Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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Cancer Research 67, 3229-3238, April 1, 2007. doi: 10.1158/0008-5472.CAN-06-2759
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Peroxisome Proliferator-Activated Receptor {gamma}–Independent Suppression of Androgen Receptor Expression by Troglitazone Mechanism and Pharmacologic Exploitation

Chih-Cheng Yang1, Yu-Chieh Wang1, Shuo Wei1, Li-Fang Lin1, Chang-Shi Chen1, Cheng-Chun Lee2, Cheng-Chieh Lin2 and Ching-Shih Chen2

1 Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio and 2 China Medical University Hospital, Taichung, Taiwan

Requests for reprints: Ching-Shih Chen, Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Parks Halls, 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
 
Previously, we showed that the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist troglitazone at high doses was able to suppress androgen receptor (AR) expression in LNCaP prostate cancer cells independently of PPAR{gamma}. Pharmacologic exploitation of this finding led to STG28, a PPAR{gamma}-inactive analogue of troglitazone with substantially higher potency in AR repression. Considering the pivotal role of AR in prostate tumorigenesis, this study investigates the mechanism by which troglitazone and derivatives suppress AR expression in LNCaP cells. Reverse transcription-PCR and reporter gene assays indicate that this drug-induced AR repression occurs at both mRNA and protein levels. Evidence suggests that troglitazone and derivatives mediate the transcriptional repression of AR by facilitating the ubiquitin-dependent proteasomal degradation of the transcriptional factor Sp1. These agents also cause the proteolysis of two proteins that regulate Sp1-mediated transcription (i.e., the TATA-binding protein–associated factor TAFII250 and cyclin D1). However, their involvement in the transcriptional repression of AR is refuted by the finding that small interfering RNA knockdown of these two regulatory proteins does not cause AR down-regulation. STG28 does not cause significant reduction in Sp1 or AR expression in normal prostate epithelial cells. This discriminatory effect underscores the differential susceptibility of malignant versus normal cells to the inhibitory effect of STG28 on cell viability. From a translational perspective, STG28 provides a proof of principle that potent AR-ablative agents could be developed through structural modifications of troglitazone. Moreover, as the control of Sp1 degradation remains unclear, STG28 represents a unique pharmacologic probe to investigate the ubiquitin-proteasome system that regulates Sp1 proteolysis. [Cancer Res 2007;67(7):3229–38]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A major challenge in the management of patients with prostate cancer is the treatment of hormone-refractory tumors (HRPC), a hallmark of incurable and lethal prostate cancer progression. To date, chemotherapeutic regimens provide substantive benefits through palliation but yield no definitive enhancements in survival. A clear need exists for novel strategies that will improve the treatment of prostate cancer and ultimately increase the survival of prostate cancer patients. Recent advances have identified key pathways that contribute to the development of HRPC, among which androgen receptor (AR) gene amplification and mutations play a crucial role in facilitating androgen-refractory progression (1–7). These molecular defects enhance AR sensitivity and permit AR activation by antiandrogens, respectively, thereby allowing prostate cancer cells to acquire a resistant phenotype to androgen withdrawal-induced apoptosis (8, 9).

Considering the clinical relevance of AR in androgen-refractory prostate cancer, identification of small-molecule agents that interfere with AR expression/function has been the focus of many investigations (3, 10–14). Previously, data from this and other laboratories indicate that the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonist troglitazone might interfere with AR function through two PPAR{gamma}-independent mechanisms (15, 16). At concentrations ~10 µmol/L, troglitazone and its PPAR{gamma}-inactive derivative {Delta}2TG repressed prostate-specific antigen (PSA) through the inhibition of AR recruitment to the PSA promoter. At substantially higher concentrations (≥40 µmol/L), troglitazone and {Delta}2TG were able to suppress AR expression through a yet unidentified mechanism (16). From a therapeutic perspective, separation of AR repression from PPAR{gamma} agonist activity provides a molecular rationale for the pharmacologic exploitation of {Delta}2TG to develop AR-ablative agent, of which the proof of principle was shown by STG28, a structurally optimized derivative. Here, we report that troglitazone, {Delta}2TG, and STG28 inhibit AR expression at the transcriptional level by facilitating the proteasomal degradation of the transcription factor Sp1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Troglitazone and the proteasome inhibitor MG132 were purchased from Sigma (St. Louis, MO). {Delta}2TG {5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methoxy-benzylidene]-2,4-thiazolidine-dione} and STG28 {5-[(4-{[(S)-6-(allyloxy)-3,4-dihydro-2,5,7,8-tetramethyl-2H-chromen-2yl]-methoxy}-3-methoxyphenyl)-methylene]-thiazolidine-2,4-dione} were synthesized according to a procedure reported elsewhere (17). The identity and purity (≥99%) of these synthetic agents were verified by nuclear magnetic resonance, high-resolution mass spectrometry, and elemental analysis. These agents were dissolved in DMSO and added to cells in medium at various concentrations with a final DMSO concentration of 0.1%. Mouse antibodies against AR, PSA, {alpha}-tubulin, cyclin D1, and p300 and rabbit antibodies against Sp1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against poly(ADP-ribose) polymerase (PARP) and cyclic AMP (cAMP)-responsive element binding protein (CREB) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The proteasome inhibitor epoxomicin was a kind gift from Dr. Kyung Bo Kim (University of Kentucky, Lexington, KY). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugates and rabbit anti-mouse IgG-HRP conjugates were from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cell culture. LNCaP androgen-dependent (p53+/+) and PC3 androgen-nonresponsive (p53–/–) prostate cancer cells were purchased from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS). Normal prostate epithelial cells (PrEC) were obtained from Cambrex Bioscience-Walkersville, Inc. (Walkersville, MD) 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. Cells in log-phase growth were harvested by trypsinization for use in viability assays.

