Tirapazamine cytotoxicity for neuroblastoma is p53 dependent

Relapse of neuroblastoma commonly occurs in hypoxic tissues, and is associated with an acquired and sustained high-level drug resistance, often due to p53 loss of function. Abrogating p53 function with HPV16 E6 transduction in drug-sensitive neuroblastoma cell lines caused high-level drug resistance. Tirapazamine (TPZ) is a bioreductive agent that forms a toxic free radical in hypoxia. We determined in six neuroblastoma cell lines the cytotoxicity of TPZ using DIMSCAN, a digital imaging fluorescence assay, apoptosis and mitochondrial membrane potential (Delta Psi(m)) by flow cytometry, and protein expression by immunoblotting. TPZ exhibited high cytotoxicity, especially in hypoxia (2% O-2), for all four p53-functional neuroblastoma cell lines, achieving >3 logs of cell kill (LC99 <= 0.7 mu g/mL). In p53-nonfunctional neuroblastoma cell lines, all TPZ LC99 values were >3.0 mu g/mL (average clinically achievable level). TPZ (24 hours) induced apoptosis in >46% of cells in p53-functional cell lines but failed to cause apoptosis in p53 nonfunctional cell lines. Induction of p53 and p21 expression by TPZ was observed in a p53-functional cell line (SMS-SAN) but not in a p53-nonfunctional cell line (CHLA-90). Significant Delta Psi(m) loss and glutathione (GSH) depletion in response to TPZ was observed in p53-functional cell lines (SMS-SAN, SMS-SAN EV, and CHLA-15) but not in p53-nonfunctional cell lines (SMS-SAN E6 and CHLA-90). N-Acetylcysteine inhibited TPZ-mediated Delta Psi(m) loss and GSH depletion, but neither N-acetylcysteine nor Boc-d-fmk inhibited apoptosis caused by TPZ. In response to TPZ, Delta Psi(m) loss preceded apoptosis. Thus, TPZ cytotoxicity for neuroblastoma cell lines in hypoxia occurred via a p53-dependent mitochondrial pathway that caused induction of p53 and p21, Delta Psi(m) decrease, GSH depletion, and apoptosis. These data further define the mechanism of action of TPZ and suggest that as a single agent, TPZ would only have clinical activity against p53-functional neuroblastomas.