Hypoxia increases chemoresistance in human medulloblastoma DAOY cells via hypoxia-inducible factor 1-mediated downregulation of the CYP2B6, CYP3A4 and CYP3A5 enzymes and inhibition of cell proliferation

Medulloblastomas are among the most frequently diagnosed pediatric solid tumors, and drug resistance remains as the principal cause of treatment failure. Hypoxia and the subsequent activation of hypoxia-inducible factor 1 (HIF-1) are considered key factors in modulating drug antitumor effectiveness, but the underlying mechanisms in medulloblastomas have not yet been clearly understood. The aim of the present study was to determine whether hypoxia induces resistance to cyclophosphamide (CPA) and ifosfamide (IFA) in DAOY medulloblastoma cells, whether the mechanism is dependent on HIF-1, and whether involves the modulation of the expression of cytochromes P450 (CYP)2B6, 3A4 and 3A5 and the control of cell proliferation. Monolayer cultures of DAOY medulloblastoma cells were exposed for 24 h to moderate (1% O-2) or severe (0.1% O-2) hypoxia, and protein expression was evaluated by immunoblotting. Cytotoxicity was studied with the MTT assay and by Annexin V/PI staining and flow cytometry. Cell proliferation was determined by the trypan-blue exclusion assay and cell cycle by propidium iodide staining and flow cytometry. Hypoxia decreased CPA and IFA cytotoxicity in medulloblastoma cells, which correlated with a reduction in the protein levels of CYP2B6, CYP3A4 and CYP3A5 and inhibition of cell proliferation. These responses were dependent on hypoxia-induced HIF-1 activation, as evidenced by chemical inhibition of its transcriptional activity with 2-methoxyestradiol (2-ME), which enhanced the cytotoxic activity of CPA and IFA and increased apoptosis. Our results indicate that by stimulating HIF-1 activity, hypoxia downregulates the expression of CYP2B6, CYP3A4 and CYP3A5, that in turn leads to decreased conversion of CPA and IFA into their active forms and thus to diminished cytotoxicity. These results support that the combination of HIF-1 inhibitors and canonical antineoplastic agents provides a potential therapeutic alternative against medulloblastoma.