Cell viability assay. Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in six replicates (96-well format). Cancer cells and PrECs were seeded at 2,500 to 3,000 and 8,000 per well, respectively, in 96-well flat-bottomed plates and incubated for 24 h in 10% FBS-supplemented RPMI 1640 or 10% FBS-supplemented prostate epithelial growth medium. The cells were then treated with STG28 at the indicated concentrations. Controls received DMSO at a concentration equal to that in drug-treated cells. After 48 h, one-tenth volume of 10x MTT (5 mg/mL) was added to each well and cells were incubated at 37°C for 2 h. Medium was removed and the reduced MTT dye was solubilized in 200 µL/well DMSO. Absorbance was determined at 570 nm.

Cell cycle analysis. LNCaP cells were exposed to STG28 at indicated concentrations in 10% FBS-supplemented RPMI 1640 for 48 h, collected by trypsinization, washed twice with PBS, and then fixed in ice-cold 40% ethanol at –20°C overnight. Cells were then centrifuged for 5 min at 400 x g at room temperature, and after decanting the ethanol without disturbing the pellet, the cells were stained with propidium iodide (50 µg/mL) and RNase A (100 units/mL) in PBS. Cell cycle phase distributions were determined on a FACScort flow cytometer and analyzed by the ModFitLT V3.0 program.

RNA isolation and reverse transcription-PCR analysis. LNCaP cells were subjected to total RNA isolation by using an RNeasy mini kit (Qiagen, Valencia, CA). RNA concentrations were determined by measuring absorption at 260 nm in a spectrophotometer. Aliquots of 6 µg of total RNA from each sample were reverse transcribed to cDNA using an Omniscript RT kit (Qiagen) according to the manufacturer's instructions. PCR primers used were as follows: AR, 5'-ACACATTGAAGGCTATGAATGTC-3' and 5'-TCACTGGGTGTGGAAATAGATGGG-3'; human protease-activated receptor-1 (hPar-1), 5'-GCCAGAATCAAAAGCAACAA-3' and 5'-GAGATGAATGCAGGAAGTTGTTT-3'; fibroblast growth factor 8 (FGF8), 5'-AAAGGCAAGGACTGCGTCTTCACG-3' and 5'-CGTGAAGGGCGGGTAGTTGAG-3'; and ß-actin, 5'-TCTACAATGAGCTGCGTGTG-3' and 5'-GGTCAGGATCTTCATGAGGT-3'. PCR primers for Sp1, epidermal growth factor receptor (EGFR), and TAFII250 were purchased from Santa Cruz Biotechnology. PCR products were separated electrophoretically by 1.2% agarose gel and visualized by ethidium bromide staining.

Transfection and luciferase assay. The 3.6-kb AR promoter-linked reporter plasmid p-3600ARCAT was kindly provided by Dr. Chawnshang Chang (University of Rochester Medical Center, Rochester, NY). The AR promoter gene (–3,600 to +550) encompassing the transcription start site was isolated by using PCR to generate hAR-luc with the following primers: 5'-TACAGGTACCGGTATCTCGACCTGCAGGTC-3' and 5'-TGTTAGATCTTGCTGAAGCCGCTCCCCAGT-3'. The fragment was subcloned into the pGL3 luciferase reporter vector (Promega, Madison, WI) at KpnI and BglII in the multiple cloning site. The PPRE-x3-TK-Luc reporter vector contains three copies of the PPAR response element (PPRE) upstream of the thymidine kinase promoter-luciferase fusion gene and was kindly provided by Dr. Bruce Spiegelman (Harvard University, Cambridge, MA). The pCMVSp1 plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD). LNCaP or PC3 cells were transfected with 5 µg of individual plasmids in an Amaxa Nucleofector using a cell line–specific nucleofector kit according to the manufacturer's protocol (Amaxa Biosystems, Cologne, Germany) and then seeded in six-well plates at 5 x 105 per well for 48 h. The transfection efficiency was determined to be 70% to 80% by transfecting cells with 2 µg of pmaxGFP plasmid followed by fluorescence microscopy to measure green fluorescent protein expression. For each transfection, herpes simplex virus (HSV) thymidine kinase promoter-driven Renilla reniformis luciferase was used as an internal control for normalization.

