Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro

Siim, Bronwyn G.; Zijl, Pierre L van; Brown, J. Martin

doi:10.1038/bjc.1996.187

Cited by 59 publications

(60 citation statements)

References 34 publications

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“…It is of interest to note that SR4317 is not metabolized when incubated with breast cancer cell lysates in hypoxia (EC Chinjie and H Barham, unpublished data) and that SR4330, the four-electron reduced product was not detected in the metabolism studies reported here or by Patterson et al (1995) or Siim et al (1996). SR4317 can also be formed by direct two-electron reduction of tirapazamine by DT-diaphorase (Riley and Workman, 1992), thus bypassing the tirapazamine radical.…”

Section: Discussionmentioning

confidence: 96%

“…This strongly implicates P450 reductase-driven activation of tirapazamine to its one-electron reduced radical as an important contributory factor to the hypoxic toxicity of this drug, and this is consistent with our previous studies using a range of breast cancer cell lines. In contrast, Siim et al (1996) have exam-ined the hypoxic metabolism of tirapazamine by a variety of human and rodent cell lines of diverse histological type and showed that SR4317 formation correlated poorly with toxicity, whereas loss of tirapazamine was significantly related to drug potency. However, the reductive metabolic routes that could result in loss of tirapazamine and generate a radical (to give DNA strand breaks and toxicity) without subsequent production of SR4317…”

Section: Discussionmentioning

confidence: 99%

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Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069

Patterson

Saunders²,

Chinje³

et al. 1997

Br J Cancer

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Summary P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine Br J Cancer 72: 1144-1150. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.Keywords: P450 reductase; hypoxia, bioreductive drugs; tirapazamine; RSU 1069; breast cancer It is now well established that regions of low oxygen tension (hypoxia) can exist in many human solid tumours and that this hypoxia can sometimes predispose to failure of some treatments with radiotherapy (Hoeckel et al, 1996). One of the reasons for this failure is likely to be the inherent radiation resistance of hypoxic cells. One strategy being developed to overcome this problem is via the use of drugs that will selectively kill hypoxic cells Stratford, 1986, 1994;Zeman et al, 1986;Kennedy, 1987;Brown and Siim, 1996;Denny et al, 1996). This approach involves the exploitation of various biochemicla processes that, at low oxygen tensions, can result in selective reductive activation of a prodrug to give cytotoxic species (Workman and Stratford, 1993 enzymes. Thus, it has been proposed that hypoxic cells could be more effectively targeted if differences in the levels of the reductases in various cell types were taken into account to direct appropriate agents to particular human tumours based on their enzymology Workman and Stratford, 1993). Patterson et al (1995) examined the possible application of the enzyme-directed approach to the development of tirapazamine. The metabolism of this agent was suggested to be dependent on the presence of P450 reductase (Walton et al, , 1992. In a panel of six breast cancer cell lines with a sixfold range in P450 red...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069

Patterson

Saunders²,

Chinje³

et al. 1997

Br J Cancer

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“…Both sets of data are compatible with the hypothesis that NOSII activity is involved with the hypoxic activation of TPZ. It has been suggested that the mechanisms implicated in aerobic and hypoxic cytotoxicity are distinct, and a wealth of experimental data indicate that DNA is an important cellular target for TPZ (Biedermann et al, 1991;Siim et al, 1996;Peters et al, 2001). However, the exact nature of the chemical species responsible for TPZ-mediated strand cleavage remains unresolved.…”

Section: Discussionmentioning

confidence: 99%

Non-Nuclear Localized Human NOSII Enhances the Bioactivation and Toxicity of Tirapazamine (SR4233) in Vitro

