1521-009X/43/5/660–668$25.00 http://dx.doi.org/10.1124/dmd.114.062380DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:660–668, May 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Induction of Human UDP-Glucuronosyltransferase 2B7 GeneExpression by Cytotoxic Anticancer Drugs in Liver Cancer

HepG2 Cells

Dong Gui Hu, Peter I. Mackenzie, Lu Lu, Robyn Meech, and Ross A. McKinnon

Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University School of Medicine, FlindersMedical Centre, Bedford Park, Australia (D.G.H., P.I.M., L.L., R.M., R.A.M.)

Received December 1, 2014; accepted February 23, 2015

ABSTRACT

We recently reported induction ofUGT2B7 by its substrate epirubicin,a cytotoxic anthracycline anticancer drug, via activation of p53 andsubsequent recruitment of p53 to the UGT2B7 promoter in hepato-cellular carcinoma HepG2 cells. Using the same HepG2 model cellline, the present study assessed the possibility of a similar inductionof UGT2B7 by several other cytotoxic drugs. We first demonstratedby reverse transcriptase quantitative real-time polymerase chainreaction that, as observed with epirubicin, nine cytotoxic drugsincluding three anthracyclines (doxorubicin, daunorubicin, andidarubicin) and six nonanthracyclines (mitomycin C, 5-fluorouracil,camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, andetoposide) significantly increased UGT2B7mRNA levels. To investigatea potential involvement of p53 in this induction, we conducted furtherexperiments with four of the nine drugs (doxorubicin, daunorubicin,

idarubicin, and mitomycin C). The cytotoxic drugs studied increasedp53 and UGT2B7 protein levels. Knockdown of p53 expression bysmall interfering RNA reduced cytotoxic drug-induced UGT2B7expression. Luciferase reporter assays showed activation of theUGT2B7 promoter by cytotoxic drugs via a previously reported p53site. Finally, chromatin immunoprecipitation assays demonstrated p53recruitment to the UGT2B7 p53 site upon exposure tomitomycin C, themost potent UGT2B7 inducer among the nine tested drugs. Takentogether, these results provide further evidence supportingUGT2B7 asa p53 target gene. The cytotoxic drug-induced UGT2B7 activity intarget liver cancer cells or possibly in normal liver cells may affect thetherapeutic efficacy of co-administered cytotoxic drugs (e.g., epirubi-cin) and noncytotoxic drugs (e.g., morphine), which are UGT2B7substrates.

Introduction

UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymesresponsible for the glucuronidation of numerous endogenous andexogenous small lipophilic compounds, including bilirubin, bile acids,fatty acids, steroid hormones, and carcinogens, as well as therapeuticdrugs; the resulting glucuronide products are generally inactive andwater soluble, thus promoting their excretion from the body (Mackenzieet al., 1997, 2005). For example, UGT2B7 displays high activity towarda variety of endogenous compounds such as bile acids, sex hormonesand their metabolites, mineralocorticoid and glucocorticoid hormones,fatty acids, and retinoic acids (Hu et al., 2014b). UGT2B7 has also beenshown to conjugate many therapeutic drugs, such as morphine(Coffman et al., 1997), epirubicin (Innocenti et al., 2001), valproic acid(Argikar and Remmel, 2009), mycophenolic acid (Picard et al., 2005),and nonsteroid anti-inflammatory drugs (Jin et al., 1993). UGT2B7 iswidely expressed in human tissues, including liver, small intestine,kidney, and breast (Hu et al., 2014b). The broad substrate spectrum ofUGT2B7 combined with its hepatic and extrahepatic expression highlightsits physiological and pharmacological importance in metabolism ofendogenous bioactive molecules and therapeutic drugs. Regulation

of UGT2B7 expression and activity in human tissues, especially inthe liver and gastrointestinal tract, is thus critical for determiningsubstrate detoxification and clearance. As we have recently reviewed,studies to date have investigated the regulation of UGT2B7 using liverand colon cancer cell lines and transgenic mice and revealed regulationby a number of tissue-specific and ligand-dependent transcription factors(Hu et al., 2014b).Chemotherapy using cytotoxic drugs continues to play an important

role in treating most human cancers; however, both intrinsic resistanceand acquired resistance limit their clinical value (Holohan et al., 2013).The cytotoxic drugs that have been frequently incorporated inchemotherapy protocols include DNA alkylating agents (e.g.,mitomycin C), platinum coordination complexes, topoisomerases I(e.g., irinotecan and topotecan) and II (e.g., doxorubicin, epirubicin,and etoposide) inhibitors, microtubule-damaging drugs, and anti-metabolites [e.g., 5-fluorouracil (5-FU)] (Pavelic, 2014). Multiplemechanisms have been shown to be involved in emergence of cancerresistance to cytotoxic drugs, including intratumoral overexpressionof drug efflux transporters, drug-metabolizing enzymes, and drug-targeting molecules such as DNA topisomerases (Szakács et al.,2006; Meijerman et al., 2008). Specifically, overexpression ofcytotoxic drug-metabolizing enzymes in tumor cells enhances theinactivation of cytotoxic drugs, and thus reduces sensitivity tochemotherapy or even promotes resistance. For example, severalcytotoxic drugs and/or their metabolites are primarily inactivated by

This work was supported by funding from the National Health and MedicalResearch Council of Australia [ID1020931].

dx.doi.org/10.1124/dmd.114.062380.

ABBREVIATIONS: 5-FU, 5-fluorouracil; MT, mutant; PCR, polymerase chain reaction; si, small interfering; SN-38, 7-ethyl-10-hydroxycamptothecin;UGT, UDP-glucuronosyltransferase.

