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 HPV 16 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 (DeltaPsim) by flow cytometry, and protein expression by immunoblotting. TPZ exhibited high cytotoxicity, especially in hypoxia (2% O2), for all four p53-functional neuroblastoma cell lines, achieving >3 logs of cell kill (LC99 < or = 0.7 microg/mL). In p53-nonfunctional neuroblastoma cell lines, all TPZ LC99 values were >3.0 microg/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 DeltaPsim 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 DeltaPsim loss and GSH depletion, but neither N-acetylcysteine nor Boc-d-fmk inhibited apoptosis caused by TPZ. In response to TPZ, DeltaPsim 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, DeltaPsim 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.
Yang
Bo
B
Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children's Hospital Los Angeles, California.
Reynolds
C Patrick
CP
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001593:Benzyl Compounds / Q000494:pharmacology; D015153:Blotting, Western; D061945:Caspase Inhibitors; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D000077704:Tirapazamine; D014227:Triazines / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D001593:Benzyl Compounds; C476029:Boc-D-FMK; C493956:CDKN1A protein, human; D061945:Caspase Inhibitors; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006845:Hydrocarbons, Fluorinated; D019253:Proto-Oncogene Proteins c-bcl-2; D014227:Triazines; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000077704:Tirapazamine; D005978:Glutathione