Research Article

Induction of Retinoid X Receptor Activity and ConsequentUpregulation of p21WAF1/CIP1 by Indenoisoquinolines inMCF7 Cells

Eun-Jung Park1, Tamara P. Kondratyuk1, Andrew Morrell2, Evgeny Kiselev2, Martin Conda-Sheridan2,Mark Cushman2, Soyoun Ahn3, Yongsoo Choi3, Jerry J. White3, Richard B. van Breemen3, andJohn M. Pezzuto1

AbstractRetinoid X receptor (RXR) has been targeted for the chemoprevention and treatment of cancer. To

discover potential agents acting through RXRs, we utilized an RXR response element (RXRE)-luciferase

reporter gene assay. Following extensive screening, 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-

11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities.

AM6-36 inhibited COX-2 expression and anchorage-independent growth with 12-O-tetradecanoylphorbol

13-acetate-stimulated JB6 Cl41 cells, induced the expression of CD38 in HL-60 cells, and attenuated the

growth of N-methyl-N-nitrosourea–induced mammary tumors in rats. Consistent with other reports

describing the antiproliferative effects of RXR agonists in breast cancers, AM6-36 showed growth inhibition

with cultured MCF7 breast cancer cells, accompanied by G2/M-phase arrest at lower concentrations and

enhanced S-phase arrest at higher concentrations. On the basis of DNA microarray analysis, AM6-36

upregulated the expression of CDKN1A, a target gene of RXR, by 35-fold. In accord with this response, the

expression of the corresponding protein, p21WAF1/CIP1, was increased in the presence of AM6-36. Induction

of p21 by AM6-36 was abrogated following transient knockdown of RXRa, demonstrating that the effect of

AM6-36 on the expression of p21 is closely related to modulation of RXRa transcriptional activity.

Intestinal permeability was suggested with Caco-2 cells and limitedmetabolism resulted when AM6-36 was

incubated with human livermicrosomes. Oral administration with rats resulted in 0.8 mg/mL, 4.3 mg/g, and0.3 mg/g in serum, liver, and mammary gland, respectively. In sum, these data suggest that AM6-36 is a

promising lead for the treatment or prevention of breast cancer and provide a strong rationale for testing in

more advanced antitumor systems. Cancer Prev Res; 4(4); 592–607. �2011 AACR.

Introduction

Retinoid X receptor (RXR), a member of the nuclearreceptor superfamilies, was identified as an orphan recep-tor with a high sequence homology to retinoic acid receptor(RAR), and a specific responsiveness to vitamin A meta-bolites (1). Three subtypes of RXRs, RXRa, RXRb and RXRg ,have been identified, and essential roles of RXRs have been

reported in various physiologic processes includingembryonic development, metabolic processes, differentia-tion, and apoptosis. Similar to other nuclear receptors,RXRs require specific ligands to be functionally activated.Whereas ligand-free RXRs are sequestered by specificnuclear receptor corepressors; on ligand binding, RXRsundergo conformational changes, leading to the dissocia-tion of corepressors and the association of coactivators (2).Consequently, ligand-activated RXR dimers recognize thecorresponding cis-acting element in the promoter region oftarget genes, called hormone response elements (HRE),and are capable of triggering transcription. HREs consist of2 direct repeats (DR) of the consensus hexamers (AGGTCA)in tandem, separated by 1 to 5 nucleotides, which aredesignated as DR1 to DR5, respectively. In particular,RXR homodimers are known to interact with DR1, RXRresponse element (RXRE; ref. 3).

As mentioned above, specific ligands for RXRs generallyplay key roles in the activation of RXR signaling and severalsubstances are known to function as endogenous ligandsincluding 9-cis-retinoic acid (9-cis-RA), docosahexaenoic

Authors' Affiliations: 1College of Pharmacy, University of Hawaii at Hilo,Hilo, Hawaii; 2Department of Medicinal Chemistry and Molecular Pharma-cology, College of Pharmacy, and the Purdue Center for Cancer Research,Purdue University, West Lafayette, Indiana; and 3Department of MedicinalChemistry and Pharmacognosy, University of Illinois College of Pharmacy,Chicago, Illinois

Note: Supplementary data for this article are available at Cancer Preven-tion Research Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: John M. Pezzuto, College of Pharmacy, Univer-sity of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720. Phone: 808-933-2909; Fax: 808-933-2981; E-mail: pezzuto@hawaii.edu

doi: 10.1158/1940-6207.CAPR-10-0004

�2011 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 4(4) April 2011592

acid, and other unsaturated fatty acids (4). Besides putativeendogenous ligands of RXRs, additional compounds havebeen reported as RXR agonists, so-called rexinoids includ-ing AGN194204, CD3254, LG100268, LGD1069, andSR11237. Although the mechanisms underlying their sup-pressive and inhibitory actions against cancerous processeshave not been fully clarified, both naturally occurring andsynthetic rexinoids have shown promise in the preventionand treatment of cancer (5). For example, 9-cis-RA,LG100268, and LGD1069 have shown efficacy in N-methyl-N-nitrosourea (MNU)-induced breast cancer ani-mal models (6–8). Furthermore, 9-cis-RA and LGD1069have been approved by the Food and Drug Administration(FDA) for the treatment of Kaposi’s sarcoma and cutaneousT-cell lymphoma, respectively (9, 10).In searching for novel cancer chemopreventive and ther-

apeutic agents capable of functioning as RXR agonists, weutilized an RXRE-luciferase reporter gene assay. This assayassesses RXR transcriptional activity as well as binding toRXR, since trans-activation occurs only after ligand binding.During the course of testing over 5,000 natural productextracts and compounds, only a single active lead wasidentified: 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride(AM6-36; Fig. 1). On the basis of the unexpected yet uniqueability of AM6-36 to serve as an RXR agonist, 18 structuralrelatives were tested, but AM6-36 still demonstrated thegreatest activity (Table 1).Using MCF7 breast cancer cells as a model, AM6-36 was

found to suppress proliferation and this was accompaniedby cell-cycle arrest. RXRa transcriptional activity was upre-gulated with subsequent expression of RXR target geneCDKN1A. Moderate absorption and slow metabolismwas demonstrated. Strong indications of cancer chemopre-ventive potential were observed with HL-60 cells, 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated JB6 Cl41cells, and rats bearing mammary tumors. Finally, a syn-thetic procedure for large-scale production is reported thatwill enable additional antitumor evaluations.

Materials and Methods

Reagents9-cis-RA, TPA, trichloroacetic acid (TCA), sulforhoda-

mine B (SRB), paraformaldehyde, IgG1-fluorescein isothio-cyanate (FITC) isotype control frommurine myeloma, and7-hydroxystaurosporine (UCN-01) were purchased fromSigma-Aldrich, Inc. Bexarotene was purchased from LCLaboratories. The translucent reporter vector carrying fireflyluciferase under the control of RXRE (50-AGGT-CACAGGTCACAGGTCACAGGTCACAGGTCA-30; pRXRE)was purchased from Panomics. pBABE-puro vector encod-ing the cDNA for human RXRa (phRXRa) was purchasedfrom Addgene Inc. Renilla reniformis luciferase vector (pRL)and Dual-Luciferase Reporter Assay System were purchasedfrom Promega. Dulbecco’s modified Eagle’s medium(DMEM), Eagle’s minimal essential medium (MEM), fetalbovine serum (FBS) and antibiotics-antimycotics solution

(100�), Lipofectamine 2000, and Trizol reagent were pur-chased from Invitrogen. FITC-conjugated CD38 antibody(anti–CD38-FITC), and nuclear isolation medium 40,6-dia-midino-2-phenylindole (NIM-DAPI) were purchased fromBeckman Coulter, Inc. Rabbit polyclonal anti-RXRa anti-body, mouse monoclonal anti-p21WAF1/CIP1 (p21) anti-body, mouse monoclonal anti-p53 antibody, humanRXRa small interfering RNA (siRNA), human p53 siRNA,human p21 siRNA, and control siRNA-A were purchasedfrom Santa Cruz Biotechnology. Rabbit polyclonal anti–b-actin, rabbit polyclonal anti–COX-2, Cell lysis buffer(10�), and LumiGLO chemiluminescent detection kit werefrom Cell Signaling Biotechnology. RT2 First Strand Kit (C-03) and RT2 Profiler PCR Array related to human cell cyclewere purchased from SABiosciences. Cell transformationdetection assay kit was purchased from Millipore.