660

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

UGTs, including etoposide and 7-ethyl-10-hydroxycamptothecin(SN-38) (the active metabolite of irinotecan) by UGT1A1 (O’Dwyer andCatalano, 2006; Wen et al., 2007) and epirubicin by UGT2B7 (Innocentiet al., 2001). Basseville et al. (2011) have recently shown that SN-38stimulates UGT1A1 expression via the PXR in colorectal cancer LS180cells and HepG2 cells, suggesting a potential role in conferring irinotecanresistance. In support of this view, Takahata et al. (2008) havedemonstrated that overexpression of UGT1A1 reduced the sensitivityof hepatocellular cancer cells to irinotecan. We recently reported theinduction of UGT2B7 by epirubicin via activation of p53 and thesubsequent recruitment of p53 to a p53 response element (59-TGGCATGTCCATACAAGATC-39, core sequence of p53 motifunderlined) at the UGT2B7 proximal promoter in liver cancer HepG2 cells(Hu et al., 2014c). Epirubicin has been administered alone (mono-therapy), in combination with cisplatin or mitomycin C (double therapy),or together with cisplatin and mitomycin C (triple therapy) for treatingpatients with nonresectable advanced hepatocellular carcinoma (Marelliet al., 2007). Therefore, it is believed that the epirubicin-inducedUGT2B7 glucuronidation activity promotes epirubicin metabolismand thus reduces therapeutic efficacy or may even contribute to the

development of cancer resistance to epirubicin-containing hepatocellularcarcinoma chemotherapy (Hu et al., 2014c).That UGT2B7 is a p53 target gene is also supported by a recent

finding that silencing of p53 expression by stably expressing an anti-p53 short-hairpin RNA reduces basal UGT2B7 mRNA levels inHepG2 cells (Goldstein et al., 2013). In addition to epirubicin, manyother drugs that belong to different classes of cytotoxic drugs havealso been shown to activate p53 in cancer cells, such as doxorubicin,camptothecin, etoposide, 5-FU, and mitomycin C (Fritsche et al.,1993; Tishler et al., 1993, 1995; Müller et al., 1997). The presentstudy hypothesized that, similar to epirubicin, these p53-activatingcytotoxic drugs would also be able to stimulate the expression ofUGT2B7 in HepG2 cells, and presents several lines of evidence tosupport this hypothesis.

Materials and Methods

Chemical compounds of analytical grade were purchased from Sigma-Aldrich (St. Louis, MO), including doxorubicin hydrochloride, epirubicinhydrochloride, daunorubicin hydrochloride, idarubicin hydrochloride, nutlin-3a,mitomycin C, dimethyl sulfoxide, 5-FU, (S)-(+)-camptothecin, SN-38, topotecan

Fig. 1. Anthracyclines elevate both UGT2B7 mRNA andprotein levels in the liver cancer HepG2 cell line. HepG2cells were treated in triplicate (A and B) or duplicate (C) for24 hours with vehicle (0.1% ethanol) or one of fouranthracyclines at 1 mM as indicated. (A) Data shown are thefold induction (i.e., mean 6 S.D. of three independentexperiments) in target gene mRNA levels in drug-treatedcells over those in vehicle-treated cells (set to a value of 1).(B) Shown are immunosignals (top) and calculated foldinduction in UGT2B7 protein levels in drug-treated cellsover those in vehicle-treated cells (set to a value of 1)following background subtraction and normalizing to thecontrol calnexin protein levels (bottom) from a representativeexperiment of two independent experiments as describedin Materials and Methods. (C) Whole cell lysates wereprepared from treated HepG2 cells as indicated and subjectedto Western blotting assays (;30 mg each sample). Blots weresequentially probed with an anti-p53 antibody and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) anti-body as described in Materials and Methods. Shown areimmunosignals from one experiment performed in duplicate.This experiment was repeated and similar results were obtained.Eth, ethanol; EPI, epirubicin; Dox, doxorubicin; Dau,daunorubicin; Ida, idarubicin. Statistical analyses of datapresented in (A) and (B) used one-way analysis of variancefollowed by Tukey’s post hoc multiple comparison test. *, 0.05,** , 0.01, *** , 0.001.

Induction of UGT2B7 by Cytotoxic Drugs 661

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

hydrochloride hydrate, and etoposide. Primers were synthesized by Sigma-Genosys (Castle Hill, NSW, Australia).

Cell Culture, Drug Treatment, RNA Extraction, and Reverse Tran-scriptase Quantitative Real-Time Polymerase Chain Reaction (PCR). TheHepG2 cell line was purchased from ATCC (Manassas, VA) and maintained in

Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at37�C in a 5% CO2 atmosphere. For drug treatment, cells were plated in 6-wellplates and cultured for 3 to 4 days to reach 100% confluence. Cells were thentreated with drugs in triplicate at intervals of time as indicated in the figures1–7. The concentrations of drugs used (i.e., anthracyclines at 1 mM, mitomycin

Fig. 2. siRNAs against p53 reduce anthracycline-inducedUGT2B7 mRNA levels in HepG2 cells. Briefly, cells wereplated and transfected in 6-well plates in four wells at100 nM with either p53 siRNA or nontargetting siRNA(neg-siRNA). Forty-eight hours after transfection, cells wereharvested from one well of each transfection to preparewhole cell lysates for Western blotting assays using anantibody recognizing p53 (top) or glyceraldehyde-3-phosphatedehydrogenase (GAPDH) (bottom) (A), and the remainingthree wells of each transfection were treated for 24 hours withone of the four anthracyclines at 1 mM as indicated, followedby quantitative real-time PCR to quantify the mRNA levels ofthree genes (UGT2B7, UGT2B10, and p21) (B), as describedinMaterials and Methods. Shown are the immunosignals froma representative experiment (A) and the calculated (i.e., mean6 S.D. of three independent experiments) target gene mRNAlevels in anthracycline-treated and p53 siRNA-transfectedHepG2 cells compared with those in anthracycline-treated andneg-siRNA-transfected cells (set to a value of 100%) (B).Statistical analyses used two-way analysis of variancefollowed by independent t tests with Bonferroni correction.** P , 0.01, *** , 0.001.

