点击显示 收起
【关键词】 Methyl 2-Cyano-3
Methyl 2-cyano-3,11-dioxo-18β-olean-1,12-diene-30-oate (β-CDODA-Me) is a synthetic analog of the naturally occurring triterpenoid glycyrrhetinic acid, which contains a 2-cyano substituent in the A-ring. β-CDODA-Me was a potent inhibitor of LNCaP prostate cancer cell growth (IC50 1 µM) and activated peroxisome proliferator-activated receptor (PPAR), whereas analogs without the cyano group were weakly cytotoxic and did not activate PPAR. β-CDODA-Me induced p21 and p27, down-regulated cyclin D1 protein expression, and induced two other proapoptotic proteins, namely nonsteroidal anti-inflammatory drug-activated gene-1 and activating transcription factor-3. However, induction of these responses by β-CDODA-Me was PPAR-independent and due to activation of phosphatidylinositol-3-kinase, mitogen-activated protein kinase, and jun N-terminal kinase pathways by this compound. In contrast, β-CDODA-Me also decreased androgen receptor (AR) and prostate-specific antigen (PSA) mRNA and protein levels through kinase-independent pathways. β-CDODA-Me repressed AR mRNA transcription, whereas decreased PSA mRNA levels were dependent on protein synthesis and were reversed by cycloheximide. Thus, potent inhibition of LNCaP cell survival by β-CDODA-Me is due to PPAR-independent activation of multiple pathways that selectively activate growth-inhibitory and proapoptotic responses.
Peroxisome proliferator-activated receptors (PPARs) are a subfamily of the nuclear receptor superfamily of ligand-activated receptors (Mangelsdorf et al., 1995). The three members of this family, PPAR, PPAR, and PPARβ/, are lipid sensors and play a key role in regulating tissue-specific lipid homeostasis and metabolism (Lee et al., 2003). PPARs also play an important role in many diseases, particularly those related to obesity, metabolic disorders, cancer, and atherogenesis (Escher and Wahli, 2000; Lee et al., 2003). Endogenous ligands for PPARs include fatty acid-derived compounds and 15-deoxy-12,14-prostaglandin J2 (PGJ2), which exhibits high affinity for PPAR; however, PGJ2 may not be the endogenous ligand for this receptor because of the low cellular expression of this metabolite. Synthetic PPAR agonists, such as the thiazolidinediones rosiglitazone and pioglitazone, are insulin-sensitizing drugs that are widely used for clinical treatment of type II diabetes.
Several different structural classes of PPAR agonists have been characterized, and these include flavones, various indole derivatives, and triterpenoids such as 2-cyano-3,12-dioxo-17-olean-1,9-dien-28-oic acid (CDDO) and related compounds (Honda et al., 1998; Rieusset et al., 2002; Berger et al., 2003; Qin et al., 2004; Schopfer et al., 2005). PPAR is overexpressed in many tumor types and cancer cell lines (Ikezoe et al., 2001), and PPAR agonists show promise for the clinical treatment of various types of tumors (Kubota et al., 1998; Chang and Szabo, 2000; Gupta et al., 2003; Qin et al., 2004). Ligands for this receptor typically inhibit G0/G1-to S-phase progression, and this is accompanied by down-regulation of cyclin D1 expression and induction of the cyclin-dependent kinase inhibitors p27 or p21. Research from our laboratory has identified a series of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes [methylene-substituted diindolyl-methanes (C-DIMs)], which inhibit cancer cell and tumor growth (Chintharlapalli et al., 2004, 2005a, 2007b; Hong et al., 2004; Qin et al., 2004; Abdelrahim et al., 2006) through both receptor-dependent and independent pathways, and similar observations have been reported for other PPAR agonists (Clay et al., 2002; Konopleva et al., 2004; Samudio et al., 2005; Yang et al., 2006).
A new class of synthetic PPAR agonists has been derived from glycyrrhetinic acid (GA), a major triterpenoid acid found in licorice extracts. Methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (β-CDODA-Me) is a 2-cyano derivative of GA and has the same oleanolic acid pentacyclic triterpene backbone structure as CDDO, which is also a 2-cyano derivative of oleanolic acid (Honda et al., 1998). However, there are major structural differences between CDODA and CDDO with respect to the position of the carboxylic acid group in the E-ring, the position of the double bonds and keto group in the C-ring. We reported recently that the 18 and 18β isomers of CDODA-Me activate PPAR in colon cancer cells and induced both caveolin-1 and Krüppel-like Factor-4 through receptor-dependent pathways (Chintharlapalli et al., 2007a). In this study, we investigated the effects of β-CDODA-Me on the proliferation of LNCaP prostate cancer cells, and the IC50 value for growth inhibition was approximately 1 µM. In contrast to studies in colon cancer cells, β-CDODA-Me had minimal effects on caveolin-1 or Krüppel-like Factor-4 expression in LNCaP cells. The proapoptotic and growth-inhibitory effects of β-CDODA-Me in LNCaP cells were associated with the induction of p21 and p27 expression, down-regulation of cyclin D1, and induction of nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1). β-CDODA-Me also decreased androgen receptor (AR) and prostate-specific antigen (PSA) protein and RNA expression, and all of these responses were PPAR-independent. Thus, β-CDODA-Me, a PPAR agonist, inhibited growth and induced apoptosis in LNCaP cells through activation of multiple receptor-independent pathways, including ablation of AR gene expression.
Cell Lines. LNCaP, PC3, and DU145 human prostate carcinoma cells were obtained from American Type Culture Collection (Manassas, VA). Fetal bovine serum was obtained from JRH Biosciences (Lenexa, KS). LNCaP, PC3, and DU145 cells were maintained in RPMI 1640 medium (Sigma Chemical, St. Louis, MO) supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 0.45% glucose, 0.24% HEPES, 10% FBS, and 10 ml/l 100x antibiotic/antimycotic solution (Sigma). Cells were maintained at 37°C in the presence of 5% CO2.
