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Cancer Research 67, 7815, August 15, 2007. doi: 10.1158/0008-5472.CAN-07-0794
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

A Potent Indole-3-Carbinol–Derived Antitumor Agent with Pleiotropic Effects on Multiple Signaling Pathways in Prostate Cancer Cells

Jing-Ru Weng1,2, Chen-Hsun Tsai3, Samuel K. Kulp3, Dasheng Wang3, Chia-Hui Lin3, Hsiao-Ching Yang3, Yihui Ma3, Aaron Sargeant3, Chang-Fang Chiu2, Ming-Hsui Tsai2 and Ching-Shih Chen3

1 Department of Biological Science and Technology, China Medical University; 2 China Medical University Hospital, Taichung, Taiwan; and 3 Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio

Requests for reprints: Ching-Shih Chen, Division of Medicinal Chemistry, College of Pharmacy, Parks Halls, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210. Phone: 614-688-4008; Fax: 614-688-8556; E-mail: chen.844{at}osu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Indole-3-carbinol has emerged as a promising chemopreventive agent due to its in vivo efficacy in various animal models. However, indole-3-carbinol exhibits weak antiproliferative potency and is unstable in acidic milieu. Thus, this study was aimed at exploiting indole-3-carbinol to develop potent antitumor agents with improved chemical stability. This effort culminated in OSU-A9 {[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol}, which is resistant to acid-catalyzed condensation, and exhibits 100-fold higher apoptosis-inducing activity than the parent compound. Relative to indole-3-carbinol, OSU-A9 displays a striking qualitative similarity in its effects on the phosphorylation or expression of multiple signaling targets, including Akt, mitogen-activated protein kinases, Bcl-2 family members, survivin, nuclear factor-{kappa}B, cyclin D1, p21, and p27. The ability of OSU-A9 to concurrently modulate this broad range of signaling targets underscores its in vitro and in vivo efficacy in prostate cancer cells. Nevertheless, despite this complex mode of mechanism, normal prostate epithelial cells were less susceptible to the antiproliferative effect of OSU-A9 than PC-3 and LNCaP prostate cancer cells. Treatment of athymic nude mice bearing established s.c. PC-3 xenograft tumors with OSU-A9 at 10 and 25 mg/kg i.p. for 42 days resulted in a 65% and 85%, respectively, suppression of tumor growth. Western blot analysis of representative biomarkers in tumor lysates revealed significant reductions in the intratumoral levels of phosphorylated (p-) Akt, Bcl-xL, and RelA, accompanied by robust increases in p-p38 levels. In conclusion, the ability of OSU-A9 to target multiple aspects of cancer cell survival with high potency suggests its clinical value in prostate cancer therapy. [Cancer Res 2007;67(16):7815–24]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The chemopreventive potential of indole-3-carbinol, a naturally occurring phytochemical in cruciferous vegetables (1), has received much attention in light of its shown in vivo efficacy to protect against chemically induced carcinogenesis in animals (2–7). Moreover, the clinical benefits of indole-3-carbionol have also been shown in human trials for cervical dysplasia (8), breast cancer (9, 10), and vulvar intraepithelial neoplasia (11). Despite these advances in translational research, the mechanism by which indole-3-carbinol inhibits tumorigenesis remains inconclusive, which, in part, might be attributable to its metabolic instability and complicated pharmacologic properties. In an acidic milieu, indole-3-carbinol undergoes spontaneous dehydration and condensation to form diindoylmethane and a series of oligomeric products (12, 13), all of which exhibit in vitro antiproliferative activities against cancer cells (14–18). Furthermore, mechanistic evidence indicates that indole-3-carbinol facilitates growth arrest and apoptosis by targeting a broad range of signaling pathways pertinent to cell cycle regulation and survival, including those mediated by Akt, nuclear factor-{kappa}B (NF-{kappa}B), Bcl-2, mitogen-activated protein (MAP) kinases, the cyclin-dependent kinase (CDK) inhibitors p21 and p27, and cyclin D1 (reviews: refs. 19–23). However, as these signaling targets often operate in a cell-specific fashion, it remains in dispute whether any of them could solely account for the effect of indole-3-carbinol on growth arrest and apoptosis in tumor cells (24).

From a mechanistic perspective, the ability of indole-3-carbinol and its metabolites to target a broad spectrum of signaling pathways underlies their antiproliferative effects. However, these agents suffer from metabolic instability, unpredictable pharmacokinetic properties, and low in vitro antiproliferative potency, which render therapeutic concentrations difficult to achieve in the body. Consequently, recent years have witnessed the use of diindoylmethane as a scaffold to carry out structural modifications, which has led to three distinct antitumor agents with higher potency reported in the literature: (p-substituted phenyl)-diindoylmethanes [peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists; refs. 18, 25–28], SR13668 (an Akt inhibitor; refs. 29, 30), and an indole-3-carbinol tetrameric derivative (a CDK6 inhibitor; ref. 31; Fig. 1A ). These novel agents exhibit µmol/L potency in inducing apoptosis or cell cycle arrest, however, through signaling pathways distinct from that affected by diindoylmethane.


Figure 1
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  		Figure 1. Use of indole-3-carbinol and diindoylmethane as scaffolds for developing new anticancer agents. A, schematic diagram of the acid-catalyzed condensation of indole-3-carbinol to form diindoylmethane (DIM) and structures of the diindoylmethane-derived antitumor agents. B, structures and potencies for inducing apoptotic death in PC-3 and LNCaP cells of the indole-3-carbinol derivatives A1 to A24. The general structures of these compounds (I, II, III) are shown above the data for each of their respective series of derivatives. The reported IC50 values are concentrations at which PC-3 or LNCaP cell death measures 50% relative to DMSO control after 48-h exposure in 5% FBS–containing RPMI 1640 in 96-well plates. Cell viability was assessed by MTT assays with six replicates.