Fig. 3. Anthracylines activate the UGT2B7 promoter inHepG2 cells via a reported p53 site in its proximal promoter.The UGT2B7-575/-1 plasmid contains the proximal 575base pair (bp) UGT2B7 promoter fragment cloned in thepromoterless pGL3-basic vector; UGT2B7-575/-1/MT3 plas-mid contains the 575 bp promoter with a mutated p53 site (A);UGT2B7-575/-1/MT5 contains the 575 bp promoter withmutation of a sequence located 7 bp downstream from the p53site (A). HepG2 cells were transfected with these constructsand subsequently treated for 24 hours with vehicle [0.1%ethanol (ETH) for anthracyclines or 0.1% dimethylsulfoxide(DMSO) for nutlin-3a], the p53 activator nutlin-3a (10 mM),or one of the anthracyclines [i.e., epirubicin (EPI), doxorubicin(DOX), daunorubicin (DAU), and idarubicin (IDA), all at1 mM]. After treatment, cells were harvested and assayed forluciferase activity as described in Materials and Methods. (A)Shown is the UGT2B7 promoter sequence between nucleotides-278 and -238 containing the p53 site (underlined and coresequence in bold) and both the positions and sequences of thetwo introduced mutations (MT3 and MT5). (B) Data arepresented as fold induction of the activities of promoterconstructs relative to those of the promotorless pGL3-basicvector (set at a value of 1). Data shown are mean 6 S.D. ofthree independent experiments performed in triplicate. Statis-tical analyses used one-way analysis of variance followedby Tukey’s post hoc multiple comparison test. ** P , 0.01,*** P , 0.001.

662 Hu et al.

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

C at 10 mM, 5-FU at 300 ng/ml, camptothecin at 0.5 mM, SN-38 at 10 ng/ml,topotecan at 0.1 mg/ml, and etoposide at 50 mM) are clinically relevant and/orapproximate those that have been frequently used in similar cell culture studies(Italia et al., 1983; Piret et al., 2006; Roudier et al., 2006; Liedtke et al., 2007;L’Espérance et al., 2008; Malmlof et al., 2008; Cosse et al., 2010; Saunderset al., 2010; Basseville et al., 2011; Hu et al., 2014c). Total RNA was extractedusing the RNeasy Mini Kit (QIAGEN, Valencia, CA) and converted to cDNAusing Invitrogen reverse transcription reagents (Mulgrave, VIC, Australia) aspreviously reported (Hu and Mackenzie, 2009). Real-time PCR was performedusing a RotorGene 3000 instrument (Corbett Research, NSW, Australia) andeither QuantiTect SYBR Green PCR master mix (QIAGEN) or GoTaqqPCRmaster Mix (Promega, Madison, WI) in a 20 ml reaction containing ;60 ng ofcDNA sample and previously published primers (Hu and Mackenzie, 2009; Huet al., 2014c). The mRNA levels of target genes (i.e., UGT2B7, UGT2B10, andp21) relative to those of 18S rRNA were quantified using the 22DDCT method(Livak and Schmittgen, 2001).

Transient Transfection and Luciferase Reporter Assay. Three pGL3luciferase constructs containing the proximal 575 base pair UGT2B7 promoterfragment used in this study have been reported previously (Hu et al., 2014c). Asshown in Fig. 3A, the UGT2B7-575/-1 construct contained a wild-type p53 site,the UGT2B7-575/-1/MT3 (where MT denotes mutant) carried a mutated p53 site,and the UGT2B7/-575/-1/MT5 construct had a mutation in the sequence that is7 base pairs downstream from the p53 site. Transient transfection of theseconstructs together with the internal control pRL-null vector into HepG2 cells andthe subsequent drug treatment and luciferase assays using the Dual-LuciferaseReporter Assay System (Promega) were conducted essentially as reportedpreviously (Hu et al., 2014c).

Small Interfering RNA (siRNA) Knockdown Experiments. On-TARGETplusSMARTpool siRNA against p53 and On-TARGETplus non-targeting pool siRNA (neg-siRNA) were purchased from Dharmacon RNAiTechnologies (Lafayette, CO). HepG2 cells were transfected in either 6- or 24-well plates at a concentration of 100 nM using Lipofectamine2000 (Invitrogen) asrecently reported (Hu et al., 2014c). 48 hour post transfection, cells were treatedwith drugs at the concentrations and time indicated in the figures 2 and 6. Aftertreatment, cells were then harvested either for total RNA, followed by quantitativereal-time PCR to quantify target mRNA levels as described previously or forwhole cell lysates for Western blotting analysis as described subsequently.

Western Blotting. Whole cell lysates were prepared from drug- or vehicle-treated HepG2 cells or p53 siRNA (or neg-siRNA)–transfected HepG2 cells inradioimmunoprecipitation assay buffer [50 mM Tris-HCl, pH 8.0, 1% nonidetp40 substitute (Astral Scientific, Gymea, NSW, Australia), 150 mM sodiumchloride, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate].Protein concentrations were determined using the Bradford Protein Assay (Bio-Rad) (Gladeville, NSW, Australia), and 30 mg protein was separated on SDS-polyacrylamide gels (10%) and transferred to nitrocellulose membranes forimmunodetection. The development and specificity of the rabbit anti-UGT2B7antibody has been reported previously (Kerdpin et al., 2009; Hu et al., 2014a).Other primary antibodies used were anti-calnexin antibodies obtained fromSigma-Aldrich, anti-p53 antibodies (FL-393 or DO-1) obtained from SantaCruz Biotechnology (Dallas, Texas), and anti-glyceraldehyde-3-phosphatedehydrogenase antibodies (G9545 or G8795) obtained from Sigma-Aldrich.Membranes were incubated first with the primary antibodies at supplier-recommended concentrations and then with a horseradish peroxidase-conjugateddonkey anti-rabbit (or anti-mouse) secondary antibody (NeoMarkers) (Fremont,CA). Immunosignals were detected with the SuperSignalWest Pico Chemilumi-nescent kit (Thermo-Fisher Scientific, Waltham, MA) and an ImageQuant LAS4000 luminescent image analyzer (GE Healthcare, Chalfont St. Giles, UnitedKingdom). Quantitation of band intensity and background subtraction wasperformed using NIH ImageJ analysis (Qin et al., 2011; Schneider et al., 2012).

