Cancer Chemother Pharmacol (2007) 60:713723 DOI 10.1007/s00280-007-0417-8ORIGINAL ARTICLE
Role of NADPH cytochrome P450 reductase in activation of RH1
Asher Begleiter Marsha K. Leith Daywin Patel Brian B. HasinoV
Received: 28 April 2006 / Accepted: 3 January 2007 / Published online: 26 January 2007 Springer-Verlag 2007
AbstractPurpose RH1 is a new bioreductive agent that is anexcellent substrate for the two-electron reducingenzyme, NAD(P)H quinone oxidoreductase 1 (NQO1).RH1 may be an eVective NQO1-directed antitumoragent for treatment of cancer cells having elevatedNQO1 activity. As some studies have indicated thatRH1 may also be a substrate for the one-electron reduc-ing enzyme, NADPH cytochrome P450 reductase (P450Red), P450 Red may contribute to the activation of RH1where NQO1 activities are low and P450 Red activitiesare high. The mean P450 Red activity in the humantumor cell line panel used by NCI for evaluation of newanticancer agents is 14.8 nmol min1 mg prot1, whilethe mean NQO1 activity in these cell lines is
199.5 nmol min1 mg prot1. Thus, we investigatedwhether P450 Red could play a role in activating RH1.Methods Reduction of RH1 by puriWed human P450Red was investigated using electron paramagnetic res-onance and spectroscopic assays. The ability of RH1 toproduce DNA damage following reduction by P450Red was studied using gel assays. To determine therole of P450 Red in activation of RH1 in cells, cellgrowth inhibition studies with inhibitors of P450 Redand NQO1 were carried out in T47D human breastcancer cells and T47D cells transfected with the humanP450 Red gene (T47D-P450) that have P450 Red activ-ities of 11.5 and 311.8 nmol min1 mg prot1, respec-tively.Results Reduction studies using puriWed P450 Redand NQO1 conWrmed that RH1 can be reduced byboth enzymes, but redox cycling was slower followingreduction by NQO1. RH1 produced DNA strandbreaks and crosslinks in isolated DNA after reduc-tion by either P450 Red or NQO1. DPIC, an inhibi-tor of P450 Red, had no eVect on cell growthinhibition by RH1 in T47D cells, and had only a smalleVect on cell growth inhibition by RH1 in the pres-ence of the NQO1 inhibitor, dicoumarol, in T47D-P450 cells.Conclusions These results demonstrated thatP450 Red does not contribute to the activation ofRH1 in cells with normal P450 Red activity and playsonly a minor role in activating this agent in cells withhigh levels of this enzyme. These studies conWrmedthat P450 Red could activate RH1 and provided theWrst direct evidence that RH1 could produce bothDNA strand breaks and DNA crosslinks after reduc-tion by P450 Red. However, the results strongly sug-gest that P450 Red does not play a signiWcant role in
Supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the CancerCare Manitoba Foundation to A.B., by grants from the Canadian Institutes of Health Research to B.B.H. and by a Canada Research Chair in Drug Development to B.B.H.
A. Begleiter (&) M. K. LeithManitoba Institute of Cell Biology, CancerCare Manitoba, Department of Internal Medicine, University of Manitoba, 675 McDermot Avenue, R3E 0V9 Winnipeg, MB, Canadae-mail: firstname.lastname@example.org
A. BegleiterManitoba Institute of Cell Biology, CancerCare Manitoba, Department of Pharmacology & Therapeutics, University of Manitoba, 675 McDermot Avenue, R3E 0V9 Winnipeg, MB, Canada
