Cancer Research The Future of Cancer Research: Science and Patient Impact Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ramji, S.
Right arrow Articles by Riddick, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ramji, S.
Right arrow Articles by Riddick, D. S.
[Cancer Research 63, 6914-6919, October 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Human NADPH-Cytochrome P450 Reductase Overexpression Does Not Enhance the Aerobic Cytotoxicity of Doxorubicin in Human Breast Cancer Cell Lines1

Shairoz Ramji, Chunja Lee, Tadanobu Inaba, Adam V. Patterson and David S. Riddick2

Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8 Canada [S. R., C. L., T. I., D. S. R.], and Auckland Cancer Society Research Centre, University of Auckland, Auckland 1000, New Zealand [A. V. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Doxorubicin is a useful antineoplastic drug with multiple mechanisms of cytotoxicity. One such mechanism involves the reductive bioactivation of the quinone ring to a semiquinone radical, which can exert direct toxic effects and/or undergo redox cycling. We hypothesized that human NADPH-cytochrome P450 reductase (CYPRED) catalyzes doxorubicin reduction and that overexpression of this enzyme sensitizes human breast cancer cell lines to the aerobic cytotoxicity of doxorubicin. cDNA-expressed human CYPRED catalyzed doxorubicin reduction, measured as the rate of doxorubicin-stimulated NADPH consumption. Using a bank of 17 human liver microsomal samples, the rate of doxorubicin reduction correlated with CYPRED catalytic activity and CYPRED protein immunoreactivity. Diphenyliodonium chloride, a mechanism-based inactivator of CYPRED, inhibited CYPRED activity and doxorubicin reduction in human liver microsomes with similar concentration dependence. Stably transfected clones of MDA231 human breast cancer cells overexpressing human CYPRED immunoreactive protein and catalytic activity showed enhanced sensitivity to the aerobic cytotoxicity of tirapazamine, a bioreductive drug known to be activated by CYPRED; however, no sensitization to the cytotoxic effects of doxorubicin was observed. Although human CYPRED is an important catalyst of doxorubicin reduction, overexpression of this enzyme does not confer enhanced sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Doxorubicin is an antineoplastic drug with a wide range of clinical activity against several solid and hematological malignancies (1) . Several mechanisms appear to contribute to the cytotoxic effect of doxorubicin and related anthracyclines. These include DNA intercalation, DNA alkylation and cross-linking, direct membrane effects, initiation of DNA damage via inhibition of topoisomerase II, and free radical production with consequent DNA damage and lipid peroxidation (2) .

Free radical formation from anthracyclines such as doxorubicin generally results from processes of drug biotransformation, and these compounds display complex metabolic patterns. The major metabolite in humans is doxorubicinol, produced via cytosolic carbonyl reductase-catalyzed reduction of the ketone at C-13 of doxorubicin (3) . Although this is generally viewed as a detoxication step, there is some evidence that doxorubicinol contributes to the dose-limiting cardiotoxicity that characterizes the clinical use of doxorubicin (4) . Reductive deglycosylation of doxorubicin to the 7-deoxyaglycone and two-electron reduction of the quinone moiety to the hydroquinone catalyzed by cytosolic NAD(P)H:quinone oxido-reductase are generally viewed as detoxication pathways (5) . Free radical production by doxorubicin involves the one-electron reductive bioactivation of the quinone ring to a semiquinone radical (6) , which can exert direct toxic effects and/or undergo redox cycling. Under aerobic conditions, the semiquinone radical reacts rapidly with oxygen to generate superoxide anion, which in turn may produce ROS3 such as hydrogen peroxide and hydroxyl radical (7) . This may result in oxidative stress leading to cell death, events that seem to be particularly important in doxorubicin cardiotoxicity (8 , 9) ; however, the role of free radical formation and oxidative stress in tumor cell killing remains controversial (2) . Under hypoxic conditions, the unstable and reactive semiquinone radical and/or subsequent breakdown products may contribute to cytotoxic outcomes via covalent binding to cellular macromolecules (10, 11, 12) . In addition, the iron-chelating ability of doxorubicin contributes to the production of formaldehyde during oxidative stress, and doxorubicin-formaldehyde conjugates contribute to DNA covalent binding (13 , 14) .

Our main interest is enzyme-catalyzed reductive bioactivation of doxorubicin to free radicals and subsequent redox cycling under aerobic conditions and whether these events can be manipulated to make human cancer cells more sensitive to the cytotoxic effects of anthracyclines. Although several enzymes can potentially catalyze the one-electron reductive bioactivation of doxorubicin to the semiquinone radical, our focus is on CYPRED (EC 1.6.2.4). Rat and rabbit CYPRED catalyze the one-electron reduction of doxorubicin to the semiquinone radical (15, 16, 17) . Furthermore, purified rat CYPRED enhances doxorubicin toxicity toward human breast cancer cells via a mechanism that involves covalent binding to macromolecules (18 , 19) . Transfection of human CYPRED cDNA into Chinese hamster ovary cells enhances doxorubicin cytotoxicity by 1.8–3.3-fold (20) , whereas a similar manipulation in V79 Chinese hamster fibroblasts had no effect on doxorubicin toxicity (21) . In addition to CYPRED, the major phenobarbital-inducible CYP in rat liver, CYP2B1, can also catalyze the one-electron reduction of doxorubicin to the semiquinone radical (22) . Phenobarbital-inducible CYPs play a role in the cytotoxicity of doxorubicin in rat hepatocytes (23 , 24) , but not in V79 Chinese hamster fibroblasts genetically engineered to overexpress CYP2B1 (24) . When considering the effects of CYPRED on doxorubicin cytotoxicity, it is important to keep in mind that CYPRED also plays an important role in the detoxication of this anthracycline via the reductive deglycosylation pathway. Consistent with this, it was shown that decreased CYPRED expression (achieved via both pharmacological and antisense approaches) in human lung cancer and lymphoma cell lines resulted in enhanced doxorubicin toxicity (25) .