Synthesis of indenoisoquinolinesAs outlined in Figure 1, scalable conditions were devel-

oped for the preparation of AM6-36 (compound 3) andrelated indenoisoquinolines. Azidopropyl indenoisoqui-noline 2 was obtained following previously described pro-cedures (11, 12). We have previously prepared AM6-36 onsmall scale from compound 1 according to method A.However, this method was found impractical for the pre-paration of multigram quantities of the target compound.Therefore, an alternative approach for the reduction ofcompound 2 or conversion of compound 1 to the desiredamount of AM6-36 was necessary. After a series of attemptsto reduce both nitro and azido groups of compound 2stepwise using various conditions, the reduction with ironpowder in the presence of ammonium chloride solutionwas found to be the best method (method B). Tosylatedanalogues of AM6-36 12–15 were prepared by treatment ofthe corresponding alkoxy derivatives 21–24 with 4-tolue-nesulfonyl chloride in dichloromethane in the presence of4-dimethylaminopyridine and triethylamine. Compound 5was prepared by condensation of 5-nitrohomophthalicanhydride (24) withN-benzylidenemethylamine, followedby treatment of the product with thionyl chloride, intra-molecular Friedel–Crafts reaction, and reduction of thenitro group (11). Compounds 17 and 18 were obtainedas the products of Sandmeyer reaction of 5. Compounds 6to 11 and 21were prepared as previously described (11–13,14, 15). Melting points were determined using capillarytubes with a Mel-Temp apparatus and are uncorrected.Proton nuclear magnetic resonance (1H NMR) spectrawere recorded using ARX300 300 MHz and DRX500500 MHz Bruker NMR spectrometers. IR spectra wererecorded using a Perkin-Elmer 1600 series FTIR spectro-meter. Combustion microanalyses were performed at thePurdue University Microanalysis Laboratory and thereported values were within 0.4% of the calculated values.High-performance liquid chromatography (HPLC) ana-lyses were performed on a Waters 1525 binary HPLC

pump/Waters 2487 dual l absorbance detector system.Analytic thin-layer chromatography was carried out onBaker-flex silica gel IB2-F plates and compounds were

RXRE Transcriptional Activity by Indenoisoquinoline

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visualized with short-wavelength UV light. Silica gel flashchromatography was performed using 230 to 400 meshsilica gel. Physical and spectral data are reported asSupplementary Method S1.

Cell cultureAll cell lines were grown in media supplemented with

penicillin G (100 units/mL), streptomycin (100 mg/mL),and heat-inactivated FBS at 37�C in 5% CO2 in a humidi-fied incubator. COS-1 African green monkey kidney fibro-blast cells and MCF7 human breast cancer cells were

maintained in DMEM containing 10% FBS. JB6 Cl41mouse epidermal cells were grown in MEM containing5% FBS. HL-60 human promyelocytic leukemia cells weregrown in RPMI 1640 supplemented with 10% FBS. Thesecell lines were obtained from the American Type CultureCollection (ATCC). Stock cultures were prepared and ali-quots were stored in liquid nitrogen. During the course ofthe study, cells were used below a passage number of 20and no significant changes were noted in morphology orgrowth characteristics. The cell lines were not authenticatedby the authors.

N

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Synthesis of AM6-36 analogues

( )n( )n

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2.KI, dioxane, H2O, 0°C

5726252

5+

7182 18

n = 4 n = 4

Figure 1. Synthesis of AM6-36 and structurally related indenoisoquinolines.

Park et al.

Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research594

RXRE-luciferase reporter gene assayCOS-1 cells (1 � 104 cells/well) were plated in a 96-well

culture plate and incubated for 24 hours. Then, 100 ng ofpRXRE, 50 ng of phRXRa, and 3 ng of pRL were transientlycotransfected into COS-1 cells in each well by using Lipo-fectamine 2000 according to the manufacturer’s protocol.After 24 hours of transfection, cells were treated withcompounds and further incubated for 24 hours. Cells werethen lysed and the RXRE transcriptional activities weredetermined by measuring the reporter luciferase activitiesusing the Dual-Luciferase Reporter Assay System.

Evaluation of antiproliferative potentialMCF7 cells (1 � 104 cells/well) were seeded in 96-well

plates and incubated with compounds at 37�C in a humi-dified atmosphere with 5% CO2 for 24, 48, and 72 hours.After the incubation, cell viability was estimated by the SRBcellular protein staining method as previously described(16).

Analysis of cell-cycle distributionCell-cycle distribution was assessed by staining DNA

content with NIM-DAPI solution according to the man-ufacturer’s instructions. Briefly, the media was discardedfrom MCF7 cells after 24, 48, and 72 hours of incubationwith various concentrations of AM6-36. Cells were har-vested and stained with NIM-DAPI solution just before themeasurement using Cell Lab Quanta SC flow cytometer(Beckman Coulter; ref. 17).

Evaluation of protein expressionCells were lysed by using 1� cell lysis buffer diluted from

10� cell lysis buffer according to the manufacturer’s pro-tocol. Cell lysates were centrifuged at 14,000 � g for 10minutes at 4�C and the resultant supernatants were col-lected and stored at�80�C until use. After quantification ofprotein by the Bradford method (18), equal amounts oftotal protein in each cell lysate were resolved using 12%SDS-PAGE and electrotransferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were incu-bated with 5% skimmed milk in 0.1% Tris-buffered saline(TBS) containing Tween 20 (TBST) for 1 hour at roomtemperature to block nonspecific protein binding. Then,membranes were incubated overnight at 4�C with corre-sponding primary antibodies in 3% skimmed milk in TBSfollowed by the incubation with horseradish peroxidase(HRP)-conjugated secondary antibodies and visualizedusing a LumiGLO chemiluminescent detection kit underGeliance 1000 imager (Perkin Elmer, Inc.).

Profile of altered gene expressionTotal RNA was extracted from cells using Trizol reagent

according to the method of Chomczynski and Sacchi (19).Isolated RNA was dissolved in RNase-free water and thequality and quantity were measured using a NanoDropND-1000 spectrophotometer (Thermo Fisher ScientificInc.). Total RNA (1 mg) was incubated with genomicDNA elimination mixture at 42�C for 5 minutes followed

by reverse transcription to cDNA under the condition of42�C for 15 minutes and 95�C for 5 minutes using an RT2

First Strand Kit (C-03) on an ABI 7300 thermocycler(Applied Biosystems Inc.). cDNA was applied to RT2 Pro-filer PCR Array related to human cell-cycle according to amanufacturer’s instructions. Samples were run in quadru-plicate to ensure amplification integrity. The expressionlevels of 84 cell-cycle–related genes in cDNA of eachsample were analyzed (20).

siRNA transient transfectionMCF7 cells were plated at 10� 104 cells per well in 6-well

culture plates and incubated for 24 hours. Cells in eachwellwere transfected with 50 pmol of human RXRa siRNA,human p53 siRNA, human p21 siRNA, or control siRNA-Ausing Lipofectamine 2000 for 24 hours followed by sampletreatment for additional 24 hours. To examine the expres-sion of RXRa and p21, Western blot analysis was per-formed as described above.

Expression of COX-2 in TPA-treated JB6 Cl41 cellsJB6 Cl41 cells were seeded in 6-well plates (2� 105 cells/

well) and incubated at 37�C in a humidified atmospherewith 5% CO2 for 24 hours. After incubation, cells wereexposed to TPA (10 ng/mL) with vehicle (0.1% dimethyl-sulfoxide, DMSO) alone or various concentrations of AM6-36. After 15 hours of further incubation, protein of eachwell was extracted and subjected toWestern blot analysis asdescribed in the "Evaluation of protein expression" section.

