Nuclear Localization of NADPH:Cytochrome<i>c</i>(P450) Reductase Enhances the Cytotoxicity of Mitomycin C to Chinese Hamster Ovary Cells

Seow, Helen A.; Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Tomasz, Maria; Rockwell, Sara; Sartorelli, Alan C.

doi:10.1124/mol.104.004929

Cited by 10 publications

(11 citation statements)

References 30 publications

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“…Consistent with many previous studies that demonstrate the important role of POR in MMC bioactivation (Belcourt et al, 1996;Seow et al, 2005), Flp-In CHO cells expressing wild-type human POR showed a remarkable increase in MMC-induced toxicity in comparison to the cells transfected with the expression vector alone (containing no POR cDNA). Microscopic examination showed that 24-h treatment of MMC (20 M) in the cells expressing wild-type POR caused abnormal changes in cell morphology and a significant reduction in the number of cells (Fig.…”

Section: Resultssupporting

confidence: 75%

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Wang¹,

Han²,

He³

et al. 2006

Drug Metab Dispos

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ABSTRACT:The importance of genetic variation in clinical response to various drugs is now well recognized. Identification of genetic biomarkers that can predict efficacy and toxicity of chemotherapeutic drugs in cancer patients holds great promise in treatment improvement and cost reduction. Mitomycin C (MMC) is a common anticancer drug used for the treatment of numerous types of tumors. Metabolismmediated activation, by either one-electron or two-electron reduction, plays a critical role in the chemotherapeutic action of MMC. NADPH-cytochrome P450 (oxido)reductase (POR) is a major enzyme responsible for MMC activation through the one-electron reductive pathway, which leads to the production of semiquinone anion radicals and subsequent DNA damage in the cells. Recently, a total of six naturally occurring human POR variants with single amino acid changes (Y181D, A287P, R457H, V492E, C569Y, and V608F) have been identified. Although the catalytic efficiency of these variants in reduction of cytochrome c was reported to be altered, their capability in activating MMC, a direct substrate of POR, has not been examined. In the present study, we demonstrated that except for the C569Y variant, MMC-induced toxicity assayed as cell viability and proliferative capability was significantly decreased in the Flp-In Chinese hamster ovary cells stably expressing all the other POR variants in comparison with the cells expressing wild-type human POR. Cells expressing the V608F and Y181D variants had a complete loss of the capability to activate MMC. Our finding suggests that these functional POR genetic variations may serve as a potential biomarker to predict the chemotherapeutic response to MMC.

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Section: Resultssupporting

confidence: 75%

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Wang¹,

Han²,

He³

et al. 2006

Drug Metab Dispos

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“…Differences between our findings and previous reports using CHO cells, with respect to sensitivity to MMC and cytochrome P450 reductase expression, may be due to methods used to assess cytotoxicity. While our growth inhibition assays showed no differences in MMC sensitivity between CHO-WT and CHO-OR cells following either a 3 h or a 4 day exposure, Belcourt et al (13) and Seow et al (14) reported increased sensitivity of their cytochrome P450 reductase overexpressing CHO cells when treated with MMC for 1 h. One can speculate that the sensitivity of the different cells to MMC may be due to differences in uptake and/or metabolism of the drug during the exposure. Belcourt et al (13) and Seow et al (14) also used clonogenic assays to assess cytotoxicity and it may be that this assay selects for cells with altered sensitivity to the drug.…”

Section: Discussioncontrasting

confidence: 53%

“…While our growth inhibition assays showed no differences in MMC sensitivity between CHO-WT and CHO-OR cells following either a 3 h or a 4 day exposure, Belcourt et al (13) and Seow et al (14) reported increased sensitivity of their cytochrome P450 reductase overexpressing CHO cells when treated with MMC for 1 h. One can speculate that the sensitivity of the different cells to MMC may be due to differences in uptake and/or metabolism of the drug during the exposure. Belcourt et al (13) and Seow et al (14) also used clonogenic assays to assess cytotoxicity and it may be that this assay selects for cells with altered sensitivity to the drug. Selected clones may express other enzymes reported to activate MMC such as DT-diaphorase (17, 35), NRH:quinone oxidoreductase 2 (37, 38), NADPH-ferrodoxin reductase (39) or cytochrome b5 reductase (9); they may also express antioxidants that detoxify and protect against ROS-induced damage.…”