 
In this study, we report the use of indole-3-carbinol as a lead compound to develop antitumor agents with improved chemical stability and apoptosis-inducing potency. This structural optimization effort culminated in the generation of OSU-A9, a novel, acid-stable analogue with two orders of magnitude higher apoptosis-inducing potency than the parental indole-3-carbinol. Moreover, OSU-A9 retained the pleiotropic effects of indole-3-carbinol on multiple signaling targets associated with growth arrest and apoptosis. Equally important, despite this broad spectrum of pharmacologic activities, normal prostate epithelial cells were less sensitive to the antiproliferative effect of OSU-A9 relative to prostate cancer cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. 1H-indole-3-carbaldehyde (Sigma-Aldrich) was used as the starting material to synthesize 1H-indole-3-carbinol and series I to III agents (Fig. 1A), of which the synthesis will be published elsewhere. The identity and purity (≥99%) of these synthetic derivatives were verified by proton nuclear magnetic resonance, high-resolution mass spectrometry, and elemental analysis. For in vitro experiments, these agents at various concentrations were dissolved in DMSO, and were added to cells in medium with a final DMSO concentration of 0.1%. Rabbit polyclonal antibodies against various biomarkers were obtained from the following sources: phosphorylated (p-)473Ser Akt, p-Bad, Bad, phosphorylated extracellular signal-regulated kinases (p-ERK), phosphorylated c-Jun-NH2-kinase (p-JNK), JNK, p-p38, p38, cyclin D1, and NF-{kappa}B, Cell Signaling Technologies; Akt, ERKs, p27, p21, Bax, Bcl-2, Bcl-xL, and AR, Santa Cruz Biotechnology; survivin, R&D Systems; and ß-actin, Sigma-Aldrich. Mouse monoclonal anti-PARP antibody was purchased from PharMingen. The enhanced chemiluminescence system for detection of immunoblotted proteins was from GE Healthcare Bioscience. Other chemicals and biochemistry reagents were obtained from Sigma-Aldrich unless otherwise mentioned.

Cell culture. LNCaP androgen-responsive (p53+/+) and PC-3 androgen-nonresponsive (p53–/–) human prostate cancer cells were purchased from the American Type Tissue Collection and cultured in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies). Normal human prostate epithelial cells were obtained from Cambrex Bioscience-Walkersville, and maintained in the vendor's recommended defined prostate epithelial growth medium. All cell types were cultured at 37°C in a humidified incubator containing 5% CO2.

Cell viability analysis. The effect of test agents on cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in 6 to 12 replicates. Cancer cells and prostate epithelial cells were grown in 5% FBS–supplemented RPMI 1640 or 5% FBS–supplemented prostate epithelial growth medium, respectively, in 96-well, flat-bottomed plates for 24 h, and then exposed to various concentrations of test agents in the same medium for the indicated time intervals. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. At the end of the treatment, the medium was removed, replaced by 200 µL of 0.5 mg/mL of MTT in the same medium, and cells were incubated in the CO2 incubator at 37°C for 2 h. Supernatants were removed from the wells, and the reduced MTT dye was solubilized in 200 µL/well DMSO. Absorbance at 570 nm was determined on a plate reader.

Cell proliferation. PC-3 and LNCaP cells were seeded into six-well plates at ~200,000 per well in 5% FBS–containing RPMI 1640. Following a 24 h attachment period, cells were treated in triplicate with the indicated concentrations of test agent or DMSO vehicle in 5% FBS–containing RPMI 1640. At different time intervals, cells were harvested by trypsinization and counted using a Coulter counter (Model Z1 D/T, Beckman Coulter).

Apoptosis analysis. Three apoptosis biomarkers were used to assess drug-induced apoptosis by Western blot analysis: cytochrome c release, caspase-3 activation, and PARP cleavage. Cytosolic-specific, mitochondria-free lysates for cytochrome c analysis was prepared according to an established procedure (32). After drug treatments, both the incubation medium and adherent cells in T-75 flasks were collected and centrifuged at 600 x g for 5 min. The pellet fraction was recovered, placed on ice, and triturated with 300 µL of a chilled hypotonic lysis solution [50 mmol/L PIPES-KOH (pH 7.4), containing 220 mmol/L mannitol, 68 mmol/L sucrose, 50 mmol/L KCl, 5 mmol/L EDTA, 2 mmol/L MgCl2, 1 mmol/L DTT, and a mixture of protease inhibitors including 100 µmol/L AEBSF, 80 nmol/L aprotinin, 5 µmol/L bestatin, 1.5 µmol/L E-64 protease inhibitor, 2 µmol/L leupeptin, and 1 µmol/L pepstatin A]. After a 45-min incubation on ice, the mixture was centrifuged at 200 x g for 10 min. The supernatant was collected in a microcentrifuge tube and centrifuged at 14,000 rpm for 30 min. Equivalent amount of protein (50 µg) from each supernatant was subject to immunoblotting with anti–cytochrome c antibodies, as described below.

With regard to the other two apoptosis biomarkers, collected drug-treated cells were washed with ice-cold PBS and resuspended in lysis buffer containing 20 mmol/L Tris-HCl (pH 8), 137 mmol/L NaCl, 1 mmol/L CaCl2, 10% glycerol, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 100 µmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride, leupeptin at 10 µg/mL, and aprotinin at 10 µg/mL. Soluble cell lysates were collected after centrifugation at 10,000 x g for 5 min. Equivalent amounts of proteins (60–100 µg) from each lysate were subject to immunoblotting with antibodies against caspase-3 or PARP as described below.

Immunoblotting. Biomarkers of apoptosis and signaling components associated with cell survival and growth arrest were assessed by Western immunoblotting as follows. Treated cells were washed in PBS, resuspended in SDS sample buffer, sonicated for 5 s, and then boiled for 5 min. After brief centrifugation, equivalent amounts of proteins from the soluble fractions of cell lysates were resolved in 10% SDS-polyacrylamide gels on a Minigel apparatus, and transferred to a nitrocellulose membrane using a semidry transfer cell. The transblotted membrane was washed thrice with TBS containing 0.05% Tween 20 (TBST). After blocking with TBST containing 5% nonfat milk for 60 min, the membrane was incubated with an appropriate primary antibody at 1:500 dilution (with the exception of anti–ß-actin antibody, 1:2,000) in TBST–5% low fat milk at 4°C for 12 h, and was then washed thrice with TBST. The membrane was probed with goat anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugates (1:2,500) for 90 min at room temperature, and washed thrice with TBST. The immunoblots were visualized by enhanced chemiluminescence.