D. Patel B. B. HasinoVFaculty of Pharmacy, University of Manitoba, R3T 2N2 Winnipeg, MB, Canada123
714 Cancer Chemother Pharmacol (2007) 60:713723activating RH1 in cells with normal P450 Redactivity.
Keywords RH1 NADPH cytochrome P450 reductase Reduction Activation
-6-methyl-1,4-benzoquinoneP450 Red NADPH cytochrome P450 reductaseNQO1 NAD(P)H quinone oxidoreductase 1T47D-P450 T47D cells transfected with the
human NADPH cytochrome P450 reductase gene
EPR Electron paramagnetic resonanceDPIC Diphenyliodonium chlorideIC50 Concentration of drug that reduced the
relative absorbance fraction to 0.5
The bioreductive drug, 2,5-diaziridinyl-3-(hydroxym-ethyl)-6-methyl-1,4-benzoquinone (RH1) has demon-strated signiWcant antitumor activity in vitro [31, 37]and in vivo [7, 16]. Bioreductive agents are a class ofanticancer drugs that require activation by reductiveenzymes like the one-electron reducing enzyme,NADPH cytochrome P450 reductase (P450 Red) (EC22.214.171.124) [23, 28] or the two-electron reducing enzymeNAD(P)H quinone oxidoreductase 1 (NQO1; DT-diaphorase) (EC 126.96.36.199) [27, 29]. The relative con-tribution of these enzymes to the reductive activationof bioreductive agents is dependent on the drug,enzyme, oxygen level and pH [2, 13, 18, 27, 32]. RH1is a very eYcient substrate for NQO1  and reduc-tion by NQO1 results in activation of the aziridinegroups and subsequent DNA alkylation with inter-strand cross-links formed preferentially at 5 GCC 3sequences [4, 7]. Because RH1 is a very good sub-strate for NQO1, this agent was considered ideal foruse in an enzyme-directed tumor targeting strategy totreat tumors, like non-small cell lung cancer, thathave high NQO1 activity . RH1 is currently in aclinical trial .
Although RH1 is an excellent substrate for NQO1and this enzyme plays an important role in activatingthis agent [37, 38], Cenas [6, 20] showed that RH1could be a substrate for both NQO1 and P450 Red(Fig. 1). However, Kim  found no increase inRH1 activity in MDA-MB231 cells transfected withP450 Red compared with parent cells. MDA-MB231cells have no NQO1 activity due to an inactivating
NQO1 mutation. Tudor  studied the toxicity ofRH1 in the NCIs 60 tumor cell line panel and foundthat although the sensitivity of RH1 was marked incell lines expressing NQO1, there was no correlationbetween RH1 cytotoxicity and NQO1 activity. Theyalso found a high sensitivity to RH1 in leukemia andlymphoma cell lines with low or absent NQO1expression. While studies suggest that NQO1 is themost important activating enzyme for RH1, there isevidence that P450 Red can activate this bioreduc-tive antitumor agent. Thus, it is possible that P450Red may contribute to the activation of RH1 in cir-cumstances where NQO1 activities are low and P450Red activities are high. P450 Red activities in thehuman tumor cell line panel used by NCI for evalua-tion of new anticancer agents range from approxi-mately 2.563 nmol min1 mg prot1 with a mean of14.8 nmol min1 mg prot1, while NQO1 activities inthese cell lines range from approximately 05,000nmol min1 mg prot1 with a mean of 199.5 nmolmin1mg prot1 . Furthermore, DNA damage pro-duced by RH1 following reduction by P450 Red hasnot been studied in detail. In this study we investi-gated the reduction of RH1 by P450 Red and its rolein the DNA damaging activities and cell growthinhibition of RH1.
Materials and methods
RH1 was obtained from Professor David Ross (Uni-versity of Colorado Health Sciences Center, Denver,CO, USA). Streptonigin was obtained from Sigma (St.Louis, MO, USA) and tirapazamine was prepared asdescribed previously . T47D human breast cancercells and T47D cells transfected with the human P450Red gene, (T47D-P450) were obtained from ProfessorIan Stratford (University of Manchester, Manchester,UK). Both cell lines were grown in 10% FBS in HamsF12:DMEM 1:1 media and the T47D-P450 cells weregrown in the presence of puromycin. PuriWed humanP450 Red was purchased from BD Biosciences (SanJose, CA, USA), pBR322 plasmid DNA was from NewEngland Biolabs (Pickering, ON, Canada) and humanNQO1 was provided by Dr. Sushu Pan (University ofPittsburgh, Pittsburgh, PA, USA).