Anchorage-independent cell transformationThe effect of AM6-36 on TPA-induced neoplastic trans-

formation was investigated with JB6 Cl41 cells cultivated insoft agar according to the manufacturer’s protocol. Briefly,500 exponentially growing cells were suspended in 50 mL0.4% agar complete MEM medium over 100 mL 0.8% agarcomplete MEM medium in a 96-well plate. Cells wereexposed to 5 nmol/L TPA, with or without AM6-36, at37�C in a humidified atmosphere with 5%CO2 for 28 days.After incubation, cell quantification solution was added toeach well and further incubated at 37�C for 4 hours. TPA-treated control wells turned to burgundy-brown. Theabsorbance was measured at 490 nm and data wereexpressed as % transformation activity relative to TPA-treated control.

Expression of cell surface marker, CD38HL-60 cells (2 � 105 cells/well) were seeded in 12-well

plates and incubated with samples at 37�C in a humidifiedatmosphere with 5% CO2 for 24 hours. Following themanufacturer’s instructions, cells were harvested and cen-trifuged at 300 � g for 5 minutes, then washed withphosphate-buffered saline (PBS) once. Cells were incu-bated with anti–CD38-FITC for 30 minutes in the dark.Similarly, autofluorescence control (cells only) and nega-tive staining control (cells þ FITC-conjugated, isotype-matched nonspecific mouse immunoglobulin) wereprepared. After the incubation, cells were washed with

RXRE Transcriptional Activity by Indenoisoquinoline

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diluents and centrifuged at 500 � g for 10 minutes. Pre-cipitated cells were fixed with 2% cold paraformaldehydesolution and analyzed by flow cytometry.

Metabolism of AM6-36 by human liver microsomesThe metabolic stability of AM6-36 was determined using

human livermicrosomes. Incubations were performedwithpooled human liver microsomes containing 0.5 mg/mL ofmicrosomalprotein, 10or50mmol/LAM6-36, and1mmol/L NADPH in 50 mmol/L phosphate buffer (pH¼ 7.4), in atotal volume of 0.4 mL. The reactions were initiated byaddition of NADPH and, after a 5-minute preincubation,were performed at 37�C for 40 minutes. The incubationswere terminated by adding 1.6 mL of an ice-cold mixture ofacetonitrile-ethanol (1:1, v/v) and processed high resolu-tion liquid chromatography-mass spectrometry (LC-MS)and liquid chromatography-tandem mass spectrometry(LC-MS-MS) analysis as described previously (21).

Determination of intestinal epithelial monolayerpermeability

To evaluate the absorption of AM6-36 in intestinalepithelial cells, a Caco-2 cell monolayer assay was per-formed. In preparation for the Caco-2 cell monolayerassays, the cell culture medium was removed from boththe apical (AP) and basolateral (BL) chambers. Thecells were washed 3 times and preincubated withHanks’ balanced salt solution (HBSS) containing 25mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES), pH ¼ 7.4, for 30 minutes at 37�C on ashaker bath at 50 rpm. A 20 mmol/L stock solution ofAM6-36 in DMSO was diluted to different concentrationsin HBSS/HEPES buffer (the final concentration of DMSOwas >1%). These test solutions containing AM6-36 wereadded to either the apical chambers (for AP ! BL mea-surement) or the basolateral chambers (for BL ! APassay), and blank HBSS/HEPES buffer was added to theother side. As a test of the integrity of themonolayer and asa marker of low permeability, sucrose (50 mmol/L finalconcentration) was added to the apical chambers. Propra-nolol (10 mmol/L final concentration) was also added as amarker for compounds that are highly permeable. Thetotal volumes of solution in the apical and basolateralchambers were 1.5 and 2.6 mL, respectively. The solutionsin the recipient compartments were removed and replacedwith fresh medium. The samples were stored at �20�Cuntil analysis of AM6-36 by LC-MS-MS as described belowfor the analysis of extracts of serumand tissue. At the endofeach experiment, basolateral samples were also analyzedby LC-MS for sucrose and propranolol. The apparentpermeability coefficients (Papp; �10�6 cm/s � SD) werethen calculated. The Papp of sucrose from the apical to thebasolateral side of each well was also measured andremained acceptably low at 0.16 � 10�6 cm/s (22).

Evaluation of absorption and distribution in ratsA preliminary evaluation was performed to assess the

absorption and distribution of AM6-36. Female Sprague

Dawley rats were obtained at 4 weeks of age (Harlan, Inc.;virus-free colony 202A) and placed on Teklad diet. At77 days of age, the rats were treated with AM6-36 [40mg/kg; 0.5 mL; ethanol:polyethylene glycol (PEG) 400;10:90, v/v] by gavage, and the treatment was continued ona daily basis for 3 days. Blood samples were collected(jugular vein) 3 hours after the first treatment. Three hoursafter the final treatment, the animals were sacrificed, andblood, mammary tissue, liver, and perirenal fat were col-lected. After clotting, the blood samples were centrifuged at100 � g for 20 minutes. Serum and tissue samples werestored at �80�C until analysis. Rat liver was homogenizedin 0.05 mol/L phosphate buffer (pH ¼ 7.4) and mammarygland and perirenal fat tissues were homogenized in amixture (50:50; v/v) of phosphate buffer and methanolto give homogenates containing 0.2 g tissue per mL. As aninternal standard for quantitative analysis, UCN-01 at 1 ng/mL was added to each homogenate and serum sample.Proteins in the homogenized tissue and serum sampleswere precipitated with 4 volumes of ice-cold acetonitrile.After centrifugation at 10,000 � g for 5 minutes, eachsupernatant was removed, evaporated to dryness, andreconstituted in 100 mL of methanol/water (50:50; v/v)for analysis using LC-MS-MS (23). The injection volumewas 10 mL. Briefly, LC-MS-MS analyses were carried outusing a Shimadzu LC-20AD prominence UFLC pump andSIL-20AC HT prominence autosampler interfaced to anApplied Biosystems API 4000 triple quadruple mass spec-trometer. A Waters XTerra MS C18 column (2.1 mm �100 mm, 3.5 mm) was used for HPLC separation with a 13-minute linear gradient from 10% to 80% acetonitrile in0.1% aqueous formic acid at a flow rate of 0.25 mL/min.Positive ion electrospray was used for ionization andproduct ion mass spectra were recorded using collision-induced dissociation and selected reaction monitoring(SRM) of the transition of m/z 320.4 to 303.2 and m/z483.2 to 130.0 (300 ms per transition) for measurement ofAM6-36 and internal standard, respectively. Calibrationcurves (linear from 0.5–500 ng/mL AM6-36; R2 ¼0.996) were prepared using blank serum or homogenizedtissue from control rats.

Effect of AM6-36 on MNU-induced mammary tumorsA preliminary study was performed as described pre-

viously (24). Female Sprague Dawley rats at 28 days ofage were obtained from Harlan Sprague Dawley and accli-mated under the controlled animal quartersmaintained at a12-hour light or 12-hour dark cycle and 22�C� 2�C. MNU(National Cancer Institute Chemical Carcinogen Reposi-tory)was dissolved in sterile-acidified saline (pH¼ 5.0) andinjected via the jugular vein of rats at 50 days of age with adosage of 75 mg/kg body weight. When an animal devel-oped a palpablemammary tumor (approximately 150–200mm2), treatment with AM6-36 (10 mg/kg body weight/d;ethanol:PEG 400, 10:90, v/v) or vehicle was initiated (10rats/group).Over aperiodof 6weeks, the largest diameter ofthe cancer was measured and this value was multipliedby the perpendicular diameter (size expressed as mm2).

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Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research596

Measurements were performed twice a week during theperiod of the study, the average tumor size in the controland treatment groupswere plotted as a functionof time, anddata were analyzed using the Mann–Whitney test.

Statistical analysesResults are presented as means � SD. Unless otherwise

indicated, statistical comparisons were performed by theuse of Student’s t test and the level of P < 0.05 wasconsidered as significantly different.

