Tirapazamine cytotoxicity for neuroblastoma is p53 dependent
1Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children's Hospital Los Angeles, California.
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.
Insights
Tirapazamine (TPZ) shows high cytotoxicity against p53-functional neuroblastoma cells in hypoxia, acting through a p53-dependent mitochondrial pathway. TPZ is ineffective against p53-nonfunctional neuroblastoma, suggesting limited clinical activity as a single agent.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Neuroblastoma relapse often occurs in hypoxic tissues, linked to drug resistance from p53 loss.
- Human Papillomavirus 16 E6 transduction in neuroblastoma cells confers high-level drug resistance by abrogating p53 function.
- Tirapazamine (TPZ) is a hypoxia-activated bioreductive agent generating toxic free radicals.
Purpose of the Study:
- To investigate the cytotoxicity of TPZ in neuroblastoma cell lines under hypoxic conditions.
- To elucidate the p53-dependent mechanisms underlying TPZ's action, including apoptosis and mitochondrial pathways.
- To determine the potential clinical activity of TPZ as a single agent based on p53 functionality.
Main Methods:
- Cytotoxicity assays (DIMSCAN) were performed on six neuroblastoma cell lines.
- Apoptosis and mitochondrial membrane potential (DeltaPsim) were assessed by flow cytometry.
- Protein expression (p53, p21) and glutathione (GSH) levels were analyzed by immunoblotting and other assays.
Main Results:
- TPZ demonstrated high cytotoxicity (>3 logs cell kill) in hypoxia against p53-functional cell lines (LC99 ≤ 0.7 μg/mL).
- TPZ showed significantly lower cytotoxicity (LC99 > 3.0 μg/mL) in p53-nonfunctional cell lines.
- TPZ induced apoptosis, p53/p21 expression, DeltaPsim loss, and GSH depletion in a p53-dependent manner, with DeltaPsim loss preceding apoptosis.
Conclusions:
- TPZ's cytotoxicity in hypoxic neuroblastoma cells is mediated by a p53-dependent mitochondrial pathway involving p53/p21 induction, DeltaPsim decrease, GSH depletion, and apoptosis.
- These findings suggest TPZ as a single agent may only be effective against neuroblastomas with functional p53.
- The study defines TPZ's mechanism of action and highlights the importance of p53 status in predicting treatment response.
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