Chromatin Immunoprecipitation Assay and Quantitative Real-TimePCR. The chromatin immunoprecipitation assay and quantitative PCR wereperformed essentially as reported previously (Hu et al., 2014c). In brief, HepG2cells were treated with vehicle, 1mM epirubicin, 10 mM mitomycin C, or 10 mMnutlin-3a for 24 hours and then crosslinked using 1% formadehyde for 30minutes at 37�C, followed by quenching using 125 mM glycine solution. Cellswere lysed, sonicated, and then subjected to immunoprecipitation with 8 mg ofthe rabbit preimmune IgG control (sc-2027, Santa Cruz Biotechnology) orequivalent amounts of the anti-p53 antibody (FL-393). Precipitated chromatin

Fig. 4. Nonanthracycline cytotoxic drugs enhance UGT2B7 mRNA levels in theliver cancer HepG2 cell line. Nearly 100% confluent HepG2 cells in 6-well plateswere treated for 24 hours with vehicle [water for mitomycin C or 0.1%dimethylsulfoxide (DMSO) for the remaining five drugs] or one of the six drugs,namely, mitomycin C (10 mM), 5-FU (300 ng/ml), camptothecin (0.5 mM), SN-38(10 ng/ml), topotecan (0.1 mg/ml), and etoposide (50 mM). Total RNA was extractedand reverse transcribed to generate cDNA, followed by quantitative real-time PCRto quantify the mRNA levels of three genes (UGT2B7, UGT2B10, and p21) asdescribed in Materials and Methods. Data are presented as the fold induction in targetgene mRNA levels in drug-treated cells over those in vehicle-treated cells (set as a valueof 1). Data shown are mean6 S.D. from a representative experiment of two independentexperiments performed in triplicate. Statistical analysis used one-way analysis ofvariance followed by Tukey’s post hoc multiple comparison test. ** P , 0.01,*** , 0.001.

Induction of UGT2B7 by Cytotoxic Drugs 663

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

was captured by Protein A Sepharose CL-4B beads (GE Healthcare), andsubsequently eluted from the beads as described previously (Hu and Mackenzie,2009). Crosslinking was reversed by heating the eluates at 65�C overnight. Theresulting DNA/protein precipitates were digested with proteinase K, followed byphenol-chloroform extraction and ethanol precipitation. The DNA pellets wereresuspended in 100 ml of Tris-EDTA buffer. Using 3 ml of each of the resultantDNA samples as the template, real-time quantitative PCR was conducted toquantify three UGT2B7 promoter regions, namely, the UGT2B7 p53 site(nucleotides -355/-209 relative to the translation start site) spanning the recentlyreported p53 recognition element (Hu et al., 2014c), and two negative controlregions located at -3026/-2907 (negative control 1 region) and -7906/-7747(negative control 2 region) relative to the UGT2B7 translation start site. Inaddition, the CYP3A4 promoter region covering the known p53 site (termed theCYP3A4 p53 site) was quantified as a positive control (Goldstein et al., 2013).Data from negative control region 2 were used to normalize the starting amountsof immunoprecipitated DNA added to each PCR. Enrichment of p53 binding atthe UGT2B7 and CYP3A4 p53 sites and negative control region 1 were quantifiedrelative to negative control region 2 using the 22DDCT method (Livak andSchmittgen, 2001).

Morphine Glucuronidation Assay. HepG2 cells were plated in T75 flasksand cultured until they reached 90% confluence. Cells were then treated intriplicate with vehicle (water) or mitomycin C at 5, 10, or 20 mM for 24 hours.Cells were harvested, washed twice with 1� phosphate-buffered saline, andthen lysed in 250 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6),followed by protein concentration determination using the Bradford Protein

Assay (Bio-Rad). Morphine glucuronidation assays using these samples wereconducted essentially the same as previously reported (Hu et al., 2014c).

Statistical Analysis. Log transformed data met the assumption of equalvariance at a significance level of less than 0.01 (Levene’s test). Statisticalanalyses of the transformed data were conducted by one- or two-way analysisof variance with the Tukey’s post hoc test or independent t test using theIBM SPSS program (version 22) (Chicago, IL) as detailed in the legendsof Figures 1–7. A P value of less than 0.05 was considered statisticallysignificant.

Results

Induction of UGT2B7 by Anthracyclines. Our recent demonstra-tion of induction of UGT2B7 by epirubicin in HepG2 cells (Hu et al.,2014c) prompted us to test whether other anthracyclines could alsostimulate UGT2B7 expression. As shown in Fig. 1A, the anthracy-clines, doxorubicin, epirubicin, daunorubicin, and idarubicin, at 1mMsignificantly elevated UGT2B7 mRNA levels in HepG2 cells fol-lowing a 24 hour treatment, with epirubicin showing a significantlygreater induction than the three other anthracylines. In contrast,UGT2B10 mRNA levels remained unchanged. Western blot analysisdemonstrated that all four anthracyclines significantly increasedUGT2B7 protein levels in HepG2 cells following a 24 hour exposureto these drugs (Fig. 1B). Epirubicin and doxorubicin showed a similar

Fig. 5. Mitomycin C increases the protein levels of both UGT2B7 and p53, and the enzymatic activity of UGT2B7 in HepG2 cells in a dose-dependent manner. HepG2 cellswere treated for 24 hours in triplicate with vehicle (water) or mitomycin C at three concentrations (i.e., 5, 10, and 20 mM). After treatment, whole cell lysates were preparedfrom treated cells and subjected to Western blotting assays using antibodies recognizing UGT2B7, p53, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or assayedfor morphine glucuronidation activity as described in Materials and Methods. Shown are the immunosignals (A) and the fold induction (i.e., mean 6 S.D.) in the proteinlevels of UGT2B7 (B, left graph) and p53 (B, right graph), or the fold induction (i.e., mean 6 S.D.) in morphine glucuronidation activity (B, middle graph) in mitomycinC–treated cells compared with those in vehicle-treated cells (set at a value of 1) from a representative experiment of two independent experiments performed in triplicate.Statistical analyses used one-way analysis of variance followed by Tukey’s post hoc multiple comparison test. *** P , 0.001.