Although it is clear that rat and rabbit CYPRED catalyze the reductive bioactivation of doxorubicin to the semiquinone radical, the role that CYPRED plays in modulating the cytotoxicity of doxorubicin remains controversial. We are particularly interested in the role that human CYPRED plays in the reductive bioactivation of doxorubicin and whether enhanced expression of this enzyme can sensitize human breast cancer cells to the cytotoxic effects of this anthracycline. Because many human tumors and cancer cell lines display low levels of CYPRED and associated CYP enzymes (26 , 27) , these studies may suggest a future treatment strategy combining conventional chemotherapy with a gene therapy approach designed at selectively enhancing drug bioactivation within tumor cells. In this regard, it is particularly desirable to characterize human enzymes to minimize clinical complications that could result from immune responses directed toward enzymes of foreign origin (28) .

In the present investigation, we have demonstrated that human CYPRED is an important catalyst of doxorubicin reduction. To examine the potential chemotherapeutic significance of this finding, we analyzed stable transfectants of the estrogen receptor-negative human breast cancer cell line MDA231 overexpressing human CYPRED protein. Overexpression of human CYPRED in the MDA231 cell line confers enhanced sensitivity to the bioreductive drug tirapazamine (SR4233; Ref. 29 ), a positive control in the present study. Although human CYPRED is an important catalyst of doxorubicin reduction, our results suggest that overexpression of this enzyme does not confer enhanced sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Source of Chemicals.
Doxorubicin hydrochloride, diethylenetriaminepentaacetic acid, cytochrome c, MTT, puromycin, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). DPIC was obtained from Aldrich Chemical Co. (Milwaukee, WI). Tirapazamine was a kind gift from Dr. A. M. Rauth (Ontario Cancer Institute, Toronto, Ontario, Canada). Fetal bovine serum was purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). A goat polyclonal antibody raised against rat CYPRED was obtained from BD Gentest (Woburn, MA). ECL detection reagents were obtained from Amersham Biosciences (Piscataway, NJ). Electrophoresis and blotting equipment and reagents were obtained from Bio-Rad Laboratories Inc. (Hercules, CA).

Source of Microsomes.
Microsomes from human lymphoblastoid cells transfected with human CYPRED cDNA or vector were purchased from BD Gentest.

Human liver microsomes were prepared from 17 individual frozen liver samples that were maintained in a tissue bank at the University of Toronto. The livers were originally obtained at the time of kidney donation, and information on the donors has been reported (30) . Each liver is designated with a code K (for kidney donor) plus a number; in this investigation, the following specific liver samples were used: K10; K12; K14; K15; K16; K18; K19; K20; K21; K22; K23; K24; K25; K26; K27; K28; and K29. Approximately 10 g of each liver were homogenized in 1 volume of Tris-buffered KCl [1.15% KCl and 50 mM Tris (pH 7.4)], and microsomes were isolated by differential centrifugation. Microsomes were suspended in storage buffer [50 mM Tris and 10% sucrose (pH 7.4)], frozen in liquid nitrogen, and stored at -70°C until use. Microsomal protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL).

Cell Lines.
The derivation of stably transfected clones of the human breast cancer cell line MDA231 overexpressing human CYPRED has been described previously (29) . These transfectants were maintained as monolayer cultures in RPMI 1640 supplemented with 2 mM glutamine, 10% fetal bovine serum, and 2 µg/ml puromycin. All cell culture experiments were performed in an atmosphere of 5% CO2 and 95% air at 37°C. Microsomes were isolated from cell homogenates by differential centrifugation. Microsomes were suspended in storage buffer [10 mM Tris, 20% glycerol, and 1 mM EDTA (pH 7.4)], frozen in liquid nitrogen, and stored at -70°C until use. Microsomal protein concentration was determined by the method of Lowry et al. (31) .

Cytotoxicity Assays.
Sensitivity of the cell lines to doxorubicin or tirapazamine was assessed using a microplate colorimetric assay that measures the ability of viable cells to reduce a soluble tetrazolium salt (MTT) to an insoluble purple formazan precipitate (32) . Cells in the logarithmic phase of growth were plated in 96-well microtiter plates at 5000 cells/well in 200 µl. After a 24-h incubation, cells were exposed to various concentrations of doxorubicin (1 nM to 6 µM) or tirapazamine (0.1–500 µM) for an additional 72 h, at which time 50 µl of MTT (1 mg/ml) were added to each culture well. After a 4-h incubation at 37°C, medium was removed, and the formazan crystals were solubilized by adding 150 µl of DMSO and by mixing on an orbital shaker for 5 min. Absorbance at 540 nm was measured on a Labsystems Multiskan LS microplate reader (Franklin, MA). Fractional absorbance was expressed as a percentage of the absorbance displayed by cells exposed only to vehicle. The IC50 (drug concentration required to reduce fractional absorbance by 50%) was determined by interpolation from a plot of fractional absorbance versus log10[drug].