Antibodies and Reagents. Antibodies for poly(ADP-ribose) polymerase, cyclin D1, p27, FKBP51, AR, ATF3, Akt, and caveolin-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PSA was obtained from Dako Denmark A/S (Glostrup, Denmark); NAG-1 was purchased from Upstate Biotechnology (Charlottesville, VA); and EGR-1, pAKT, phosphorylated extracellular signal-regulated kinase, extracellular signal-regulated kinase, pJNK, and JNK were obtained from Cell Signaling Technology Inc. (Danvers, MA). Monoclonal β-actin antibody and dihydrotesterone were purchased from Sigma-Aldrich. Reporter lysis buffer and luciferase reagent for luciferase studies were purchased from Promega (Madison, WI). β-Galactosidase reagent was obtained from Tropix (Bedford, MA), and Lipofectamine reagents were supplied by Invitrogen (Carlsbad, CA). Western blotting chemiluminescence reagents were from PerkinElmer Life and Analytical Sciences (Waltham, MA). The PPAR antagonist T007 was prepared in this laboratory, and the synthesis of the GA derivatives has been described previously (Chintharlapalli et al., 2007a).
Cell Proliferation and DNA Fragmentation Assays. LNCaP prostate cancer cells (2 x 104/well) were added to 12-well plates and allowed to attach for 24 h. The medium was then changed to DMEM/Ham's F-12 media containing 2.5% charcoal-stripped FBS, and either vehicle (DMSO) or the indicated compounds were added. Fresh medium and indicated compounds were added every 48 h, and cells were then trypsinized and counted after 2, 4, and 6 days using a Coulter Z1 cell counter (Beckman Coulter, Fullerton, CA). Each experiment was done in triplicate, and results are expressed as means ± S.E. for each set of three experiments. The DNA fragmentation assay was performed using an Apoptotic DNA ladder extraction kit (BioVision, Mountain View, CA) according to the manufacturer's protocol.
Transfections. The Gal4 reporter construct containing 5x Gal4 response elements (pGal4) was kindly provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). The Gal4DBD-PPAR construct was a gift of Dr. Jennifer L. Oberfield (Glaxo SmithKline, Research Triangle Park, NC). The PPRE-luc construct contains three tandem PPREs with a minimal TATA sequence linked to the luciferase gene in pGL2. The AR-luc construct containing the -5400 to +580 region of the androgen receptor promoter was provided by Dr. Donald J. Tindall (Mayo Clinic, Rochester, MN), and the PSA-luc construct containing the 5.8-kilobase region of the PSA promoter was provided by Dr. Hong-Wu Cheng (University of California, Davis, CA). LNCaP cells (1 x 105) were seeded in 12-well plates in DMEM/Ham's F-12 media supplemented with 2.5% charcoal-stripped FBS and grown overnight. Transient transfections were performed using Lipofectamine reagent (Invitrogen) according to the protocol provided by the manufacturer. Transfection studies were performed using 0.4 µg of Gal4Luc, 0.04 µg of β-galactosidase, 0.04 µg of Gal4DBD-PPAR, 0.4 µg of AR-luc, and 0.3 µg of PSA-luc. Six hours after transfection, the transfection mix was replaced with complete media containing either vehicle (DMSO) or the indicated ligand for 20 to 22 h. Cells were then lysed with 100 µl of 1x reporter lysis buffer, and 30 µl of cell extract was used for luciferase and β-galactosidase assays. A Lumicount luminometer (PerkinElmer Life and Analytical Sciences) was used to quantify luciferase and β-galactosidase activities, and the luciferase activities were normalized to β-galactosidase activity.
Fluorescence-Activated Cell Sorting Analysis. LNCaP cells were treated with either the vehicle (DMSO) or the compound for 24 h. Cells were trypsinized, centrifuged, and resuspended in staining solution containing 50 µg/ml propidium iodide, 4 mM sodium citrate, and 30 U/ml RNase. After incubation at room temperature for 1 h, cells were analyzed on a FACSVantage SE DiVa (BD Biosciences, San Jose, CA), using BD FACSDiva software, version 4.1.1. Propidium iodide (PI) fluorescence was collected through a 610SP bandpass filter, and list mode data were acquired on a minimum of 50,000 single cells defined by a dot plot of PI width versus PI area. Data analysis was performed in BD FACSDiva software version 4.1.1 using PI width versus PI area to exclude cell aggregates.
Real-Time PCR. Total RNA was isolated using the RNeasy Protect Mini kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. RNA was eluted with 30 µl of RNase-free water and stored at -80°C. RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol. cDNA was prepared from the LNCaP cell line using a combination of oligodeoxythymidylic acid and dNTP mix (Applied Biosystems, Foster City, CA) and Superscript II (Invitrogen). Each PCR was carried out in triplicate in a 25-µl volume using SYBR Green Master mix (Applied Biosystems) for 15 min at 95°C for initial denaturing, followed by 40 cycles of 95°C for 30 s and 60°C for 1 min in the ABI Prism 7700 sequence detection system (Applied Biosystems). The ABI Dissociation Curves software was used after a brief thermal protocol (95°C for 15 s and 60°C for 20 s, followed by a slow ramp to 95°C) to control for multiple species in each PCR amplification. The comparative CT method was used for relative quantitation of samples. Values for each gene were normalized to expression levels of TATA-binding protein. Primers were purchased from Integrated DNA Technologies (Coralville, IA). The sequences of the primers used for reverse transcription-PCR were as follows: AR: forward, 5'-GTA CCC TGG CGG CAT GGT-3', and reverse, 5'-CCC ATT TCG CTT TTG ACA CA-3'; PSA: forward, 5'-GCA TTG AAC CAG AGG AGT TCT TG-3', and reverse, 5'-TTG CGC ACA CAC GTC ATT G-3'; and TATA-binding protein: forward, 5'-TGC ACA GGA GCC AAG AGT GAA-3', and reverse, 5'-CAC ATC ACA GCT CCC CAC CA-3'.
Western Blot Analysis. Cells were seeded in DMEM/Ham's F-12 media containing 2.5% charcoal-stripped FBS for 24 h and then treated with either the vehicle (DMSO) or the indicated compounds. Cells were collected by scraping in 150 µl of high-salt lysis buffer [50 mM HEPES, 0.5 M NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, and 5 µl/ml Protease Inhibitor Cocktail (Sigma)]. The lysates were incubated on ice for 1 h with intermittent vortexing followed by centrifugation at 20,000g for 10 min at 4°C. Before electrophoresis, samples were boiled for 3 min at 100°C; the amount of protein was determined, and 60 µg of protein was applied per lane. Samples were subjected to SDS-polyacrylamide gel electrophoresis on 10% gel at 120 V for 3 to 4 h. Proteins were transferred on to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA) at 0.9 V for 90 min at 4°C in 1x transfer buffer (48 mM Tris-HCl, 39 mM glycine, and 0.025% SDS). The membranes were blocked for 30 min with 5% TBST-Blotto (10 mM Tris-HCl, 150 mM NaCl, pH 8.0, 0.05% Triton X-100, and 5% nonfat dry milk) and incubated in fresh 5% TBST-Blotto with primary antibody overnight with gentle shaking at 4°C. After washing with TBST for 10 min, the polyvinylidene difluoride membrane was incubated with secondary antibody (1:5000) in 5% TBST-Blotto for 2 to 3 h. The membrane was washed with TBST for 10 min and incubated with 10 ml of chemiluminescence substrate (PerkinElmer Life Sciences) for 1.0 min and exposed to ImageTeK-H medical imaging film (Eastman American X-Ray Supply, Inc., Rochester, NY).