For the reporter gene assay, after transfection, cells were cultured in 24-well plates in 10% FBS-supplemented RPMI 1640 for 48 h, subjected to different treatments for the indicated times, collected, and lysed with passive lysis buffer (Promega). Aliquots of the lysates (50 µL) were added to 96-well plates, and luciferase activity was monitored after adding 100 µL of luciferase substrate (Promega) each well by using a MicroLumatPlus LB96V luminometer (Berthold Technologies, Oak Ridge, TN) with the WinGlow software package. All transfection experiments were carried out in six replicates.

Immunoblotting. Cells in T-75 flasks were collected by scraping, and cell lysates were prepared using M-PER lysis buffer (Pierce, Rockford, IL) in the presence of a protease inhibitor cocktail (Calbiochem, San Diego, CA) consisting of 100 mmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride, 80 nmol/L aprotinin, 5 mmol/L bestatin, 1.5 mmol/L E-64 protease inhibitor, 2 mmol/L leupeptin, and 1 mmol/L pepstatin A. After centrifugation for 20 min, 2 µL of the suspension were taken for protein determination using a Bradford assay kit (Bio-Rad, Hercules, CA). To the remaining solution was added the same volume of 2x SDS-PAGE sample loading buffer [100 mmol/L Tris-HCl (pH 6.8), 4% SDS, 5% ß-mercaptoethanol, 20% glycerol, 0.1% bromphenol blue] and boiled for 5 min. Equal amounts of proteins were resolved in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes using a semidry transfer cell. The transblotted membrane was washed twice with TBS containing 0.1% Tween 20 (TBST). After blocking with TBST containing 5% nonfat milk for 40 min, the membrane was incubated with the appropriate primary antibody in TBST-1% nonfat milk at 4°C overnight. All primary antibodies were diluted 1:1,000 in 1% nonfat milk–containing TBST. After treatment with the primary antibody, the membrane was washed thrice with TBST for a total of 15 min followed by incubation with goat anti-rabbit or anti-mouse IgG-HRP conjugates (diluted 1:5,000) for 1 h at room temperature and three washes with TBST for a total of 1 h. The immunoblots were visualized by enhanced chemiluminescence.

Small interfering RNA transfection. Small interfering RNA (siRNA) duplexes for TAFII250, Sp1, cyclin D1, and scrambled siRNA were purchased from Santa Cruz Biotechnology. LNCaP cells were cultured in six-well plates and transfected with 6 µL of transfection reagent (Santa Cruz Biotechnology) and the indicated amount of siRNA according to the manufacturer's instructions.

Immunoprecipitation. LNCaP cells were transfected with 5 µg of hemagglutinin (HA)-ubiquitin plasmid (kindly provided by Dr. Hung-Wen Chen, Academia Sinica, Taipei, Taiwan) in an Amaxa Nucleofector using a LNCaP-specific nucleofector kit according to the manufacturer's protocol and then seeded in six-well plates at 5 x 105 per well. After 48-h incubation, cells were treated with 10 µmol/L STG28 alone or in combination with a proteasome inhibitor (MG132 or epoxomicin) for 12 h and lysed by radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology) with freshly added phosphatase and protease inhibitors consisting of 100 µmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride, 80 nmol/L aprotinin, 5 µmol/L bestatin, 1.5 µmol/L E-64 protease inhibitor, 2 µmol/L leupeptin, 1 µmol/L pepstatin A, 2 mmol/L imidazole, 1 mmol/L sodium fluoride, 1 mmol/L sodium molybdate, 1 mmol/L sodium orthovanadate, and 4 mmol/L sodium tartrate dihydrate. After centrifugation at 13,000 x g for 15 min, the supernatant was collected, preincubated with protein A-agarose (Santa Cruz Biotechnology) for 15 min, and centrifuged at 1,000 x g for 5 min. Supernatant (10 µL) was stored away at 4°C to be used as input, whereas the remainder was exposed to anti-Sp1 antibody-agarose conjugates at 4°C for 12 h. After brief centrifugation, immunoprecipitates were collected, washed with the aforementioned lysis buffer twice, suspended in 2x SDS sample buffer, and subjected to Western blot analysis with antibodies against HA (Roche, Indianapolis, IN) and Sp1 (Santa Cruz Biotechnology).

