic and inflammatory conditions associated with the development of chemotherapy resistance. Under these conditions, HIF-1α and ABCB1 (P-glycoprotein), a major ATP-dependent drug efflux transporter implicated in multidrug resistance, were upregulated in a concentration-dependent manner, and this hypoxia-associated response persisted following co-treatment with the chemotherapeutic agents doxorubicin (DOX) and 5-fluorouracil (5-FU). Notably, cells exposed to moderate hypoxia (200 µM CoCl₂) together with DOX/5-FU exhibited the greatest activation of apoptosis, as demonstrated by increased caspase-3 expression accompanied by reduced GLUT-1 expression, indicating enhanced chemotherapy-induced cytotoxicity under controlled hypoxic conditions. To evaluate these findings in vivo, mammary tumors were induced in rats using 7,12-dimethylbenz[a]anthracene (DMBA), a well-established model that recapitulates key pathological and molecular features of human breast cancer. Intermittent mild hypoxia (13% O₂, cyclic exposure) alone increased tumor size approximately twofold and upregulated HIF-1α, ABCB1 (P-glycoprotein), and BCL-2 compared with rats bearing DMBA-induced tumors maintained under normoxic conditions, consistent with activation of hypoxia-associated pro-survival pathways. In contrast, combining intermittent hypoxia with DOX/5-FU chemotherapy significantly reduced tumor growth compared with both the hypoxia-only and chemotherapy-only groups and produced the strongest caspase-3-associated apoptotic response. Kaplan–Meier survival analysis demonstrated a significant survival benefit of chemotherapy irrespective of oxygenation status (log-rank p = 0.0387), while body weight measurements indicated no evidence of treatment-related systemic toxicity. Collectively, these findings demonstrate that although mild intermittent hypoxia alone promotes pro-survival signaling characteristic of the hypoxic tumor microenvironment, its combination with chemotherapy enhances apoptotic cell death and suppresses tumor growth. These results support the concept that controlled hypoxic conditioning can remodel the tumor microenvironment in a manner that improves responsiveness to cytotoxic therapy, providing a potential strategy for overcoming hypoxia-associated chemoresistance in breast cancer.</p>