Electron paramagnetic resonance (EPR) studies
A freshly prepared 15:l aliquot of the drug in thereaction mixture (10 mM RH1, 2 mM NADPH,123
Cancer Chemother Pharmacol (2007) 60:713723 71516 g prot ml1 P450 Red in 50 mM Tris buVer at pH7.4 containing 10% DMSO for P450 Red; or 1 mMRH1, 97:M FAD, 0.4 mM NADH, 0.0005% Tween 20,1.1 g prot ml1 NQO1 in 0.5 mM Tris buVer at pH 7.4containing 1% DMSO for NQO1) was injected into an8 cm length of gas-permeable TeXon tubing (ZeusIndustrial Products, Raritan, NJ, USA) which was thenfolded at both ends, inserted into a quartz EPR tubeopen at both ends and placed in the EPR cavity as pre-viously described [1, 12]. The EPR spectra wererecorded with a Bruker (Milton, Canada) EMX EPRspectrometer. Pre-puriWed grade thermostated (37C)argon (400 l h1) or air as indicated was Xowed contin-uously over the sample while the spectra wererecorded. Recording of the Wrst-derivative EPR spec-tra was started approximately 2 min after the samplewas prepared. A total of 10 spectra (42 s/scan) wererecorded over 7 min and their signals were averaged.For recording of the spectra the instrument settingswere: microwave power 20 mW, modulation frequency100 kHz, microwave frequency 9.24 GHz, modulationamplitude 2.0 G, time constant 0.02 s, gain 20,000,1,024 data points/scan, magnetic Weld centered at3315 G, and a 50 G scan range.
BuVers (0.05 M phosphate pH 7.8 for P450 Red or0.05 M Tris pH 7.4 for NQO1) were pre-incubated inrubber septum-sealed Starna cuvettes (Starna Cells,Atascadero, CA, USA) with 100% nitrogen or air bub-bled into the buVer via 2 20 gauge needles for 2 h.RH1 (50 M Wnal concentration), NADPH (200 MWnal concentration) and enzyme (1.2 g prot ml1 Wnalconcentration for P450 Red or 0.22 g prot ml1 Wnalconcentration for NQO1) were added via a gas-tightsyringe and the cuvettes were transferred to the spec-trophotometer. Reductions were monitored using thescanning kinetics module in a Varian Cary 1 (Mississa-uga, ON, Canada) spectrophotometer and kineticscontinuums at 370 nm were calculated .
DNA strand breaks
Reaction mixtures in buVers (0.05 M phosphate pH 7.8for P450 Red or 0.05 M Tris pH 7.4 for NQO1) wereRH1 (50 M), NADPH (4 mM), pBR322 plasmidDNA and enzyme (4.8 g prot ml1 for P450 Red or0.88 g prot ml1 for NQO1). Following a 2 h incuba-tion in air, the reactions were terminated by addition of1 mM diphenyliodonium chloride (DPIC) (to inhibitP450 Red) or 10 M dicoumarol (to inhibit NQO1).DNA strand breaks were determined using a modiWca-tion of a gel-based assay we have used previously .BrieXy, following isolation of the DNA by centrifuga-tion, the DNA pellets were dissolved in 15 l loadingdye (30% sucrose in 10 mM TrisHCl (pH 8)-1 mMEDTA) and were then run on a 1% agarose gel with noethidium bromide for 45 min at 75 V. DNA strandbreaks are presented as the amount of relaxed DNA asa percent of the total DNA. All values were correctedfor a background level of strand breaks in the plasmidDNA of approximately 15%. Control experimentscontaining RH1 but no P450 Red or NQO1 producedno DNA stand breaks above the background.
Similar experiments were carried out under hypoxicconditions in a hypoxia chamber (Forma ScientiWcAnaerobic System, Model 1025, Fisher ScientiWc, Win-nipeg, Canada) maintained under 95% nitrogen:5%carbon dioxide. Reaction buVers were incubated in thehypoxia chamber for 5 days prior to the initiation ofthe reaction. All other reagents were incubated in thehypoxia chamber for 0.5 h prior to initiation of thereaction. Following 2 h incubation in the hypoxiachamber, the reactions were terminated by addition of1 mM DPIC (to inhibit P450 Red) or 10 M dicouma-rol (to inhibit NQO1). DNA strand break formationwas analyzed as described above.
