Induction of Retinoid X Receptor Activity and Consequent Upregulation of P21waf1/CIP1 by Indenoisoquinolines in MCF7 Cells

Cancer Prevention Research Article Research

Induction of Retinoid X Receptor Activity and Consequent Upregulation of p21WAF1/CIP1 by Indenoisoquinolines in MCF7 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, and John M. Pezzuto1

Abstract Retinoid 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 limited metabolism resulted when AM6-36 was incubated with human liver microsomes. Oral administration with rats resulted in 0.8 mg/mL, 4.3 mg/g, and 0.3 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 reported in various physiologic processes including embryonic development, metabolic processes, differentia- Retinoid X receptor (RXR), a member of the nuclear tion, and apoptosis. Similar to other nuclear receptors, receptor superfamilies, was identified as an orphan recep- RXRs require specific ligands to be functionally activated. tor with a high sequence homology to retinoic acid receptor Whereas ligand-free RXRs are sequestered by specific (RAR), and a specific responsiveness to vitamin A meta- nuclear receptor corepressors; on ligand binding, RXRs bolites (1). Three subtypes of RXRs, RXRa, RXRb and RXRg, undergo conformational changes, leading to the dissocia- have been identified, and essential roles of RXRs have been tion of corepressors and the association of coactivators (2). Consequently, ligand-activated RXR dimers recognize the corresponding cis-acting element in the promoter region of 1 Authors' Affiliations: College of Pharmacy, University of Hawaii at Hilo, target genes, called hormone response elements (HRE), Hilo, Hawaii; 2Department of Medicinal Chemistry and Molecular Pharma- cology, College of Pharmacy, and the Purdue Center for Cancer Research, and are capable of triggering transcription. HREs consist of Purdue University, West Lafayette, Indiana; and 3Department of Medicinal 2 direct repeats (DR) of the consensus hexamers (AGGTCA) Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, Chicago, Illinois in tandem, separated by 1 to 5 nucleotides, which are designated as DR1 to DR5, respectively. In particular, Note: Supplementary data for this article are available at Cancer Preven- tion Research Online (http://cancerprevres.aacrjournals.org/). RXR homodimers are known to interact with DR1, RXR Corresponding Author: John M. Pezzuto, College of Pharmacy, Univer- response element (RXRE; ref. 3). sity of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720. Phone: 808-933- As mentioned above, specific ligands for RXRs generally 2909; Fax: 808-933-2981; E-mail: [email protected] play key roles in the activation of RXR signaling and several doi: 10.1158/1940-6207.CAPR-10-0004 substances are known to function as endogenous ligands 2011 American Association for Cancer Research. including 9-cis-retinoic acid (9-cis-RA), docosahexaenoic

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acid, and other unsaturated fatty acids (4). Besides putative (100), Lipofectamine 2000, and Trizol reagent were pur- endogenous ligands of RXRs, additional compounds have chased from Invitrogen. FITC-conjugated CD38 antibody been reported as RXR agonists, so-called rexinoids includ- (anti–CD38-FITC), and nuclear isolation medium 40,6-dia- ing AGN194204, CD3254, LG100268, LGD1069, and midino-2-phenylindole (NIM-DAPI) were purchased from SR11237. Although the mechanisms underlying their sup- Beckman Coulter, Inc. Rabbit polyclonal anti-RXRa anti- pressive and inhibitory actions against cancerous processes body, mouse monoclonal anti-p21WAF1/CIP1 (p21) anti- have not been fully clarified, both naturally occurring and body, mouse monoclonal anti-p53 antibody, human synthetic rexinoids have shown promise in the prevention RXRa small interfering RNA (siRNA), human p53 siRNA, and treatment of cancer (5). For example, 9-cis-RA, human p21 siRNA, and control siRNA-A were purchased LG100268, and LGD1069 have shown efficacy in N- from Santa Cruz Biotechnology. Rabbit polyclonal anti– methyl-N-nitrosourea (MNU)-induced breast cancer ani- b-actin, rabbit polyclonal anti–COX-2, Cell lysis buffer mal models (6–8). Furthermore, 9-cis-RA and LGD1069 (10), and LumiGLO chemiluminescent detection kit were have been approved by the Food and Drug Administration from Cell Signaling Biotechnology. RT2 First Strand Kit (C- (FDA) for the treatment of Kaposi’s sarcoma and cutaneous 03) and RT2 Profiler PCR Array related to human cell cycle T-cell lymphoma, respectively (9, 10). were purchased from SABiosciences. Cell transformation In searching for novel cancer chemopreventive and ther- detection assay kit was purchased from Millipore. apeutic agents capable of functioning as RXR agonists, we utilized an RXRE-luciferase reporter gene assay. This assay Synthesis of indenoisoquinolines assesses RXR transcriptional activity as well as binding to As outlined in Figure 1, scalable conditions were devel- RXR, since trans-activation occurs only after ligand binding. oped for the preparation of AM6-36 (compound 3) and During the course of testing over 5,000 natural product related indenoisoquinolines. Azidopropyl indenoisoqui- extracts and compounds, only a single active lead was noline 2 was obtained following previously described pro- identified: 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11- cedures (11, 12). We have previously prepared AM6-36 on dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride small scale from compound 1 according to method A. (AM6-36; Fig. 1). On the basis of the unexpected yet unique However, this method was found impractical for the pre- ability of AM6-36 to serve as an RXR agonist, 18 structural paration of multigram quantities of the target compound. relatives were tested, but AM6-36 still demonstrated the Therefore, an alternative approach for the reduction of greatest activity (Table 1). compound 2 or conversion of compound 1 to the desired Using MCF7 breast cancer cells as a model, AM6-36 was amount of AM6-36 was necessary. After a series of attempts found to suppress proliferation and this was accompanied to reduce both nitro and azido groups of compound 2 by cell-cycle arrest. RXRa transcriptional activity was upre- stepwise using various conditions, the reduction with iron gulated with subsequent expression of RXR target gene powder in the presence of ammonium chloride solution CDKN1A. Moderate absorption and slow metabolism was found to be the best method (method B). Tosylated was demonstrated. Strong indications of cancer chemopre- analogues of AM6-36 12–15 were prepared by treatment of ventive potential were observed with HL-60 cells, 12-O- the corresponding alkoxy derivatives 21–24 with 4-tolue- tetradecanoylphorbol 13-acetate (TPA)-treated JB6 Cl41 nesulfonyl chloride in dichloromethane in the presence of cells, and rats bearing mammary tumors. Finally, a syn- 4-dimethylaminopyridine and triethylamine. Compound 5 thetic procedure for large-scale production is reported that was prepared by condensation of 5-nitrohomophthalic will enable additional antitumor evaluations. anhydride (24) with N-benzylidenemethylamine, followed by treatment of the product with thionyl chloride, intra- Materials and Methods molecular Friedel–Crafts reaction, and reduction of the nitro group (11). Compounds 17 and 18 were obtained Reagents as the products of Sandmeyer reaction of 5. Compounds 6 9-cis-RA, TPA, trichloroacetic acid (TCA), sulforhoda- to 11 and 21 were prepared as previously described (11–13, mine B (SRB), paraformaldehyde, IgG1-fluorescein isothio- 14, 15). Melting points were determined using capillary cyanate (FITC) isotype control from murine myeloma, and tubes with a Mel-Temp apparatus and are uncorrected. 7-hydroxystaurosporine (UCN-01) were purchased from Proton nuclear magnetic resonance (1HNMR)spectra Sigma-Aldrich, Inc. Bexarotene was purchased from LC were recorded using ARX300 300 MHz and DRX500 Laboratories. The translucent reporter vector carrying firefly 500MHzBrukerNMRspectrometers.IRspectrawere luciferase under the control of RXRE (50-AGGT- recorded using a Perkin-Elmer 1600 series FTIR spectro- CACAGGTCACAGGTCACAGGTCACAGGTCA-30; pRXRE) meter. Combustion microanalyses were performed at the was purchased from Panomics. pBABE-puro vector encod- Purdue University Microanalysis Laboratory and the ing the cDNA for human RXRa (phRXRa) was purchased reported values were within 0.4% of the calculated values. from Addgene Inc. Renilla reniformis luciferase vector (pRL) High-performance liquid chromatography (HPLC) ana- and Dual-Luciferase Reporter Assay System were purchased lyses were performed on a Waters 1525 binary HPLC from Promega. Dulbecco’s modified Eagle’s medium pump/Waters 2487 dual l absorbance detector system. (DMEM), Eagle’s minimal essential medium (MEM), fetal Analytic thin-layer chromatography was carried out on bovine serum (FBS) and antibiotics-antimycotics solution Baker-flex silica gel IB2-F plates and compounds were