Molecular modeling. Molecular structures of A7, OSU-A9, and A12 were subjected to energy minimization using the Merck Molecular Force Field program available as part of the Macromodel 7.0 software package (Schrodinger4). The minimum conformations were then fully optimized at a density functional theory level of B3LYP/6-31G* basis set using Gaussian 03 (Gaussian, Inc.). All of the fully optimized structures were confirmed by normal mode analysis; no negative frequencies were found. Computations for electron density and electrostatic potential were then carried out for each of the fully optimized structures with a grid of 216,000 points using Gaussian 03. Molecular electrostatic potential maps for each compound were generated with the electrostatic potential mapped onto the electron density. The electron density isosurface value was 0.002 (electron/Å) with a range of –0.03 to 0.03 for the electrostatic potential. All molecular modeling calculations and manipulations were done on Silicon Graphics O2 (Silicon Graphics, Inc.).

Nuclear magnetic resonance analysis of acid stability. Indole-3-carbinol and OSU-A9, 20 mg each, were dissolved in 1 mL of CD3OD. To each solution, 100 µL of deuterium-labeled HCl were added, and nuclear magnetic resonance (NMR) spectra were recorded in a 300 MHz NMR spectrometer at room temperature at different time intervals.

Luciferase assay for PPAR{gamma} activation. The PPAR-response element (PPRE)-x3-TK-Luc reporter vector contains three copies of the PPRE upstream of the thymidine kinase promoter-luciferase fusion gene and was kindly provided by Dr. Bruce Spiegelman (Harvard University, Cambridge, MA). The reporter gene assay was carried out as previously described (33). In brief, PC-3 cells were cultured in a 100-mm plate in phenol red–free RPMI 1640 containing 10% FBS until the achieved 50% to 70% confluency, after which they were transfected with 6 µg of the plasmid using Fugene 6 (Roche) in RPMI 1640. For each transfection, herpes simplex virus-thymidine kinase promoter–driven Renilla luciferase (phRL-TK) was used as an internal control for normalization. Following transfections, cells were treated as indicated in RPMI 1640 containing 10% charcoal-stripped FBS. Cells were then collected into Passive Lysis Buffer (Promega), and luciferase activities in the cell lysates were determined by luminometry. All transfection experiments were carried out in triplicate plates and repeated separately at least thrice.

In vivo studies. Intact male NCr athymic nude mice (5–7 weeks of age) were obtained from the National Cancer Institute. The mice were group housed under conditions of constant photoperiod (12 h light/12 h dark) with ad libitum access to sterilized food and water. All experimental procedures using these mice were done in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University.

Each mouse was inoculated s.c. in the right flank with 5 x 105 PC-3 cells in a total volume of 0.1 mL serum-free medium containing 50% Matrigel (BD Biosciences) under isoflurane anesthesia. As tumors became established (mean starting tumor volume, 109 ± 10 mm3), mice were randomized to three groups (n = 7) that received the following treatments: (a) OSU-A9 at 10 mg/kg body weight qd, (b) OSU-A9 at 25 mg/kg qd, and (c) DMSO vehicle. Mice received treatments by i.p. injection (50 µL/mouse) for the duration of the study. Tumors were measured weekly using calipers and their volumes calculated using a standard formula, as follows: width2 x length x 0.52. Body weights were measured weekly. At terminal sacrifice, a complete necropsy was done on all mice and PC-3 tumors were harvested. A portion of each tumor was snap-frozen in liquid nitrogen and stored at –80°C until needed for Western blot analysis of relevant biomarkers, and the remainder was fixed in 10% formalin. All other tissues were fixed overnight in 10% formalin then transferred to 70% ethanol. Four-micrometer-thick, paraffin-embedded tissue sections were stained with H&E by standard procedures. A core list of tissues from three mice per group were evaluated microscopically animal by animal by a veterinary pathologist in accordance with Society of Toxicologic Pathology–proposed guidelines for repeat-dose toxicity studies (34), with the exception of spinal cord and thymus. Blood from each mouse was submitted to The Ohio State University Veterinary Clinical Laboratory Services for evaluation of serum chemistry and hematologic variables routinely evaluated in a clinical setting (35).

Statistical analysis. Differences in relative PPAR{gamma} activation in vitro and among group means of tumor volume in vivo were analyzed for statistical significance using one-way ANOVA followed by the Neuman-Keuls test for multiple comparisons. Differences were considered significant at P < 0.05. Statistical analyses were done using SPSS for Windows (SPSS, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Structure-activity relationship. The intrinsic instability of indole-3-carbinol in acidic conditions arises from the vinyl hemiaminal moiety of the indole ring, which renders the molecule highly susceptible to acid-catalyzed dehydration and condensation to form oligomeric products (13). Mechanistically, this acid lability could be circumvented by blocking the vinyl hemiaminal function through substitution. Accordingly, to test the premise that indole-3-carbinol was amenable to structural optimization, we synthesized three series of N-substituted analogues: (1-aryloyl-1H-indol-3-yl)-methanols (I), (1-arylsulfonyl-1H-indol-3-yl)-carbaldehydes (II), and (1-arylsulfonyl-1H-indol-3-yl)-methanols (III; Fig. 1B).

These agents (A1–A24) were evaluated for their ability to reduce cell viability of PC-3 (p53–/–) androgen-nonresponsive and LNCaP (p53+/+) androgen-responsive prostate cancer cells after 24-h exposure by MTT assay. Although the IC50 values of all carboxamide derivatives (series I) were >50 µmol/L, some of the derivatives in the II and III series showed improved antitumor activities vis-à-vis indole-3-carbinol. Especially noteworthy is the compound A9 {[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol; designated as OSU-A9}, which exhibited IC50 values of 2 and 3.8 µmol/L for PC-3 and LNCaP cells, respectively (Fig. 1B). This antitumor potency was two orders of magnitude higher than that of indole-3-carbinol (IC50, 512 and 267 µmol/L, respectively; Fig. 2A ). Moreover, assessment of effects on nonmalignant cells revealed that prostate epithelial cells exhibited a 2.2- to 4.5-fold lower sensitivity to OSU-A9 (IC50, 9 µmol/L) than the prostate cancer cells (Fig. 2A).