664 Hu et al.

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

induction in UGT2B7 protein levels despite the fact that epirubicinelicited a greater induction in UGT2B7 mRNA levels compared withdoxorubicin (Fig. 1, A and B).Induction of UGT2B7 by epirubicin was previously found to be

mediated by p53 activation and subsequent binding of p53 toa recognition element in the UGT2B7 proximal promoter (Hu et al.,2014c). As shown in Fig. 1C, Western blot analyses using whole celllysates of HepG2 cells treated with four anthracyclines for 24 hoursdemonstrated that, similar to epirubicin, all three other anthracylinessignificantly increased p53 protein levels. Daunorubicin gave thelowest level of induction while the three other anthracyclines exhibiteda similar level of induction. This activation of p53 was functionallyrelevant because all four anthracyclines increased the mRNA levels ofp21, a p53 target gene (el-Deiry et al., 1993) (Fig. 1A). Knockdown ofp53 by transfection of p53 siRNA into HepG2 cells was conducted toconfirm a role for p53 in anthracyline-induced UGT2B7 expression.As shown in Fig. 2A, p53 siRNA significantly reduced the levels ofendogenously expressed p53 protein; this reduction significantlydecreased the anthracyline-elevated mRNA levels of both UGT2B7and p21 but did not affect UGT2B10 mRNA levels (Fig. 2B).Furthermore, transient luciferase reporter assays demonstrated that,similar to epirubicin and the p53-activator nutlin-3a, doxorubicin andidarubicin activated the UGT2B7 promoter and this activation wasabolished by mutating the p53 site (MT3) but not by a mutation (MT5)that changed the sequence immediately downstream from the p53 site(Fig. 3, A and B). Daunorubicin was not able to stimulate the UGT2B7promoter, suggesting that factors additional to p53 are required toregulate its effect on UGT2B7 gene expression and that other UGT2B7cis regulatory elements may be involved. The reduction of basalUGT2B7 promoter activity resulting from mutation of the p53 site(MT3) is indicative of a role for p53 in controlling constitutiveexpression of UGT2B7 as previously reported (Goldstein et al., 2013;Hu et al., 2014c). Taken collectively, our results demonstrate that

classic anthracyclines stimulate the expression of UGT2B7 in HepG2cells via a p53-signaling pathway.Induction of UGT2B7 by Nonanthracycline Cytotoxic Drugs.

Nonanthracycline cytotoxic drugs have also been shown to be ableto activate p53, including DNA topoisomerase II inhibitors (e.g.,etoposide), DNA topoisomerase I inhibitors (e.g., camptothecin, SN-38, andtopotecan), DNA alkylating agents (e.g., mitomycin C), and antimetaboliteanticancer drugs (e.g., 5-FU) (Fritsche et al., 1993; Tishler et al., 1993,1995; Müller et al., 1997; Inoue et al., 2001; Daoud et al., 2003). To testthe possibility of induction of UGT2B7 by nonanthracycline p53-activating cytotoxic drugs, we treated HepG2 cells for 24 hours with sixnonanthracycline cytotoxic drugs (i.e., mitomycin C, 5-FU, camptothecin,SN-38, topotecan, and etoposide) at concentrations as detailed in theFig. 4 legend. Results from these experiments demonstrated that allsix cytotoxic drugs significantly elevated UGT2B7 mRNA levels withmitomycin C showing the highest induction. In control experiments, allsix drugs enhanced the mRNA levels of p21 (a p53-target gene) butonly SN-38 had any effect on the mRNA levels of UGT2B10 (a smallbut significant reduction). These results demonstrated the capacity ofnonanthracycline p53-activating cytotoxic drugs to induce UGT2B7expression.To investigate the involvement of p53 in the above-described

induction of UGT2B7 by nonanthracycline cytotoxic drugs, we carriedout further experiments in HepG2 cells using mitomycin C, the mostpotent inducer of UGT2B7 among the tested six nonanthracyclinecytotoxic drugs (Fig. 4). First, Western blot assays demonstrated thatmitomycin C elevated the protein levels of both p53 and UGT2B7 ina dose-dependent manner but did not alter the control glyceraldehyde-3-phosphate dehydrogenase protein levels (Fig. 5, A and B); UGT2B7activity, using morphine as the substrate was also increased bymitomycin C in a dose-dependent manner (Fig. 5B, middle panel).Second, knockdown of p53 protein levels by anti-p53 siRNA (Fig.6A) significantly reduced the mitomycin C–induced expression of

Fig. 6. siRNAs against p53 reduce mitomycin C–inducedUGT2B7 mRNA levels in HepG2 cells. Briefly, cells weretransfected at 100 nM with either p53 siRNA or nontarget siRNA(neg-siRNA). Forty-eight hours after transfection, cells weretreated for 24 hours with mitomycin C (10 mM) or the p53activator nutlin-3a (10 mM), followed by quantitative real-timePCR to quantify the mRNA levels of UGT2B7 and p21 asdescribed in Materials and Methods. Shown are the immunosig-nals (A) and the fold induction (i.e., mean 6 S.D) (B) in targetgene mRNA levels in drug-treated and p53 siRNA-transfectedHepG2 cells compared with those in drug-treated and neg-siRNA–transfected cells (set as a value of 100%) from arepresentative experiment of two independent experimentsperformed in triplicate. GAPDH, glyceraldehyde-3-phosphatedehydrogenase. Statistical analyses used two-way analysis ofvariance followed by independent t tests with Bonferronicorrection, * , 0.05, ** P , 0.01.

Induction of UGT2B7 by Cytotoxic Drugs 665

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

both UGT2B7 and p21 (Fig. 6B). As expected, this strategy reduced theinduction of both genes by the p53-activator nutlin-3a. Third, transientluciferase reporter assays showed that mitomycin C significantly increasedthe activity of the wild-type UGT2B7 promoter (UGT2B7-575/-1) but notthe p53 site-mutated UGT2B7 promoter (UGT2B7-575/-1/MT3) (Fig.7A). Fourth, similar to epirubicin and the p53 activator nutlin-3a,chromatin immunoprecipitation analysis showed that mitomycin Crecruited p53 to the region covering the recently reported UGT2B7 p53site (Hu et al., 2014c) or the CYP3A4 p53 site (Goldstein et al., 2013)but not to the region that is approximately 3 kb upstream of theUGT2B7 p53 site (Fig. 7B). Taken together, these results demonstratedthat, similar to epirubicin, mitomycin C stimulates UGT2B7 expressionvia p53 activation and subsequent binding of p53 to the reported p53recognition element in the UGT2B7 promoter.