Immunocytochemical analysis. LNCaP cells were cultured on slides in six-well plates (500,000 per well) in 10% FBS-supplemented phenol red–free RPMI 1640, exposed to 10 µmol/L STG28 for different intervals, washed with Dulbecco's PBS, fixed with 4% paraformaldehyde for 30 min at 37°C, and then washed with PBS twice. For costaining of Sp1 and AR, cells were treated with mouse monoclonal anti-AR (1:250 dilution) in PBS containing 0.1% Triton X-100 and 0.2% bovine serum albumin at 4°C for 5 h, washed with PBS, and then treated with rabbit polyclonal anti-Sp1 (1:250 dilution) in PBS at 4°C overnight. For fluorescent microscopy, Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (1:400 dilution; Molecular Probes, Eugene, OR) were used for conjugating Sp1 and AR, respectively. The nuclear counterstaining was done using a 4',6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories, Burlingame, CA) before examination. Images of immunocytochemically labeled samples were observed using a Nikon (Melville, NY) microscope (Eclipse TE300).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pharmacologic exploitation of the PPAR{gamma}-independent effect of troglitazone on AR repression to develop AR-ablative agents. Our previous report indicates that troglitazone and its PPAR{gamma}-inactive analogue {Delta}2TG were able to suppress the expression of AR in LNCaP cells irrespective of their PPAR{gamma} activities (16). However, the concentration of either agent required for AR repression was substantially higher than that required for PSA down-regulation (40 versus 10 µmol/L), indicating that the mechanisms underlying the repression of AR and PSA differed. From a translational perspective, dissociation of these two pharmacologic activities (AR repression versus PPAR{gamma} activation) provided a molecular basis to use {Delta}2TG as a scaffold to develop novel AR-ablative agents. Accordingly, structure-activity analysis of a series of {Delta}2TG derivatives via Western blotting identified an optimal agent, STG28, which exhibited multifold higher potency than troglitazone and {Delta}2TG in AR repression in 10% FBS-supplemented medium (Fig. 1A ). Nevertheless, the potency in repressing PSA was comparable among these three agents. The PPRE-luciferase reporter assay indicates that STG28, like its parent compound {Delta}2TG, lacked appreciable activity in PPAR{gamma} transactivation (Fig. 1B).


Figure 1
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  		Figure 1. PPAR{gamma}-independent effect of troglitazone, {Delta}2TG, and STG28 on the suppression of AR expression in LNCaP cells and normal PrECs. A, structures of troglitazone, {Delta}2TG, and STG28 and their dose-dependent effects on AR and PSA protein expression in LNCaP cells. Cells were exposed to DMSO vehicle or individual agents at the indicated concentrations in 10% FBS-supplemented medium for 48 h, and the expression levels of AR and PSA were analyzed by Western blot analysis. The values in percentage 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 ß-actin. Each value represents the average of two independent experiments. B, dose-dependent effect of troglitazone, {Delta}2TG, and STG28 on PPAR{gamma} activation in PC3 cells. PC3 cells were transiently transfected with PPRE-x3-TK-Luc reporter vector and then exposed to individual test agents or DMSO vehicle in 10% FBS-supplemented RPMI 1640 for 48 h. Analysis of luciferase activity was carried out as described in Materials and Methods. Columns, mean (n = 6); bars, SD. C, top, time-dependent effects of 10 µmol/L STG28 on AR protein expression in PrECs. Cells were exposed to 10 µmol/L STG28 at the indicated time intervals, and the expression levels of AR were analyzed by Western blot analysis. Bottom, effect of STG28 on the viability of PrECs in comparison with LNCaP cells at the indicated concentrations in 10% FBS-supplemented prostate epithelial growth and RPMI 1640, respectively, in 96-well plates for 48 h, and cell viability was assessed by MTT assays. Points, mean (n = 6); bars, SD. D, flow cytometry analysis LNCaP cells after treatment with DMSO vehicle or the indicated concentrations of STG28 for 48 h. Each data point represents the mean of two independent determinations.

 
Relative to LNCaP cells, normal PrECs were resistant to the effect of STG28 on suppressing AR expression and cell viability (Fig. 1C). As shown, exposure of PrECs to 10 µmol/L STG28 did not cause appreciable reduction in AR expression level throughout the course of investigation (Fig. 1C, top). This discriminatory effect underscored the differential susceptibility of LNCaP cells versus PrECs to the inhibitory effect of STG28 on cell viability (Fig. 1C, bottom). For example, treatment of LNCaP cells and PrECs to 20 µmol/L STG28 in 10% FBS-supplemented medium for 48 h caused 18% and 51% cell death, respectively.

To investigate the role of cell cycle arrest versus apoptosis in STG28-mediated suppression of cell viability, cell cycle analysis was carried out after exposing LNCaP cells to different doses of STG28 for 48 h (Fig. 1D). As shown, STG28 caused G1 arrest at ≤5 µmol/L, accompanied by decreases in the S phase. Meanwhile, treatment with STG28 at ≥10 µmol/L led to a dose-dependent increase in apoptotic cells (sub-G1). These data suggest that both cell cycle arrest and apoptosis contributed to the antiproliferative effects of STG28.

Together, STG28 provides a proof of principle that the unique ability of troglitazone and {Delta}2TG in AR repression could be pharmacologically exploited to develop a novel class of AR-ablative agents.

Troglitazone and PPAR{gamma}-inactive analogues inhibit AR expression at the mRNA and protein levels in LNCaP cells. Two lines of evidence indicate that this decrease in AR protein contents was attributable to the transcriptional repression of AR gene expression. First, reverse transcription-PCR (RT-PCR) analysis of the mRNA transcript of AR gene in LNCaP cells showed a dose-dependent decrease after exposure to individual agents for 48 h (Fig. 2A ). Second, the AR promoter-luciferase reporter gene assay confirmed that these agents were able to inhibit AR gene transcription in a dose-dependent manner (Fig. 2B).