Immunoblot Analysis.
Equal amounts of microsomal protein from human livers (10 µg) or human breast cancer cell lines (5 µg) were resolved by SDS-PAGE (33) and transferred to nitrocellulose (Hybond-ECL; Amersham Biosciences; Ref. 34 ). For detection of CYPRED protein, a goat antirat CYPRED polyclonal antibody (BD Gentest), known to cross-react with human CYPRED, was used at a 1:5000 dilution, followed by a rabbit antigoat immunoglobulin-horseradish peroxidase conjugate (Sigma) at a 1:5000 dilution. The ECL system (Amersham Biosciences) was used for protein detection. Films were scanned on a SuperVista S-12 scanner (UMAX Technologies Inc., Fremont, CA), and relative quantitation of the human liver microsomal samples was performed using IPLab Gel for the Power Macintosh v1.5e (Signal Analytics, Vienna, VA). Quantitative analyses were performed under conditions that yielded a linear relationship between microsomal protein and immunoreactive signal intensity. Microsomes from human lymphoblastoid cells overexpressing human CYPRED (BD Gentest) were run as a positive control on immunoblots.

Catalytic Activity Assays.
CYPRED activity was assayed under aerobic conditions at 30°C in 1-ml incubation mixtures containing 300 mM potassium phosphate (pH 7.7), 70 µM cytochrome c, and microsomal protein (100 µg for human liver, 25 µg for human lymphoblastoid cell lines, and 100 µg for human breast cancer cell lines). Reactions were initiated by the addition of 1 mM NADPH, and the rate of cytochrome c reduction was determined spectrophotometrically at 550 nm based on {epsilon} = 21 mM-1 cm-1 (35) . The rate of the enzyme-catalyzed reaction was determined by subtracting the rate of the reaction occurring in the absence of microsomes. Product formation was linear with respect to protein concentration and incubation time.

Doxorubicin reduction was measured indirectly as the doxorubicin-stimulated microsomal oxidation of NADPH, based on the method of Goeptar et al. (22) . NADPH oxidation was assayed under aerobic conditions at 37°C in 1-ml incubation mixtures containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.5 mM diethylenetriaminepentaacetic acid, 100 µM doxorubicin, and microsomal protein (500 µg for human liver and 200 µg for human lymphoblastoid cell lines). Reactions were initiated by the addition of 0.125 mM NADPH, and the rate of NADPH oxidation was determined spectrophotometrically at 340 nm based on {epsilon} = 6.22 mM-1 cm-1. Microsomal consumption of NADPH in the absence of doxorubicin was calculated by subtracting the rate of the reaction observed in samples lacking both microsomes and doxorubicin from the rate of the reaction observed in samples lacking doxorubicin. Microsomal consumption of NADPH in the presence of doxorubicin was calculated by subtracting the rate of the reaction observed in samples lacking microsomes from the rate of the reaction observed in the complete incubation mixture. Finally, doxorubicin-stimulated microsomal NADPH consumption was calculated by subtraction of microsomal NADPH consumption in the absence of doxorubicin from microsomal NADPH consumption in the presence of doxorubicin, and this final parameter is referred to as doxorubicin reduction. Product formation was linear with respect to protein concentration and incubation time.

To assess inhibition of human liver microsomal CYPRED activity and doxorubicin reduction, DPIC was added to incubation mixtures at various concentrations (0–2.5 mM) before initiation of the reactions by the addition of NADPH. The IC50 (DPIC concentration required to reduce enzyme activity by 50%) was determined by interpolation from a plot of activity versus [DPIC].

Statistical Analysis.
Where appropriate, data are presented as mean ± SD. Comparisons of two independent treatment groups were carried out by Student’s t test. Comparisons of three or more independent treatment groups were carried out by randomized design one-way ANOVA followed by post hoc Newman-Keuls test. Statistical significance of linear correlation data was determined by the Pearson correlation test. In all cases, a result was considered to be statistically significant at P <= 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To study the role of human CYPRED in doxorubicin reduction under aerobic conditions, we used a combination of "reaction phenotyping" approaches. As described previously (22 , 36) , we measured doxorubicin-stimulated NADPH consumption as an indirect indicator of doxorubicin reductive bioactivation to the semiquinone radical. First, microsomes from human lymphoblastoid cells overexpressing human CYPRED displayed 17-fold higher CYPRED catalytic activity compared with microsomes from vector-transfected cells (1075 ± 126 versus 63 ± 3 nmol/min/mg microsomal protein; P <= 0.01, Student’s t test) and 11-fold higher doxorubicin reduction activity compared with microsomes from vector-transfected cells (22.0 ± 5.0 versus 2.0 ± 1.0 nmol/min/mg microsomal protein; P <= 0.01, Student’s t test).

Next, experiments were conducted to determine whether human CYPRED is an important contributor to doxorubicin reduction in a complex subcellular fraction such as human liver microsomes, which contain several enzymes active in drug biotransformation. Using a bank of 17 human liver microsome samples, CYPRED catalytic activity varied approximately 2.4-fold (range, 66–155 nmol/min/mg protein), and doxorubicin reduction varied approximately 4.4-fold (range, 2.04–8.94 nmol/min/mg protein). Doxorubicin reduction correlated significantly with both CYPRED catalytic activity (Fig. 1A)Citation and CYPRED immunoreactivity (Fig. 1B)Citation . DPIC, a mechanism-based inactivator of CYPRED (37) , caused concentration-dependent inhibition of CYPRED activity and doxorubicin reduction in human liver microsomes (Fig. 2)Citation . Both reactions showed a similar maximal inhibition by DPIC ({approx}80%), and the sensitivity of both reactions to DPIC was similar (IC50 {approx} 0.6 mM).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. Correlation between doxorubicin reduction and CYPRED catalytic activity (A) and CYPRED immunoreactivity (B), using a bank of 17 human liver microsome samples. CYPRED activity was determined spectrophotometrically as the rate of cytochrome c reduction. Doxorubicin reduction was assayed spectrophotometrically as the rate of doxorubicin-stimulated microsomal NADPH consumption. Relative CYPRED immunoreactivity was determined by immunoblot analysis. The equations of the lines of best fit were generated by least square linear regression analysis. Statistical significance of the correlations was determined by the Pearson correlation test.