Fig. 1 Structure and reduction pathways for RH1
P450 Red P450 Red
O2. O2.- -
716 Cancer Chemother Pharmacol (2007) 60:713723DNA crosslinking
Reaction mixtures in cell free buVers (0.05 M phosphatepH 7.8 for P450 Red or 0.05 M Tris pH 7.4 for NQO1)were RH1 (100 M), NADPH (400 M), linearized 32Pend-labeled pBR322 DNA and enzyme (1.2 g prot ml1
for P450 Red or 0.22 g prot ml1 for NQO1). BuVerswere gassed with 100% nitrogen for 1 h prior to additionof the other reaction mixture components. Following45 min incubation under hypoxic conditions, the reac-tions were terminated by addition of 1 mM DPIC (toinhibit P450 Red) or 10 M dicoumarol (to inhibitNQO1). DNA crosslinks were determined using a modi-Wcation of a gel-based assay we have used previously .BrieXy, following isolation of the DNA by centrifugation,the DNA pellets were dissolved in 20 l strand separa-tion loading dye (35% DMSO, 1 mM Na2EDTA pH 7,10 mM Tris pH 7, 0.05% bromophenol blue, 0.05%xylene cyanol). DNA strand separation consisted of incu-bation of the DNA in a 70C water bath for 3 min, fol-lowed by immediate cooling in an ice-water bath for30 min. The DNA was then run on a 1% agarose gel withno ethidium bromide for 45 min at 75 V. DNA crosslinksare presented as the amount of double stranded DNA asa percent of total DNA. Control experiments containingRH1 but no P450 Red or NQO1 produced no DNAcrosslinks.
P450 Red activities in cells were determined in cell soni-cates using a standard method  with the addition ofpotassium cyanide . P450 Red activity is reported asnmol cytochrome c reduced per minute per milligram ofprotein. NQO1 activities in cells were determined in cellsonicates as previously described . NQO1 activity isreported as nmol 2,6-dichlorophenolindophenolreduced per minute per milligram of protein.
Cell growth inhibition
Cells under aerobic conditions were treated withbuVer, 0.1 mM DPIC alone, 0.2 mM dicoumarol aloneor 0.1 mM DPIC and 0.2 mM dicoumarol for 1 h andthen with varying concentrations of RH1, streptonigrinor tirapazamine for an additional 1 h in the presence ofthe inhibitors. Cell growth inhibition was determinedby MTT assay  and is presented as the relativeabsorbance calculated from the ratio of the absorbancefrom drug treated cells to absorbance from untreatedcells. IC50 values were deWned as the concentration ofdrug that reduced the relative absorbance to 0.5 asdetermined from the linear regression lines of RH1
dose versus the logarithm of the relative absorbancecurves. The cell growth inhibition activities were com-pared statistically using a t test comparing the signiW-cance of the diVerence of the slopes of the dose versusthe logarithm of the relative absorbance curves.
Formation of RH1 semiquinone free radical followingreduction by P450 Red or NQO1 was monitored byEPR. Reduction of RH1 by P450 Red under hypoxicconditions resulted in a semiquinone free radical EPRsignal at g 2.0 due to the RH1 semiquinone (Fig. 2a),
Fig. 2 EPR spectra following reduction of RH1 by P450 Red orNQO1. A freshly prepared aliquot of RH1 in the reaction mixturecontaining P450 Red or NQO1 was injected into an 8 cm length ofgas-permeable TeXon which was then folded at both ends, insertedinto a quartz EPR tube open at both ends and placed in the EPRcavity. The EPR spectra were recorded with a Bruker EMX EPRspectrometer. Argon or air as indicated was Xowed continuouslyover the sample while the spectra were recorded. Recording of theWrst-derivative...