Statistical Analysis. Statistical differences between different groups were determined by analysis of variance and Scheffe's test for significance. The data are presented as mean ± S.E. for at least three separate determinations for each treatment group.
Cytotoxicity of β-CDODA-Me and Activation of PPAR. β-DODA or 1,2-dehydro-GA exhibited minimal inhibition of LNCaP cell growth with an IC50 value >15 µM, whereas the IC50 value for the corresponding methyl ester derivative was between 10 and 15 µM (Fig. 1A). Introduction of a 2-cyano group to give β-CDODA-Me increased the cytotoxicity by at least an order of magnitude, and the IC50 value was approximately 1 µM in LNCaP cells (Fig. 1A). These results were similar to those observed in colon cancer cells (Chintharlapalli et al., 2007a) and demonstrate the importance of 2-cyano substituents in mediating the cytotoxicity of GA derivatives. Figure 1B shows that decreased LNCaP cell survival induced by β-CDODA-Me was not affected after cotreatment with the PPAR antagonist T007, suggesting that the cytotoxicity of this compound was receptor-independent. β-CDODA-Me was also cytotoxic to androgen-insensitive PC3 and DU145 prostate cancer cells (Fig. 1C). The induction of PPAR-dependent transactivation by β-CDODA-Me was also investigated in LNCaP cells transfected with PPAR-GAL4/GAL4-Luc or PPRE3-Luc constructs and treated with 1 to 5 µM concentrations. β-CDODA-Me significantly induced luciferase activity (Fig. 1D), and in cells cotreated with β-CDODA-Me plus 10 µM T007 (a PPAR antagonist), there was significant inhibition of induced transactivation. In contrast, β-DODA-Me did not activate PPAR (data not shown), demonstrating the requirement for the 2-cyano substituent to confer PPAR agonist activity on the GA derivative.
Fig. 1. Effects of β-CDODA-Me and related compounds on LNCaP cell survival and activation of PPAR. Cell survival of LNCaP (A and B), PC3, and DU145 (C) cells. Prostate cancer cells were treated with different concentrations of β-DODA, β-DODA-Me, or β-CDODA-Me alone or in combination with 5 µM T007 (C) for 96 h, and the percentage of cell survival relative to DMSO (solvent control set at 100%) was determined as described under Materials and Methods. Results are expressed as means ± S.E. for three separate determinations for each treatment group, and significantly (p < 0.05) decreased survival is indicated (*). D, β-CDODA-Me activates PPAR. LNCaP cells were treated with β-CDODA, T007, or their combination, transfected with PPAR-GAL4/pGAL4 or PPRE-luc, and luciferase activity determined as described under Materials and Methods. Results are expressed as means ± S.E. for three replicate determinations for each treatment group, and significant (p < 0.05) induction by β-CDODA-Me (*) and inhibition after cotreatment with T007 (**) are indicated.
PPAR agonists typically modulate the expression of one or more of the cell cycle proteins p27, p21, and cyclin D1, and Fig. 2A illustrates the effects of 1 to 5 µM β-CDODA-Me on the expression of these proteins in LNCaP cells. There was a concentration-dependent induction of p27 and p21 and a decrease in cyclin D1 proteins and retinoblastoma protein phosphorylation in cells treated with β-CDODA-Me alone, and similar results were observed in cells cotreated with the PPAR antagonist T007 and β-CDODA-Me (Fig. 2B), suggesting that these responses were PPAR-independent. FACS analysis of LNCaP cells treated with different concentrations of β-CDODA-Me indicated that 1 µM β-CDODA-Me did not affect the percentage of cells in G0/G1, S, or G2/M, whereas at higher concentrations, the percentage of cells in these phases of the cell cycle decreased (Fig. 2C). β-CDODA-Me induced a concentration-dependent increase in the percentage of apoptotic sub-G1 LNCaP cells, and the proapoptotic effects of this compound were also observed in PC3 and DU145 cells (Fig. 2D), in which β-CDODA-Me induced caspase-dependent PARP cleavage. Thus, the overall cytotoxicity of β-CDODA-Me in prostate cancer cells was associated with both inhibition of growth and induction of apoptosis.
Fig. 2. β-CDODA-Me modulates the cell cycle and cell cycle genes and induces apoptosis in prostate cancer cells. Modulation of cell cycle genes by β-CDODA-Me alone (A) and in combination with T007 (B). Cells were treated as indicated for 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods. C, cell cycle progression. LNCaP cells were treated with DMSO and different concentrations of β-CDODA-Me for 24 h and analyzed for the percentage of distribution of cells in different phases of the cell cycle by FACS analysis as described under Materials and Methods. Results are expressed at means ± S.E. for three replicate determinations, and significant (p < 0.05) changes (compared with DMSO group) are indicated by an asterisk. D, induction of PARP cleavage. PC3 and DU145 cells were treated for 24 h with different concentrations of β-CDODA-Me, and whole-cell lysates were analyzed by Western blots as described under Materials and Methods.