Results

AM6-36 induced the RXRE transcriptional activity inCOS-1 cells and MCF7 cellsTo investigate the effect of samples on the functional

role of RXRa protein as a transcriptional activator on

RXRE cis-acting element in the promoter region of targetgene, an RXRE-luciferase reporter gene assay (RXRE assay)was performed. As described above, ligand-bound RXRhomodimer interacts with DR1 and initiates the tran-scription of target genes. To discover RXR modulators,COS-1 cells were transiently transfected with pRXRE. Atthe same time, due to the poor expression of endogenousRXRs in COS-1 cells, phRXRa was cotransfected (Fig. 2A).pRL was used as an internal control for normalization. Asshown in Figure 2B, 9-cis-RA induced the relative lucifer-ase activity up to about 20-fold in cells cotransfectedincluding phRXRa, whereas there was no marked increasein cells without phRXRa transfection. Therefore, theexpression of RXRa is necessary for the RXRE-mediatedtranscriptional activation by rexinoids, and it is requiredto enhance the expression of RXRa in COS-1 cell line-based assay.

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Figure 2. Effect of 9-cis-RA and AM6-36 on RXR transcriptional activity. A, MCF7 cells were transfected with empty vector (control) or phRXRa (phRXRa-TF)for 24 and 48 hours. Total protein lysate (35 mg) of each sample was separated on 12% SDS-PAGE and immunoblotted with anti-RXRa antibody or anti-GAPDH antibody. The mean � SD of the band density of RXRa relative to internal control GAPDH of each group (n ¼ 3) is shown as a bar graph.Representative Western blot data are presented under the graph. B–D, cells were transiently transfected with plasmids for 24 hours, then, further incubatedwith 9-cis-RA or AM6-36 for 24 hours. Cells were lysed and dual luciferase activity was measured. Results were expressed as mean� SD (n¼ 4). Each meanvalue was calculated by fold change over control after normalizing ratios of relative light units (RLU) produced by firefly luciferase to RLU produced by Renillaluciferase (firefly Luc/Renilla Luc). B, COS-1 cells cotransfected with pRXRE (100 ng/well) and pRL (5 ng/well), or pRXRE (100 ng/well), phRXRa (50 ng/well),and pRL (7.5 ng/well) were treated with various concentrations of 9-cis-RA. C, COS-1 cells cotransfected with pRXRE (100 ng/well), phRXRa (50 ng/well), andpRL (7.5 ng/well) were treated with various concentrations of 9-cis-RA or AM6-36. D, MCF7 cells cotransfected with pRXRE (100 ng/well) and pRL (5 ng/well)were incubated with the indicated concentrations of 9-cis-RA or AM6-36. *, value of P < 0.05 was considered statistically significant.

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www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 597

Next, we tested several thousand natural products andsynthetic compounds. Through the process, only one activelead was found, AM6-36. As illustrated in Figure 2C, AM6-36 greatly induced relative luciferase activities in a dose-dependent manner. Although the compound was lesspotent than 9-cis-RA, prominent induction was observedat higher concentrations (10 mmol/L, 4.1-fold; 15 mmol/L,31.1-fold; 20 mmol/L, 133.2-fold). In comparison, bexar-otene, a RXR-specific agonist, increased relative luciferaseactivities by 13.2 � 2.8-, 16.8 � 2.9-, 25.7 � 3.5-, and 48.9� 17.6-fold at concentrations of 12.5, 25, 50, and 100mmol/L, respectively.

On the basis of these data, further studies wereperformed with MCF7 cells, since RXRa has beenshown to have a role in the inhibition of cell prolifera-tion and induction of apoptosis in breast cancer cells(25). We further determined the effect of AM6-36 onRXR–RXRE transcriptional regulation. In MCF7 cells, itis not necessary to cotransfect phRXRa, owing to theexpression of RXRs (25). As shown in Figure 2D, treat-ment of transiently transfected MCF7 cells with 9-cis-RAincreased the relative luciferase activity, in a dose-dependent manner, with 3.5-, 3.7-, and 6.1-fold induc-tion at 40, 120, and 400 nmol/L, respectively. Likewise,AM6-36 induced relative luciferase activities by 1.5-,3.2-, and 16.8-fold at 312.5, 625, and 1,250 nmol/L,respectively. Therefore, with MCF7 cells, AM6-36 clearlyincreased RXR transcriptional activity. Nonetheless, toconfirm the effect of AM6-36 on RXR protein–RXREDNA sequence binding, an electrophoretic mobilityshift assay was performed, similar to 9-cis-RA, AM6-36 enhanced the binding of RXRs to RXRE in MCF7cells (Supplementary Fig. S2).

Induction of RXRE transcriptional activity andinhibition of MCF7 cell proliferation by AM6-36derivatives

On the basis of the response observed with AM6-36,additional indenoisoquinolines were assessed to deter-mine if a superior lead could be easily generated, and tolearn something about structure–activity relationships. Assummarized in Table 1, a small collection of structuralderivatives were evaluated in the RXRE assay with COS-1cells and the SRB assay with MCF7 cells. AM6-36mediated the strongest induction of RXRE transcriptionalactivity, as well as the strongest growth inhibitory effectwith MCF7 cells. The only other test compounds showingenhanced transcriptional activity were 5 and 6, and theseresponses correlated with growth inhibition. In detail,compound 5 substituted with an amino group at R1 and amethyl group at the R2 position and compound 6 sub-stituted with a nitro group at R1 and a dimethylamino-propyl group at R2, exhibited 3.0- and 3.3-fold induction,respectively. Even though compounds 5 and 6 were lessactive than AM6-36, they were capable of exerting induc-tive effects on RXRE activity in a dose-dependent manner(data not shown). Along with RXRE activity, compounds

5 and 6 showed significant antiproliferative effects withMCF7 cells, yielding IC50 values of 0.47 and 0.34 mmol/L,respectively.

AM6-36 inhibited proliferation in MCF7 cells by cell-cycle arrest in the G2/M phase at lower concentrationsand S phase at higher concentrations concomitantwith increased expression of CDKN1A

The potential of AM6-36 to inhibit the proliferation ofMCF7 cells was examined. As shown in Figure 3A, vehicle(0.1% DMSO)-treated control cells exhibited over a 4-fold increase in cell proliferation, compared with that ofstarting time point (0 hours). However, in the presence ofAM6-36, MCF7 cell proliferation was reduced in a con-centration-dependent manner, yielding IC50 values of1.09, 0.85, and 0.42 mmol/L at 24, 48, and 72 hours,respectively. At these respective incubation times, bexar-otene inhibited the cell proliferation with IC50 values of82.3, 55.2, and 43.4 mmol/L.

To better understand the antiproliferative effect of AM6-36, DNA content and cell-cycle distribution were analyzedby flow cytometric analysis. As shown in Figure 3B, treat-ment with 0.625 mmol/L AM6-36 caused cells to accumu-late in the G2/M phase, whereas treatment with higherconcentrations, such as 2.5 mmol/L, induced S-phase arrest.This was confirmed by treatment with 0.3125 mmol/L or2.5 mmol/L AM6-36 for 24 hours, as shown in Figure 3C. Tofurther elucidate the mechanism underlying the antiproli-feration and cell-cycle arrest induced by AM6-36, a cDNAmicroarray was employed. For this analysis, MCF7 cellswere incubated in the presence or absence of AM6-36 (1mmol/L; IC50 value after 24 hours of treatment), then RNAwas isolated and reverse transcribed to cDNA. The sameamount of cDNA was subjected to the microarray plate forreal-time PCR reaction. The results were analyzed and geneswhich were up- or downregulated with a value of P < 0.05in comparison with control were considered as signifi-cantly altered by the treatment (SupplementaryTable S1). As shown in Figure 3D, the expression of 26of 84 genes was altered by treatment of 1 mmol/L AM6-36.Specifically, the expression of 7 genes, including CCNG2,CDC34, CDK5RAP1, CDKN1A, GADD45A, RAD1, andSERTAD1, were significantly increased, whereas those of19 genes, including BIRC5, BRCA2, CCNB2, CCNF, CDC2,CDC20, CDK2, CDKN3, CHEK1, DDX11, GTSE1,MAD2L1, MCM2, MCM3, MCM5, MKI67, PCNA, RBL1,and SKP2, were significantly downregulated. Among these,CDKN1A and MKI67 were most highly up- and down-regulated, respectively.