Figure 2
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  		Figure 2. Antiproliferative effects of OSU-A9 and indole-3-carbinol in two prostate cancer cell lines and prostate epithelial cells. A, effect of OSU-A9 on the viability of prostate epithelial cells (PrEC), and PC-3 and LNCaP prostate cancer cells versus that of indole-3-carbinol in PC-3 and LNCaP cells. Cells were treated with OSU-A9 or indole-3-carbinol at the indicated concentrations in 5% FBS–supplemented RPMI 1640 in 96-well plates for 48 h, and cell viability was assessed by MTT assays. Points, mean; bars, SD (n = 6). B, dose-dependent antiproliferative effect of indole-3-carbinol (DMSO vehicle; 100, 200, 300, 400, and 500 µmol/L) versus OSU-A9 (DMSO vehicle; 0.5, 1, 2.5, and 5 µmol/L) in PC-3 and LNCaP cells. Cells were seeded onto six-well plates (200,000 per well) and exposed to the test agent at the indicated concentrations in 5% FBS–supplemented RPMI 1640. At different time intervals, cells were harvested and counted using a Coulter counter. Values were obtained from triplicates.

 
The in vitro efficacy of OSU-A9 vis-à-vis indole-3-carbinol in inhibiting the proliferation of PC-3 and LNCaP cells was also examined by direct counting of drug-treated cells (Fig. 2B). OSU-A9, at 2.5 µmol/L and above, completely suppressed cell proliferation and cause a net decrease in cell numbers in both cell lines, indicative of cytocidal activity. In line with the cell viability data, a concentration of at least 300 µmol/L was required for indole-3-carbinol to attain the same level of efficacy.

Molecular modeling analysis. The structure-activity analysis revealed a stringent structural requirement for the high potency of OSU-A9. Any change in the structure of the compound, such as substitution of the sulfonyl (-SO2-) linker with a carbonyl (-CO-) function (i.e., A7) or replacement of the nitro (-NO2) substituent with an amino (-NH2) moiety (i.e., A12), resulted in a substantial loss of antitumor activity (Fig. 1B). To shed light onto the structural basis underlying this subtle structure-activity relationship, we analyzed the configuration and surface electrostatic potential of OSU-A9 versus those of A7 and A12 via modeling analysis (Fig. 3A ).


Figure 3
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  		Figure 3. Molecular modeling and chemical stability analyses of OSU-A9. A, molecular modeling of A7, OSU-A9, and A12. The surface electrostatic potential of individual areas is color coded: blue and red, negative and positive electrostatic potentials, respectively. Transitions in electrostatic potential from negative to positive (or vice versa) are shown in other colors (green and yellow). The nitro and amino groups of OSU-A9 (center) and A12 (right) are coded red and blue, respectively, in the chemical structures and indicated by arrows in the electrostatic potential images. B, proton NMR analysis of the chemical stability of OSU-A9 versus indole-3-carbinol in an acid environment. OSU-A9 and indole-3-carbinol, 20 mg each, were dissolved in 1 mL of CD3OD. To each solution, 100 µL of deuterium-labeled HCl were added, and NMR spectra were recorded in a 300 MHz NMR spectrometer. Treatment of indole-3-carbinol with HCl led to oligomerization to form an acid reaction mixture (12, 13) consisting of diindoylmethane and other oligomeric products, as evidenced by an immediate shift of the CH2 signal (* in the chemical structure) from 4.73 to 4.66 ppm. In contrast, treatment of OSU-A9 with HCl gave rise to no appreciable change in the spectrum.

 
As shown, OSU-A9 and A7 assumed vastly different configurations resulting from the structural differences between the tetrahedron-like sulfonyl (-SO2-) and the triagonal-planar carbonyl (-CO-) linkers. Equally important, the electropositive nature of the nitrosubstituent on the appended phenyl ring was crucial to the ability of OSU-A9 to induce apoptosis. Replacement of the electron-withdrawing nitro group with an electron-donating amino function, as in A12, changed the regional surface potential, resulting in the loss of apoptosis-inducing activity.

OSU-A9 resists acid-catalyzed dimerization. We used a NMR technique to analyze the chemical stability of OSU-A9 versus indole-3-carbinol in 0.1 N HCl by monitoring changes in the proton signal associated with CH2OH (Fig. 3B). Individual compounds (20 mg) were dissolved in 1 mL of deuterium-labeled methanol (CD3OD). The NMR spectra revealed signals for the methylene protons (indicated by *) at 4.73 and 4.74 ppm for indole-3-carbinol and OSU-A9, respectively (top spectra, left and right). Addition of 100 µL of 0.1 N deuterium-labeled HCl to indole-3-carbinol resulted in an immediate shift of the CH2 signal from 4.73 to 4.66 ppm (t = 5 min), indicating the chemical transformation of indole-3-carbinol to an acid reaction mixture consisting of diindoylmethane and other oligomeric products (12, 13). On the other hand, no appreciable change in the spectrum was noted after exposure of OSU-A9 to HCl for up to 8 h, indicating its significantly greater chemical stability.

OSU-A9 facilitates apoptosis by targeting multiple signaling pathways identical to that of indole-3-carbinol. Evidence suggest that the antiproliferative effect of OSU-A9 was, at least in part, attributable to apoptosis, reminiscent of that of indole-3-carbinol. Western blot analysis showed a dose-dependent effect of both agents on cytochrome c release into the cytoplasm, caspase-3 activation, and PARP cleavage (Fig. 4A ). The effects of indole-3-carbinol and OSU-A9 on these two apoptosis-related biomarkers were qualitatively similar, albeit with a 100-fold difference in potency.