Induction of Proapoptotic Responses by β-CDODA-Me. NAG-1 and ATF-3 are proapoptotic proteins induced by PPAR agonists, and results in Fig. 3A show that 1 to 5 µM β-CDODA-Me induced NAG-1 and ATF-3, which are often coinduced, and this was accompanied by caspase-dependent PARP cleavage, DNA fragmentation, and decreased bcl2 expression in LNCaP cells. In addition, the DNA laddering response was not affected after cotreatment with the PPAR antagonist T007 (Fig. 3B). In LNCaP cells cotreated with β-CDODA-Me plus T007 (Fig. 3C), the induced responses were not inhibited by the PPAR antagonist, indicating that induction of these proapoptotic responses was receptor-independent. Previous studies show that different structural classes of PPAR agonists down-regulate AR expression in LNCaP cells, and this response can also result in the activation of apoptosis (Yang et al., 2006; Chintharlapalli et al., 2007b). Figure 3D summarizes the effects of β-CDODA-Me on AR expression in the presence or absence of 10 nM DHT and on the expression of FKBP51 and PSA, two androgen-responsive genes in LNCaP cells. DHT increases expression of AR as a result of stabilization of the receptor and induces both androgen-responsive FKBP51 and PSA genes; in cells treated with 1 to 5 µM β-CDODA-Me, there was a concentration-dependent decrease in AR, PSA, and FKBP51 expression in the presence or absence of DHT. In addition, down-regulation of AR, PSA, and FKBP51 proteins in LNCaP cells treated with β-CDODA-Me was not affected by cotreatment with the PPAR antagonist T007 or the proteasome inhibitor MG132 (Fig. 3E). In contrast, β-CDODA-Me-dependent degradation of cyclin D1 was inhibited after cotreatment with MG132, and these observations are similar to those reported for other PPAR agonists that induce proteasome-dependent degradation of cyclin D1 (Chintharlapalli et al., 2004, 2005a,b). These results clearly show that β-CDODA-Me decreases expression of androgen-responsive genes and AR through PPAR-independent pathways. The down-regulation of AR in cells treated with β-CDODA-Me is consistent with the induction of apoptosis by this compound because decreased AR expression by small inhibitory RNAs in LNCaP cells also induces apoptosis (Liao et al., 2005).
Fig. 3. β-CDODA induces apoptotic pathways and decreases androgen-responsiveness in LNCaP cells. β-CDODA-Me alone (A) and in combination with T007 (B and C) induces proapoptotic pathways. LNCaP cells were treated as indicated for 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods. β-CDODA-Me-induced DNA fragmentation (A and B) was also determined as described. Effects of β-CDODA-Me alone and in combination with DHT or T007 (D) or MG132 (E) on AR and androgen-responsive proteins. LNCaP cells were treated with DMSO or the various compounds for 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods.
Fig. 4. β-CDODA-Me induces proapoptotic proteins and kinases. Induction of NAG-1, ATF3, and Egr-1 (A) and kinases (B) by β-CDODA-Me. LNCaP cells were treated with 2.5 µM β-CDODA-Me, and whole-cell lysates isolated at different times after treatment were analyzed by Western blot analysis as described under Materials and Methods. Effects of kinase inhibitors on proapoptotic responses (C) and quantitation of NAG-1 and ATF3 expression (D). LNCaP cells were treated with 2.5 µM β-CDODA alone or in combination with various kinase inhibitors, and after 24 h, whole-cell lysates were analyzed by Western blot analysis. Levels of NAG-1 and ATF3 proteins (normalized to β-actin) (D) are means ± S.E. for three separate determinations for each treatment group, and significantly (p < 0.05) decreased levels after cotreatment with a kinase inhibitor are indicated (**).
Fig. 5. β-CDODA-Me induction of p21 is MAPK-dependent. A, effects of kinase inhibitors on induction of p21. LNCaP cells were treated with DMSO, 2.5 µM β-CDODA-Me alone or in combination with kinase inhibitors for 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods. B, β-CDODA-Me activates p21 promoter constructs. LNCaP cells were transfected with p21 promoter constructs, treated with DMSO or different concentrations of β-CDODA-Me, and luciferase activity was determined as described under Materials and Methods. Results are means ± S.E. for three separate determinations for each treatment group, and significant (p < 0.05) induction of activity is indicated (*). C, inhibition by PD98059. Cells were transfected with p21-luc(101), treated with DMSO or β-CDODA-Me alone or in combination with 10 µM PD98059. Results are expressed as means ± S.E. for three separate determinations for each treatment group, and significant (p < 0.05) induction by β-CDODA-Me (*) and inhibition after cotreatment with PD98059 (**) are indicated.
β-CDODA-Me Induced Kinase-Dependent Activation of Proapoptotic/Growth Inhibitory Pathways. Previous studies show that NAG-1 is induced by some PPAR agonists and other cytotoxic compounds in colon cancer cells (Baek et al., 2003, 2005; Chintharlapalli et al., 2005a) through PI3K-dependent activation of EGR-1, which acts as a transacting factor to induce NAG-1 expression. Figure 4A summarizes the time-dependent induction of EGR-1, ATF-3, and NAG-1 by 2.5 µM β-CDODA-Me, and the induction responses followed a similar time course, whereas EGR-1-dependent induction of NAG-1 in colon cancer cells is associated with the increased expression of EGR-1 before induction of NAG-1 (Baek et al., 2005; Chintharlapalli et al., 2005a). Previous studies show that NAG-1 induction is kinase-dependent (Baek et al., 2005; Chintharlapalli et al., 2005a), and results in Fig. 4B show that 2.5 µM β-CDODA-Me induces activation of the JNK (pJNK), PI3K (pAkt), and MAPK (pErk) pathways. Maximal activation of JNK and PI3K was observed after 8 and 8 to 12 h, respectively, whereas pErk activation remained elevated for 24 h. The effects of inhibitors of MAPK (PD98059), PI3K (LY294002), protein kinase C (GF109203X), and JNK (SP600125) on induction of NAG-1 and ATF3, and decreased expression of AR, PSA, and FKBP51 was also investigated in LNCaP cells treated with 2.5 µM β-CDODA-Me (Fig. 4C). Both PD98059 and LY294002 inhibited the induction of NAG-1 by β-CDODA-Me. However, the JNK inhibitor SP600125 was the most potent inhibitor of ATF-3 induction (Fig. 4, C and D). In contrast, decreased expression of AR, PSA, and FKBP51 in LNCaP cells treated with β-CDODA-Me was unaffected by kinase inhibitors. CDDO and related compounds also induce activation of JNK in leukemia cells through increased reactive oxygen species (Ikeda et al., 2003), and Fig. 4E illustrates the effects of β-CDODA-Me on activation of JNK and ATF3 after cotreatment with the antioxidant NAC. Induction of JNK phosphorylation and ATF3 by β-CDODA-Me was inhibited after cotreatment with NAC, demonstrating that like CDDO (Ikeda et al., 2003), β-CDODA-Me activates JNK through increased oxidative stress.