Bexarotene (100 mmol/L; approximate IC50 value after24 hours of treatment) was tested in parallel experiments.Consequently, the expression of 9 of 84 genes was alteredsignificantly (upregulated genes: CDKN1A, CDKN2B, andGADD45A; downregulated genes: BCL2, BIRC5, CDK2,GTSE1, MCM2, and PCNA). In particular, BIRC5, CDK4,CDKN1A, GADD45A, GTSE1, MCM2, and PCNA werealtered by both AM6-36 and bexarotene. Of the 2 genes

Park et al.

Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research598

Table 1. Effects of indenoisoquinolines in RXRE assay and cell survival

Compound R1 R2 R3 R4 Biological activities

RXRE assay SRB assayb

Control 1.00 ± 0.20

1 NO2 H H 0.85 ± 0.04 >40

2 NO2 H H 0.72 ± 0.05 >40

3 NH2 H H 5.49 ± 0.34 0.20

4 NH2 H H 0.88 ± 0.15 5.09

5 NH2 CH3 H H 3.04 ± 1.52 0.47

6 NO2 H H 3.27 ± 1.08 0.34

7 NO2 H OCH3 0.85 ± 0.24 1.54

8 NO2 OCH3 H 1.12 ± 0.13 >40

9 NO2 H F 0.58 ± 0.04 1.45

10 NO2 F H 1.26 ± 0.77 6.09

11 NO2 H H 0.77 ± 0.04 >40

12 H

H H 0.68 ± 0.21 >40

13 H

H H 1.43 ± 0.95 >40

14 H H H 0.69 ± 0.03 >40

15 H

H H 1.62 ± 0.81 >40

16 H H H 1.09 ± 0.13 15.74

17 H CH3 H H 0.93 ± 0.54 4.11

18 I CH3 H H 0.59 ± 0.47 >40

9-cis-RA 14.61 ± 2.10 Bexarotene 48.91 ± 17.55

Camptothecin 1.41 (nmol/L)

Cl

N3

N3

NH2

N

N3

N3

N3

N3

Br

SO

O O

SO

O O

SO

O O

SO

O O

OH

N

O

O

R2

R3

R1

R4

a

c

c

d

aCOS-1 cells were transiently transfected with pRXRE, phRXRa, and pRL for 24 hours. Then cells were treated with 40 mmol/Lindenoisoquinolines (compounds 1-18) for 24 hours and subjected to the dual luciferase assay. Results were expressed as relativevalues calculatedby fold increaseover vehicle (0.5%DMSO)-treated controls after normalizing ratios of relative light units (RLU) producedby firefly luciferase activity to RLU produced by R. reniformis luciferase activity (firefly Luc/Renilla Luc).bMCF7 breast cancer cells were diluted at a density of 5� 104 cells/mL and 190 mL of cell suspension were plated into each well of 96-well cell culture plates which contained 10 mL of diluted compounds and incubated for 72 hours. Cell proliferation was assessed by theSRB assay and the percentage of survival of sample-treated cells relative to vehicle (0.5% DMSO)-treated control cells wascalculated. Results were presented as IC50 (mmol/L) values.c9-cis-RA (100 nmol/L) and bexarotene (100 mmol/L) were used as positive controls in the RXRE assay.dCamptothecin was used as a positive control in the SRB assay.

RXRE Transcriptional Activity by Indenoisoquinoline

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0 24 48 72

Incubation time (hour)

Dis

trib

utio

n of

cel

ls in

cel

l-cyc

le (

%)

AM6-36 (µmol/L)

41.9

19.641.4 35.2

31.3

25.1

25.451.0

21.2

50.229.1

9.4

51.1

24.0

20.3

18.2

35.9

44.524.1

46.6

24.3

11.9

45.5

38.7

58.1

17.5

17.6

15.9

25.5

52.732.8

40.8

21.9

15.4

39.8

37.2

Dis

trib

utio

n of

cel

ls (

%)

Sub-G0 G0/G1 S G2/M

Bexarotene 1 2 3 4 5 6 7 8 9 10 11 12

A ABL1 ANAPC2 ANAPC4 DIRAS3 ATM ATR BAX BCCIP BCL2 BIRC5 BRCA1 BRCA21.32 0.98 0.9 1.83 1.26 0.98 1.14 0.78 0.13 0.26 0.35 0.36

B CCNB1 CCNB2 CCNC CCND1 CCND2 CCNE1 CCNF CCNG1 CCNG2 CCNH CCNT1 CCNT20.6 0.5 1.18 0.33 0.24 0.63 0.4 1.12 1.88 1.21 1.14 1.24

C CDC16 CDC2 CDC20 CDC34 CDK2 CDK4 CDK5R1 CDK5RAP1 CDK6 CDK7 CDK8 CDKN1A1.27 0.32 0.54 1.56 0.28 1.19 0.7 0.99 0.47 1.98 1.4 5.46

D CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CHEK2 CKS1B CKS2 CUL1 CUL2 CUL3 DDX110.83 1.07 2.58 0.52 0.36 0.31 0.93 0.85 1.25 0.82 0.99 0.44

E DNM2 E2F4 GADD45A GTF2H1 GTSE1 HERC5 HUS1 KNTC1 KPNA2 MAD2L1 MAD2L2 MCM21.08 1.04 13.68 1.57 0.3 1.37 1.18 0.31 0.56 0.35 0.85 0.2

F MCM3 MCM4 MCM5 MKI67 MNAT1 MRE11A NBN PCNA RAD1 RAD17 RAD51 RAD9A0.27 0.24 0.31 0.23 1.05 0.33 0.8 0.2 0.55 1.55 0.31 1.02

G RB1 RBBP8 RBL1 RBL2 RPA3 SERTAD1 SKP2 SUMO1 TFDP1 TFDP2 TP53 UBA10.71 0.96 0.27 0.69 0.51 1.47 0.24 0.83 0.44 1.14 1.37 0.95

Control

AM6-36 0.3125 μmol/L

AM6-36 2.5 μmol/L

24 hour

48 hour

72 hour

-12.676 0 12.676

Magnitude of log2 (fold change)

-33.554 0 33.554

Magnitude of log2 (fold change)

AM6-36 1 2 3 4 5 6 7 8 9 10 11 12

A ABL1 ANAPC2 ANAPC4 DIRAS3 ATM ATR BAX BCCIP BCL2 BIRC5 BRCA1 BRCA20.88 1.56 1.08 0.8 0.55 0.65 1.69 1.11 0.22 0.07 0.18 0.09

B CCNB1 CCNB2 CCNC CCND1 CCND2 CCNE1 CCNF CCNG1 CCNG2 CCNH CCNT1 CCNT20.37 0.18 0.98 1.3 0.96 0.58 0.19 1.7 2.81 0.96 0.99 0.94

C CDC16 CDC2 CDC20 CDC34 CDK2 CDK4 CDK5R1 CDK5RAP1 CDK6 CDK7 CDK8 CDKN1A1.03 0.09 0.11 2.22 0.28 0.53 0.63 1.95 0.51 1.61 0.8 34.55

D CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CHEK2 CKS1B CKS2 CUL1 CUL2 CUL3 DDX110.92 1.68 1.9 0.16 0.04 0.32 0.6 1.34 1.44 1.65 1.18 0.17

E DNM2 E2F4 GADD45A GTF2H1 GTSE1 HERC5 HUS1 KNTC1 KPNA2 MAD2L1 MAD2L2 MCM20.81 1.11 12.09 1.41 0.09 2.36 0.46 0.29 0.43 0.11 1.21 0.15