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Method A O O O O

H2-Pd/C NaN3, DMSO 1.P(OEt)3, PhH THF 70–100°C 2.MeOH, HCl N Cl N Cl N N3 N NH2 O2N H2N H2N H2N O O O O 1 91 4 3 (AM6-36) Method B O

Fe, NH4Cl

EtOH, H2O N N3 O2N O 2

SfSynthesis of AM6-36 analogues

O O O

NH2(CH2)nOH TsCl, DMAP, Et 3N CHCl O 3 N OH CH2Cl2,RT, 14 h N O ( )n ( )n S O O O O O 02 12 , n = 3 12, n = 3 22, n = 4 13,13 n = 4 23, n = 5 14, n = 5 24, n = 6 15, n = 6

O O CO2H O 1.SOCl2, PhH 100˚C Fe, NH Cl + 4 O 2.AlCl3, PhNO2 O N CHCl3 N EtOH, H2O 2 100˚C N N N O2N O O2N H2N O O O 52 62 72 5

O O O

1.NaNO2, HBr, 1.NaNO2, HCl, dioxane, H O, 0°C dioxane, H O, 0°C + 2 5 2

N N 2.CuBr, dioxane, H2O, 0°C 2.KI, dioxane, H2O, 0°C N Br I

O O O 82 71 18

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

visualized with short-wavelength UV light. Silica gel flash maintained in DMEM containing 10% FBS. JB6 Cl41 chromatography was performed using 230 to 400 mesh mouse epidermal cells were grown in MEM containing silica gel. Physical and spectral data are reported as 5% FBS. HL-60 human promyelocytic leukemia cells were Supplementary Method S1. grown in RPMI 1640 supplemented with 10% FBS. These cell lines were obtained from the American Type Culture Cell culture Collection (ATCC). Stock cultures were prepared and ali- All cell lines were grown in media supplemented with quots were stored in liquid nitrogen. During the course of penicillin G (100 units/mL), streptomycin (100 mg/mL), the study, cells were used below a passage number of 20 and heat-inactivated FBS at 37 Cin5%CO2 in a humidi- and no significant changes were noted in morphology or fied incubator. COS-1 African green monkey kidney fibro- growth characteristics. The cell lines were not authenticated blast cells and MCF7 human breast cancer cells were by the authors.

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RXRE-luciferase reporter gene assay by reverse transcription to cDNA under the condition of COS-1 cells (1 104 cells/well) were plated in a 96-well 42C for 15 minutes and 95C for 5 minutes using an RT2 culture plate and incubated for 24 hours. Then, 100 ng of First Strand Kit (C-03) on an ABI 7300 thermocycler pRXRE, 50 ng of phRXRa, and 3 ng of pRL were transiently (Applied Biosystems Inc.). cDNA was applied to RT2 Pro- cotransfected into COS-1 cells in each well by using Lipo- filer PCR Array related to human cell-cycle according to a fectamine 2000 according to the manufacturer’s protocol. manufacturer’s instructions. Samples were run in quadru- After 24 hours of transfection, cells were treated with plicate to ensure amplification integrity. The expression compounds and further incubated for 24 hours. Cells were levels of 84 cell-cycle–related genes in cDNA of each then lysed and the RXRE transcriptional activities were sample were analyzed (20). determined by measuring the reporter luciferase activities using the Dual-Luciferase Reporter Assay System. siRNA transient transfection MCF7 cells were plated at 10 104 cells per well in 6-well Evaluation of antiproliferative potential culture plates and incubated for 24 hours. Cells in each well MCF7 cells (1 104 cells/well) were seeded in 96-well were transfected with 50 pmol of human RXRa siRNA, plates and incubated with compounds at 37C in a humi- human p53 siRNA, human p21 siRNA, or control siRNA-A dified atmosphere with 5% CO2 for 24, 48, and 72 hours. using Lipofectamine 2000 for 24 hours followed by sample After the incubation, cell viability was estimated by the SRB treatment for additional 24 hours. To examine the expres- cellular protein staining method as previously described sion of RXRa and p21, Western blot analysis was per- (16). formed as described above.

Analysis of cell-cycle distribution Expression of COX-2 in TPA-treated JB6 Cl41 cells Cell-cycle distribution was assessed by staining DNA JB6 Cl41 cells were seeded in 6-well plates (2 105 cells/ content with NIM-DAPI solution according to the man- well) and incubated at 37C in a humidified atmosphere ufacturer’s instructions. Briefly, the media was discarded with 5% CO2 for 24 hours. After incubation, cells were from MCF7 cells after 24, 48, and 72 hours of incubation exposed to TPA (10 ng/mL) with vehicle (0.1% dimethyl- with various concentrations of AM6-36. Cells were har- sulfoxide, DMSO) alone or various concentrations of AM6- vested and stained with NIM-DAPI solution just before the 36. After 15 hours of further incubation, protein of each measurement using Cell Lab Quanta SC flow cytometer well was extracted and subjected to Western blot analysis as (Beckman Coulter; ref. 17). described in the "Evaluation of protein expression" section.