Fig. 6. β-CDODA-Me decreases AR gene expression. Effects of T007 (A) and cycloheximide (B) on β-CDODA-Me-dependent effects on AR gene expression. LNCaP cells were treated with β-CDODA-Me alone or in combination with T007 or cycloheximide for 18 h, and AR mRNA levels were determined by real-time PCR as described under Materials and Methods. Similar results were observed after treatment for 12 h (data not shown). C, β-CDODA-Me decreases AR promoter activity. LNCaP cells were transfected with AR-luc, treated with DMSO or β-CDODA-Me, and luciferase activity was determined as described under Materials and Methods. Results are means ± S.E. for three separate experiments for each treatment group, and a significant (p < 0.05) decrease in activity is indicated (*). D, time-dependent effects of β-CDODA-Me on AR, Sp1, and PARP (cleaved). LNCaP cells were treated with DMSO or β-CDODA-Me for up to 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods.
These results suggest that the underlying pathways associated with the growth-inhibitory/proapoptotic pathways induced by β-CDODA-Me in LNCaP cells are due in part to activation of kinases. Therefore, the effects of kinase inhibitors on modulation of cell cycle proteins by β-CDODA-Me were also investigated, and the down-regulation of cyclin D1 and induction of p21 were partially blocked in cells cotreated with the MAPK inhibitor PD98059 (Fig. 5A); MAPK-dependent activation of p21 has been observed previously (De Siervi et al., 2004). Results in Fig. 5B show that the 1 to 5 µM β-CDODA-Me also induces luciferase activity in LNCaP cells transfected with constructs containing -2325 to +8 [p21-Luc (Fl)], -124 to +8 [p21-Luc (-124)], -101 to +8 [p21-Luc (-101)], and -60 to +8 [p21-Luc (-60)] p21 promoter inserts. The latter three constructs contain the six proximal GC-rich sites (VI-I), and the results of the transfection studies suggest that these GC-rich sites are necessary for β-CDODA-Me-induced transactivation. Deletion analysis of the p21 promoter indicates that loss of inducibility [i.e., p21-luc(60)] is associated with loss of GC-rich sites IV and III, which are essential for MAPK-dependent activation of p21 by β-CDODA-Me. The role of MAPK in activation of the p21 promoter was confirmed in LNCaP cells transfected with p21-luc(101); β-CDODA-Me induced luciferase activity, and cotreatment with the MAPK inhibitor PD98059 inhibited this response (Fig. 5C). These results show that the induction of p21 and the proapoptotic NAG-1 protein by β-CDODA-Me were related to the activation of MAPK and PI3K but were independent of PPAR (Fig. 2, B and C).
β-CDODA-Me Differentially Decreased AR and PSA Gene Expression in LNCaP Cells. β-CDODA-Me decreases expression of AR, PSA, and FKBP51 protein levels through proteasome and PPAR-independent pathways (Fig. 3, D and E), and these responses are also not modulated by kinase inhibitors (Fig. 4C). The results in Fig. 6A show that β-CDODA-Me also decreases AR mRNA levels after treatment for 18 h, and cotreatment with the PPAR antagonist T007 did not affect mRNA levels confirming the β-CDODA-Me-induced down-regulation of AR mRNA levels was also PPAR-independent. Similar results were obtained in LNCaP cells treated with β-CDODA-Me alone or in the presence of the protein synthesis inhibitor cycloheximide (10 µg/ml) (Fig. 6B); cycloheximide did not modulate the effects of β-CDODA-Me, suggesting that an induced inhibitory protein(s) does not mediate the effects of β-CDODA-Me on AR mRNA expression. β-CDODA-Me also decreased luciferase activity in LNCaP cells transfected with the AR-Luc construct that contains the -5400 to +580 region of the AR promoter linked to the luciferase gene (Fig. 6C). The results indicate that β-CDODA-Me inhibits AR transcription without the parallel induction of inhibitory transacting factors. Recent studies suggest that AR down-regulation of a PPAR-inactive thiazolidinedione analog was due to down-regulation of Sp1 protein (Yang et al., 2007). Results in Fig. 6D show that β-CDODA-Me induces a time-dependent induction of PARP cleavage and a decrease of both AR and Sp1.
Fig. 7. β-CDODA-Me decreases PSA expression. Effects of T007 (A) and cycloheximide (B) on β-CDODA-Me-dependent effects on PSA gene expression. LNCaP cells were treated with β-CDODA-Me alone or in combination with T007 or cycloheximide for 18 h, and PSA mRNA levels were determined by real-time PCR as described under Materials and Methods. Similar results were observed after treatment for 12 h (data not shown). β-CDODA-Me decreases PSA promoter (C) and DHT-induced (D) PSA promoter activity. LNCaP cells were transfected with PSA-luc, treated with DMSO, β-CDODA-Me, DHT, and β-CDODA-Me plus DHT (combined), and luciferase activity determined as described under Materials and Methods. Results are means ± S.E. for three replicate determinations for each treatment group, and significantly (p < 0.05) decreased basal or DHT-induced luciferase activity by β-CDODA-Me is indicated (*).
PSA protein expression is also decreased in LNCaP cells treated with β-CDODA-Me (Fig. 3D), and similar effects were observed for PSA mRNA levels after treatment for 18 h; these responses were not inhibited after cotreatment with the PPAR antagonist T007 (Fig. 7A). However, β-CDODA-Me-induced down-regulation of PSA mRNA levels after treatment for 18 h was significantly inhibited after cotreatment with cycloheximide (Fig. 7B). In addition, β-CDODA-Me inhibited transactivation in LNCaP cells transfected with the PSA-Luc construct (contains 5.85 kilobases of the PSA promoter insert) (Fig. 7C), and similar results were obtained for DHT-induced luciferase activity (Fig. 7D). Thus, in contrast to results obtained for AR, β-CDODA-Me inhibits PSA expression through induction of inhibitory transacting factors, and the mechanisms associated with the decreased PSA expression and the cis-elements important for this response are currently being investigated.
PPAR agonists have been investigated extensively in both in vitro and in vivo cancer models for their potential applications in cancer chemotherapy (Honda et al., 1998; Escher and Wahli, 2000; Berger et al., 2003; Qin et al., 2004). PPAR agonists inhibit prostate cancer cell and tumor growth (Kubota et al., 1998; Moretti et al., 2001; Segawa et al., 2002; Chintharlapalli et al., 2007b), and the fact that approximately 40% of patients with prostate cancer carry hemizygous deletions of PPAR (Mueller et al., 2000) suggests that this receptor may serve as a tumor suppressor gene for prostate cancer. However, in animal studies using the transgenic adenocarcinoma mouse prostate model with hemizygous deletion in PPAR, it was shown that the loss of receptor expression did not enhance or inhibit prostate tumor development in these animals (Saez et al., 2003). Thus, at least in the transgenic adenocarcinoma mouse prostate mouse model, PPAR does not seem to act as a tumor suppressor gene.