F MCM3 MCM4 MCM5 MKI67 MNAT1 MRE11A NBN PCNA RAD1 RAD17 RAD51 RAD9A0.17 0.28 0.2 0.03 1.05 0.18 0.62 0.2 2.4 1.64 0.33 0.72

G RB1 RBBP8 RBL1 RBL2 RPA3 SERTAD1 SKP2 SUMO1 TFDP1 TFDP2 TP53 UBA10.91 0.38 0.12 1.44 0.64 3.27 0.06 1.35 0.44 0.94 0.83 1.57

AM6-36 (µmol/L)

G2/MSG0/G1Sub-G0

G2/MSG0/G1Sub-G0

G2/MSG0/G1Sub-G0

A B

C

D

Figure 3. Induction of RXRE-luciferase activity and inhibition of cell proliferation accompanied with cell-cycle arrest by indenoisoquinolines accompanied bysignificant changes in cell-cycle regulatory genes by AM6-36 treatment in MCF7 breast cancer cells. A, the proliferative state of cells was evaluated with the SRBassay.Cellswere incubated for24,48,and72hours.Resultsarepresentedas fold increasesofabsorbanceat515nmovercontrol atastartingpoint (0hour).BandC,MCF7 cells were seeded at a density of 50 � 104 cells in a 10-cm cell culture dish and incubated for 48 hours. After incubation with AM6-36 for each timeperiod, 10,000of cellswereanalyzed forDNAcontentand thedistributionof cells ineachphasewasestimatedbyusingModFit LT (VeritySoftwareHouse).D,MCF7cells were seeded at a density of 50 � 104 cells in a 10-cm cell culture dish and incubated for 48 hours. Cells were treated with vehicle (DMSO) or seriallydiluted AM6-36 for 24 hours. RNA was extracted from cells and reverse transcribed to cDNA. Then, cDNA was mixed with RT2 qPCRMaster Mix and 25 mL wereplaced in each well of the array plate. Real-time quantitative PCR was performed using SyBR Green detection system. The thermal cycling condition was at95�C for 10minutesand then40cyclesat95�C for 15seconds followedby60�C for 1minute.On thebasisof theDDCtmethod, the foldchangesbetween thecontrolgroupandsample-treatedgroupswereanalyzedusing theWeb-basedsoftwareprovidedbySABiosciences (RT2ProfilerPCRArrayDataAnalysis). Foldchangesof84 genes induced by AM6-36 or bexarotene were determined and presented as heatmaps (Red: upregulation, green: downregulation).

Park et al.

Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research600

significantly altered by bexarotene but not AM6-36 (BCL2,downregulated; CDKN2B, upregulated), there was clearly asimilar trend ofmodulation by both compounds. Of the 14genes significantly downregulated by AM6-36 but notbexarotene, 9 were also downregulated by bexarotene atthe P � 0.083 level.Supplementary Figure S1 illustrates the connectivity of

the main genes used in this array that were affected byAM6-36 or bexarotene. Notably, expression levels of severalgenes, which are transcriptionally activated by p53 (the pro-tein of the TP53 gene), were significantly upregulated byAM6-36 and bexarotene. Prominent examples are CDKN1A(P ¼ 0.00029, AM6-36; P ¼ 0.00014, bexarotene) andGADD45A (P¼ 0.00031,AM6-36;P¼ 0.00011, bexarotene).

p53 and p21 were increased after AM6-36 treatmentand RXRa protein knockdown by RXRa siRNAabrogated the effect of AM6-36 on the induction ofp21 expressionAlthough the expression of TP53 was not modulated by

AM6-36 with MCF7 cells, the potential effect on proteinlevel was examined by Western blot analyses. As shown inFigure 4A, treatment with AM6-36 caused an increase ofp53 at 8 hours, followed by decreases at 24 and 48 hours.Even though the protein level of p53 was diminished atlater time points, it still remained at higher levels than thecontrol. Also, since CDKN1A levels were most highlyelevated by treatment with AM6-36, its protein(p21CIP1/WAF1) levels were determined. Expression ofp21 in control cells was low at all time points, whereastreatment of AM6-36 caused the moderate increase at 8hours, and incremental expression was observed at 24and 48 hours. To establish a direct correlation betweenthe transcriptional activity of RXRa and the induction ofp21 expression, the expression of RXRa protein wastransiently depleted in MCF7 cells by introducing RXRasiRNA into the cells. As shown in Figure 4B, siRNAtargeted to RXRa markedly reduced RXRa expression,and consequently affected the upregulating effect ofAM6-36 on the protein expression of p21. Also, transfec-tion of p53 siRNA resulted in the abrogation of the p21expression induced by AM6-36 (Fig. 4C). However, p53knockdown did not influence the expression of RXRa(Fig. 4D). These data suggested that AM6-36 was not ableto induce p21 expression in the absence of RXRa or p53(i.e., RXRa and p53 are required for AM6-36–mediatedmRNA upregulation of p21).

Effects of AM6-36 on JB6 Cl41 cellsTo begin assessment of the chemopreventive potential

of AM6-36, studies were performed with TPA-treated JB6Cl41 cells. As shown in Figure 5A, the expression of COX-2 was induced after exposure to TPA (10 ng/mL) for 15hours. In contrast to control, however, cotreatment withAM6-36 attenuated COX-2 protein expression in a con-centration-dependent manner. Next, anchorage-indepen-dent cell transformation assay was performed. After28 days of incubation in the presence or absence of

AM6-36, cells were stained and quantified. As shownin Figure 5B, as judged by growth in soft agar, treatmentwith AM6-36 reduced cell transformation by approxi-mately 70%.

Expression of cell surface marker, CD38As an additional test of chemopreventive potential, the

ability of AM6-36 to affect the expression of CD38 wasassessed with HL-60 cells. As shown in Figure 5C, withthis model, treatment with 9-cis-RA can induce theexpression of CD38 in about 95% of the cell populationat higher concentrations (2,000, 200, and 20 nmol/L). Ata concentration of 2 nmol/L, the 9-cis-RA–treated cellpopulation moved to the left with a decreased percentageof FL-1–positive cells (86.7%), and at 0.2 nmol/L,further reduction in expression was observed. Whentreated with 2,000 or 200 nmol/L, AM6-36, cells exhib-ited a 72% or 58% FL-1–positive response (CD38-posi-tive), respectively; although, the fluorescence intensitywas lower in comparison with 2,000 nmol/L 9-cis-RA–treated cells.

Evaluation of AM6-36 permeability and metabolicstability

Judged by the Caco-2 cell permeability model, the appar-ent permeability coefficient for AM6-36 in the AP ! BL,2.75 � 10�6 cm/second, was found to be 10-fold slowerthan that of the high permeability standard, propranolol,but 17-fold faster than the low permeability standard,sucrose (Table 2). These measurements indicate that theintestinal absorption of AM6-36 should proceed at a mod-erate rate following oral administration. Since the apparentpermeability coefficients in the AP ! BL and in the BL !AP were not significantly different, AM6-36 does notappear to be a substrate for efflux proteins that mightreduce its bioavailability. During incubation of AM6-36with human liver microsomes, 1 abundant and 3 minormetabolites were observed. In the absence of eitherNADPH or microsomes, no metabolites were detected.Therefore, we assume that all 4 metabolites are producedin a cytochrome P450- and NADPH-dependent manner.The structures of these metabolites are currently underinvestigation and will be reported later.

Preliminary studies conducted with ratsTo assess absorption and metabolism, AM6-36 (40 mg/

kg body weight) was administered to rats, by gavage, over aperiod of 3 days. Blood was collected on days 1 and 3, andtissues were collected at the end of the study. As summar-ized in Table 3, the concentration of AM6-36 in the ratserum was approximately 0.83 mg/mL. The concentrationin liver (4.28 mg/g) was around 5 times higher. Appreciablequantities were also found in the mammary gland (0.29mg/g) and perirenal fat (0.28 mg/g). These data are consis-tent with moderate oral absorption and slow metabolism/excretion.