Evaluation of protein expression Anchorage-independent cell transformation Cells were lysed by using 1 cell lysis buffer diluted from The effect of AM6-36 on TPA-induced neoplastic trans- 10 cell lysis buffer according to the manufacturer’s pro- formation was investigated with JB6 Cl41 cells cultivated in tocol. Cell lysates were centrifuged at 14,000 g for 10 soft agar according to the manufacturer’s protocol. Briefly, minutes at 4C and the resultant supernatants were col- 500 exponentially growing cells were suspended in 50 mL lected and stored at 80C until use. After quantification of 0.4% agar complete MEM medium over 100 mL 0.8% agar protein by the Bradford method (18), equal amounts of complete MEM medium in a 96-well plate. Cells were total protein in each cell lysate were resolved using 12% exposed to 5 nmol/L TPA, with or without AM6-36, at SDS-PAGE and electrotransferred to polyvinylidene 37 C in a humidified atmosphere with 5% CO2 for 28 days. difluoride (PVDF) membranes. The membranes were incu- After incubation, cell quantification solution was added to bated with 5% skimmed milk in 0.1% Tris-buffered saline each well and further incubated at 37C for 4 hours. TPA- (TBS) containing Tween 20 (TBST) for 1 hour at room treated control wells turned to burgundy-brown. The temperature to block nonspecific protein binding. Then, absorbance was measured at 490 nm and data were membranes were incubated overnight at 4C with corre- expressed as % transformation activity relative to TPA- sponding primary antibodies in 3% skimmed milk in TBS treated control. followed by the incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized Expression of cell surface marker, CD38 using a LumiGLO chemiluminescent detection kit under HL-60 cells (2 105 cells/well) were seeded in 12-well Geliance 1000 imager (Perkin Elmer, Inc.). plates and incubated with samples at 37C in a humidified atmosphere with 5% CO2 for 24 hours. Following the Profile of altered gene expression manufacturer’s instructions, cells were harvested and cen- Total RNA was extracted from cells using Trizol reagent trifuged at 300 g for 5 minutes, then washed with according to the method of Chomczynski and Sacchi (19). phosphate-buffered saline (PBS) once. Cells were incu- Isolated RNA was dissolved in RNase-free water and the bated with anti–CD38-FITC for 30 minutes in the dark. quality and quantity were measured using a NanoDrop Similarly, autofluorescence control (cells only) and nega- ND-1000 spectrophotometer (Thermo Fisher Scientific tive staining control (cells þ FITC-conjugated, isotype- Inc.). Total RNA (1 mg) was incubated with genomic matched nonspecific mouse immunoglobulin) were DNA elimination mixture at 42C for 5 minutes followed prepared. After the incubation, cells were washed with

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diluents and centrifuged at 500 g for 10 minutes. Pre- Dawley rats were obtained at 4 weeks of age (Harlan, Inc.; cipitated cells were fixed with 2% cold paraformaldehyde virus-free colony 202A) and placed on Teklad diet. At solution and analyzed by flow cytometry. 77 days of age, the rats were treated with AM6-36 [40 mg/kg; 0.5 mL; ethanol:polyethylene glycol (PEG) 400; Metabolism of AM6-36 by human liver microsomes 10:90, v/v] by gavage, and the treatment was continued on The metabolic stability of AM6-36 was determined using a daily basis for 3 days. Blood samples were collected human liver microsomes. Incubations were performed with (jugular vein) 3 hours after the first treatment. Three hours pooled human liver microsomes containing 0.5 mg/mL of after the final treatment, the animals were sacrificed, and microsomal protein, 10 or 50 mmol/L AM6-36, and 1 mmol/ blood, mammary tissue, liver, and perirenal fat were col- L NADPH in 50 mmol/L phosphate buffer (pH ¼ 7.4), in a lected. After clotting, the blood samples were centrifuged at total volume of 0.4 mL. The reactions were initiated by 100 g for 20 minutes. Serum and tissue samples were addition of NADPH and, after a 5-minute preincubation, stored at 80C until analysis. Rat liver was homogenized were performed at 37C for 40 minutes. The incubations in 0.05 mol/L phosphate buffer (pH ¼ 7.4) and mammary were terminated by adding 1.6 mL of an ice-cold mixture of gland and perirenal fat tissues were homogenized in a acetonitrile-ethanol (1:1, v/v) and processed high resolu- mixture (50:50; v/v) of phosphate buffer and methanol tion liquid chromatography-mass spectrometry (LC-MS) to give homogenates containing 0.2 g tissue per mL. As an and liquid chromatography-tandem mass spectrometry internal standard for quantitative analysis, UCN-01 at 1 ng/ (LC-MS-MS) analysis as described previously (21). mL was added to each homogenate and serum sample. Proteins in the homogenized tissue and serum samples Determination of intestinal epithelial monolayer were precipitated with 4 volumes of ice-cold acetonitrile. permeability After centrifugation at 10,000 g for 5 minutes, each To evaluate the absorption of AM6-36 in intestinal supernatant was removed, evaporated to dryness, and epithelial cells, a Caco-2 cell monolayer assay was per- reconstituted in 100 mL of methanol/water (50:50; v/v) formed. In preparation for the Caco-2 cell monolayer for analysis using LC-MS-MS (23). The injection volume assays, the cell culture medium was removed from both was 10 mL. Briefly, LC-MS-MS analyses were carried out the apical (AP) and basolateral (BL) chambers. The using a Shimadzu LC-20AD prominence UFLC pump and cells were washed 3 times and preincubated with SIL-20AC HT prominence autosampler interfaced to an Hanks’ balanced salt solution (HBSS) containing 25 Applied Biosystems API 4000 triple quadruple mass spec- mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic trometer. A Waters XTerra MS C18 column (2.1 mm acid (HEPES), pH ¼ 7.4, for 30 minutes at 37Cona 100 mm, 3.5 mm) was used for HPLC separation with a 13- shaker bath at 50 rpm. A 20 mmol/L stock solution of minute linear gradient from 10% to 80% acetonitrile in AM6-36 in DMSO was diluted to different concentrations 0.1% aqueous formic acid at a flow rate of 0.25 mL/min. in HBSS/HEPES buffer (the final concentration of DMSO Positive ion electrospray was used for ionization and was >1%). These test solutions containing AM6-36 were product ion mass spectra were recorded using collision- added to either the apical chambers (for AP ! BL mea- induced dissociation and selected reaction monitoring surement) or the basolateral chambers (for BL ! AP (SRM) of the transition of m/z 320.4 to 303.2 and m/z assay), and blank HBSS/HEPES buffer was added to the 483.2 to 130.0 (300 ms per transition) for measurement of other side. As a test of the integrity of the monolayer and as AM6-36 and internal standard, respectively. Calibration a marker of low permeability, sucrose (50 mmol/L final curves (linear from 0.5–500 ng/mL AM6-36; R2 ¼ concentration) was added to the apical chambers. Propra- 0.996) were prepared using blank serum or homogenized nolol (10 mmol/L final concentration) was also added as a tissue from control rats. marker for compounds that are highly permeable. The total volumes of solution in the apical and basolateral Effect of AM6-36 on MNU-induced mammary tumors chambers were 1.5 and 2.6 mL, respectively. The solutions A preliminary study was performed as described pre- in the recipient compartments were removed and replaced viously (24). Female Sprague Dawley rats at 28 days of with fresh medium. The samples were stored at 20C age were obtained from Harlan Sprague Dawley and accli- until analysis of AM6-36 by LC-MS-MS as described below mated under the controlled animal quarters maintained at a for the analysis of extracts of serum and tissue. At the end of 12-hour light or 12-hour dark cycle and 22C 2C. MNU each experiment, basolateral samples were also analyzed (National Cancer Institute Chemical Carcinogen Reposi- by LC-MS for sucrose and propranolol. The apparent tory) was dissolved in sterile-acidified saline (pH ¼ 5.0) and 6 permeability coefficients (Papp; 10 cm/s SD) were injected via the jugular vein of rats at 50 days of age with a then calculated. The Papp of sucrose from the apical to the dosage of 75 mg/kg body weight. When an animal devel- basolateral side of each well was also measured and oped a palpable mammary tumor (approximately 150–200 remained acceptably low at 0.16 10 6 cm/s (22). mm2), treatment with AM6-36 (10 mg/kg body weight/d; ethanol:PEG 400, 10:90, v/v) or vehicle was initiated (10 Evaluation of absorption and distribution in rats rats/group). Over a period of 6 weeks, the largest diameter of A preliminary evaluation was performed to assess the the cancer was measured and this value was multiplied absorption and distribution of AM6-36. Female Sprague by the perpendicular diameter (size expressed as mm2).