 


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Concentration-dependent inhibition of CYPRED activity and doxorubicin reduction by DPIC in human liver microsomes. CYPRED activity was determined spectrophotometrically as the rate of cytochrome c reduction. Doxorubicin reduction was assayed spectrophotometrically as the rate of doxorubicin-stimulated microsomal NADPH consumption. Enzyme activity is expressed as a percentage of the activity observed in the absence of DPIC. Human liver microsome sample K15 was used in this experiment, and control values (100%) for CYPRED activity and doxorubicin reduction were 103.3 and 5.1 nmol/min/mg microsomal protein, respectively. Each point represents the mean ± SD of triplicate determinations. *, significantly different from enzyme activity in the absence of DPIC, based on a randomized design one-way ANOVA and post hoc Newman-Keuls test (P <= 0.01). The chemical structure of DPIC is shown as an inset.

 
To determine whether CYPRED overexpression enhances the sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin, we used two previously characterized stably transfected clones of MDA231 overexpressing human CYPRED. The Rd-16 and Rd-42 lines were originally found to display 16- and 42-fold elevations, respectively, in CYPRED catalytic activity compared with parental MDA231 cells, as assessed in cell lysates (29) . Under the conditions used in the present study, both Rd-16 and Rd-42 showed microsomal CYPRED catalytic activity that was 9-fold higher than parental MDA231 cells (Fig. 3B)Citation and pronounced overexpression of CYPRED immunoreactive protein (Fig. 3A)Citation .



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3. Overexpression of CYPRED immunoreactive protein (A) and catalytic activity (B) in microsomes from human breast cancer cell lines stably transfected with human CYPRED cDNA. MDA231 human breast cancer cell lines were either not transfected (Parental) or transfected with vector containing the human CYPRED cDNA (Rd). A, immunoblot analysis of microsomal protein (5 µg) using a polyclonal antibody directed against rat CYPRED. The number on the left indicates the size of a molecular mass marker, and the arrow indicates the position of immunoreactive CYPRED protein (~77 kDa). B, microsomal CYPRED activity was determined spectrophotometrically as the rate of cytochrome c reduction. Data are expressed as mean ± SD of quintuplicate determinations. *, significantly different from MDA231 parental cells, based on a randomized design one-way ANOVA and post hoc Newman-Keuls test (P <= 0.001).

 
Based on MTT assays, doxorubicin produced a concentration-dependent cytotoxic response in parental MDA231 cells with an IC50 {approx} 0.4 µM (Fig. 4A)Citation . CYPRED transfectants, Rd-16 and Rd-42, displayed sensitivities to doxorubicin that were similar to the parental line (Fig. 4A)Citation . As a positive control, we also tested the MDA231 cell lines for their sensitivity to tirapazamine, a drug that is known to be bioactivated via CYPRED-catalyzed one-electron reduction (29) . Tirapazamine produced a concentration-dependent cytotoxic response in parental MDA231 cells with an IC50 {approx} 50 µM (Fig. 4B)Citation , and the Rd-16 and Rd-42 lines were approximately 8-fold more sensitive to the cytotoxic effects of this bioreductive agent (Fig. 4B)Citation .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Concentration-dependent doxorubicin (A) and tirapazamine (B) cytotoxicity in human breast cancer cell lines stably transfected with human CYPRED cDNA. MDA231 human breast cancer cell lines were either not transfected (MDA231/Parental) or transfected with vector containing the human CYPRED cDNA (MDA231/Rd-16 and MDA231/Rd-42). Cells were treated for 72 h with the concentrations of doxorubicin (A) or tirapazamine (B) indicated on the abscissa. Cytotoxicity was assessed using a microplate MTT colorimetric assay. Fractional absorbance is expressed as a percentage of the absorbance displayed by cells exposed only to vehicle. Each point represents the mean ± SD of duplicate 96-well microplates with sextuplicate determinations per plate. Similar results were obtained in a total of two to three independent experiments. The chemical structure of tirapazamine is shown as an inset.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that human CYPRED is an important contributor to the reductive biotransformation of doxorubicin; however, overexpression of human CYPRED does not sensitize human breast cancer cells to the aerobic cytotoxicity of doxorubicin.

A limitation of the in vitro biochemical component is that we used an indirect assay of doxorubicin reduction, which measures the rate of doxorubicin-stimulated microsomal NADPH oxidation. Although we did not directly measure the drug metabolites produced, in particular the doxorubicin semiquinone radical, the indirect assay was validated as a simple surrogate indicator of the reductive bioactivation of doxorubicin to the redox-cycling radical species (22 , 36) . This validation was performed by electron spin resonance measurements of doxorubicin semiquinone production in rat liver microsomes and reconstituted systems under anaerobic conditions (22) .