Finally, to obtain an early indication of chemopreventivepotential, a short-term mammary tumor model was used.

RXRE Transcriptional Activity by Indenoisoquinoline

www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 601

Time (h) 0 8 24 48 8 24 48

Control AM6-36 (1 µmol/L)

RXRα

p53

p21WAF1/CIP1

β-Actin

RXRα

p21WAF1/CIP1

β-Actin

Control AM6-36 9-cis-RA Control AM6-36 9-cis-RA

Control siRNA RXRα siRNA

Control AM6-36 9-cis-RA Control AM6-36 9-cis-RA

Control siRNA p21 siRNA

RXRα

p53

p21WAF1/CIP1

GAPDH

AM6-36 0 0.625 1.25 0 0.625 1.25 0 0.625 1.25

(µmol/L) Control siRNA RXRα siRNA p53 siRNA

A

B

C

D

RXRα

p21WAF1/CIP1

β-Actin

Figure 4. Increased protein expression of p53 and p21 by AM6-36, and altered expression of p21 by knockdown of RXRa or p53 in MCF7 cells. A, MCF7cells were seeded at a density of 10 � 104 cells in 6-well culture plates and incubated for 24 hours. Cells were treated with 1 mmol/L AM6-36 for theindicated time points. Cell lysates were prepared and subjected to Western blot analysis. B–D, after transient transfection with the indicated siRNAs,MCF7 cells were treated with vehicle (0.1% DMSO), AM6-36 (1 mmol/L, B and C; 0.625, 1, or 1.25 mmol/L, D) or 9-cis-RA (1 mmol/L, B and C) for an additional24 hours. Cell lysates were prepared and subjected to Western blot analysis.

Park et al.

Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research602

As shown in Figure 5D, at a dose of 10 mg/kg bodyweight per day, AM6-36 clearly attenuated tumor growth(P ¼ 0.1). It will now be necessary to evaluate largernumbers of animals with various treatment regimens toestablish statistical significance, but the trend demonstratespromising antitumor activity.

Discussion

RXR agonists have shown promise for the treatment orprevention of cancer. 9-cis-RA and LGD1069 are keyexamples of agents in clinical use. In addition, unlikeother nuclear receptors, RXRs are expressed in humancarcinomas and overexpression of RXR has enhanced thetranscriptional response after binding of ligands (26). Onthe basis of these considerations, we introduced a cellline–based RXRE-luciferase reporter gene assay to evalu-

ate the RXRa transcriptional activity of various test sub-stances. As the result of an extensive search, AM6-36 wasfound to be active. Since the COS-1 cells used for thisdiscovery were transiently transfected with pRXRE andphRXRa, a response is expected to be due to the interac-tion of a test substance with RXR–RXR homodimers.Further investigation is required to determine the poten-tial of any lead, including AM6-36, to interact with othernuclear receptors, such as RXR–PPAR (27). The goal ofthis work, however, was to investigate the biologicalramifications of the highly unique response mediatedby AM6-36 with this model system.

Indenoisoquinolines were first synthesized in 1978 (28)and studied as topoisomerase I inhibitors with advanta-geous properties compared with camptothecins, known astopoisomerase I inhibitors. Additional activities are alsoknown, such as induction of cell-cycle arrest, overcoming

%%

600

l l l l l l l l

600

ol l l l l l l l o

D

––––

D

60

90

120 COX-2

GAPDH

Con

trol

* *

A B

0

30AM6-36 (µmol/L)

0 0 1.25 2.5 5

TPA (10 ng/mL)

- + + + + AM6-36 (nmol/L)

0 50 100

C D200 –

–

100–

–

l l l l

200 –

–

100–

–

200 –

–

100–

–

Control

Sample-treated

FL-1 (+)

Control : 4.93% Autofluorescence control: 2.17%

Negative staining control : 6.17%

300

400

500

r siz

e (m

ea

n,m

m2 ) Control AM6-36-treated

0 101 102 1030 101 102 103 0 101 102 103

200 –

–

100–

–

200 –

–

100–

–

200 –

–

100–

–

200 –

–

100–

–

2,000 nmol/L 9-cis-RA: 95.17%

200 nmol/L 9-cis-RA: 96.16%

20 nmol/L 9-cis-RA: 93.19%

2 nmol/L 9-cis-RA: 86.7%

0.2 nmol/L 9-cis-RA: 25.12%

200 –

–

100–

–

0

100

200

0 7 14 21 28 35 42

Ma

mm

ary

tum

l l l l0 101 102 103

l l l l0 101 102 103

l l l l0 101 102 103

l l l l0 101 102 103

200 –

–

100–

200 –

–

100–

200 –

–

100–

200 –

–

100–

200 –

–

100–

2,000 nmol/LAM6-36: 72.24%

200 nmol/L AM6-36: 58.47%

20 nmol/LAM6-36: 10.31%

2 nmol/LAM6-36: 8.15%

0.2 nmol/LAM6-36: 8.29%

l l l l0 101 102 103

ays

l l l l0 101 102 103

l l l l0 101 102 103

l l l l0 101 102 103

l l l l0 101 102 103

l l l l0 101 102 103

Figure 5. Evaluation of chemopreventive potential of AM6-36. A, JB6 Cl41 cells were stimulated by treatment with TPA (10 ng/mL) in the presenceor absence of AM6-36 for 15 hours. Whole cell lysates were prepared and COX-2 expression was determined by Western blot analysis. b-Actin wasused as an internal control. B, JB6 Cl41 cells were suspended in 50 mL of 0.4% agar complete MEM medium over 100 mL of 0.8% agar completeMEMmedium. Cells were exposed to 5 nmol/L TPA with or without AM6-36 at 37�C in a humidified atmosphere with 5%CO2 for 28 days. At the end of theincubation, cell quantification solution was added to each well and further incubated at 37�C for 4 hours. Absorbance was measured at 490 nmand the data are shown as % transformation activity relative to TPA-treated control. C, HL-60 cells were incubated with serially diluted 9-cis-RA orAM6-36 (2,000, 200, 20, 2, 0.2 nmol/L) for 96 hours. Cells were stained with anti–CD38-FITC the intensity of FITC was measured using flow cytometry.FL-1 (þ) indicates % of cell population which possess FITC fluorescence, the expression of CD38. D, effect of AM6-36 (10 mg/kg per day) on MNU-induced mammary tumors in Sprague Dawley rats. The plots summarize average tumor size in control (closed triangles) and AM6-36–treated (closedsquares) animals (n ¼ 10). P ¼ 0.1; Mann–Whitney test.

RXRE Transcriptional Activity by Indenoisoquinoline

www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 603

multidrug-resistance, and enhancement of the differentia-tion-inducing activity of retinoids (29–31). AM6-36mediated only modest inhibition of the DNA religationreaction (data not shown), but unexpectedly, RXRE-luci-ferase activity was highly upregulated. On the basis of theresponse induced by AM6-36, a small group of structuralrelatives were evaluated to determine whether activitycould be further enhanced. As shown in Table 1, com-pounds 5 and 6 induced the RXR transcriptional activitiesas well as antiproliferative effects toward MCF7 cells, butAM6-36 was still the most active compound.

The indenoisoquinoline AM6-36 is an atypical rexinoidsince it does not contain a carboxylic acid and it does notstructurally resemble a retinoic acid. In comparison withknown rexinoids, most of which contain carboxylic acids(32–34), the indenoisoquinoline rexinoids constitute afundamentally different type of RXR ligand. The fact that

indenoisoquinolines are structurally unique rexinoidsoffers the possibility of them having unique pharmaco-logic properties that could be clinically useful. Consider-ing these factors as a whole, AM6-36 was selected forfurther evaluation.