Discussion

Recent studies have shown that a novel group of genes involved indrug metabolism are induced by the cytotoxic drug-activated p53-signaling pathway, including CYP enzymes (Goldstein et al., 2013)and UGTs (Dellinger et al., 2012; Hu et al., 2014c). This enhanceddrug metabolism activity promotes drug clearance and may be relevantto reduced chemotherapeutic efficacy or the development of drugresistance. A recent example is the induction of UGT2B7 expressionby epirubicin in the liver cancer HepG2 cell line via a p53 site in theproximal UGT2B7 promoter (Hu et al., 2014c). Because epirubicin is

a substrate of UGT2B7 (Innocenti et al., 2001), this induction enhancesepirubicin inactivation via glucuronidation. The present study demon-strates that, similar to epirubicin, several other cytotoxic drugs, includingthree anthracyclines (i.e., doxorubicin, daunorubicin, and idarubicin) andsix nonanthracyclines (i.e., mitomycin C, 5-FU, camptothecin, SN-38,topotecan, and etoposide), stimulated UGT2B7 expression via the p53pathway. UGT2B7 is not involved in the metabolism of these ninecytotoxic drugs, and thus this enhanced UGT2B7 activity would notaffect the therapeutic efficacy of these cytotoxic drugs themselves;however, cytotoxic drugs with different modes of action are frequentlyused in combination (termed combinatorial chemotherapy) to enhancechemotherapy efficacy and/or to prevent/delay development of che-motherapy resistance. Frequently, two of these nine cytotoxic drugs areused in combinatorial chemotherapy for treating various cancers; forexample, the double (epirubicin/mitomycin C) and triple (epirubicin/mitomycin C/cisplatin) chemotherapeutic regimens for treating advancedhepatocellular carcinoma (Marelli et al., 2007). Thus, if induction ofUGT2B7 by mitomycin C occurs in vivo the enhanced glucuronidationof epirubicin may reduce the overall efficacy of regimens that containboth of these drugs. In addition to the cytotoxic drugs tested in thepresent study, other cytotoxic and noncytotoxic drugs have also beenshown to be able to activate the p53-signaling pathway, such asmethotrexate, cisplatin, cyclophosphamide, bleomycin, taxol, vinblas-tine, nocodazole, arsenite, and deferoxamine mesylate (Fritsche et al.,1993; Tishler et al., 1993, 1995; Müller et al., 1997; Appella andAnderson, 2001; Pluquet and Hainaut, 2001). The capacity of these

Fig. 7. Mitomycin C activates the UGT2B7promoter and recruits p53 to the UGT2B7promoter encompassing the previously reportedp53 site in HepG2 cells. (A) HepG2 cells weretransfected with the internal control pRL-nullvector and one of the three luciferase con-structs (pGL3-basic vector, UGT2B7-575/-1,and UGT2B7-575/-1/MT3); 24–48 hours aftertransfection, cells were treated for 24 hourswith vehicle (water) or mitomycin C (5 mM).After treatment, cells were harvested andassayed for luciferase activity as described inMaterials and Methods. Data are presented asthe fold induction of the activities of promoterconstructs relative to those of the promotorlesspGL3-basic vector (set to a value of 1). Datashown are mean 6 S.D. from a representativeexperiment of two independent experimentsperformed in triplicate. (B) Cells were treatedfor 24 hours with vehicle, 1 mM epirubicin,10 mM nutlin-3a, or 10 mM mitomycin C, andthen subjected to chromatin immunoprecipitationassays, followed by real-time PCR to quantifythe amount of precipitated DNA correspondingto (1) the previously defined UGT2B7 p53-bindingsite; (2) a region located approximately 3 kbupstream of the UGT2B7 p53 site (negativecontrol 1); and (3) the reported CYP3A4 p53 site(positive control). Data were normalized as de-scribed in Materials and Methods using amplifica-tion values for the negative control 2 locus, andwere then expressed as the fold enrichment in DNAsamples precipitated with the anti-p53 antibody(FL393) compared with that in the control DNAsamples precipitated with equivalent amounts ofpreimmune IgG serum (IgG) (set as a value of 1).Data shown are mean 6 S.D. from one represen-tative experiment of two independent experimentsperformed in triplicate. Statistical analyses usedtwo-way analysis of variance followed by Tukey’spost hoc multiple comparison test. *** P , 0.001.

666 Hu et al.

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

drugs to stimulate UGT2B7 expression and activity remains to beinvestigated. In addition to cytotoxic drugs, the p53-signaling pathwayhas also been shown to be activated by other genotoxic stress, includinghypoxia, UV light, radiation, and nucleotide depletion (Appella andAnderson, 2001; Pluquet and Hainaut, 2001; Resnick-Silverman andManfredi, 2006; Riley et al., 2008). It remains to be determined whetherUGT2B7 is induced upon exposure to these diverse genotoxic stimuli.Because the liver is the major glucuronidation site in the human body,

hepatic UGT expression and activity governs systemic metabolism andclearance of UGT substrates. As reviewed recently (Hu et al., 2014c),UGT2B7 displays high activity toward a variety of noncytotoxic drugs.In fact, it was estimated that 20 of the top 200 prescribed drugs in 2002were UGT substrates, and seven of these 20 drugs were UGT2B7substrates (Williams et al., 2004). Therefore, co-administration ofnoncytotoxic drugs that are UGT2B7 substrates (e.g., morphine) withUGT2B7-inducing cytotoxic drugs for treating liver cancer andnonhepatic cancers may induce the expression and activity of UGT2B7in normal liver tissue, and thus enhance systemic metabolism andclearance of cytotoxic and noncytotoxic drugs that are primarilyinactivated by UGT2B7. This represents a novel mechanism that maylead to cytotoxic drug-related drug-drug interactions. Table 1 lists themajor types of human cancers with reported chemotherapeutic regimenscontaining cytotoxic drugs that have been shown in the present study tobe able to induce UGT2B7 expression in a hepatic cellular context.Taken together, this possible hepatic induction of UGT2B7 by p53-activating cytotoxic drugs could promote systemic metabolism ofcytotoxic drugs themselves (e.g., epirubicin) and co-administerednoncytotoxic drugs that are UGT2B7 substrates (e.g., morphine), thusleading to overall reduced efficacy of therapy in cancer patients. Thispossibility requires testing in patient populations.