"Reaction phenotyping" involves the use of a combination of complementary experimental approaches to determine the enzyme(s) that make important contributions to the biotransformation of a chemical. In addition to our direct demonstration that heterologously expressed human CYPRED has the intrinsic ability to catalyze doxorubicin reduction, we also used multiple approaches to characterize the biotransformation of doxorubicin in human liver microsomes. Using a bank of 17 human liver microsome samples, CYPRED catalytic activity varied approximately 2.4-fold, and the amount of immunoreactive CYPRED protein varied approximately 3-fold (Fig. 1)Citation . These findings are consistent with those of other investigators who reported very little variation (i.e., less than 3-fold) in the range of hepatic CYPRED activity, protein expression, and mRNA levels in human populations (38, 39, 40, 41) . Despite this rather limited variation in CYPRED levels/activity across human liver samples, we found a clear correlation between CYPRED and doxorubicin reduction (Fig. 1)Citation , providing strong evidence that human CYPRED is an important catalyst of the reductive biotransformation of doxorubicin in hepatic microsomes. This conclusion is further supported by our chemical inhibition studies using DPIC (Fig. 2)Citation , a mechanism-based inactivator of CYPRED, and other flavoenzymes that function as one-electron donors (37) . CYPRED reduces DPIC to a reactive phenyl radical, which binds covalently to the flavin cofactor or adjacent amino acids important in catalysis, resulting in inhibition of CYPRED that is irreversible and time and concentration dependent. Although DPIC can inhibit several other flavoenzymes that catalyze one-electron reduction (e.g., NADPH oxidase, NADH dehydrogenase, xanthine oxidase, and nitric oxide synthase; Ref. 37 ), CYPRED is the major target for inhibition by this compound in hepatic microsomes.

Our data are supportive of a direct role for CYPRED in doxorubicin reduction; in addition, Goeptar et al. (22) showed that CYPRED can also participate in the reduction of doxorubicin to the semiquinone radical catalyzed by CYP enzymes in rat liver microsomes. It will be important to assess the contributions of various human CYP enzymes to the reductive biotransformation of doxorubicin under aerobic and anaerobic conditions.

We also found that doxorubicin inhibits CYPRED activity in a concentration-dependent manner in human liver microsomes (data not shown), supporting a direct interaction of doxorubicin with CYPRED. However, the pharmacological significance of this is not clear because high concentrations of doxorubicin (200–600 µM) were required, and only partial CYPRED inhibition was achieved. In summary, many enzymes in addition to CYPRED can catalyze the reductive bioactivation of doxorubicin to the semiquinone radical: xanthine oxidase; NADH dehydrogenase; ferredoxin reductase; NADH-cytochrome b5 reductase; and nitric oxide synthase (reviewed in Ref. 42 ). Based on the subcellular localization and cofactor requirements for these enzymes, none is expected to contribute to the NADPH-dependent microsomal reduction of doxorubicin examined in this study. Collectively, our data demonstrate that human CYPRED is an important catalyst of doxorubicin reduction, and therefore, this enzyme became the focus of our attempts to enhance the aerobic cytotoxicity of doxorubicin in human breast cancer cell lines.

It is generally accepted that bioactivation of doxorubicin to free radicals and subsequent generation of ROS are involved in cardiotoxicity (8 , 9) . Thus, under specific cellular conditions where free radical generation overwhelms antioxidant defenses, enzymatic bioactivation of doxorubicin to the semiquinone radical can clearly contribute to cytotoxicity. Although the role that this free radical mechanism plays in tumor cell killing by doxorubicin is debated (2) , the above suggests that it should be possible to manipulate the tumor cell environment in ways that permit the free radical mechanism to augment the cytotoxic effects of doxorubicin. Together with the important role of CYPRED as a catalyst of doxorubicin reduction, this provided the rationale for our studies of human breast cancer cells stably transfected with CYPRED cDNA.

Many studies using human breast cancer cell lines, in particular the estrogen receptor-positive MCF-7 line and doxorubicin-resistant clones, suggest that doxorubicin redox cycling and ROS generation are an important mechanism of tumor cell cytotoxicity. Cellular changes such as elevation of selenium-dependent glutathione peroxidase, which lead to enhanced antioxidant defense, contribute to doxorubicin resistance (43, 44, 45, 46) . To examine the role of human CYPRED in this cytotoxic mechanism in breast cancer cells, we made use of the parental MDA231 cell line and transfectants stably overexpressing CYPRED protein (29) .

Despite demonstrating human CYPRED protein overexpression in the MDA231 transfectants (Fig. 3)Citation , enhanced aerobic doxorubicin toxicity was not observed (Fig. 4A)Citation . The one-electron reduction potential of doxorubicin is -292 mV (47) , and this is a key determinant of the rate of enzymatic reduction of the drug. This reduction potential is sufficiently high that we anticipated that reductive metabolism and redox cycling would occur in the breast cancer cells. There are several possible reasons why CYPRED overexpression did not enhance doxorubicin cytotoxicity. First, MTT assays are less sensitive indicators of cell survival than clonogenic assays, and thus small changes in drug sensitivity may be overlooked. Second, the CYPRED expression level achieved in the cells may not be adequate to promote significant free radical generation. Both explanations seem unlikely because our MTT assay could clearly detect enhanced tirapazamine cytotoxicity with the levels of CYPRED expression achieved in the MDA231 transfectants (Fig. 4B)Citation . Third, the subcellular localization of the overexpressed CYPRED protein in the endoplasmic reticulum membranes may not be optimal in relation to critical cellular targets for doxorubicin. Fourth, these cell lines may possess adequate antioxidant defenses to counteract any doxorubicin free radicals and ROS that may be generated; measurement of doxorubicin metabolite formation, covalent binding to macromolecules, ROS generation, and biological responses such as lipid peroxidation in the CYPRED transfectants would assist with interpretation of future studies.