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Measurements were performed twice a week during the RXRE cis-acting element in the promoter region of target period of the study, the average tumor size in the control gene, an RXRE-luciferase reporter gene assay (RXRE assay) and treatment groups were plotted as a function of time, and was performed. As described above, ligand-bound RXR data were analyzed using the Mann–Whitney test. homodimer interacts with DR1 and initiates the transcription of target genes. To discover RXR modulators, Statistical analyses COS-1 cells were transiently transfected with pRXRE. At Results are presented as means SD. Unless otherwise thesametime,duetothepoorexpressionofendogenous indicated, statistical comparisons were performed by the RXRs in COS-1 cells, phRXRa was cotransfected (Fig. 2A). use of Student’s t test and the level of P < 0.05 was pRL was used as an internal control for normalization. As considered as significantly different. showninFigure2B,9-cis-RA induced the relative luciferase activity up to about 20-fold in cells cotransfected Results including phRXRa, whereas there was no marked increase in cells without phRXRa transfection. Therefore, the AM6-36 induced the RXRE transcriptional activity in expression of RXRa is necessary for the RXRE-mediated COS-1 cells and MCF7 cells transcriptional activation by rexinoids, and it is required To investigate the effect of samples on the functional to enhance the expression of RXRa in COS-1 cell line- role of RXRa protein as a transcriptional activator on based assay.

A B * pRXRE+pRL pRXRE+phRXRα+pRL * Control phRXRα-TF * Luc) expression

α * /GAPDH) Renilla α (RXR * Relative RXR Relative (firefly Luc/ (firefly Relative luciferaseRelative activity RXRα 06.2525100 GAPDH 9-cis-RA (nmol/L) Control phRXRα-TF Control phRXRα-TF CD24 hour transfection 48 hour transfection * * Luc) Luc) Renilla Renilla

* * * * * *

* * Luc/ (firefly * (firefly Luc/ (firefly Relative luciferase activity Relative

Relative luciferase activity * * * * * * * 1.6 3.1 6.3 12.5 25 50 100 200 1.3 2.5 5 10 15 20 40 120 400 312.5 625 1250 Control Control 9-cis-RA (nmol/L) AM6-36 (µmol/L) 9-cis-RA (nmol/L) AM6-36 (nmol/L)

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 incubated with 9-cis-RA or AM6-36 for 24 hours. Cells were lysed and dual luciferase activity was measured. Results were expressed as mean SD (n ¼ 4). Each mean value was calculated by fold change over control after normalizing ratios of relative light units (RLU) produced by firefly luciferase to RLU producedbyRenilla luciferase (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), and pRL (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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Next, we tested several thousand natural products and 5 and 6 showed significant antiproliferative effects with synthetic compounds. Through the process, only one active MCF7 cells, yielding IC50 values of 0.47 and 0.34 mmol/L, lead was found, AM6-36. As illustrated in Figure 2C, AM6- respectively. 36 greatly induced relative luciferase activities in a dose- dependent manner. Although the compound was less AM6-36 inhibited proliferation in MCF7 cells by cell- potent than 9-cis-RA, prominent induction was observed cycle arrest in the G2/M phase at lower concentrations at higher concentrations (10 mmol/L, 4.1-fold; 15 mmol/L, and S phase at higher concentrations concomitant 31.1-fold; 20 mmol/L, 133.2-fold). In comparison, bexar- with increased expression of CDKN1A otene, a RXR-specific agonist, increased relative luciferase The potential of AM6-36 to inhibit the proliferation of activities by 13.2 2.8-, 16.8 2.9-, 25.7 3.5-, and 48.9 MCF7 cells was examined. As shown in Figure 3A, vehicle 17.6-fold at concentrations of 12.5, 25, 50, and 100 (0.1% DMSO)-treated control cells exhibited over a 4- mmol/L, respectively. fold increase in cell proliferation, compared with that of On the basis of these data, further studies were starting time point (0 hours). However, in the presence of performed with MCF7 cells, since RXRa has been AM6-36, MCF7 cell proliferation was reduced in a con- shown to have a role in the inhibition of cell prolifera- centration-dependent manner, yielding IC50 values of tion and induction of apoptosis in breast cancer cells 1.09, 0.85, and 0.42 mmol/L at 24, 48, and 72 hours, (25). We further determined the effect of AM6-36 on respectively. At these respective incubation times, bexar- RXR–RXRE transcriptional regulation. In MCF7 cells, it otene inhibited the cell proliferation with IC50 values of is not necessary to cotransfect phRXRa, owing to the 82.3, 55.2, and 43.4 mmol/L. expression of RXRs (25). As shown in Figure 2D, treat- To better understand the antiproliferative effect of AM6- ment of transiently transfected MCF7 cells with 9-cis-RA 36, DNA content and cell-cycle distribution were analyzed increased the relative luciferase activity, in a dose- by flow cytometric analysis. As shown in Figure 3B, treat- dependent manner, with 3.5-, 3.7-, and 6.1-fold induc- ment with 0.625 mmol/L AM6-36 caused cells to accumu- tion at 40, 120, and 400 nmol/L, respectively. Likewise, late in the G2/M phase, whereas treatment with higher AM6-36 induced relative luciferase activities by 1.5-, concentrations, such as 2.5 mmol/L, induced S-phase arrest. 3.2-, and 16.8-fold at 312.5, 625, and 1,250 nmol/L, This was confirmed by treatment with 0.3125 mmol/L or respectively. Therefore, with MCF7 cells, AM6-36 clearly 2.5 mmol/L AM6-36 for 24 hours, as shown in Figure 3C. To increased RXR transcriptional activity. Nonetheless, to further elucidate the mechanism underlying the antiproli- confirm the effect of AM6-36 on RXR protein–RXRE feration and cell-cycle arrest induced by AM6-36, a cDNA DNA sequence binding, an electrophoretic mobility microarray was employed. For this analysis, MCF7 cells shift assay was performed, similar to 9-cis-RA, AM6- were incubated in the presence or absence of AM6-36 (1 36 enhanced the binding of RXRs to RXRE in MCF7 mmol/L; IC50 value after 24 hours of treatment), then RNA cells (Supplementary Fig. S2). was isolated and reverse transcribed to cDNA. The same amount of cDNA was subjected to the microarray plate for Induction of RXRE transcriptional activity and real-time PCR reaction. The results were analyzed and genes inhibition of MCF7 cell proliferation by AM6-36 which were up- or downregulated with a value of P < 0.05 derivatives in comparison with control were considered as signifi- On the basis of the response observed with AM6-36, cantly altered by the treatment (Supplementary additional indenoisoquinolines were assessed to deter- Table S1). As shown in Figure 3D, the expression of 26 mine if a superior lead could be easily generated, and to of 84 genes was altered by treatment of 1 mmol/L AM6-36. learn something about structure–activity relationships. As Specifically, the expression of 7 genes, including CCNG2, summarized in Table 1, a small collection of structural CDC34, CDK5RAP1, CDKN1A, GADD45A, RAD1, and derivatives were evaluated in the RXRE assay with COS-1 SERTAD1, were significantly increased, whereas those of cells and the SRB assay with MCF7 cells. AM6-36 19 genes, including BIRC5, BRCA2, CCNB2, CCNF, CDC2, mediated the strongest induction of RXRE transcriptional CDC20, CDK2, CDKN3, CHEK1, DDX11, GTSE1, activity, as well as the strongest growth inhibitory effect MAD2L1, MCM2, MCM3, MCM5, MKI67, PCNA, RBL1, with MCF7 cells. The only other test compounds showing and SKP2, were significantly downregulated. Among these, enhanced transcriptional activity were 5 and 6,andthese CDKN1A and MKI67 were most highly up- and down- responses correlated with growth inhibition. In detail, regulated, respectively. 1 compound 5 substituted with an amino group at R and a Bexarotene (100 mmol/L; approximate IC50 value after methyl group at the R2 position and compound 6 sub- 24 hours of treatment) was tested in parallel experiments. stituted with a nitro group at R1 and a dimethylamino- Consequently, the expression of 9 of 84 genes was altered propyl group at R2, exhibited 3.0- and 3.3-fold induction, significantly (upregulated genes: CDKN1A, CDKN2B, and respectively. Even though compounds 5 and 6 were less GADD45A; downregulated genes: BCL2, BIRC5, CDK2, active than AM6-36, they were capable of exerting induc- GTSE1, MCM2, and PCNA). In particular, BIRC5, CDK4, tive effects on RXRE activity in a dose-dependent manner CDKN1A, GADD45A, GTSE1, MCM2, and PCNA were (data not shown). Along with RXRE activity, compounds altered by both AM6-36 and bexarotene. Of the 2 genes

