Tirapazamine cytotoxicity for neuroblastoma is p53 dependent.

Abstract

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.

• Authors: Bo Yang, C Patrick Reynolds
• Journal: Clinical cancer research : an official journal of the American Association for Cancer Research (Clin Cancer Res) Vol. 11 Issue 7 Pg. 2774-80 (Apr 01 2005) ISSN: 1078-0432 [Print] United States
• PMID: 15814660 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antineoplastic Agents
• BCL2L1 protein, human
• Benzyl Compounds
• Boc-D-FMK
• CDKN1A protein, human
• Caspase Inhibitors
• Cell Cycle Proteins
• Cyclin-Dependent Kinase Inhibitor p21
• Hydrocarbons, Fluorinated
• Proto-Oncogene Proteins c-bcl-2
• Triazines
• Tumor Suppressor Protein p53
• bcl-2-Associated X Protein
• bcl-X Protein
• Tirapazamine
• Glutathione

Topics

• Antineoplastic Agents (pharmacology)
• Apoptosis (drug effects)
• Benzyl Compounds (pharmacology)
• Blotting, Western
• Caspase Inhibitors
• Cell Cycle Proteins (metabolism)
• Cell Line, Tumor
• Cell Survival (drug effects)
• Cyclin-Dependent Kinase Inhibitor p21
• Dose-Response Relationship, Drug
• Flow Cytometry
• Glutathione (metabolism)
• Humans
• Hydrocarbons, Fluorinated (pharmacology)
• Intracellular Membranes (drug effects, physiology)
• Membrane Potentials (drug effects)
• Mitochondria (drug effects, physiology)
• Neuroblastoma (metabolism, pathology)
• Proto-Oncogene Proteins c-bcl-2 (metabolism)
• Tirapazamine
• Triazines (pharmacology)
• Tumor Suppressor Protein p53 (metabolism)
• bcl-2-Associated X Protein
• bcl-X Protein