Finally, a possible interpretation of our work is that reductive bioactivation of doxorubicin catalyzed by human CYPRED does not enhance the aerobic cytotoxicity of doxorubicin in breast cancer cells. As summarized by Gewirtz (2) , it is likely that interaction of doxorubicin with topoisomerase II is the primary mechanism of tumor cell kill at drug concentrations in the nanomolar range that are likely to be achieved clinically. In contrast, many in vitro investigations of doxorubicin-induced free radical formation have been carried out at excessively high drug concentrations (>=100 µM) that are not pharmacologically relevant. Thus, free radical generation is likely to be of secondary importance compared with topoisomerase II inhibition in anthracycline cytotoxicity in tumor cells. Our cytotoxicity studies were validated by use of tirapazamine, a bioreductive drug that undergoes one-electron reduction to a cytotoxic free radical (reviewed in Ref. 48 ). The tirapazamine radical exhibits selective toxicity toward hypoxic cells; however, aerobic cytotoxicity results from redox cycling of the tirapazamine radical with subsequent ROS generation. Although several cellular reductases participate in the bioactivation of tirapazamine (48 , 49) , CYPRED is a key determinant of cytotoxicity in human breast cancer cells under both hypoxic and aerobic conditions (29 , 50) .

The conclusion of this study is that although human CYPRED is an important catalyst of doxorubicin reduction, overexpression of this enzyme does not confer enhanced sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin. This has important implications for our understanding of anthracycline cytotoxic mechanisms and for future directions in enzyme-based prodrug activation strategies. It will be desirable to characterize both CYPRED- and CYP-dependent doxorubicin reduction under aerobic and anaerobic conditions as well as the doxorubicin sensitivity of breast cancer cells overexpressing CYPRED and/or other activating enzymes under aerobic and hypoxic conditions. It will also be important to assess the relative importance of the doxorubicin free radical mechanism versus topoisomerase II inhibition in cardiac myocytes, tumor cells in exponential growth, and tumor cells that have reached plateau phase (51) ; it is likely that the relative balance of these cytotoxic mechanisms may shift depending on the proliferative state of the target cells. Finally, the transfected cell lines used in this study will be of use in the characterization of novel bioreductive agents.


   ACKNOWLEDGMENTS
 
We thank the following individuals for their contributions to this work: Dr. Mike Rauth for providing tirapazamine; Peter Beaumont for assistance with cytotoxicity assays; and Dr. Xu, Nancy Fischer, and Tommy Cheung for assistance with human liver microsome preparation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Canadian Breast Cancer Foundation. Back

2 To whom requests for reprints should be addressed, at Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8 Canada. Phone: (416) 978-0813; Fax: (416) 978-6395; E-mail: david.riddick{at}utoronto.ca Back

3 The abbreviations used are: ROS, reactive oxygen species; CYP, cytochrome P450; CYPRED, NADPH-cytochrome P450 reductase; DPIC, diphenyliodonium chloride; ECL, enhanced chemiluminescence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back