In conclusion, the present study demonstrates that similar to epirubicin,another nine cytotoxic drugs stimulate UGT2B7 expression in HepG2cells through the p53-signaling pathway, most likely via the previouslyreported UGT2B7 p53 site in the proximal promoter. These observationsfurther support our previous finding that UGT2B7 is a p53 target gene ina hepatic cellular context and shed more insight into the molecularmechanisms controlling the hepatic expression and activity of UGT2B7.This cytotoxic drug-induced UGT2B7 activity in target liver cancer cellswithin the tumor or possibly in normal liver cells could promoteintratumoural or systemic metabolism and clearance of cytotoxic drugsand other co-administered drugs that are inactivated by UGT2B7, andthus it may relate to reduced efficacy of cancer therapy or even lead todevelopment of drug resistance.

Authorship ContributionsParticipated in research design: Hu, Mackenzie, McKinnon.Conducted experiments: Hu, Lu.Performed data analysis: Hu, Mackenzie.Wrote or contributed to the writing of the manuscript: Hu, Mackenzie,

Meech, McKinnon.

References

Appella E and Anderson CW (2001) Post-translational modifications and activation of p53 bygenotoxic stresses. Eur J Biochemistry 268:2764–2772.

Argikar UA and Remmel RP (2009) Effect of aging on glucuronidation of valproic acid in humanliver microsomes and the role of UDP-glucuronosyltransferase UGT1A4, UGT1A8, andUGT1A10. Drug Metab Dispos 37:229–236.

Basseville A, Preisser L, de Carné Trécesson S, Boisdron-Celle M, Gamelin E, Coqueret O,and Morel A (2011) Irinotecan induces steroid and xenobiotic receptor (SXR) signaling todetoxification pathway in colon cancer cells. Mol Cancer 10:80.

Coffman BL, Rios GR, King CD, and Tephly TR (1997) Human UGT2B7 catalyzes morphineglucuronidation. Drug Metab Dispos 25:1–4.

Cosse JP, Rommelaere G, Ninane N, Arnould T, and Michiels C (2010) BNIP3 protects HepG2cells against etoposide-induced cell death under hypoxia by an autophagy-independent path-way. Biochem Pharmacol 80:1160–1169.

Daoud SS, Munson PJ, Reinhold W, Young L, Prabhu VV, Yu Q, LaRose J, Kohn KW,Weinstein JN, and Pommier Y (2003) Impact of p53 knockout and topotecan treatment on geneexpression profiles in human colon carcinoma cells: a pharmacogenomic study. Cancer Res 63:2782–2793.

Dellinger RW, Matundan HH, Ahmed AS, Duong PH, and Meyskens FL, Jr (2012) Anti-cancerdrugs elicit re-expression of UDP-glucuronosyltransferases in melanoma cells. PLoS ONE 7:e47696.

el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE,Kinzler KW, and Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression.Cell 75:817–825.

Fritsche M, Haessler C, and Brandner G (1993) Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 8:307–318.

Goldstein I, Rivlin N, Shoshana OY, Ezra O, Madar S, Goldfinger N, and Rotter V (2013)Chemotherapeutic agents induce the expression and activity of their clearing enzyme CYP3A4by activating p53. Carcinogenesis 34:190–198.

Holohan C, Van Schaeybroeck S, Longley DB, and Johnston PG (2013) Cancer drug resistance:an evolving paradigm. Nat Rev Cancer 13:714–726.

Hu DG and Mackenzie PI (2009) Estrogen receptor a, Fos-related antigen-2, and c-Jun co-ordinately regulate human UDP glucuronosyltransferase 2B15 and 2B17 expression in re-sponse to 17b-estradiol in MCF-7 cells. Mol Pharmacol 76:425–439.

Hu DG, Meech R, Lu L, McKinnon RA, and Mackenzie PI (2014a) Polymorphisms and hap-lotypes of the UDP-glucuronosyltransferase 2B7 gene promoter. Drug Metab Dispos 42:854–862.

Hu DG, Meech R, McKinnon RA, and Mackenzie PI (2014b) Transcriptional regulation ofhuman UDP-glucuronosyltransferase genes. Drug Metab Rev 46:421–458.

Hu DG, Rogers A, and Mackenzie PI (2014c) Epirubicin upregulates UDP glucuronosyl-transferase 2B7 expression in liver cancer cells via the p53 pathway. Mol Pharmacol 85:887–897.

Innocenti F, Iyer L, Ramírez J, Green MD, and Ratain MJ (2001) Epirubicin glucuronidation iscatalyzed by human UDP-glucuronosyltransferase 2B7. Drug Metab Dispos 29:686–692.

Inoue T, Geyer RK, Yu ZK, and Maki CG (2001) Downregulation of MDM2 stabilizes p53 byinhibiting p53 ubiquitination in response to specific alkylating agents. FEBS Lett 490:196–201.

Italia C, Paglia L, Trabattoni A, Luchini S, Villas F, Beretta L, Marelli G, and Natale N (1983)Distribution of 49epi-doxorubicin in human tissues. Br J Cancer 47:545–547.

Jin C, Miners JO, Lillywhite KJ, and Mackenzie PI (1993) Complementary deoxyribonucleic acidcloning and expression of a human liver uridine diphosphate-glucuronosyltransferase glucuronidatingcarboxylic acid-containing drugs. J Pharmacol Exp Ther 264:475–479.

Kerdpin O, Mackenzie PI, Bowalgaha K, Finel M, and Miners JO (2009) Influence of N-terminaldomain histidine and proline residues on the substrate selectivities of human UDP-glucuronosyltransferase 1A1, 1A6, 1A9, 2B7, and 2B10. Drug Metab Dispos 37:1948–1955.

L’Espérance S, Bachvarova M, Tetu B, Mes-Masson AM, and Bachvarov D (2008) Global geneexpression analysis of early response to chemotherapy treatment in ovarian cancer spheroids.BMC Genomics 9:99.

Liedtke C, Lambertz D, Schnepel N, and Trautwein C (2007) Molecular mechanism of Mito-mycin C-dependent caspase-8 regulation: implications for apoptosis and synergism withinterferon-alpha signalling. Apoptosis 12:2259–2270.

