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.