Received 2/25/03. Revised 6/24/03. Accepted 7/28/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Booser D. J., Hortobagyi G. N. Anthracycline antibiotics in cancer therapy: focus on drug resistance. Drugs, 47: 223-258, 1994.[Medline]
  2. Gewirtz D. A. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics Adriamycin and daunorubicin. Biochem. Pharmacol., 57: 727-741, 1999.[Medline]
  3. Forrest G. L., Gonzalez B. Carbonyl reductase. Chem. Biol. Interact., 129: 21-40, 2000.[Medline]
  4. Forrest G. L., Gonzalez B., Tseng W., Li X. L., Mann J. Human carbonyl reductase overexpression in the heart advances the development of doxorubicin-induced cardiotoxicity in transgenic mice. Cancer Res., 60: 5158-5164, 2000.[Abstract/Free Full Text]
  5. Cummings J., Allan L., Willmott N., Riley R., Workman P., Smyth J. F. The enzymology of doxorubicin quinone reduction in tumour tissue. Biochem. Pharmacol., 44: 2175-2183, 1992.[Medline]
  6. Bachur N. R., Gordon S. L., Gee M. V. Anthracycline antibiotic augmentation of microsomal electron transport and free radical formation. Mol. Pharmacol., 13: 901-910, 1977.[Abstract/Free Full Text]
  7. Sinha B. K., Katki A. G., Batist G., Cowan K. H., Myers C. E. Adriamycin-stimulated hydroxyl radical formation in human breast tumor cells. Biochem. Pharmacol., 36: 793-796, 1987.[Medline]
  8. Minotti G., Cairo G., Monti E. Role of iron in anthracycline cardiotoxicity: new tunes for an old song?. FASEB J., 13: 199-212, 1999.[Abstract/Free Full Text]
  9. Singal P., Li T. M., Kumar D., Danelisen I., Iliskovic N. Adriamycin-induced heart failure: mechanisms and modulation. Mol. Cell. Biochem., 207: 77-86, 2000.[Medline]
  10. Ghezzi P., Donelli M. G., Pantarotto C., Facchinetti T., Garattini S. Evidence for covalent binding of adriamycin to rat liver microsomal proteins. Biochem. Pharmacol., 30: 175-177, 1981.[Medline]
  11. Zeman S. M., Phillips D. R., Crothers D. M. Characterization of covalent Adriamycin-DNA adducts. Proc. Natl. Acad. Sci. USA, 95: 11561-11565, 1998.[Abstract/Free Full Text]
  12. Cummings J., Bartoszek A., Smyth J. F. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography: studies with doxorubicin and NADPH cytochrome P450 reductase. Anal. Biochem., 194: 146-155, 1991.[Medline]
  13. Taatjes D. J., Fenick D. J., Gaudiano G., Koch T. H. A redox pathway leading to the alkylation of nucleic acids by doxorubicin and related anthracyclines: application to the design of antitumor drugs for resistant cancer. Curr. Pharm. Des., 4: 203-218, 1998.[Medline]
  14. Taatjes D. J., Fenick D. J., Koch T. H. Nuclear targeting and nuclear retention of anthracycline-formaldehyde conjugates implicates DNA covalent bonding in the cytotoxic mechanism of anthracyclines. Chem. Res. Toxicol., 12: 588-596, 1999.[Medline]
  15. Bachur N. R., Gordon S. L., Gee M. V., Kon H. NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc. Natl. Acad. Sci. USA, 76: 954-957, 1979.[Abstract/Free Full Text]
  16. Berlin V., Haseltine W. A. Reduction of Adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J. Biol. Chem., 256: 4747-4756, 1981.[Abstract/Free Full Text]
  17. Komiyama T., Kikuchi T., Sugiura Y. Generation of hydroxyl radical by anticancer quinone drugs, carbazilquinone, mitomycin C, aclacinomycin A and Adriamycin, in the presence of NADPH-cytochrome P450 reductase. Biochem. Pharmacol., 31: 3651-3656, 1982.[Medline]
  18. Bartoszek A., Wolf C. R. Enhancement of doxorubicin toxicity following activation by NADPH cytochrome P450 reductase. Biochem. Pharmacol., 43: 1449-1457, 1992.[Medline]
  19. Bartoszek A. Metabolic activation of Adriamycin by NADPH-cytochrome P450 reductase: overview of its biological and biochemical effects. Acta Biochim. Pol., 49: 323-331, 2002.[Medline]
  20. Sawamura A. O., Aoyama T., Tamakoshi K., Mizuno K., Suganuma N., Kikkawa F., Tomoda Y. Transfection of human cytochrome P-450 reductase cDNA and its effect on the sensitivity to toxins. Oncology (Basel), 53: 406-411, 1996.
  21. Schmalix W. A., Lang D., Schneider A., Bocker R., Greim H., Doehmer J. Stable expression and coexpression of human cytochrome P450 oxidoreductase and cytochrome P450 1A2 in V79 Chinese hamster cells: sensitivity to quinones and biotransformation of 7-alkoxyresorufins and triazines. Drug Metab. Dispos., 24: 1314-1319, 1996.[Abstract]
  22. Goeptar A. R., Koppele J. M. T., Lamme E. K., Pique J. M., Vermeulen N. P. E. Cytochrome P450-2B1-mediated one-electron reduction of Adriamycin: a study with rat liver microsomes and purified enzymes. Mol. Pharmacol., 44: 1267-1277, 1993.[Abstract]
  23. Goeptar A. R., Groot E. J., Scheerens H., Commandeur J. N. M., Vermeulen N. P. E. Cytotoxicity of mitomycin C and Adriamycin in freshly isolated rat hepatocytes: the role of cytochrome P450. Cancer Res., 54: 2411-2418, 1994.[Abstract/Free Full Text]
  24. Goeptar A. R., Tekoppele J. M., Glatt H. R., Groot E. J., Seidel A., Barrenscheen M., Wolfel C., Doehmer J., Vermeulen N. P. E. The cytotoxicity of mitomycin C and Adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes. Chem. Biol. Interact., 97: 149-168, 1995.[Medline]
  25. Niitsu N., Kasukabe T., Yokoyama A., OkabeKado J., Yamamoto-Yamaguchi Y., Umeda M., Honma Y. Anticancer derivative of butyric acid (pivalyloxymethyl butyrate) specifically potentiates the cytotoxicity of doxorubicin and daunorubicin through the suppression of microsomal glycosidic activity. Mol. Pharmacol., 58: 27-36, 2000.[Abstract/Free Full Text]
  26. Fitzsimmons S. A., Workman P., Grever M., Paull K., Camalier R., Lewis A. D. Reductase enzyme expression across the National Cancer Institute tumor cell line panel: correlation with sensitivity to mitomycin C and E09. J. Natl. Cancer Inst. (Bethesda), 88: 259-269, 1996.[Abstract/Free Full Text]