598 Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research

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

R4 O

R3 N R1 R2 O Biological activities Compound R1 R2 R3 R4 RXRE assay a SRB assayb Control 1.00 ± 0.20 1 NO H H 0.85 ± 0.04 >40 2 Cl

2 NO2 N3 H H 0.72 ± 0.05 >40

3 NH2 NH2 H H 5.49 ± 0.34 0.20

4 NH2 N3 H H 0.88 ± 0.15 5.09

5 NH2 CH3 H H 3.04 ± 1.52 0.47

6 NO2 N H H 3.27 ± 1.08 0.34

7 NO H OCH 0.85 ± 0.24 1.54 2 N3 3

8 NO2 N3 OCH3 H 1.12 ± 0.13 >40

9 NO2 N3 H F 0.58 ± 0.04 1.45

10 NO2 N3 F H 1.26 ± 0.77 6.09

11 NO2 Br H H 0.77 ± 0.04 >40

12 H H H 0.68 ± 0.21 >40 O S O O

13 H H H 1.43 ± 0.95 >40 O S O O

14 H O H H 0.69 ± 0.03 >40 S O O

15 H H H 1.62 ± 0.81 >40 O S O O 16 H H H 1.09 ± 0.13 15.74 OH

17 H CH3 H H 0.93 ± 0.54 4.11

18 I CH3 H H 0.59 ± 0.47 >40 9-cis-RAc 14.61 ± 2.10 Bexarotenec 48.91 ± 17.55 Camptothecind 1.41 (nmol/L)

aCOS-1 cells were transiently transfected with pRXRE, phRXRa, and pRL for 24 hours. Then cells were treated with 40 mmol/L indenoisoquinolines (compounds 1-18) for 24 hours and subjected to the dual luciferase assay. Results were expressed as relative values calculated by fold increase over vehicle (0.5% DMSO)-treated controls after normalizing ratios of relative light units (RLU) produced by 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 the SRB assay and the percentage of survival of sample-treated cells relative to vehicle (0.5% DMSO)-treated control cells was

calculated. 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. www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 599

AB 24 hour 9.4 21.2 29.1 50.2 AM6-36 (µmol/L) 31.3 51.0 25.4 G2/M S 25.1 41.9 G0/G1 41.4 35.2 19.6 Sub-G0

48 hour 11.9 20.3 24.1 44.5 24.0 45.5

46.6 G2/M 0244872 35.9 S 51.1 G /G Incubation time (hour) 38.7 0 1 24.3 Sub-G C 18.2 0

Control 72 hour Distribution of cells in cell-cycle (%) cell-cycle in of cells Distribution AM6-36 0.3125 μmol/L 17.6 15.4 32.8 AM6-36 2.5 μmol/L 17.5 52.7 39.8 G /M 40.8 2 S 58.1 25.5 37.2 G0/G1 15.9 21.9 Sub-G0 Distribution of cells (%)

AM6-36 (µmol/L) Sub-G0 G0/G1 SG2/M D AM6-36 123456789101112 ABL1 ANAPC2 ANAPC4 DIRAS3 ATM ATR BAX BCCIP BCL2 BIRC5 BRCA1 BRCA2 A 0.88 1.56 1.08 0.8 0.55 0.65 1.69 1.11 0.22 0.07 0.18 0.09 CCNB1 CCNB2 CCNC CCND1 CCND2 CCNE1 CCNF CCNG1 CCNG2 CCNH CCNT1 CCNT2 B 0.37 0.18 0.98 1.3 0.96 0.58 0.19 1.7 2.81 0.96 0.99 0.94 CDC16 CDC2 CDC20 CDC34 CDK2 CDK4 CDK5R1 CDK5RAP1 CDK6 CDK7 CDK8 CDKN1A C 1.03 0.09 0.11 2.22 0.28 0.53 0.63 1.95 0.51 1.61 0.8 34.55 CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CHEK2 CKS1B CKS2 CUL1 CUL2 CUL3 DDX11 D 0.92 1.68 1.9 0.16 0.04 0.32 0.6 1.34 1.44 1.65 1.18 0.17 DNM2 E2F4 GADD45A GTF2H1 GTSE1 HERC5 HUS1 KNTC1 KPNA2 MAD2L1 MAD2L2 MCM2 E 0.81 1.11 12.09 1.41 0.09 2.36 0.46 0.29 0.43 0.11 1.21 0.15 MCM3 MCM4 MCM5 MKI67 MNAT1 MRE11A NBN PCNA RAD1 RAD17 RAD51 RAD9A F 0.17 0.28 0.2 0.03 1.05 0.18 0.62 0.2 2.4 1.64 0.33 0.72 RB1 RBBP8 RBL1 RBL2 RPA3 SERTAD1 SKP2 SUMO1 TFDP1 TFDP2 TP53 UBA1 G 0.91 0.38 0.12 1.44 0.64 3.27 0.06 1.35 0.44 0.94 0.83 1.57