Figure 4
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  		Figure 4. Evidence of apoptosis for indole-3-carbinol– and OSU-A9–induced cell death. A, dose-dependent effect of indole-3-carbinol and A9 of cytochrome c release into the cytoplasm (top), caspase-3 activation, and PARP cleavage (bottom) in PC-3 cells after 48-h exposure in 5% FBS–supplemented RPMI 1640 medium. Cytosolic-specific, mitochondria-free lysates for cytochrome c analysis and cell lysates for the other two biomarkers were prepared as described in Materials and Methods. B, pharmacologic evidence that the apoptotic effect of OSU-A9 in PC-3 cells is independent of PPAR{gamma} activation. PC-3 cells were transfected with PPRE-X3-TK-Luc and treated with 10 µmol/L rosiglitazone (Rosi) or the indicated concentrations of OSU-A9 for 24 h, and luciferase activity was determined as described in Materials and Methods. Columns, mean; bars, SD (n = 3). **, P < 0.01.

 
From a translational perspective, the mechanism by which OSU-A9 mediated antiproliferative effects warranted investigation. The finding that many indole derivatives, including diindoylmethanes, exhibited PPAR{gamma} agonist activity (18, 25–28, 36, 37) raised the possibility that PPAR{gamma} activation might contribute to the apoptosis-inducing effect of OSU-A9. Accordingly, we used an established PPRE-luciferase reporter assay (33) to assess the ability of OSU-A9 to transactivate PPAR{gamma} in PC-3 cells. However, even at 20 µmol/L, OSU-A9 lacked appreciable activity in PPAR{gamma} transactivation, whereas the PPAR{gamma} agonist rosiglitazone induced significant activation of the reporter gene (Fig. 4B).

Indole-3-carbinol has been reported to target an array of signaling pathways to induce apoptosis and cell cycle arrest in cancer cells (reviews: refs. 19–23). Consequently, we examined the dose-dependent effect of OSU-A9 vis-à-vis indole-3-carbinol in PC-3 cells on the phosphorylation and/or expression status of a series of molecular targets reported for indole-3-carbinol in the literature. These targets comprised three categories of biomarkers: (a) phosphorylation of signaling kinases: Akt and its downstream effector GSK3ß, ERKs, JNKs, and p38; (b) phosphorylation/expression of Bcl-2 family members: Bad, Bax, Bcl-2, Bcl-xL, and Mcl-1; (c) expression of other apoptosis and cell-cycle regulatory proteins: survivin, NF-{kappa}B/RelA, cyclin D1, p27, and p21 (Fig. 5 ).


Figure 5
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  		Figure 5. Dose-dependent effects of OSU-A9 versus indole-3-carbinol on the phosphorylation of Akt, ERKs, JNK, p38, GSK3ß, and Bad, and the expression levels of Bax, Bcl-2, Bcl-xL, Mcl-1, survivin, NF-{kappa}B, cyclin D1, p27, and p21 in PC-3 cells. Cells were treated with the indicated concentrations of indole-3-carbinol or OSU-A9 in 5% FBS–supplemented RPMI 1640 for 48 h, and cell lysates were immunoblotted as described in Materials and Methods. The values in percentage or fold denote the relative intensity of protein bands of drug-treated samples to that of the respective DMSO vehicle–treated control after being normalized to the respective internal reference (total respective protein or ß-actin). Each value represents the average of two independent experiments.

 
Despite a 100-fold difference in antitumor potency, the pharmacologic profiles of OSU-A9 and indole-3-carbinol in interfering with these target proteins were virtually identical. As shown, both agents facilitated dose-dependent dephosphorylation of Akt and its substrates GSK3ß and Bad, accompanied by increased phosphorylation of ERKs, JNK, and p38. Moreover, the dose-dependent effects of OSU-A9 on suppressing the expression of Bcl-2, Bcl-xL, Mcl-1, survivin, NF-{kappa}B, and cyclin D1, and on up-regulating the expression of Bax, p27, and p21 paralleled those of indole-3-carbinol.

Together, these data suggest that OSU-A9 retains the pleiotropic effects of indole-3-carbinol with regard to the activating apoptosis machinery by targeting multiple signaling pathways. Pursuant to these findings, the in vivo antitumor potential of OSU-A9 was further assessed in a PC-3 xenograft animal model.

OSU-A9 suppresses PC-3 tumor xenograft growth in vivo. The maximum tolerated dose of OSU-A9 in athymic nude mice was determined by i.p. injection at 5, 10, 25, and 50 mg/kg/d (n = 3) continuously for 14 days (data not shown). No more than 10% weight loss was noted at doses up to 25 mg/kg (Fig. 6A, inset ).


Figure 6
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  		Figure 6. Effects of OSU-A9 at 10 or 25 mg/kg/d, administered i.p., on the growth of established PC-3 tumors in nude mice and the expression of intratumoral biomarkers of drug activity. Subcutaneous PC-3 tumor xenografts were established and treatments were administered as described in Materials and Methods. A, mean tumor volumes for each treatment group as a function of day of treatment. Inset, mean body weights for each treatment group as a function of day of treatment. Points, mean tumor volume; bars, SE (n = 7). B, Western blot analysis of intratumoral biomarkers of drug activity in the homogenates of three representative PC-3 tumors from each treatment group and their respective final volumes. C, relative expression levels of p-Akt, p-p38, Bcl-xL, and RelA. Amounts of immunoblotted proteins from three representative tumor lysates from each treatment group were quantitated by densitometry and normalized to that of ß-actin. Columns, mean (n = 3); bars, SD. D, diagram depicting the pleiotropic effects of OSU-A9 on multiple signaling targets that regulate cell cycle and apoptosis at multiple levels, including transcriptional activation of gene expression, cell cycle checkpoint control, intracellular kinase signaling, mitochondrial integrity, and caspase activation.

 
To examine the in vivo antitumor efficacy of OSU-A9, athymic nude mice bearing established s.c. PC-3 tumor xenografts (mean tumor volume, 109 ± 10 mm3) were treated i.p. for 42 days with OSU-A9 at 10 or 25 mg/kg daily, or with vehicle. As shown in Fig. 6A, treatment of mice with 10 and 25 mg/kg of OSU-A9 significantly inhibited PC-3 tumor growth by 66% and 85%, respectively, relative to vehicle-treated controls (P < 0.01). Importantly, mice seemed to tolerate treatment with OSU-A9 without overt signs of toxicity, without significant loss of body weight compared with vehicle-treated controls (Fig. 6A, inset), and with normal hematologic and serum chemistry findings. The sole lesions observed at necropsy in all OSU-A9–treated mice were mild to moderate amounts of fibrous adhesions that were limited to the peritoneal surfaces of tissues, including the colon, seminal vesicles, and body wall, and did not seem to compromise function. Microscopically, there were no treatment-associated lesions in the array of tissues examined in the OSU-A9–treated mice, with the exception of the affected peritoneum, which was variably expanded by mixtures of granulation and fibrous tissue and activated macrophages. No lesions were observed in the vehicle-treated controls.