Section: Discussioncontrasting

confidence: 53%

“…This includes breast tumor cells in culture and maintained as xenographs (15, 16), as well as variant CHO cell lines overexpressing cytochrome P450 reductase (13, 14). Differences between our findings and previous reports using CHO cells, with respect to sensitivity to MMC and cytochrome P450 reductase expression, may be due to methods used to assess cytotoxicity.…”

Section: Discussionmentioning

confidence: 99%

“…For example, Belcourt et al (13) have shown that overexpression of cytochrome P450 reductase in Chinese hamster ovary (CHO) cells enhances their sensitivity to MMC under both oxygenated and hypoxic conditions. Increased sensitivity to MMC under these conditions has also been described in CHO cells engineered to express nuclear cytochrome P450 reductase (14). Similarly, viral delivery of cytochrome P450 reductase increases the sensitivity of human breast cancer cells to MMC, although this is only evident under oxygenated conditions (15).…”

Section: Introductionmentioning

confidence: 88%

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Distinct Roles of Cytochrome P450 Reductase in Mitomycin c Redox Cycling and Cytotoxicity

Wang

Gray

Mishin

et al. 2010

Molecular Cancer Therapeutics

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Mitomycin c (MMC), a quinone-containing anticancer drug, is known to redox cycle and generate reactive oxygen species. A key enzyme mediating MMC redox cycling is cytochrome P450 reductase, a microsomal NADPH-dependent flavoenzyme. In the present studies, CHO cells overexpressing this enzyme (CHO-OR cells) and corresponding control cells (CHO-WT cells) were used to investigate the role of cytochrome P450 reductase in the actions of MMC. In lysates from both cell types, MMC was found to redox cycle and generate H2O2; this activity was greater in CHO-OR cells (Vmax = 1.2 ± 0.1 nmol H2O2/min/mg protein in CHO-WT cells vs. 32.4 ± 3.9 nmol H2O2/min/mg protein in CHO-OR cells). MMC was also more effective in generating superoxide anion and hydroxyl radicals in CHO-OR cells, relative to CHO-WT cells. Despite these differences in MMC redox cycling, MMC-induced cytotoxicity, as measured by growth inhibition, was similar in the two cell types (IC50 = 72 ± 20 nM for CHO-WT and 75 ± 23 nM for CHO-OR cells), as was its ability to induce G2/M and S phase arrest. Additionally, in 9 different tumor cell lines, although a strong correlation was observed between MMC-induced H2O2 generation and cytochrome P450 reductase activity, there was no relationship between redox cycling and cytotoxicity. Hypoxia, which stabilizes MMC radicals generated by redox cycling, also had no effect on the sensitivity of tumor cells to MMC-induced cytotoxicity. These data indicate that NADPH cytochrome P450 reductase-mediated MMC redox cycling is not involved in cytotoxicity of this chemotherapeutic agent.

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Cytochrome b5 reductase, a plasma membrane redox enzyme, protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics

Hyun

Lee

2015

AGE

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The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD(+)/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H2O2 (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H2O2 and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD(+)/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases.

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Nuclear Localization of NADPH:Cytochromec(P450) Reductase Enhances the Cytotoxicity of Mitomycin C to Chinese Hamster Ovary Cells

Cited by 10 publications

References 30 publications

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity

Distinct Roles of Cytochrome P450 Reductase in Mitomycin c Redox Cycling and Cytotoxicity

Cytochrome b5 reductase, a plasma membrane redox enzyme, protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics

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