Figure 2
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  		Figure 2. Troglitazone, {Delta}2TG, and STG28 suppress AR expression at the mRNA level in LNCaP cells. A, RT-PCR analysis of the dose-dependent effects of troglitazone, {Delta}2TG, and STG28 on reducing AR mRNA. Cells were exposed to individual agents at the indicated concentrations in 10% FBS-supplemented medium for 48 h. RNA isolation and RT-PCR analysis were carried out according to that described in Materials and Methods. The values in percentage denote the relative intensity of mRNA bands of drug-treated samples to that of the respective DMSO vehicle-treated control after both being normalized to the respective internal reference ß-actin. Each value represents the average of two independent experiments. B, dose-dependent effect of troglitazone, {Delta}2TG, and TG28 on AR promoter reporter activity. LNCaP cells were transiently transfected with an AR promoter-linked luciferase reporter plasmid and exposed to individual agents at the indicated concentrations in 10% FBS-supplemented medium for 48 h. Analysis of luciferase activity was carried out as described in Materials and Methods. Columns, mean (n = 6); bars, SD. C, time-dependent effect of 10 µmol/L STG28 on suppressing the transcription of the AR downstream target genes EGFR, hPar-1, and FGF8. LNCaP cells were exposed to DMSO vehicles or 10 µmol/L STG28 for the indicated time. RNA isolation and RT-PCR analysis were carried out according to that described in Materials and Methods. The values in percentage denote the relative intensity of mRNA bands of drug-treated samples to that of the respective DMSO vehicle-treated control after both being normalized to the respective internal reference ß-actin. Each value represents the average of two independent experiments.

 
STG28 causes the transcriptional repression of AR downstream target genes. To validate the above finding, we examined the effect of STG28 on the transcriptional repression of AR downstream target genes, including those encoding EGFR, hPar-1, and FGF8. These genes contain the androgen response element in their promoter regions and are regulated by AR. It is well understood that these AR-regulated genes play a key role in prostate tumorigenesis and cancer progression (18–20). RT-PCR analysis indicates that exposure of LNCaP cells to STG28 in 10% FBS-supplemented medium led to a dose-dependent decrease in the mRNA levels of these genes (Fig. 2C).

Putative targets for the drug-induced transcriptional repression of the AR. We hypothesized that troglitazone and derivatives mediated the inhibition of AR mRNA expression by targeting the transcriptional regulators of the AR promoter, such as Sp1, CREB, and the TATA-binding protein–associated factor TAFII250 (21–24). In addition, our previous study showed that troglitazone and derivatives were able to repress cyclin D1 by activating the proteasome-facilitated proteolysis (25). Considering a possible role of cyclin D1 in regulating Sp1-mediated transcription through the interaction with TAFII250 (26), we examined the effect of troglitazone, {Delta}2TG, and STG28 on the protein level of these putative targets. Moreover, the effect on p300, an AR coactivator involved in the regulation of PSA expression (27), was tested to examine the specificity of the drug action.

As shown, troglitazone, {Delta}2TG, and STG28 caused a dose-dependent reduction in the proteins expression of cyclin D1, TAFII250, and Sp1 in LNCaP cells in 10% FBS-supplemented medium, whereas the levels of CREB and p300 remained unaffected (Fig. 3A ). Figure 3B depicts the time course of the effect of 10 µmol/L STG28 on the expression levels of this panel of proteins compared with that of AR (Fig. 3B, left). Among them, cyclin D1 exhibited the highest susceptibility to the drug-induced ablation, which was completely depleted in drug-treated cells within 24 h. In contrast, TAFII250, Sp1, and AR were degraded in a more gradual manner, and no decrease in the intracellular levels of CREB or p300 was noted.


Figure 3
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  		Figure 3. Differential effects of troglitazone, {Delta}2TG, and STG28 on the expression of various transcriptional regulators of the AR promoter. A, dose-dependent effects of troglitazone, {Delta}2TG, and STG28 on suppressing the expression of cyclin D1. TAFII250, Sp1, CREB, and p300 in LNCaP cells. Cells were exposed to individual agents at the indicated concentrations in 10% FBS-supplemented medium for 48 h, and the lysates were subjected to Western blot analysis. The values in percentage denote the relative intensity of protein bands of drug-treated samples to that of the respective DMSO vehicle-treated control after both being normalized to the internal reference ß-actin. Each value represents the average of two independent experiments. B, left, time course of the effects of STG28 on the repression of cyclin D1, TAFII250, Sp1, CREB, p300, and AR in LNCaP cells. Cells were exposed to 10 µmol/L STG28 in 10% FBS-supplemented medium at different intervals, and the lysates were subjected to Western blot analysis. Right, immunocytochemical analysis of the time-dependent effect of STG28 on the repression of Sp1 and AR in LNCaP cells. Cells were cultured on slides in six-well plates in 10% FBS-supplemented phenol red–free RPMI 1640, exposed to 10 µmol/L STG28 for different intervals, fixed, and subjected to immunochemical staining. The nuclear counterstaining was done using a DAPI-containing mounting medium. C, time course of the effects of STG28 on PARP cleavage in LNCaP cells. Cells were treated with 10 µmol/L STG28 in 10% FBS-supplemented RPMI 1640 for the indicated time, and cell lysates were subjected to Western blotting with anti-PARP. D, time course of the effect of 10 µmol/L STG28 on Sp1 expression in PrECs. Cells were treated with 10 µmol/L STG28 in prostate epithelial growth medium for the indicated time, and cell lysates were subjected to Western blotting with anti-Sp1.