To correlate this in vivo tumor-suppressive response to mechanisms identified in vitro, the effects of OSU-A9 on six representative intratumoral biomarkers of drug activity were evaluated by immunoblotting of PC-3 tumor homogenates collected after 42 days of treatment. These biomarkers included the phosphorylation status of Akt and p38, and expression levels of Bcl-xL, and NF-{kappa}B/RelA. As shown in Fig. 6B and C, the effects of OSU-A9 on these biomarkers were qualitatively similar to those observed in vitro, and reflect the dose-dependent tumor suppression in vivo. Treatment with OSU-A9 i.p. at 10 and 25 mg/kg/d induced marked reductions in intratumoral levels of p-Akt (83 ± 8% and 94 ± 1% reductions, respectively, compared with vehicle-treated controls), Bcl-xL (34 ± 8% and 86 ± 4%, respectively), and RelA (60 ± 7% and 77 ± 7%, respectively), accompanied by increases in intratumoral levels of p-p38 (9.0 ± 3.5-fold and 10.1 ± 2.4-fold, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the course of tumor progression, cancer cells constitutively up-regulate cell proliferation– and cell survival–regulatory signaling mechanisms, thereby overcoming genomic instability and/or acquiring a drug-resistant phenotype. From a clinical perspective, it is desirable to concomitantly target these molecular abnormalities by using a combination therapy or an agent with pleiotropic effects to optimize therapeutic outcomes (38). This rationale constitutes the molecular basis for structurally optimizing indole-3-carbinol to develop potent antitumor agents. This effort has culminated in the generation of OSU-A9, which provides considerable therapeutic advantages over indole-3-carbinol with respect to chemical stability and antitumor potency.

The introduction of a (3-chloro-2-nitrobenzene)sulfonyl substituent onto the indole nucleus endowed OSU-A9 with resistance to acid-catalyzed dehydration, and, equally important, a 100-fold higher apoptosis-inducing potency. The unique stereoelectronic property of this substituent allowed OSU-A9 to interact more effectively with target proteins compared with indole-3-carbinol. Meanwhile, this structural modification might also affect the kinetic behavior of drug absorption in different cell lines, as suggested by a crossover in the relative sensitivity to OSU-A9 versus indole-3-carbinol between PC-3 and LNCaP cells; that is, LNCaP cells were more sensitive to indole-3-carbinol–induced apoptosis than PC-3 cells, whereas a reversal in the relative susceptibility was noted with OSU-A9.

Relative to indole-3-carbinol, OSU-A9 displayed a striking similarity in the effects on modulating the phosphorylation or expression of a multitude of molecular targets, including Akt and its downstream effectors GSK3ß and Bad; the MAP kinases ERKs, p38, and JNK; the Bcl-2 family members Bax, Bcl-2, Bcl-xL, and Mcl-1; the inhibitor of apoptosis protein survivin; NF-{kappa}B; cyclin D1; and the CDK inhibitors p21 and p27 (Fig. 6D). Among these targets, the ability of OSU-A9 and indole-3-carbinol to facilitate Akt dephosphorylation in conjunction with increased phosphorylation of ERKs, JNK, and p38 is mechanistically intriguing. This finding is reminiscent to that reported for a number of molecules, including thrombin (39), sphingosine 1-phosphate (40), and kainate (41), in different cell systems. Moreover, one study with thrombin in human endothelial cells suggests a mechanistic link between the activation of Rho/Rho-kinase signaling and drug-induced divergent effects on the phosphorylation status of Akt and MAP kinases (39), which is currently under investigation in OSU-A9–treated prostate cancer cells.

Together, the aforementioned signaling kinases/proteins regulate cell cycle and apoptosis at multiple levels, including transcriptional activation of gene expression, cell cycle checkpoint control, intracellular kinase signaling, mitochondrial integrity, and caspase activation, all of which are clinically relevant to the tumorigenesis and progression of prostate cancer. This broad range of antitumor activities underscores the in vitro and in vivo efficacy of OSU-A9 in prostate cancer cells. It is especially noteworthy that, despite this complicated mode of drug action, normal prostate epithelial cells were less susceptible to the antiproliferative effect of OSU-A9, reflecting the in vivo tolerance of this drug in tumor-bearing nude mice.

Assessment of the in vivo efficacy in tumor-bearing nude mice indicate that daily i.p. injection of OSU-A9 at 10 and 25 mg/kg/d for 42 days resulted in a 65% and 85%, respectively, suppression of established PC-3 xenograft tumor growth. Western blot analysis of the tumor lysates revealed that the extent of changes in the six representative biomarkers paralleled the dose-dependent tumor-suppressive activity of OSU-A9. The concerted action on these molecular targets underscores the therapeutic potential of OSU-A9 to be developed into a potent antitumor agent not only as a single agent, but also potentially in combination with other chemotherapeutic drugs. Previously, indole-3-carbinol was shown to sensitize prostate and breast cancer cells to cisplatin (19) and tamoxifen (42), respectively. Use of OSU-A9 as a chemosensitizing agent in prostate cancer cells is currently under way in this laboratory.

OSU-A9 was well tolerated by tumor-bearing mice after 6 weeks of repeat dosing. The absence of gross and microscopic lesions in major organs together with normal clinical and hematologic findings indicated that the intra-abdominal fibrous adhesions observed in the OSU-A9–treated animals were likely nonspecific reactions associated with chronic irritation induced by i.p. injection of the drug. Although concerns regarding potential toxicities arising from the use of drugs with pleiotropic actions warrant consideration, our findings suggest that, at least in the case of OSU-A9, such adverse effects are not an obligatory consequence.