Magnitude of log2 (fold change)

-33.554 0 33.554 Bexarotene 123456789101112 ABL1 ANAPC2 ANAPC4 DIRAS3 ATM ATR BAX BCCIP BCL2 BIRC5 BRCA1 BRCA2 A 1.32 0.98 0.9 1.83 1.26 0.98 1.14 0.78 0.13 0.26 0.35 0.36 CCNB1 CCNB2 CCNC CCND1 CCND2 CCNE1 CCNF CCNG1 CCNG2 CCNH CCNT1 CCNT2 B 0.6 0.5 1.18 0.33 0.24 0.63 0.4 1.12 1.88 1.21 1.14 1.24 CDC16 CDC2 CDC20 CDC34 CDK2 CDK4 CDK5R1 CDK5RAP1 CDK6 CDK7 CDK8 CDKN1A C 1.27 0.32 0.54 1.56 0.28 1.19 0.7 0.99 0.47 1.98 1.4 5.46 CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CHEK2 CKS1B CKS2 CUL1 CUL2 CUL3 DDX11 D 0.83 1.07 2.58 0.52 0.36 0.31 0.93 0.85 1.25 0.82 0.99 0.44 DNM2 E2F4 GADD45A GTF2H1 GTSE1 HERC5 HUS1 KNTC1 KPNA2 MAD2L1 MAD2L2 MCM2 E 1.08 1.04 13.68 1.57 0.3 1.37 1.18 0.31 0.56 0.35 0.85 0.2 MCM3 MCM4 MCM5 MKI67 MNAT1 MRE11A NBN PCNA RAD1 RAD17 RAD51 RAD9A F 0.27 0.24 0.31 0.23 1.05 0.33 0.8 0.2 0.55 1.55 0.31 1.02 RB1 RBBP8 RBL1 RBL2 RPA3 SERTAD1 SKP2 SUMO1 TFDP1 TFDP2 TP53 UBA1 G 0.71 0.96 0.27 0.69 0.51 1.47 0.24 0.83 0.44 1.14 1.37 0.95

Magnitude of log2 (fold change)

-12.676 0 12.676

Figure 3. Induction of RXRE-luciferase activity and inhibition of cell proliferation accompanied with cell-cycle arrest by indenoisoquinolines accompanied by significant 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 SRB assay.Cells were incubated for 24, 48, and 72hours.Results arepresentedasfoldincreasesofabsorbance at515 nmovercontrol ata starting point (0hour). B and C, 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 time period, 10,000 of cells were analyzed for DNA content and the distribution of cells in each phase was estimated by using ModFit LT (Verity Software House). D, MCF7 cells 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 serially diluted AM6-36 for 24 hours. RNA was extracted from cells and reverse transcribed to cDNA. Then, cDNA was mixed with RT2 qPCR Master Mix and 25 mLwere placed in each well of the array plate. Real-time quantitative PCR was performed using SyBR Green detection system. The thermal cycling condition was at 95 C for 10 minutes and then 40 cycles at 95 C for 15 seconds followed by 60 C for 1 minute. On the basis of the DDCt method, the fold changes between the control group and sample-treated groups were analyzed using the Web-based software provided by SABiosciences (RT2 Profiler PCR Array Data Analysis). Fold changes of 84 genes induced by AM6-36 or bexarotene were determined and presented as heatmaps (Red: upregulation, green: downregulation).

600 Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research

significantly altered by bexarotene but not AM6-36 (BCL2, AM6-36, cells were stained and quantified. As shown downregulated; CDKN2B, upregulated), there was clearly a in Figure 5B, as judged by growth in soft agar, treatment similar trend of modulation by both compounds. Of the 14 with AM6-36 reduced cell transformation by approxi- genes significantly downregulated by AM6-36 but not mately 70%. bexarotene, 9 were also downregulated by bexarotene at the P 0.083 level. Expression of cell surface marker, CD38 Supplementary Figure S1 illustrates the connectivity of As an additional test of chemopreventive potential, the the main genes used in this array that were affected by ability of AM6-36 to affect the expression of CD38 was AM6-36 or bexarotene. Notably, expression levels of several assessed with HL-60 cells. As shown in Figure 5C, with genes, which are transcriptionally activated by p53 (the pro- this model, treatment with 9-cis-RA can induce the tein of the TP53 gene), were significantly upregulated by expression of CD38 in about 95% of the cell population AM6-36 and bexarotene. Prominent examples are CDKN1A at higher concentrations (2,000, 200, and 20 nmol/L). At (P ¼ 0.00029, AM6-36; P ¼ 0.00014, bexarotene) and a concentration of 2 nmol/L, the 9-cis-RA–treated cell GADD45A (P ¼ 0.00031, AM6-36; P ¼ 0.00011, bexarotene). population moved to the left with a decreased percentage of FL-1–positive cells (86.7%), and at 0.2 nmol/L, p53 and p21 were increased after AM6-36 treatment further reduction in expression was observed. When and RXRa protein knockdown by RXRa siRNA treated with 2,000 or 200 nmol/L, AM6-36, cells exhib- abrogated the effect of AM6-36 on the induction of ited a 72% or 58% FL-1–positive response (CD38-posi- p21 expression tive), respectively; although, the fluorescence intensity Although the expression of TP53 was not modulated by was lower in comparison with 2,000 nmol/L 9-cis-RA– AM6-36 with MCF7 cells, the potential effect on protein treated cells. level was examined by Western blot analyses. As shown in Figure 4A, treatment with AM6-36 caused an increase of Evaluation of AM6-36 permeability and metabolic p53 at 8 hours, followed by decreases at 24 and 48 hours. stability Even though the protein level of p53 was diminished at Judged by the Caco-2 cell permeability model, the appar- later time points, it still remained at higher levels than the ent permeability coefficient for AM6-36 in the AP ! BL, control. Also, since CDKN1A levels were most highly 2.75 10 6 cm/second, was found to be 10-fold slower elevated by treatment with AM6-36, its protein than that of the high permeability standard, propranolol, (p21CIP1/WAF1) levels were determined. Expression of but 17-fold faster than the low permeability standard, p21 in control cells was low at all time points, whereas sucrose (Table 2). These measurements indicate that the treatment of AM6-36 caused the moderate increase at 8 intestinal absorption of AM6-36 should proceed at a mod- hours, and incremental expression was observed at 24 erate rate following oral administration. Since the apparent and 48 hours. To establish a direct correlation between permeability coefficients in the AP ! BL and in the BL ! the transcriptional activity of RXRa and the induction of AP were not significantly different, AM6-36 does not p21 expression, the expression of RXRa protein was appear to be a substrate for efflux proteins that might transiently depleted in MCF7 cells by introducing RXRa reduce its bioavailability. During incubation of AM6-36 siRNA into the cells. As shown in Figure 4B, siRNA with human liver microsomes, 1 abundant and 3 minor targeted to RXRa markedly reduced RXRa expression, metabolites were observed. In the absence of either and consequently affected the upregulating effect of NADPH or microsomes, no metabolites were detected. AM6-36 on the protein expression of p21. Also, transfec- Therefore, we assume that all 4 metabolites are produced tion of p53 siRNA resulted in the abrogation of the p21 in a cytochrome P450- and NADPH-dependent manner. expression induced by AM6-36 (Fig. 4C). However, p53 The structures of these metabolites are currently under knockdown did not influence the expression of RXRa investigation and will be reported later. (Fig. 4D). These data suggested that AM6-36 was not able to induce p21 expression in the absence of RXRa or p53 Preliminary studies conducted with rats (i.e., RXRa and p53 are required for AM6-36–mediated To assess absorption and metabolism, AM6-36 (40 mg/ mRNA upregulation of p21). kg body weight) was administered to rats, by gavage, over a period of 3 days. Blood was collected on days 1 and 3, and Effects of AM6-36 on JB6 Cl41 cells tissues were collected at the end of the study. As summar- To begin assessment of the chemopreventive potential ized in Table 3, the concentration of AM6-36 in the rat of AM6-36, studies were performed with TPA-treated JB6 serum was approximately 0.83 mg/mL. The concentration Cl41 cells. As shown in Figure 5A, the expression of COX- in liver (4.28 mg/g) was around 5 times higher. Appreciable 2 was induced after exposure to TPA (10 ng/mL) for 15 quantities were also found in the mammary gland (0.29 hours. In contrast to control, however, cotreatment with mg/g) and perirenal fat (0.28 mg/g). These data are consis- AM6-36 attenuated COX-2 protein expression in a content with moderate oral absorption and slow metabolism/ centration-dependent manner. Next, anchorage-indepen- excretion. dent cell transformation assay was performed. After Finally, to obtain an early indication of chemopreventive 28 days of incubation in the presence or absence of potential, a short-term mammary tumor model was used.