 
In light of the direct involvement of Sp1 in the transcriptional regulation of AR, we examined the temporal and spatial effect of STG28 on the repression of Sp1 and AR by immunocytochemical analysis (Fig. 3B, right). In the presence of 10% FBS, Sp1 and AR were predominantly localized to the nucleus of LNCaP cells. Exposure to 10 µmol/L STG28 led to a gradual, time-dependent decline in both protein levels in a manner similar to that observed by Western blotting. Moreover, our data show that the STG28-mediated degradation of Sp1 preceded the process of apoptotic death. As shown, a 50% decrease in Sp1 expression was noted after 12 h of exposure to 10 µmol/L STG28 in 10% FBS-containing medium. In contrast, apoptosis did not take place until 24 h after treatment as indicated by PARP cleavage (Fig. 3C). This finding suggests that STG28-facilitated Sp1 down-regulation did not result from drug-induced apoptotic death.

It is noteworthy that STG28 had no significant effect on Sp1 expression in PrECs throughout a 48-h incubation (Fig. 3D), which is in line with that observed with AR expression.

In contrast to AR, RT-PCR analysis indicates that the mRNA levels of Sp1 and TAFII250 remained unaltered after exposure to troglitazone, {Delta}2TG, and STG28 for 48 h (Fig. 4A ), which suggests that the drug-induced repression of Sp1 and TAFII250 was mediated at the protein level. Pursuant to this finding, we examined the effect of two proteasome inhibitors, MG132 and epoxomicin, on STG28-facilitated suppression of TAFII250 and Sp1 expression. As shown in Fig. 4B, MG132 and epoxomicin were able to protect against the STG28-mediated ablation of these two proteins, underscoring a mechanistic link between the repressing effect of STG28 and proteasomal degradation.


Figure 4
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  		Figure 4. Evidence that STG28 represses Sp1 and TAFII250 by facilitating proteasomal degradation. A, dose-dependent effect of troglitazone, {Delta}2TG, and STG28 on the mRNA levels of Sp1 and TAFII250 LNCaP cells. Cells were treated with 10 µmol/L STG28 in 10% FBS-containing RPMI 1640 for 48 h. Total RNA was isolated and subjected to RT-PCR analysis as described in Materials and Methods. ß-Actin mRNA served as an internal standard. The values in percentage denote the relative intensity of mRNA bands of drug-treated samples to that of the respective DMSO vehicle-treated control after being normalized to the respective internal reference ß-actin. Each value represents the average of two independent experiments. B, protective effects of MG132 and epoxomicin on STG28-mediated Sp1 repression. LNCaP cells were treated with 10 µmol/L STG28 alone or in combination with the indicated concentrations of MG132 or epoxomicin in 10% FBS-supplemented medium for 24 h, and the expression levels of Sp1 and TAFII250 were analyzed by Western blotting. The values in percentage 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 internal reference ß-actin. Each value represents the average of two independent experiments. C, Sp1 ubiquitination in STG28-treated LNCaP cells. Cells were transiently transfected with HA-ubiquitin plasmids and treated with 10 µmol/L, alone or in combination, with the indicated concentrations of MG132 or epoxomicin in 10% FBS-supplemented medium for 12 h. Cell lysates were immunoblotted with anti-Sp1 antibodies (input) or immunoprecipitated (IP) with anti-Sp1-agarose conjugates, and the immunoprecipitates were analyzed by Western blotting (WB) with anti-Sp1 (left) or anti-HA (right) as described in Materials and Methods.

 
Because proteasome-facilitated proteolysis is preceded by ubiquitination, we examined the formation of ubiquinated Sp1 in STG28-treated LNCaP cells expressing ectopic HA-ubiquitin (Fig. 4C). Cells were exposed to 10 µmol/L STG28 in the presence of 1 or 5 µmol/L of epoxomicin for 12 h, and cell lysates were immunoblotted with Sp1 antibodies (input) or immunoprecipitated by anti-Sp1 antibody-agarose conjugates. Equivalent amounts of the immunoprecipitated proteins were subjected to Western blotting with Sp1 or HA antibodies. As shown, epoxomicin not only rescued Sp1 from STG28-mediated degradation (input) but also increased the extent of Sp1 ubiquitination as indicated by a complex ladder of ubiquinated Sp1 bands (IP, anti-Sp1; WB, anti-HA; Fig. 4C, right).