Although indole-3-carbinol is considered as a chemopreventive compound, the studies described here were focused on the therapeutic efficacy of OSU-A9. Nevertheless, in light of the molecular heterogeneity of premalignant lesions and the multifactorial nature of carcinogenesis (43, 44), the pleiotropic effects of OSU-A9 on multiple signaling pathways, along with its enhanced chemical stability and lack of evident toxicity, suggest its potential in the context of chemoprevention. Using prostatic intraepithelial neoplasia (PIN) as a clinically relevant intervention point (45), we have evaluated, and continue to evaluate, the chemopreventive efficacy of other compounds developed in our laboratory in the transgenic adenocarcinoma of the mouse prostate model (46) in which the predictable occurrence of PIN lesions is a component of the multistage tumor progression of the model (47). Dysplastic lesions similar to PIN precede the occurrence of tumors in other models as well, such as the hormone-induced model of prostate carcinogenesis in the Noble rat, in which PIN was shown to be a suitable intermediate end point for chemoprevention studies (48). The chemopreventive efficacy of OSU-A9 clearly warrants investigation and studies to do so are planned.

In conclusion, our results show that the novel indole-3-carbinol–derived multitargeted agent, OSU-A9, is a potent antitumor agent that modulates multiple aspects of cancer cell cycle regulation and survival, including intracellular kinase signaling, cell cycle checkpoint control, mitochondrial integrity, and caspase activation. This broad spectrum of antitumor activities in conjunction with low toxicity underlies the translational potential of OSU-A9 and suggests its viability as part of a therapeutic strategy for prostate cancer.


   Acknowledgments
 
Grant support: National Cancer Institute grant CA112250, grants from William R. Hearst Foundation and Prostate Cancer Foundation, and the Lucius A. Wing Endowed Chair Fund at the Ohio State University (C-S. Chen); National Science Council grants NSC-93018P and NSC-94-2314B-039-033 and financial support from China Medical University CMU94-180 (J-R. Weng) in Taiwan.