TABLE 1

Cytotoxic drugs studied in this study and their indications

Drug Major Indication

Doxorubicin LymphomasBreast cancerSarcomasHepatocellular cancerLeukaemias

Epirubicin Breast cancerHepatocellular cancer

Daunorubicin Acute myelogenous leukemiaAcute lymphocytic leukemia

Idarubicin Acute myelogenous leukemiaEtoposide Testicular cancer

Small cell lung cancerOvarian cancer

Irinotecan (SN-38) Colorectal cancerPancreatic cancerOvarian cancer

Topotecan Small cell lung cancerOvarian cancerLung cancer

5-FU Colorectal cancerBreast cancerOvarian cancerHepatocellular cancerStomach cancerPancreatic cancer

Camptothecin Not approved for clinical useMitomycin C Hepatocellular cancer

Stomach cancerPancreatic cancerColon cancerBreast cancerAnal cancerLung cancer

Induction of UGT2B7 by Cytotoxic Drugs 667

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from

Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real-timequantitative PCR and the 22DDCT method. Methods 25:402–408.

Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, OwensIS, and Nebert DW (2005) Nomenclature update for the mammalian UDP glycosyltransferase(UGT) gene superfamily. Pharmacogenet Genomics 15:677–685.

Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Bélanger A, Fournel-Gigleux S,Green M, Hum DW, and Iyanagi T, et al. (1997) The UDP glycosyltransferase gene super-family: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics7:255–269.

Malmlof M, Paajarvi G, Hogberg J, and Stenius U (2008) Mdm2 as a sensitive and mechanis-tically informative marker for genotoxicity induced by benzo[a]pyrene and dibenzo[a,l]pyrene.Toxicol Sci 102:232–240.

Marelli L, Stigliano R, Triantos C, Senzolo M, Cholongitas E, Davies N, Tibballs J, Meyer T,Patch DW, and Burroughs AK (2007) Transarterial therapy for hepatocellular carcinoma:which technique is more effective? A systematic review of cohort and randomized studies.Cardiovasc Intervent Radiol 30:6–25.

Meijerman I, Beijnen JH, and Schellens JH (2008) Combined action and regulation of phase IIenzymes and multidrug resistance proteins in multidrug resistance in cancer. Cancer Treat Rev34:505–520.

Müller M, Strand S, Hug H, Heinemann EM, Walczak H, Hofmann WJ, Stremmel W, KrammerPH, and Galle PR (1997) Drug-induced apoptosis in hepatoma cells is mediated by the CD95(APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. J Clin Invest 99:403–413.

O’Dwyer PJ and Catalano RB (2006) Uridine diphosphate glucuronosyltransferase (UGT) 1A1and irinotecan: practical pharmacogenomics arrives in cancer therapy. J Clin Oncol 24:4534–4538.

Pavelic J (2014) Editorial: combined cancer therapy. Curr Pharm Des 20:6511–6512.Picard N, Ratanasavanh D, Prémaud A, Le Meur Y, and Marquet P (2005) Identification of theUDP-glucuronosyltransferase isoforms involved in mycophenolic acid phase II metabolism.Drug Metab Dispos 33:139–146.

Piret JP, Cosse JP, Ninane N, Raes M, and Michiels C (2006) Hypoxia protects HepG2 cellsagainst etoposide-induced apoptosis via a HIF-1-independent pathway. Exp Cell Res 312:2908–2920.

Pluquet O and Hainaut P (2001) Genotoxic and non-genotoxic pathways of p53 induction.Cancer Lett 174:1–15.

Qin J, Lin Y, Norman RX, Ko HW, and Eggenschwiler JT (2011) Intraflagellar transport protein122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway com-ponents. Proc Natl Acad Sci USA 108:1456–1461.

Resnick-Silverman L and Manfredi JJ (2006) Gene-specific mechanisms of p53 transcriptionalcontrol and prospects for cancer therapy. J Cell Biochem 99:679–689.

Riley T, Sontag E, Chen P, and Levine A (2008) Transcriptional control of human p53-regulatedgenes. Nat Rev Mol Cell Biol 9:402–412.

Roudier E, Mistafa O, and Stenius U (2006) Statins induce mammalian target of rapamycin(mTOR)-mediated inhibition of Akt signaling and sensitize p53-deficient cells to cytostaticdrugs. Mol Cancer Ther 5:2706–2715.

Saunders RA, Fujii K, Alabanza L, Ravatn R, Kita T, Kudoh K, Oka M, and Chin KV (2010)Altered phospholipid transfer protein gene expression and serum lipid profile by topotecan.Biochem Pharmacol 80:362–369.

Schneider CA, Rasband WS, and Eliceiri KW (2012) NIH Image to ImageJ: 25 years of imageanalysis. Nat Methods 9:671–675.

Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, and Gottesman MM (2006) Targetingmultidrug resistance in cancer. Nat Rev Drug Discov 5:219–234.

Takahata T, Ookawa K, Suto K, Tanaka M, Yano H, Nakashima O, Kojiro M, Tamura Y, TateishiT, and Sakata Y, et al. (2008) Chemosensitivity determinants of irinotecan hydrochloride inhepatocellular carcinoma cell lines. Basic Clin Pharmacol Toxicol 102:399–407.

Tishler RB, Calderwood SK, Coleman CN, and Price BD (1993) Increases in sequence specificDNA binding by p53 following treatment with chemotherapeutic and DNA damaging agents.Cancer Res 53 (Suppl):2212–2216.

Tishler RB, Lamppu DM, Park S, and Price BD (1995) Microtubule-active drugs taxol, vin-blastine, and nocodazole increase the levels of transcriptionally active p53. Cancer Res 55:6021–6025.

Wen Z, Tallman MN, Ali SY, and Smith PC (2007) UDP-glucuronosyltransferase 1A1 is theprincipal enzyme responsible for etoposide glucuronidation in human liver and intestinalmicrosomes: structural characterization of phenolic and alcoholic glucuronides of etoposideand estimation of enzyme kinetics. Drug Metab Dispos 35:371–380.

Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR,and Ball SE (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates:a pharmacokinetic explanation for typically observed low exposure (AUCI/AUC) ratios. DrugMetab Dispos 32:1201–1208.

Address correspondence to: Ross A. McKinnon, Flinders Centre for Innovationin Cancer, School of Medicine, Flinders University, Bedford Park SA 5042, Australia.E-mail: ross.mckinnon@flinders.edu.au

668 Hu et al.

at ASPE

T Journals on February 17, 2021

dmd.aspetjournals.org

Dow

nloaded from