Fig. 8. β-CDODA-Me-dependent activation of kinases and kinase-dependent genes and repression of AR and PSA.
One of the perplexing problems with PPAR agonists is that although these compounds inhibit cancer cell and tumor growth, their mechanisms of action are both receptor-dependent and -independent in different cancer cell lines. For example, PGJ2, troglitazone, and PPAR-active C-DIMs induce NAG-1 in HCT-116 colon cancer cells; however, only induction by PGJ2 is inhibited by a PPAR antagonist (Baek et al., 2003; Chintharlapalli et al., 2005a). Caveolin-1 is induced by C-DIM compounds and CDDO in colon cancer cell lines, and this response is inhibited after cotreatment with PPAR antagonists (Chintharlapalli et al., 2004, 2005b). In contrast, C-DIMs decreased caveolin-1 expression in LNCaP cells, and this response was PPAR-independent (Chintharlapalli et al., 2007b).
β-CDODA-Me is a triterpenoid acid that contains an oleanolic acid backbone structure similar to that of CDDO and CDDO-Me (methyl ester) (Honda et al., 1998), but there are important structural differences in the C-, D-, and E-rings that differentiate between these compounds. However, for both compounds, the 2-cyano group was necessary for the activation of PPAR.
In this study, we investigated the growth-inhibitory and proapoptotic effects of β-CDODA-Me in LNCaP cells and the role of PPAR in mediating these responses. β-CDODA-Me was a more potent inhibitor of LNCaP cell growth than analogs (β-DODA and β-DODA-Me) that did not contain a 2-cyano substituent (Fig. 1A). Moreover, β-CDODA-Me decreased survival of androgen-insensitive PC3 and DU145 cells (Fig. 1C) and also induced apoptosis in these cells (Fig. 2D). β-CDODA-Me activated PPAR-dependent transactivation in transient transfection studies in LNCaP cells (Fig. 1D), and compounds without the CN-group were inactive (data not shown) as reported previously for these analogs in colon cancer cells (Chintharlapalli et al., 2007a). β-CDODA-Me induced p27 expression and down-regulated levels of cyclin D1 protein (Fig. 2, A and B). Similar effects were reported previously for C-DIMs in LNCaP cells (Chintharlapalli et al., 2007b), and the effects of both compounds were receptor-independent; however, β-CDODA-Me-induced responses were observed at lower concentrations (1-2.5 µM) than the C-DIM compounds (7.5-10 µM). C-DIMs did not induce p21 in LNCaP cells (Chintharlapalli et al., 2007b), whereas β-CDODA-Me induced p21 protein, and this response was not inhibited after cotreatment with PPAR antagonist T007 (Fig. 2B). Differences between PPAR-active C-DIMs and β-CDODA-Me in their induction of p21 in LNCaP cells was due to activation of MAPK signaling by the latter compound (Fig. 4B), which was required for the induction of p21 protein (Fig. 5A) and activation of the p21 promoter (Fig. 5C). This is a novel pathway for induction of p21 in LNCaP cells; however, previous studies in other cell lines have demonstrated MAPK-dependent induction of p21 expression (De Siervi et al., 2004).
NAG-1 and ATF3 are growth-inhibitory and proapoptotic proteins (Hartman et al., 2004; Jang et al., 2006), and previous studies with PPAR agonists report both receptor-dependent and -independent induction of NAG-1 (Baek et al., 2003; Chintharlapalli et al., 2005a,b). Induction of NAG-1 and ATF3 by β-CDODA-Me in LNCaP cells was also PPAR-independent. Both PI3K and MAPK inhibitors blocked the induction of NAG-1; however, the JNK inhibitor SP600125 was the most potent inhibitor of ATF3 (but not NAG-1) induction. The inhibitory effects of SP600125 are consistent with previous studies showing that homocysteine also induces ATF3 in vascular cells through activation of JNK, which activates c-jun and ATF3 through an activator protein-1 site in the ATF3 promoter (Cheng et al., 2006). The structurally related triterpenoid CDDO induces oxidative stress in leukemia and pancreatic cancer cells (Ikeda et al., 2003; Samudio et al., 2005), and this is correlated with pro-oxidant-dependent induction of JNK phosphorylation and other responses that are inhibited by antioxidants such as NAC (Ikeda et al., 2003). Similar results were observed in this study where NAC also inhibits β-CDODA-Me-dependent activation of JNK and induction of ATF3 (Fig. 4E). The kinase-dependent induction of NAG-1 has been reported previously, and these effects are both structure- and cell context-dependent. For example, troglitazone and PPAR-active C-DIMs induce NAG-1 in HCT116 colon cancer cells through rapid activation of Egr-1, which subsequently activates NAG-1 through direct interaction with the proximal region of the NAG-1 promoter (Chintharlapalli et al., 2005a). However, this induction response is MAPK-dependent for troglitazone and PI3K-dependent for the C-DIM compound. In this study, the time-dependent induction of both EGR-1 and NAG-1 are similar in LNCaP cells (Fig. 4A), and inhibition of NAG-1 expression is observed with both PI3K and MAPK inhibitors (Fig. 4B). This may involve cooperative interactions of both kinase pathways for the induction of NAG-1 by β-CDODA-Me in LNCaP cells, and mechanisms for these responses are currently being investigated. It is interesting that induction of NAG-1 by PPAR-active C-DIMs in LNCaP cells was inhibited only by the MAPK inhibitor PD98059 (Chintharlapalli et al., 2007b), suggesting differences between β-CDODA-Me and C-DIMs in the same cell line. Thus, induction of both NAG-1 and ATF3 in LNCaP cells is differentially induced by two PPAR agonists, C-DIMs and β-CDODA-Me, through receptor-independent activation of different kinase pathways.
