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Metabolism of tirapazamine by multiple reductases in the nucleus
Y M Delahoussaye1, J W Evans, J M Brown
1Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University Medical School, Stanford, CA 94305-5152, USA.
Abstract:
Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, SR4233, Tirazone), a bioreductive drug currently in clinical trials, is selectively toxic to hypoxic cells commonly found in solid tumors. The toxicity results from the intracellular metabolism of TPZ to a highly toxic radical. When oxygen levels are low, the TPZ radical reacts with cellular molecules, producing DNA damage and cell death. The much lower toxicity towards aerobic cells results from the back-oxidation of the TPZ radical by oxygen. A major unresolved aspect of the mechanism of TPZ is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form. We have studied both the metabolism of the drug using HPLC and the formation of the TPZ radical with a fluorescence assay using dihydrorhodamine 123. We also measured DNA double- and single-strand breaks produced by TPZ, using the comet assay. We demonstrated that multiple reductases in the nucleus metabolize TPZ under hypoxia. Using the cofactor dependence of the reductases for metabolizing TPZ and of the DNA damage caused by TPZ, we show that DNA single-strand breaks after TPZ metabolism are probably caused by the most abundant source of reductase in the nucleus. DNA double-strand breaks, on the other hand, are formed by TPZ metabolism by an unknown nuclear reductase that requires only NADPH for its activity. This study is the first to characterize multiple nuclear reductases capable of activating TPZ.
Insights
Tirapazamine (TPZ) is a bioreductive drug that targets hypoxic tumor cells. This study identifies multiple nuclear reductases responsible for TPZ activation, clarifying its DNA-damaging mechanism in cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Tirapazamine (TPZ) is a bioreductive drug selectively toxic to hypoxic tumor cells.
- TPZ's toxicity stems from intracellular metabolism to a radical that damages DNA under low oxygen.
- The specific nuclear reductases responsible for TPZ activation remain largely unidentified.
Purpose of the Study:
- To identify and characterize the nuclear reductases involved in Tirapazamine (TPZ) metabolism and activation.
- To elucidate the specific mechanisms by which TPZ induces DNA damage (single- and double-strand breaks) in hypoxic cells.
- To understand the role of different nuclear reductases and cofactors in TPZ-mediated cytotoxicity.
Main Methods:
- High-performance liquid chromatography (HPLC) to study TPZ metabolism.
- Fluorescence assay using dihydrorhodamine 123 to detect TPZ radical formation.
- Comet assay to measure DNA double- and single-strand breaks induced by TPZ.
Main Results:
- Multiple nuclear reductases were found to metabolize TPZ under hypoxic conditions.
- DNA single-strand breaks are likely caused by the most abundant nuclear reductase.
- DNA double-strand breaks result from TPZ metabolism by a distinct nuclear reductase requiring NADPH.
Conclusions:
- This study is the first to characterize multiple nuclear reductases capable of activating Tirapazamine (TPZ).
- The findings clarify the distinct roles of different nuclear reductases in mediating TPZ-induced DNA damage.
- Understanding these mechanisms can inform the development of novel cancer therapies targeting hypoxic tumors.