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A RXRα

p53

p21WAF1/CIP1

β-Actin

Time (h) 0 8 24 48 8 24 48 Control AM6-36 (1 µmol/L) B RXRα

p21WAF1/CIP1

β-Actin

Control AM6-36 9-cis-RA Control AM6-36 9-cis-RA Control siRNA RXRα siRNA C RXRα

p21WAF1/CIP1

β-Actin

Control AM6-36 9-cis-RA Control AM6-36 9-cis-RA Control siRNA p21 siRNA

D 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

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, MCF7 cells 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 the indicated 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 additional 24 hours. Cell lysates were prepared and subjected to Western blot analysis.

602 Cancer Prev Res; 4(4) April 2011 Cancer Prevention Research

AB 120 COX-2 90

GAPDH 60

Control *

% * 30 AM6-36 0 0 1.25 2.5 5 (µmol/L) 0 TPA - ++++ AM6-36 050100 (10 ng/mL) (nmol/L) CD 600

200 – 200 – ) 200 – 2 Control AM6-36-treated Control : 4.93% Autofluorescence control: Negative staining control : Control 2.17% 6.17% – 500 – – Sample-treated – 100– 100– 100 400 FL-1 (+) – – – 300

l l l l l l l l llll (mean,mm size r 0101 102 103 0101 102 103 0101 102 103 o 200 200 – 200 – 200 – – 200 – 2,000 nmol/L 9-cis-RA: 200 nmol/L 9-cis-RA: 20 nmol/L 9-cis-RA: 200 2 nmol/L 9-cis-RA: 0.2 nmol/L 9-cis-RA: 95.17% 96.16% 93.19% 86.7% 25.12% – – – – – 100

100– 100– 100– 100– 100–

Mammary tum 0 – – – – – 0 7 14 21 28 35 42

llll llll llll llll llll Days 0101 102 103 0101 102 103 0101 102 103 0101 102 103 0101 102 103

– 200 – 200 – 2,000 nmol/LAM6-36: 200 – 200 nmol/L AM6-36: 200 – 20 nmol/LAM6-36: 200 2 nmol/LAM6-36: 0.2 nmol/LAM6-36: 72.24% 58.47% 10.31% 8.15% 8.29% – – – – –

100– 100– 100– 100– 100–

– – – –

llll llll llll llll llll 1 2 3 0101 102 103 0101 102 103 0101 102 103 0101 102 103 01010 10

Figure 5. Evaluation of chemopreventive potential of AM6-36. A, JB6 Cl41 cellswerestimulatedbytreatmentwith TPA (10 ng/mL) in the presence or absence of AM6-36 for 15 hours. Whole cell lysates were prepared and COX-2 expression was determined by Western blot analysis. b-Actin was used as an internal control. B, JB6 Cl41 cells were suspended in 50 mL of 0.4% agar complete MEM medium over 100 mLof0.8%agarcomplete MEM medium. 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 the incubation, cell quantification solution was added to each well and further incubated at 37C for 4 hours. Absorbance was measured at 490 nm and the data are shown as % transformation activity relative to TPA-treated control. C, HL-60 cells were incubated with serially diluted 9-cis-RA or AM6-36 (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 (closed squares) animals (n ¼ 10). P ¼ 0.1; Mann–Whitney test.

As shown in Figure 5D, at a dose of 10 mg/kg body ate the RXRa transcriptional activity of various test sub- weight per day, AM6-36 clearly attenuated tumor growth stances. As the result of an extensive search, AM6-36 was (P ¼ 0.1). It will now be necessary to evaluate larger found to be active. Since the COS-1 cells used for this numbers of animals with various treatment regimens to discovery were transiently transfected with pRXRE and establish statistical significance, but the trend demonstrates phRXRa, a response is expected to be due to the interac- promising antitumor activity. tion of a test substance with RXR–RXR homodimers. Further investigation is required to determine the poten- Discussion tial of any lead, including AM6-36, to interact with other nuclear receptors, such as RXR–PPAR (27). The goal of RXR agonists have shown promise for the treatment or this work, however, was to investigate the biological prevention of cancer. 9-cis-RA and LGD1069 are key ramifications of the highly unique response mediated examples of agents in clinical use. In addition, unlike by AM6-36 with this model system. other nuclear receptors, RXRs are expressed in human Indenoisoquinolines were first synthesized in 1978 (28) carcinomas and overexpression of RXR has enhanced the and studied as topoisomerase I inhibitors with advanta- transcriptional response after binding of ligands (26). On geous properties compared with camptothecins, known as the basis of these considerations, we introduced a cell topoisomerase I inhibitors. Additional activities are also line–based RXRE-luciferase reporter gene assay to evalu- known, such as induction of cell-cycle arrest, overcoming