Recent reports show that in LNCaP cells, AR knockdown by RNA interference results in apoptosis (Liao et al., 2005), and stable knockdown using short hairpin RNAs for AR results in decreased AR and PSA expression and inhibition of tumor growth in vivo (Cheng et al., 2006). Like β-CDODA-Me, AR and PSA expressions are also decreased by C-DIMs and troglitazone in LNCaP cells, and 3,3'-diindolylmethane (DIM) also decreases expression of both genes and proteins (Bhuiyan et al., 2006; Li et al., 2007). Troglitazone differentially decreases PSA and AR expression at relatively low (IC50 10 µM) and high (IC50 40 µM) concentrations, respectively (Yang et al., 2006). In contrast, C-DIMs decreased AR and PSA mRNA, protein and reporter gene activity in cells transfected with PSA-Luc and AR-Luc constructs over a narrow range of concentrations (7.5-10 µM) (Chintharlapalli et al., 2007b), and similar results were observed for β-CDODA-Me (1-2.5 µM) in this study (Figs. 6 and 7). Moreover, cycloheximide reversed the β-CDODA-Me- and C-DIM-dependent down-regulation of PSA but not AR mRNA levels, suggesting a similar mechanism of action for both compounds. One study reported that DIM inhibited nuclear uptake of AR in LNCaP cells (Le et al., 2003), and like β-CDODA-Me, DIM also decreased AR and PSA expression in LNCaP and androgen-insensitive C4-2B cells (Bhuiyan et al., 2006; Li et al., 2007). However, there are several differences between the pathways associated with down-regulation of these genes by β-CDODA-Me and DIM, and this includes the pivotal role for DIM as an inhibitor of phospho-Akt (Bhuiyan et al., 2006; Li et al., 2007), whereas β-CDODA-Me induces phospho-Akt (Fig. 4B), and the PI3K inhibitor LY294002 does not affect β-CDODA-dependent down-regulation of AR, PSA, or FKBP51 (Fig. 4C) or induction of p21 or p27 (Fig. 5A).
A recent report indicated that decreased AR expression in LNCaP cells treated with a PPAR-inactive thiazolidinedione derivative was due to proteasome-dependent degradation of Sp1 (Yang et al., 2007). Our results also show a parallel decrease of AR and Sp1 in LNCaP cells treated with β-CDODA-Me (Fig. 6D); however, down-regulation of AR was not reversed by a proteasome inhibitor (Fig. 3E), and the mechanism of this response is currently being investigated. Loss of AR by RNA interference results in the induction of apoptosis in LNCaP cells (Liao et al., 2005). In contrast, 2.5 µM β-CDODA-Me rapidly induces PARP cleavage and apoptosis in LNCaP cells before decreased AR expression (Fig. 6D) demonstrating that apoptotic pathways activated by β-CDODA-Me in LNCaP cells are not associated with loss of AR, and the proapoptotic mechanisms are currently being investigated.
Results of this study demonstrate that β-CDODA-Me is a potent inhibitor of LNCaP cell growth and induces proapoptotic responses through activation of kinases, which differentially activate ATF3, NAG-1, and p21. In contrast, decreased expression of AR and PSA are kinase-independent and occur through different pathways (Fig. 8). β-CDODA-Me, C-DIMs, DIM, and troglitazone exhibited both differences and similarities in their modes of action in LNCaP cells, although all of these compounds decreased expression of AR and PSA. The growth-inhibitory and proapoptotic effects of β-CDODA-Me were primarily PPAR-independent in LNCaP cells (Figs. 1B and 3). β-CDODA-Me also decreased survival and induced apoptosis in androgen-insensitive PC3 and DU145 prostate cancer cells (Figs. 1C and 2D), and similar results have been observed for other PPAR agonists. It is apparent that β-CDODA-Me decreases survival and induces apoptosis through multiple pathways, including its potent antiandrogenic activity in LNCaP cells. The successful applications of β-CDODA-Me and other such compounds for single or combined prostate cancer chemotherapies will require insights on their mechanisms of action and prostate cancer cell context-dependent similarities and differences in activating critical pathways such as those illustrated in Fig. 8.
ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; PGJ2, 15-deoxy-12,14-prostaglandin J2; TZD, thiazolidinedione; CDDO, 2-cyano-3,12-dioxo-17-olean-1,9-dien-28-oic acid; C-DIM, 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methane; GA, glycyrrhetinic acid; β-CDODA-Me, methyl 2-cyano-3,11-dioxo-18β-olean-1,12-diene-30-oate; NAG-1, nonsteroidal anti-inflammatory drug-activated gene 1; AR, androgen receptor; PSA, prostate-specific antigen; T007, N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide; DIM, 3,3'-diindolylmethane; ATF3, activating transcription factor 3; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting; PCR, polymerase chain reaction; TBST, Tris-buffered saline/Tween 20; EGR-1, early growth response-1; PARP, poly(ADP-ribose) polymerase; DHT, dihydrotestosterone; PI3K, phosphoinositol-3-kinase; NAC, N-acetyl cysteine; PI, propidium iodide; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PD98059, 2'-amino-3'-methoxyflavone; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; SP600125, 1,9-pyrazoloanthrone; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride.