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


   Footnotes
 
4 http://www.schrodinger.com Back

Received 2/27/07. Revised 5/ 7/07. Accepted 6/ 6/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beier RC. Natural pesticides and bioactive components in foods. Rev Environ Contam Toxicol 1990;113:47–137.[Medline]
  2. He YH, Friesen MD, Ruch RJ, et al. Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats. Food Chem Toxicol 2000;38:15–23.[CrossRef][Medline]
  3. Jin L, Qi M, Chen DZ, et al. Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice. Cancer Res 1999;59:3991–7.[Abstract/Free Full Text]
  4. Oganesian A, Hendricks JD, Williams DE. Long term dietary indole-3-carbinol inhibits diethylnitrosamine-initiated hepatocarcinogenesis in the infant mouse model. Cancer Lett 1997;118:87–94.[CrossRef][Medline]
  5. Bradlow HL, Michnovicz J, Telang NT, et al. Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 1991;12:1571–4.[Abstract/Free Full Text]
  6. Grubbs CJ, Steele VE, Casebolt T, et al. Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol. Anticancer Res 1995;15:709–16.[Medline]
  7. Kojima T, Tanaka T, Mori H. Chemoprevention of spontaneous endometrial cancer in female Donryu rats by dietary indole-3-carbinol. Cancer Res 1994;54:1446–9.[Abstract/Free Full Text]
  8. Bell MC, Crowley-Nowick P, Bradlow HL, et al. Placebo-controlled trial of indole-3-carbinol in the treatment of CIN. Gynecol Oncol 2000;78:123–9.[CrossRef][Medline]
  9. Reed GA, Arneson DW, Putnam WC, et al. Single-dose and multiple-dose administration of indole-3-carbinol to women: pharmacokinetics based on 3,3'-diindolylmethane. Cancer Epidemiol Biomarkers Prev 2006;15:2477–81.[Abstract/Free Full Text]
  10. Reed GA, Peterson KS, Smith HJ, et al. A phase I study of indole-3-carbinol in women: tolerability and effects. Cancer Epidemiol Biomarkers Prev 2005;14:1953–60.[Abstract/Free Full Text]
  11. Naik R, Nixon S, Lopes A, et al. A randomized phase II trial of indole-3-carbinol in the treatment of vulvar intraepithelial neoplasia. Int J Gynecol Cancer 2006;16:786–90.[CrossRef][Medline]
  12. De Kruif CA, Marsman JW, Venekamp JC, et al. Structure elucidation of acid reaction products of indole-3-carbinol: detection in vivo and enzyme induction in vitro. Chem Biol Interact 1991;80:303–15.[CrossRef][Medline]
  13. Grose KR, Bjeldanes LF. Oligomerization of indole-3-carbinol in aqueous acid. Chem Res Toxicol 1992;5:188–93.[CrossRef][Medline]
  14. Chinni SR, Li Y, Upadhyay S, et al. Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene 2001;20:2927–36.[CrossRef][Medline]
  15. Chinni SR, Sarkar FH. Akt inactivation is a key event in indole-3-carbinol-induced apoptosis in PC-3 cells. Clin Cancer Res 2002;8:1228–36.[Abstract/Free Full Text]
  16. Nachshon-Kedmi M, Yannai S, Haj A, et al. Indole-3-carbinol and 3,3'-diindolylmethane induce apoptosis in human prostate cancer cells. Food Chem Toxicol 2003;41:745–52.[CrossRef][Medline]
  17. Zhang J, Hsu BAJ, Kinseth BAM, et al. Indole-3-carbinol induces a G1 cell cycle arrest and inhibits prostate-specific antigen production in human LNCaP prostate carcinoma cells. Cancer 2003;98:2511–20.[CrossRef][Medline]
  18. Kassouf W, Chintharlapalli S, Abdelrahim M, et al. Inhibition of bladder tumor growth by 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes: a new class of peroxisome proliferator-activated receptor {gamma} agonists. Cancer Res 2006;66:412–8.[Abstract/Free Full Text]
  19. Sarkar FH, Li Y. Indole-3-carbinol and prostate cancer. J Nutr 2004;134:3493–8S.
  20. Aggarwal BB, Ichikawa H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle 2005;4:1201–15.[Medline]
  21. Kim YS, Milner JA. Targets for indole-3-carbinol in cancer prevention. J Nutr Biochem 2005;16:65–73.[CrossRef][Medline]
  22. Manson MM. Inhibition of survival signalling by dietary polyphenols and indole-3-carbinol. Eur J Cancer 2005;41:1842–53.[CrossRef][Medline]
  23. Rogan EG. The natural chemopreventive compound indole-3-carbinol: state of the science. In Vivo 2006;20:221–8.[Abstract/Free Full Text]
  24. Howells LM, Hudson EA, Manson MM. Inhibition of phosphatidylinositol 3-kinase/protein kinase B signaling is not sufficient to account for indole-3-carbinol-induced apoptosis in some breast and prostate tumor cells. Clin Cancer Res 2005;11:8521–7.[Abstract/Free Full Text]
  25. Chintharlapalli S, Smith R III, Samudio I, et al. 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induce peroxisome proliferator-activated receptor {gamma}-mediated growth inhibition, transactivation, and differentiation markers in colon cancer cells. Cancer Res 2004;64:5994–6001.[Abstract/Free Full Text]
  26. Qin C, Morrow D, Stewart J, et al. A new class of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists that inhibit growth of breast cancer cells: 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes. Mol Cancer Ther 2004;3:247–60.[Abstract/Free Full Text]
  27. Chintharlapalli S, Papineni S, Baek SJ, et al. 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes are peroxisome proliferator-activated receptor {gamma} agonists but decrease HCT-116 colon cancer cell survival through receptor-independent activation of early growth response-1 and nonsteroidal anti-inflammatory drug-activated gene-1. Mol Pharmacol 2005;68:1782–92.[Abstract/Free Full Text]
  28. Contractor R, Samudio IJ, Estrov Z, et al. A novel ring-substituted diindolylmethane,1,1-bis[3'-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane, inhibits extracellular signal-regulated kinase activation and induces apoptosis in acute myelogenous leukemia. Cancer Res 2005;65:2890–8.[Abstract/Free Full Text]
  29. Jong L, Chao WR. Analogs of indole-3-carbinol metabolites as chemotherapeutic and chemopreventive agents. US patent 7078427. 2006.
  30. Jong L, Chao WR. Analogs of indole-3-carbinol metabolites as chemotherapeutic and chemopreventive agents. US Patent 6800655. 2004.
  31. Brandi G, Paiardini M, Cervasi B, et al. A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines. Cancer Res 2003;63:4028–36.[Abstract/Free Full Text]
  32. Shiau CW, Yang CC, Kulp SK, et al. Thiazolidenediones mediate apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 functions independently of PPAR{gamma}. Cancer Res 2005;65:1561–9.[Abstract/Free Full Text]
  33. Yang CC, Ku CY, Wei S, et al. Peroxisome proliferator-activated receptor {gamma}-independent repression of prostate-specific antigen expression by thiazolidinediones in prostate cancer cells. Mol Pharmacol 2006;69:1564–70.[Abstract/Free Full Text]
  34. Bregman CL, Adler RR, Morton DG, et al. Recommended tissue list for histopathologic examination in repeat-dose toxicity and carcinogenicity studies: a proposal of the Society of Toxicologic Pathology (STP). Toxicol Pathol 2003;31:252–3.[CrossRef][Medline]
  35. Suckow MA, Danneman P, Brayton C. Important biological features. In: Suckow MA, editor. The laboratory mouse. Boca Raton: CRC Press; 2001. p. 1–12.
  36. Henke BR, Adkison KK, Blanchard SG, et al. Synthesis and biological activity of a novel series of indole-derived PPAR{gamma} agonists. Bioorg Med Chem Lett 1999;9:3329–34.[CrossRef][Medline]
  37. Dropinski JF, Akiyama T, Einstein M, et al. Synthesis and biological activities of novel aryl indole-2-carboxylic acid analogs as PPAR{gamma} partial agonists. Bioorg Med Chem Lett 2005;15:5035–8.[CrossRef][Medline]
  38. Frantz S. Drug discovery: playing dirty. Nature 2005;437:942–3.[CrossRef][Medline]
  39. Eto M, Kozai T, Cosentino F, et al. Statin prevents tissue factor expression in human endothelial cells. Role of Rho/Rho-kinase and Akt pathways. Circulation 2002;105:1756–9.[Abstract/Free Full Text]
  40. Yamashita H, Kitayama J, Shida D, et al. Sphingosine 1-phosphate receptor expression profile in human gastric cancer cells: differential regulation on the migration and proliferation. J Surg Res 2006;130:80–7.[CrossRef][Medline]
  41. Crespo-Biel N, Canudas AM, Camins A, et al. Kainate induces AKT, ERK and cdk5/GSK3ß pathway deregulation, phosphorylates{tau}protein in mouse hippocampus. Neurochem Int 2007;50:435–42.[CrossRef][Medline]
  42. Cover CM, Hsieh SJ, Cram EJ, et al. Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells. Cancer Res 1999;59:1244–51.[Abstract/Free Full Text]
  43. AACR Chemoprevention Working Group. Prevention of cancer in the next millennium: report of the chemoprevention working group to the American Association for Cancer Research. Cancer Res 1999;59:4743–58.[Free Full Text]
  44. Lippman SM, Hong WK. Cancer prevention science and practice. Cancer Res 2002;62:5119–25.[Free Full Text]
  45. O'Shaughnessy JA, Kelloff GJ, Gordon GB, et al. Treatment and prevention of intraepithelial neoplasia: an important target for accelerated new agent development. Clin Cancer Res 2002;8:314–46.[Abstract/Free Full Text]
  46. Sargeant A, Klein R, Rengel R, et al. Chemotherapeutic and bioenergetic signaling effects of PDK-1/Akt pathway inhibition in a transgenic mouse model of prostate cancer. Toxicol Pathol 2007;35:549–61.[CrossRef][Medline]
  47. Klein RD. The use of genetically engineered mouse models of prostate cancer for nutrition and cancer chemoprevention research. Mutat Res 2005;576:111–9.[Medline]
  48. Christov KT, Moon RC, Lantvit DD, et al. Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies. Eur J Cancer 2004;40:1404–11.[CrossRef][Medline]



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