Chinje

Cowen²,

Feng³

et al. 2003

Mol Pharmacol

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Tirapazamine (TPZ) is the lead member of a class of bioreductive drugs currently in phase II and III clinical trials. TPZ requires metabolic activation to give a cytotoxic free radical species, and this hypoxia-mediated process is carried out by a variety of cellular reductases, including NADPH cytochrome c (P450) reductase (P540R). Nitric-oxide synthase (NOS) is widely expressed in human tumors, and this enzyme consists of an oxidase and a reductase domain, the latter showing striking homology to P450R. Thus, in this article, we have investigated the role of one of the cytosolic isoforms of NOS [inducible NOS (NOSII)] in the bioactivation of this DNA-damaging antitumor agent. To achieve this, we have constitutively overexpressed NOSII in human breast tumor MDA231 cells by employing an optimized expression vector in which the strong human polypeptide chain elongation factor 1␣ promoter drives a bicistronic message encoding the genes for human NOSII and the puromycin-resistant gene (pac). Subcellular localization of NOSII in the stably transfected clones was determined after differential centrifugation and showed that NOSII catalytic activity was exclusively cytosolic as determined by conventional activity assay. This was confirmed by immunostaining followed by fluorescent microscopy studies. The increase in NOSII activity in a series of transfected clones was associated with an increase in TPZ metabolism and toxicity under hypoxic conditions. There was no similar increase in aerobic toxicity. These findings are of significance for two reasons. First, cellular NOSII activity, similar to that seen in human breast cancer, could contribute to TPZ toxicity; second, this will be a result of NOSderived/cytosol-associated TPZ radicals.Tirapazamine 2, SR 4233, Tirazone)] is a promising antitumor agent that has a unique spectrum of activity in that it selectively causes DNA damage in hypoxic tumor cells after one-electron bioreductive activation. Cytotoxicity results from activation by a reductive enzyme(s) that adds an electron to the parent drug to produce radical species that cause DNA single-strand breaks, double-strand breaks (DSBs), and chromosome aberrations (Wang et al

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“…Neutralization-reionization mass spectrometry (NR MS) [10] is a method allowing the generation of unusual and previously unknown neutrals in the gas phase by neutralization of their (stable) positively charged counterparts. Although the sequence of events leading to activated drug (2) in the NR MS experiment is distinct from those that take place inside cells, this experiment offers a unique method for the generation of the activated form of tirapazamine in the gas phase.…”

Section: Neutralization-reionization Experiments With M ϩ⅐ and [M ϩ Hmentioning

confidence: 99%

A mass spectrometry study of tirapazamine and its metabolites: Insights into the mechanism of metabolic transformations and the characterization of reaction intermediates

Zagorevskii

Song

Breneman

et al. 2003

J. Am. Soc. Mass Spectrom.

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Tandem mass spectrometry methods were used to study the sites of protonation and for identification of 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine), and its metabolites (3-amino-1,2,4-benzotriazine 1-oxide (3), 3-amino-1,2,4-benzotriazine 4-oxide (4), 3-amino-1,2,4-benzotriazine (5), and a related isomer 3-amino-1,2,4-benzotriazine 2-oxide (6). Fragmentation pathways of 3 and 5 indicated the 4-N-atom as the most likely site of protonation. Among the N-oxides studied, the 4-oxide (4) showed the highest degree of protonation at the oxygen atom. The differences in collision-induced dissociation of isomeric protonated 1-, 2-and 4-oxides allowed for their identification by LC/MS/MS. Gas phase and liquid phase protonation of tirapazamine occurred exclusively at the oxygen in the 4-position. A loss of OH radical from these ions (2 ϩ ) resulted in ionized 3. Neutralization-reionization mass spectrometry (NR MS) experiments demonstrated the stability of the neutral analogue of protonated tirapazamine in the gas phase in the s time-frame. A significant portion of the neutral tirapazamine radicals (2) dissociated by loss of hydroxyl radical during the NR MS event, which indicates that previously proposed mechanisms for redox-activated DNA damage are reasonable. The activation energy for loss of hydroxyl radical from activated tirapazamine (2) was estimated to be ϳ14 kcal mol Ϫ1 . Stable neutral analogues of [3 ϩ H] ϩ and [5 ϩ H] ϩ ions were also generated in the course of NR MS experiments. Structures of these radicals were assigned to the molecules having an extra hydrogen atom at one of the ring N-atoms. Quantum chemical calculations of protonated 1, 3, 4 and 5 and the corresponding neutrals were performed to assist in the interpretation of experimental results and to help identify their structures. (J Am Soc Mass Spectrom 2003, 14, 881-892)

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Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro

Cited by 59 publications

References 34 publications

Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069

Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069

Non-Nuclear Localized Human NOSII Enhances the Bioactivation and Toxicity of Tirapazamine (SR4233) in Vitro

A mass spectrometry study of tirapazamine and its metabolites: Insights into the mechanism of metabolic transformations and the characterization of reaction intermediates

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