Sp1 siRNA mimics the effect of troglitazone and derivatives on AR repression in LNCaP cells. To discern the involvement of TAFII250, cyclin D1, and Sp1 in STG28-induced transcriptional repression of AR, we used siRNA-mediated knockdown of individual proteins to examine its consequent effect on AR down-regulation. As shown, knockdown of Sp1 expression reduced AR mRNA transcription after 24 h of exposure followed by a decrease in AR protein expression at 48 h (Fig. 5A ). It is also noteworthy that siRNA-mediated Sp1 knockdown was accompanied by a transient increase in TAFII250 expression.


Figure 5
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  		Figure 5. Effect of siRNA-mediated knockdown of Sp1 (A), cyclin D1 (B), and TAFII250 (C) on the suppression of AR mRNA and protein expression in LNCaP cells. LNCaP cells were transfected with the indicated doses of the sequence-specific duplex siRNA oligonucleotide as described in Materials and Methods. After transfection, LNCaP cells were incubated in 10% FBS-supplemented RPMI 1640 for additional 24 or 48 h, collected, and subjected to Western blot and RT-PCR analyses as indicated. The values in percentage denote the relative intensity of protein or mRNA bands of drug-treated samples to that of the respective DMSO vehicle-treated control after being normalized to the respective internal reference ß-actin. Each value represents the average of two independent experiments.

 
In contrast to Sp1, siRNA-induced knockdown of cyclin D1 and TAFII250 exhibited a differential effect on AR mRNA and protein expression (i.e., decrease in cyclin D1 expression caused a marked increase in AR expression; Fig. 5B and C), whereas that of TAFII250 induced a modest elevation in AR mRNA and protein levels. These findings argued against the involvement of these two proteins in STG28-mediated AR transcriptional repression.

Ectopic Sp1 expression confers resistance to STG28-mediated AR transcriptional repression. To validate the mechanistic link between Sp1 down-regulation and STG28-facilitated AR repression, we examined the effect of ectopic Sp1 expression on rescuing the drug-induced AR down-regulation. Transient transfection of LNCaP cells with the pCMVSp1 plasmid resulted in 1.8- and 2.8-fold higher expression levels of Sp1 and AR, respectively, than those of the pcDNA-transfected cells (Fig. 6A ). Treatment of both types of transfected cells with 10 µmol/L STG28 caused a 40% to 50% decrease in Sp1 expression (Fig. 6B). However, as the Sp1 level in drug-treated pCMVSp1-transfected cells still remained high after 48-h exposure, there was only a modest decrease in AR expression levels in pCMVSp1-transfected LNCaP cells compared with that of the control, indicating the protective effect of ectopic Sp1 expression on STG28-mediated AR repression.


Figure 6
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  		Figure 6. Ectopic expression of Sp1 increases AR expression, thereby attenuating the effect of STG28 on the suppression of Sp1 and AR expression and AR promoter activity. A, LNCaP cells were transfected with 5 µg of the pcDNA3.1 (a) or pCMVSp1 (b) plasmid and incubated in 10% FBS-supplemented RPMI 1640 for 48 h. Left, Western blot analysis of Sp1 and AR expression levels in transiently transfected cells; right, relative amounts of the expression levels of Sp1 and AR in pCMVSp1-transfected cells to those of pcDNA-transfected LNCaP cells. Columns, mean (n = 3); bars, SD. B, time-dependent effect of 10 µmol/L STG28 on the protein expression levels of Sp1 and AR in pCMVSp1-transfected versus pcDNA-transfected LNCaP cells. LNCaP cells were transiently transfected with individual plasmids, incubated for 48 h, and exposed to 10 µmol/L STG28 in 10% FBS-supplemented RPMI 1640 for the indicated intervals. Cell lysates were subjected to Western blot analysis with anti-Sp1 and anti-AR antibodies. The values in percentage 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 ß-actin. Each value represents the average of two independent experiments. C, effect of ectopic Sp1 expression on rescuing STG28-mediated suppression of AR promoter activity. LNCaP cells were cotransfected with pGLC3-luciferase reporter plasmid linked to AR promoter (AR-Luc) in combination with pCMV-Sp1 expression plasmid or the control vector pcDNA for 48. In each transfection, the HSV thymidine kinase promoter-driven R. reniformis luciferase was cotransfected to monitor transfection efficacy. The transfected cells were treated with DMSO vehicles or 10 µmol/L STG28 in 10% FBS-supplemented RPMI 1640 for 24 h. Cell lysates were subjected to Western blot analysis with anti-Sp1 antibodies (left) or analyzed for luciferase activity (right). The values in percentage denote the relative intensities of protein bands of drug-treated samples to that of the respective DMSO-treated control. Each value represents the average of three independent experiments. The luciferase activity was normalized to R. reniformis luciferase. Columns, mean (n = 3); bars, SD.