  27. Yu L. J., Matias J., Scudiero D. A., Hite K. M., Monks A., Sausville E. A., Waxman D. J. P450 enzyme expression patterns in the NCI human tumor cell line panel. Drug Metab. Dispos., 29: 304-312, 2001.[Abstract/Free Full Text]
  28. Chen L., Waxman D. J. Cytochrome P450 gene-directed enzyme prodrug therapy (GDEPT) for cancer. Curr. Pharm. Des., 8: 1405-1416, 2002.[Medline]
  29. Patterson A. V., Saunders M. P., Chinje E. C., Talbot D. C., Harris A. L., Stratford I. J. Overexpression of human NADPH: cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069. Br. J. Cancer, 76: 1338-1347, 1997.[Medline]
  30. Sumida A., Kinoshita K., Fukuda T., Matsuda H., Yamamoto I., Inaba T., Azuma J. Relationship between mRNA levels quantified by reverse transcription-competitive PCR and metabolic activity of CYP3A4 and CYP2E1 in human liver. Biochem. Biophys. Res. Commun., 262: 499-503, 1999.[Medline]
  31. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275, 1951.[Free Full Text]
  32. Alley M. C., Scudiero D. A., Monks A., Hursey M. L., Czerwinski M. J., Fine D. L., Abbott B. J., Mayo J. G., Shoemaker R. H., Boyd M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res., 48: 589-601, 1988.[Abstract/Free Full Text]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.[Medline]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354, 1979.[Abstract/Free Full Text]
  35. Strobel H. W., Dignam J. D. Purification and properties of NADPH-cytochrome P-450 reductase. Methods Enzymol., 52: 89-96, 1978.[Medline]
  36. Minotti G. NADPH- and Adriamycin-dependent microsomal release of iron and lipid peroxidation. Arch. Biochem. Biophys., 277: 268-276, 1990.[Medline]
  37. O’Donnell V. B., Smith G. C. M., Jones O. T. G. Involvement of phenyl radicals in iodonium compound inhibition of flavoenzymes. Mol. Pharmacol., 46: 778-785, 1994.[Abstract]
  38. McManus M. E., Hall P. D., Stupans I., Brennan J., Burgess W., Robson R., Birkett D. J. Immunohistochemical localization and quantitation of NADPH-cytochrome P-450 reductase in human liver. Mol. Pharmacol., 32: 189-194, 1987.[Abstract]
  39. Yamano S., Aoyama T., McBride O. W., Hardwick J. P., Gelboin H. V., Gonzalez F. J. Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7. Mol. Pharmacol., 35: 83-88, 1989.
  40. Shephard E. A., Palmer C. N., Segall H. J., Phillips I. R. Quantification of cytochrome P450 reductase gene expression in human tissues. Arch. Biochem. Biophys., 294: 168-172, 1992.[Medline]
  41. Pearce R. E., McIntyre C. J., Madan A., Sanzgiri U., Draper A. J., Bullock P. L., Cook D. C., Burton L. A., Latham J., Nevins C., Parkinson A. Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch. Biochem. Biophys., 331: 145-169, 1996.[Medline]
  42. Doroshow J. H., Akman S., Chu F-F., Esworthy S. Role of the glutathione-glutathione peroxidase cycle in the cytotoxicity of the anticancer quinones. Pharmacol. Ther., 47: 359-370, 1990.[Medline]
  43. Sinha B. K., Katki A. G., Batist G., Cowan K. H., Myers C. E. Differential formation of hydroxyl radicals by Adriamycin in sensitive and resistant MCF-7 human breast tumor cells: implications for the mechanism of action. Biochemistry, 26: 3776-3781, 1987.[Medline]
  44. Sinha B. K., Mimnaugh E. G., Rajagopalan S., Myers C. E. Adriamycin activation and oxygen free radical formation in human breast tumor cells: protective role of glutathione peroxidase in adriamycin resistance. Cancer Res., 49: 3844-3848, 1989.[Abstract/Free Full Text]
  45. Mimnaugh E. G., Dusre L., Atwell J., Myers C. E. Differential oxygen radical susceptibility of Adriamycin-sensitive and -resistant MCF-7 human breast tumor cells. Cancer Res., 49: 8-15, 1989.[Abstract/Free Full Text]
  46. Doroshow J. H., Akman S., Esworthy S., Chu F. F., Burke T. Doxorubicin resistance conferred by selective enhancement of intracellular glutathione peroxidase or superoxide dismutase content in human MCF-7 breast cancer cells. Free Radic. Res. Commun., 12–13: 779-781, 1991.
  47. Svingen B. A., Powis G. Pulse radiolysis studies of antitumor quinones: radical lifetimes, reactivity with oxygen, and one-electron reduction potentials. Arch. Biochem. Biophys., 209: 119-126, 1981.[Medline]
  48. Patterson A. V., Saunders M. P., Chinje E. C., Patterson L. H., Stratford I. J. Enzymology of tirapazamine metabolism: a review. Anti-Cancer Drug Des., 13: 541-573, 1998.[Medline]
  49. Delahoussaye Y. M., Evans J. W., Brown J. M. Metabolism of tirapazamine by multiple reductases in the nucleus. Biochem. Pharmacol., 62: 1201-1209, 2001.[Medline]
  50. Patterson A. V., Barham H. M., Chinje E. C., Adams G. E., Harris A. L., Stratford I. J. Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233). Br. J. Cancer, 72: 1144-1150, 1995.[Medline]
  51. Phillips R. M., Clayton M. R. Plateau-phase cultures: an experimental model for identifying drugs which are bioactivated within the microenvironment of solid tumours. Br. J. Cancer, 75: 196-201, 1997.[Medline]



This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
C. Fang, J. Gu, F. Xie, M. Behr, W. Yang, E. D. Abel, and X. Ding
Deletion of the NADPH-Cytochrome P450 Reductase Gene in Cardiomyocytes Does Not Protect Mice against Doxorubicin-Mediated Acute Cardiac Toxicity
Drug Metab. Dispos., August 1, 2008; 36(8): 1722 - 1728.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
D. S. Riddick, C. Lee, S. Ramji, E. C. Chinje, R. L. Cowen, K. J. Williams, A. V. Patterson, I. J. Stratford, C. S. Morrow, A. J. Townsend, et al.
CANCER CHEMOTHERAPY AND DRUG METABOLISM
Drug Metab. Dispos., August 1, 2005; 33(8): 1083 - 1096.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ramji, S.
Right arrow Articles by Riddick, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ramji, S.
Right arrow Articles by Riddick, D. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online