title: Antagonism of buthionine sulfoximine cytotoxicity for human neuroblastoma cell lines by hypoxia is reversed by the bioreductive agent tirapazamine.
abstract: Relapse of neuroblastoma (NB) commonly occurs in hypoxic tissues. Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, is cytotoxic for NB cell lines in atmospheric oxygen (20% O(2)). Tirapazamine (TPZ) is a bioreductive agent that forms a toxic-free radical in hypoxia. We determined in four NB cell lines cytotoxicity using the DIMSCAN digital imaging fluorescence assay, glutathione (GSH) levels by the DTNB-GSSG reductase method, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (Delta psi(m)) by flow cytometry. Hypoxia (2% O(2)) antagonized BSO-mediated ROS, apoptosis, and cytotoxicity but not GSH depletion. TPZ synergistically enhanced BSO cytotoxicity in hypoxia for all four NB cell lines, achieving 2-4 logs of cell kill. BSO depleted GSH (8-42% of controls) in 20 and 2% O(2), whereas TPZ only decreased GSH in hypoxia. Maximal GSH depletion was induced by BSO + TPZ. N-acetylcysteine abrogated GSH depletion caused by TPZ but not by BSO. BSO increased ROS, decreased Delta psi(m), and caused apoptosis in 20% O(2) (but not in 2% O(2)). TPZ elevated ROS in 2% O(2) (but not in 20% O(2)), whereas BSO + TPZ increased ROS both in 20 and 2% O(2). In hypoxia, TPZ alone or TPZ + BSO caused an 80% decrease of Delta psi(m) at 24 h, preceding apoptosis in 74-86% of cells at 48 h. Thus, hypoxia significantly antagonizes BSO-mediated cytotoxicity for NB cell lines, but TPZ reversed the inhibition of BSO-mediated cytotoxicity in hypoxia, causing increased ROS, Delta psi(m) decrease, GSH depletion, apoptosis, and synergistic cytotoxicity. These data additionally define the role of ROS in BSO-mediated cytotoxicity and suggest that combining BSO with TPZ could have clinical activity against NB in hypoxic sites.
authors.affiliation: Division of Hematology-Oncology, Children's Hospital Los Angeles, California 90027, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Nino
authors.initials: N
authors.lastname: Keshelava
authors.forename: Clarke P
authors.initials: CP
authors.lastname: Anderson
authors.forename: C Patrick
authors.initials: CP
authors.lastname: Reynolds
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D019328:Buthionine Sulfoximine / Q000008:administration & dosage / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology; D004357:Drug Synergism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D000077704:Tirapazamine; D014227:Triazines / Q000008:administration & dosage / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D017382:Reactive Oxygen Species; D014227:Triazines; D000077704:Tirapazamine; D019328:Buthionine Sulfoximine; D005978:Glutathione; D010100:Oxygen