 
Pursuant to this finding, we examined the effect of ectopic Sp1 expression on rescuing the suppression of AR promoter-luciferase activity by STG28 (Fig. 6C). LNCaP cells were cotransfected with the AR promoter-luciferase plasmid in combination with the pCMVSp1 plasmid or the pcDNA plasmid (control) via nucleofection and exposed to STG28 (10 µmol/L) or DMSO vehicle in 10% FBS-supplemented medium for 24 h. Cell lysates were analyzed for Sp1 expression by Western blotting (Fig. 6C, left) or for luciferase activity (Fig. 6C, right). As shown, the activity of AR promoter in pCMVSp1-transfected cells was substantially higher than those of pcDNA-transfected cells due to ectopic Sp1 expression (labeled as DMSO). Although STG28 caused a 65% decrease in Sp1 expression in both types of transfected cells, there was still a significant higher level of Sp1 expression, associated with a higher AR promoter-luciferase activity (P < 0.001), in pCMVSp1-transfected cells than those of pcDNA-transfected cells. Together, these data confirm the pivotal role of Sp1 in STG28-mediated AR transcriptional repression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although PPAR{gamma} signaling plays a key role in regulating prostate epithelial differentiation and proliferation (28, 29), a growing body of evidence suggests that the antitumor effect of troglitazone might be dissociated from its original pharmacologic activity [i.e., PPAR{gamma} activation (16, 25, 30–39). In our laboratory, we have identified several "off-target" mechanisms that underlie the antiproliferative activities of troglitazone, including Bcl-2/Bcl-xL inhibition (39), proteasome-mediated proteolysis of cyclin D1 (25), and transcriptional repression of PSA (16). In the course of our investigation of the effect of troglitazone and {Delta}2TG on PSA repression, we found that these agents could also down-regulate AR expression at doses substantially higher than that required for PSA (16). Pursuant to this finding, the mechanism by which troglitazone and its PPAR{gamma}-inactive analogues suppressed AR expression represents the focus of this study. We obtained several lines of evidence that troglitazone and its PPAR{gamma}-inactive derivatives mediated transcriptional repression of AR by facilitating ubiquitin-dependent proteasomal degradation of Sp1. Although TAFII250 and cyclin D1 were also subjected to this drug-induced proteolysis, the siRNA data dispute the direct involvement of these regulatory proteins in STG28-induced AR repression.

Among the three thiazolidinediones examined, the relative potency of individual agents to mediate ubiquitin-dependent proteasomal degradation of Sp1 paralleled that of cyclin D1 (i.e., STG > {Delta}2TG > troglitazone). This finding suggests that the drug-induced proteolysis of Sp1 and cyclin D1 might be mediated by the same ubiquitin-proteasome system. However, how these small-molecule agents facilitate ubiquitination remains to be delineated. In the literature, the Skp1/Cul/F-box (SCF) ubiquitin ligase has been implicated in the degradation of cell cycle regulatory proteins, including cyclins and a series of cyclin-dependent kinase inhibitors (40). Moreover, recent reports indicate that the histone deacetylase inhibitor trichostatin A repressed cyclin D1 and estrogen receptor {alpha} expression by up-regulating Skp2, a F-box protein of the SCF complex (41, 42). In contrast, the control of Sp1 degradation is largely unknown. Based on the finding that Sp1 is rapidly degraded by proteasome in glucose-starved and cAMP-stimulated cells (43–45), it has been postulated that Sp1 degradation might be part of the cellular response to nutrient deprivation and stress (45). From a mechanistic perspective, troglitazone and derivatives represent the first class of pharmacologic agents that allow the probing of the intracellular process of the proteasomal degradation of Sp1. In light of the involvement of the SCF complex in this proteolysis of cyclin D1, the mechanistic link between this ubiquitin E3 ligase system and STG28-mediated Sp1 degradation is currently under investigation.

Also noteworthy is the translational relevance of these findings in developing therapeutic agents for prostate cancer treatment. In the literature, many natural product-based agents have been reported to suppress AR expression/function through different mechanisms, including resveratrol (10), vitamin E succinate (12), (–)-epigallocatechin-3-gallate (EGCG; ref. 11), genistein (46), quercetin (13, 47), and the curcumin derivative JC-15 (3). For example, quercetin attenuated AR function by causing changes in the post-translational modification of AR protein (13), whereas EGCG could decrease the expression and transactivation activity of Sp1 (11). The mode of action of troglitazone and derivatives in AR repression seems to differ from that of the natural product agents. Moreover, the differential ability of troglitazone, {Delta}2TG, and STG28 to facilitate proteasomal degradation of Sp1 provides a proof of principle to continue the structural optimization of STG28 to develop potent AR-ablative agents, which is currently under way.


   Acknowledgments
 
Grant support: National Cancer Institute Public Health Service grant CA112250, Department of Defense Prostate Cancer Research Program grant W81XWH-05-1-0089, William R. Hearst Foundation, and Prostate Cancer Foundation.

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.

We thank Dr. Bruce Spiegelman for providing the PPRE-x3-TK-Luc vector, Dr. Hung-Wen Chen for providing the HA-ubiquitin plasmid, and Dr. Kyung Bo Kim for providing the proteasome inhibitor epoxomicin.

Received 7/27/06. Revised 1/ 4/07. Accepted 1/30/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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