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

Abstract

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.

Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

“…WT ⌬66 exhibited a k cat /K m NADPH ϭ 220 min Ϫ1 M Ϫ1 , whereas WT holo was nearly three times more efficient with a k cat /K m NADPH ϭ 650 min Ϫ1 M Ϫ1 . Reaction rates were beneath the limit of detection when either ⌬66 or holo Y181D was assayed, in agreement with previously published findings using bacterially expressed, N-truncated proteins Huang et al, 2005;Wang et al, 2007). On the other hand, purified Y181D was shown by HPLC to retain a small proportion of bound FMN, which would hypothetically confer at least some small amount of NCR activity.…”

“…A number of studies, including the original from Arlt et al (2004) detailing the discovery of the exon 5 POR 541T3 G mutation, have shown the catalytic incompetence of Y181D-substituted CYPOR using various assays and means of recombinant protein expression (Huang et al, 2005;Wang et al, 2007;Agrawal et al, 2008). Before the discovery of POR deficiency as a cause of human disease, Shen et al (1989) showed that the orthologous Y178D variant of rat CYPOR, recombinantly expressed and purified, was unable to catalyze NCR activity because of loss of FMN.…”

“…In the same study, a bacterially expressed Y181D protein, lacking 46 N-terminal residues, was devoid of measurable NADPH-cytochrome c reductase (NCR) activity. The capacity of heterologously expressed CYPOR Y181D to support mitomycin Cinduced apoptosis in a cell culture model was further shown to be completely diminished compared with wild-type (WT) CYPOR (Wang et al, 2007).…”

The chemical and biological activities of quinones: overview and implications in analytical detection

“…Ample evidence proves that bioreductive activation of the antitumor drugs: doxorubicin (15), tirapazamine (16), and indoloquinone (EO9) (17) by the cellular oxidoreductases P450R leads to significant increases in their covalent binding to DNA and cytotoxic activity against tumour cells (Bailey et al 2001;Bartoszek and Wolf 1992;Chinje et al 1999;Cowen et al 2003;Cullinane et al 1994;Kostrzewa-Nowak et al 2005;Patterson et al 1995Patterson et al , 1997Skladanowski and Konopa 1994). The bioreduction of mitomycin C(MMC) (18) by P450R leads to the formation of free radicals which cause lipid peroxidation, protein and DNA damage, and, ultimately cell death (Belcourt et al 1998;Joseph et al 1996;Kappus 1986;Wang et al 2007). Similarly, the toxicity of 5-fluorouracil (19) is enhanced by P450R through reactive oxygen species (ROS) production and NADPH depletion in P450R-overexpressing cells (Martinez et al 2008).…”

Mitomycin C application in refractive surgery

“…The variability in scarring is the main factor involved in cases of overcorrection, undercorrection, stromal opacity, and other possible complications of these surgeries. (5) Depending on the level of desired correction, corneal scarring and stimuli for the fibrotic response may be stronger after PRK. In fact, the main effect is the structural and functional defects of the base epithelial membrane occurring when there is great irregularity of the surface, after high corrections.…”