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

Table 2. Intestinal permeability, metabolic sta- indenoisoquinolines are structurally unique rexinoids bility, and distribution of AM6-36: apparent offers the possibility of them having unique pharmaco- logic properties that could be clinically useful. Consider- permeability coefficients of AM6-36 through ing these factors as a whole, AM6-36 was selected for Caco-2 monolayers further evaluation. As shown in Figure 1, a synthetic procedure was devised P (10 6 cm/s SD)a app that is suitable for production of the lead compound in AP to BLb BL to APc multigram quantities. Since RXR agonists showed apop- totic effects in RXRa-expressed MCF7 cells (25), we AM6-36d, mmol/L examined the effect of AM6-36 using this cell line. As 5 2.75 0.78 3.95 0.11 expected, when MCF7 cells were treated with AM6-36, we 10 5.52 0.29 3.25 0.71 observed increased RXRa transcriptional activity, which 25 4.45 0.71 8.26 0.93 correlated with the response observed with transiently Standards, mmol/L transfected COS-1 cells. All of these data suggest that Propranolol (10) 27.6 MCF7 cells should be a useful model for exploring the Sucrose (50) 0.16 action of AM6-36 related to RXRa. Cell-cycle analysis revealed G2/M arrest at lower concentrations and S-phase aP app corresponds to the apparent permeability coefficients arrest at higher concentrations (Fig. 4C). A similar dose- 6 and is expressed as cm/s (10 ). related dual effect has been reported when MCF7 cells b AP to BL indicates apical to basolateral transport. were treated with adriamycin (35), and other DNA dama- cBL to AP indicates basolateral to apical transport. ging agents showed partial G1-andG2/M-phase cell-cycle d Data are expressed as mean SD, n ¼ 3. arrest at lower doses, whereas they exerted partial or total S-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. multidrug-resistance, and enhancement of the differentia- Together with significant growth inhibition, genes tion-inducing activity of retinoids (29–31). AM6-36 known as conventional proliferation markers and used mediated only modest inhibition of the DNA religation in the diagnosis of breast cancer, including MKI67 and reaction (data not shown), but unexpectedly, RXRE-luci- PCNA (37), were downregulated by treatment with AM6- ferase activity was highly upregulated. On the basis of the 36. However, a known target gene of RXR, CDKN1A (38), response induced by AM6-36, a small group of structural was the most intensively upregulated. Correspondingly, relatives were evaluated to determine whether activity p21 protein levels were increased (Fig. 4B). Treatment of could be further enhanced. As shown in Table 1, com- cells with retinoic acid can lead to elevated p21 levels (39), pounds 5 and 6 induced the RXR transcriptional activities as well as agents such as 2{[3-(2,3-dichlorophenoxy)pro- as well as antiproliferative effects toward MCF7 cells, but pyl]amino}ethanol (40) and IFN-a (41). Interestingly, AM6-36 was still the most active compound. similar to AM6-36, these agents arrest cells in the S phase The indenoisoquinoline AM6-36 is an atypical rexinoid of the cycle. In the RXRE assay, AM6-36 showed higher since it does not contain a carboxylic acid and it does not induction in RXRa transcriptional activity than 9-cis-RA. structurally resemble a retinoic acid. In comparison with The response may be augmented by enhanced expression known rexinoids, most of which contain carboxylic acids of GADD45A, a RXRa coactivator (42). It is further noted (32–34), the indenoisoquinoline rexinoids constitute a from the DNA microarray that SKP2 is greatly downregu- fundamentally different type of RXR ligand. The fact that 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 in the rata

AM6-36 Serum 1 d, Serum 3 d, Liver, Mammary Fat, mg/g (40 mg/kg) mg/mL (n ¼ 3) mg/mL (n ¼ 3) mg/g (n ¼ 3) gland, (pooled n ¼ 3) 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.

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increased protein expression of p21, since Skp2 is involved has yet to be determined, but a hypothetical structure was in the degradation of p21 (43). generated by GOLD (47) docking the indenoisoquinoline The role of p53 in these processes remains to be into the X-ray structure of RXR-a (1FBY) (48) using the further explored. Although p53 gene expression was not centroid (15.2492, 28.4959, 48.2618) of bound 9-cis-RA. increased by AM6-36, other effects, such as modified pro- As illustrated in Figure 6, AM6-36 can readily interact with tein expression and transcriptional activity, may come into the binding site of 9-cis-RA. The ligand pose with the play. In addition to upregulation of CDKN1A, other highest GOLD score and the most favorable binding energy genes associated with p53 transcriptional activity (e.g., was used for further calculations. The energy of the ligand GADD45A, CCNG2; ref. 44) showed increases in expres- and a surrounding spherical subset of atoms having a sion to some extent. Furthermore, the expression of GTSE1, radius of 6 A was minimized by the conjugate gradient which has been reported to downregulate the level and method using the MMFF94s force field and MMFF94 activity of the p53 tumor suppressor protein (45), was charges (SYBYL 8.0, Tripos International, 1699 South decreased by AM6-36. Thus, although additional studies Hanley Rd., St. Louis, Missouri, 63144, USA). The calcula- are warranted, this might explain the increase in p53 tion was terminated at a gradient of 0.05 kcal/(molA). protein level. Rexinoids are of interest due to cancer chemopreventive In any case, enhanced RXRa activity leading to highly potential (6–8), and, of course, a question of utmost elevated expression of p21, an inhibitor of CDKs which importance is the chemopreventive potential of AM6-36. negatively regulates cell-cycle progression (46), appears to Some early studies have been completed. As normal cells play a dominant role in the cellular responses orchestrated undergo neoplastic transformation, they gain the ability to by AM6-36 treatment. A direct relationship between the demonstrate "anchorage independence" and grow in soft activation of RXRa and p21 expression was further estab- agar (49). Currently, we demonstrated the potential of lished by transiently blocking the expression of RXRa.In AM6-36 to suppress TPA-induced neoplastic transforma- the absence of RXRa, the expression of p21 was abrogated tion of JB6 Cl41 cells. Furthermore, during neoplastic (Fig. 4B). The crystal structure of AM6-36 bound to RXRa transformation stimulated by TPA, JB6 Cl41 cells express

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

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COX-2, which plays pivotal roles not only in inflammation strated attenuation of MNU-induced tumorigenesis in but also in carcinogenesis. AM6-36 inhibits the expression female Sprague Dawley rats. Cleary, additional work is of COX-2 in this model system. required to assess the effect of various dose regimens An additional strategy for cancer chemoprevention is with larger numbers of animals representative of differ- the induction of differentiation (50). All-trans-retinoic ent tumor models, but this is completely feasible, since acid (ATRA) is known to induce CD38 expression AM6-36 can readily be produced on a multigram scale. through activation of RAR and RXR in myeloid progenitor AM6-36 is a surprising and unique molecule, represent- cells, which modulates cell differentiation (51, 52). As a ing a promising lead that functions through a novel further induction of cancer chemopreventive potential, mechanism. we currently demonstrated AM6-36–treated HL-60 cells express CD38 on membrane surfaces. Disclosure of Potential Conflicts of Interest Taken together, these data suggest the potential of AM6-36 to serve as a chemopreventive agent. Prelimin- No potential conflicts of interest were disclosed. ary results were also obtained with the Caco-2 cell Grant Support permeability model, suggesting that the compound would be orally absorbed. In addition, incubation This study was supported by NIH under NCI grants P01 CA48112 and with human liver microsomes indicated moderate meta- U01 CA089566. The costs of publication of this article were defrayed in part by the bolism. These data were corroborated by studies invol- payment of page charges. This article must therefore be hereby marked ving oral administration with rats. Serum and tissue advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate levels were readily achieved that should be of physio- this fact. logic relevance, based on in vitro response data. In Received January 12, 2010; revised January 10, 2011; accepted January support of this notion, a preliminary evaluation demon- 18, 2011; published online April 4, 2011.

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