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Antagonism of Buthionine Sulfoximine Cytotoxicity for Human Neuroblastoma Cell Lines by Hypoxia Is Reversed by the Bioreductive Agent Tirapazamine¹

Division of Hematology-Oncology, Children’s Hospital Los Angeles and Departments of Pediatrics [C. P. A., C. P. R.] and Pathology [C. P. R.], University of Southern California, Keck School of Medicine, Los Angeles, California 90027

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% O₂). 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 (_m) by flow cytometry. Hypoxia (2% O₂) 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% O₂, 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 _m, and caused apoptosis in 20% O₂ (but not in 2% O₂). TPZ elevated ROS in 2% O₂ (but not in 20% O₂), whereas BSO + TPZ increased ROS both in 20 and 2% O₂. In hypoxia, TPZ alone or TPZ + BSO caused an 80% decrease of _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, _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.

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