As shown in Figure 1, a synthetic procedure was devisedthat is suitable for production of the lead compound inmultigram quantities. Since RXR agonists showed apop-totic effects in RXRa-expressed MCF7 cells (25), weexamined the effect of AM6-36 using this cell line. Asexpected, when MCF7 cells were treated with AM6-36, weobserved increased RXRa transcriptional activity, whichcorrelated with the response observed with transientlytransfected COS-1 cells. All of these data suggest thatMCF7 cells should be a useful model for exploring theaction of AM6-36 related to RXRa. Cell-cycle analysisrevealed G2/M arrest at lower concentrations and S-phasearrest at higher concentrations (Fig. 4C). A similar dose-related dual effect has been reported when MCF7 cellswere treated with adriamycin (35), and other DNA dama-ging agents showed partial G1- and G2/M-phase cell-cyclearrest at lower doses, whereas they exerted partial or totalS-phase cell-cycle arrest at higher doses (36). Analo-gously, the response induced by lower doses of AM6-36 may be related to effects on DNA.

Together with significant growth inhibition, genesknown as conventional proliferation markers and usedin the diagnosis of breast cancer, including MKI67 andPCNA (37), were downregulated by treatment with AM6-36. However, a known target gene of RXR, CDKN1A (38),was the most intensively upregulated. Correspondingly,p21 protein levels were increased (Fig. 4B). Treatment ofcells with retinoic acid can lead to elevated p21 levels (39),as well as agents such as 2{[3-(2,3-dichlorophenoxy)pro-pyl]amino}ethanol (40) and IFN-a (41). Interestingly,similar to AM6-36, these agents arrest cells in the S phaseof the cycle. In the RXRE assay, AM6-36 showed higherinduction in RXRa transcriptional activity than 9-cis-RA.The response may be augmented by enhanced expressionof GADD45A, a RXRa coactivator (42). It is further notedfrom the DNA microarray that SKP2 is greatly downregu-lated by treatment with AM6-36. This might give rise to the

Table 3. Intestinal permeability, metabolic stability, and distribution of AM6-36: disposition of AM6-36 inthe rata

AM6-36(40 mg/kg)

Serum 1 d,mg/mL (n ¼ 3)

Serum 3 d,mg/mL (n ¼ 3)

Liver,mg/g (n ¼ 3)

Mammarygland,mg/g(pooled n ¼ 3)

Fat, mg/g(pooled n ¼ 3)

Rat 1 0.84 � 0.008 (2.14 mmol/L) 0.82 � 0.003 (2.09 mmol/L) 3.72 � 0.07 – –

Rat 2 0.84 � 0.01 (2.14 mmol/L) 0.84 � 0.007 (2.14 mmol/L) 5.03 � 0.23 – –

Rat 3 0.82 � 0.004 (2.09 mmol/L) 0.82 � 0.002 (2.09 mmol/L) 3.75 � 0.13 – –

Average 0.83 � 0.01 (2.12 mmol/L) 0.83 � 0.01 (2.12 mmol/L) 4.28 � 0.65 0.29 � 0.006 0.28 � 0.00

aSee text for experimental details.

Table 2. Intestinal permeability, metabolic sta-bility, and distribution of AM6-36: apparentpermeability coefficients of AM6-36 throughCaco-2 monolayers

Papp (�10�6 cm/s � SD)a

AP to BLb BL to APc

AM6-36d, mmol/L5 2.75 � 0.78 3.95 � 0.1110 5.52 � 0.29 3.25 � 0.7125 4.45 � 0.71 8.26 � 0.93

Standards, mmol/LPropranolol (10) 27.6Sucrose (50) 0.16

aPapp corresponds to the apparent permeability coefficientsand is expressed as cm/s (�10�6).bAP to BL indicates apical to basolateral transport.cBL to AP indicates basolateral to apical transport.dData are expressed as mean � SD, n ¼ 3.

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Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research604

increased protein expression of p21, since Skp2 is involvedin the degradation of p21 (43).The role of p53 in these processes remains to be

further explored. Although p53 gene expression was notincreased by AM6-36, other effects, such as modified pro-tein expression and transcriptional activity, may come intoplay. In addition to upregulation of CDKN1A, othergenes associated with p53 transcriptional activity (e.g.,GADD45A, CCNG2; ref. 44) showed increases in expres-sion to some extent. Furthermore, the expression ofGTSE1,which has been reported to downregulate the level andactivity of the p53 tumor suppressor protein (45), wasdecreased by AM6-36. Thus, although additional studiesare warranted, this might explain the increase in p53protein level.In any case, enhanced RXRa activity leading to highly

elevated expression of p21, an inhibitor of CDKs whichnegatively regulates cell-cycle progression (46), appears toplay a dominant role in the cellular responses orchestratedby AM6-36 treatment. A direct relationship between theactivation of RXRa and p21 expression was further estab-lished by transiently blocking the expression of RXRa. Inthe absence of RXRa, the expression of p21 was abrogated(Fig. 4B). The crystal structure of AM6-36 bound to RXRa

has yet to be determined, but a hypothetical structure wasgenerated by GOLD (47) docking the indenoisoquinolineinto the X-ray structure of RXR-a (1FBY) (48) using thecentroid (15.2492, 28.4959, 48.2618) of bound 9-cis-RA.As illustrated in Figure 6, AM6-36 can readily interact withthe binding site of 9-cis-RA. The ligand pose with thehighest GOLD score and themost favorable binding energywas used for further calculations. The energy of the ligandand a surrounding spherical subset of atoms having a

radius of 6 A�was minimized by the conjugate gradient

method using the MMFF94s force field and MMFF94charges (SYBYL 8.0, Tripos International, 1699 SouthHanley Rd., St. Louis, Missouri, 63144, USA). The calcula-

tion was terminated at a gradient of 0.05 kcal/(mol�A� ).Rexinoids are of interest due to cancer chemopreventive

potential (6–8), and, of course, a question of utmostimportance is the chemopreventive potential of AM6-36.Some early studies have been completed. As normal cellsundergo neoplastic transformation, they gain the ability todemonstrate "anchorage independence" and grow in softagar (49). Currently, we demonstrated the potential ofAM6-36 to suppress TPA-induced neoplastic transforma-tion of JB6 Cl41 cells. Furthermore, during neoplastictransformation stimulated by TPA, JB6 Cl41 cells express

Figure 6. Protein modeling ofAM6-36 or 9-cis-RA with RXRa. A,hypothetical structure of AM6-36(red) bound to RXRa. B, overlap ofAM6-36 with the crystal structureof 9-cis-RA-RXR complex. AM6-36 is colored red and 9-cis-RA iscolored green. The structures areprogrammed for wall-eyed(relaxed) viewing.

A

B

RXRE Transcriptional Activity by Indenoisoquinoline

www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 605

COX-2, which plays pivotal roles not only in inflammationbut also in carcinogenesis. AM6-36 inhibits the expressionof COX-2 in this model system.

An additional strategy for cancer chemoprevention isthe induction of differentiation (50). All-trans-retinoicacid (ATRA) is known to induce CD38 expressionthrough activation of RAR and RXR in myeloid progenitorcells, which modulates cell differentiation (51, 52). As afurther induction of cancer chemopreventive potential,we currently demonstrated AM6-36–treated HL-60 cellsexpress CD38 on membrane surfaces.

Taken together, these data suggest the potential ofAM6-36 to serve as a chemopreventive agent. Prelimin-ary results were also obtained with the Caco-2 cellpermeability model, suggesting that the compoundwould be orally absorbed. In addition, incubationwith human liver microsomes indicated moderate meta-bolism. These data were corroborated by studies invol-ving oral administration with rats. Serum and tissuelevels were readily achieved that should be of physio-logic relevance, based on in vitro response data. Insupport of this notion, a preliminary evaluation demon-

strated attenuation of MNU-induced tumorigenesis infemale Sprague Dawley rats. Cleary, additional work isrequired to assess the effect of various dose regimenswith larger numbers of animals representative of differ-ent tumor models, but this is completely feasible, sinceAM6-36 can readily be produced on a multigram scale.AM6-36 is a surprising and unique molecule, represent-ing a promising lead that functions through a novelmechanism.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This study was supported by NIH under NCI grants P01 CA48112 andU01 CA089566.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 12, 2010; revised January 10, 2011; accepted January18, 2011; published online April 4, 2011.

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