【参考文献】
Abdelrahim M, Newman K, Vanderlaag K, Samudio I, and Safe S (2006) 3,3'-Diindolylmethane (DIM) and derivatives induce apoptosis in pancreatic cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5. Carcinogenesis 27: 717-728.[Abstract/Free Full Text]
Baek SJ, Kim JS, Moore SM, Lee SH, Martinez J, and Eling TE (2005) Cyclooxygenase inhibitors induce the expression of the tumor suppressor gene EGR-1, which results in the up-regulation of NAG-1, an antitumorigenic protein. Mol Pharmacol 67: 356-364.[Abstract/Free Full Text]
Baek SJ, Wilson LC, Hsi LC, and Eling TE (2003) Troglitazone, a peroxisome proliferator-activated receptor (PPAR) ligand, selectively induces the early growth response-1 gene independently of PPAR. A novel mechanism for its anti-tumorigenic activity. J Biol Chem 278: 5845-5853.[Abstract/Free Full Text]
Berger JP, Petro AE, Macnaul KL, Kelly LJ, Zhang BB, Richards K, Elbrecht A, Johnson BA, Zhou G, Doebber TW, et al. (2003) Distinct properties and advantages of a novel peroxisome proliferator-activated protein selective modulator. Mol Endocrinol 17: 662-676.[Abstract/Free Full Text]
Bhuiyan MM, Li Y, Banerjee S, Ahmed F, Wang Z, Ali S, and Sarkar FH (2006) Down-regulation of androgen receptor by 3,3'-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in both hormone-sensitive LNCaP and insensitive C4-2B prostate cancer cells. Cancer Res 66: 10064-10072.[Abstract/Free Full Text]
Cai Y, Zhang C, Nawa T, Aso T, Tanaka M, Oshiro S, Ichijo H, and Kitajima S (2000) Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH2-terminal kinase and promoter response element. Blood 96: 2140-2148.[Abstract/Free Full Text]
Chang TH and Szabo E (2000) Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor in non-small cell lung cancer. Cancer Res 60: 1129-1138.[Abstract/Free Full Text]
Cheng H, Snoek R, Ghaidi F, Cox ME, and Rennie PS (2006) Short hairpin RNA knockdown of the androgen receptor attenuates ligand-independent activation and delays tumor progression. Cancer Res 66: 10613-10620.[Abstract/Free Full Text]
Chintharlapalli S, Papineni S, Baek SJ, Liu S, and Safe S (2005a) 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)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 NAG-1. Mol Pharmacol 68: 1782-1792.[Abstract/Free Full Text]
Chintharlapalli S, Papineni S, Jutooru I, McAlees A, and Safe S (2007a) Structure-dependent activity of glycyrrhetinic acid derivatives as peroxisome proliferator-activated receptor (PPAR) agonists in colon cancer cells. Mol Cancer Ther 6: 1588-1598.[Abstract/Free Full Text]
Chintharlapalli S, Papineni S, Konopleva M, Andreef M, Samudio I, and Safe S (2005b) 2-Cyano-3,12-dioxoolean-1,9-dien-28-oic acid and related compounds inhibit growth of colon cancer cells through peroxisome proliferator-activated receptor -dependent and -independent pathways. Mol Pharmacol 68: 119-128.[Abstract/Free Full Text]
Chintharlapalli S, Papineni S, and Safe SH (2007b) 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes inhibit growth, induce apoptosis, and decrease the androgen receptor in LNCaP prostate cancer cells through PPAR-independent pathways. Mol Pharmacol 71: 558-569.[Abstract/Free Full Text]
Chintharlapalli S, Smith R III, Samudio I, Zhang W, and Safe S (2004) 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes induce peroxisome proliferator-activated receptor -mediated growth inhibition, transactivation and differentiation markers in colon cancer cells. Cancer Res 64: 5994-6001.[Abstract/Free Full Text]
Clay CE, Monjazeb A, Thorburn J, Chilton FH, and High KP (2002) 15-Deoxy-(12,14)-prostaglandin J2-induced apoptosis does not require PPAR in breast cancer cells. J Lipid Res 43: 1818-1828.[Abstract/Free Full Text]
De Siervi A, Marinissen M, Diggs J, Wang XF, Pages G, and Senderowicz A (2004) Transcriptional activation of p21waf1/cip1 by alkylphospholipids: role of the mitogen-activated protein kinase pathway in the transactivation of the human p21waf1/cip1 promoter by Sp1. Cancer Res 64: 743-750.[Abstract/Free Full Text]
Escher P and Wahli W (2000) Peroxisome proliferator-activated receptors: insight into multiple cellular functions. Mutat Res 448: 121-138.
Gupta RA, Sarraf P, Mueller E, Brockman JA, Prusakiewicz JJ, Eng C, Willson TM, and DuBois RN (2003) Peroxisome proliferator-activated receptor -mediated differentiation: a mutation in colon cancer cells reveals divergent and cell type-specific mechanisms. J Biol Chem 278: 22669-22677.[Abstract/Free Full Text]
Hartman MG, Lu D, Kim ML, Kociba GJ, Shukri T, Buteau J, Wang X, Frankel WL, Guttridge D, Prentki M, et al. (2004) Role for activating transcription factor 3 in stress-induced β-cell apoptosis. Mol Cell Biol 24: 5721-5732.[Abstract/Free Full Text]
Honda T, Rounds BV, Gribble GW, Suh N, Wang Y, and Sporn MB (1998) Design and synthesis of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, a novel and highly active inhibitor of nitric oxide production in mouse macrophages. Bioorg Med Chem Lett 8: 2711-2714.
Hong J, Samudio I, Liu S, Abdelrahim M, and Safe S (2004) Peroxisome proliferator-activated receptor -dependent activation of p21 in Panc-28 pancreatic cancer cells involves Sp1 and Sp4 proteins. Endocrinology 145: 5774-5785.[Abstract/Free Full Text]
Ikeda T, Sporn M, Honda T, Gribble GW, and Kufe D (2003) The novel triterpenoid CDDO and its derivatives induce apoptosis by disruption of intracellular redox balance. Cancer Res 63: 5551-5558.[Abstract/Free Full Text]
Ikezoe T, Miller CW, Kawano S, Heaney A, Williamson EA, Hisatake J, Green E, Hofmann W, Taguchi H, and Koeffler HP (2001) Mutational analysis of the peroxisome proliferator-activated receptor gene in human malignancies. Cancer Res 61: 5307-5310.[Abstract/Free Full Text]
Jang TJ, Kim NI, and Lee CH (2006) Proapoptotic activity of NAG-1 is cell type specific and not related to COX-2 expression. Apoptosis 11: 1131-1138.
Konopleva M, Tsao T, Estrov Z, Lee RM, Wang RY, Jackson CE, McQueen T, Monaco G, Munsell M, Belmont J, et al. (2004) The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces caspase-dependent and -independent apoptosis in acute myelogenous leukemia. Cancer Res 64: 7927-7935.[Abstract/Free Full Text]
Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, and Koeffler HP (1998) Ligand for peroxisome proliferator-activated receptor (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 58: 3344-3352.[Abstract/Free Full Text]
Le HT, Schaldach CM, Firestone GL, and Bjeldanes LF (2003) Plant-derived 3,3'-diindolylmethane is a strong androgen antagonist in human prostate cancer cells. J Biol Chem 278: 21136-21145.[Abstract/Free Full Text]
Lee CH, Olson P, and Evans RM (2003) Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 144: 2201-2207.[Abstract/Free Full Text]
Li Y, Wang Z, Kong D, Murthy S, Dou QP, Sheng S, Reddy GP, and Sarkar FH (2007) Regulation of FOXO3a/β-catenin/GSK-3β signaling by 3,3'-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells. J Biol Chem 282: 21542-21550.[Abstract/Free Full Text]
Liao X, Tang S, Thrasher JB, Griebling TL, and Li B (2005) Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther 4: 505-515.[Abstract/Free Full Text]
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, et al. (1995) The nuclear receptor superfamily: the second decade. Cell 83: 835-839.
Moretti RM, Montagnani Marelli M, Motta M, and Limonta P (2001) Oncostatic activity of a thiazolidinedione derivative on human androgen-dependent prostate cancer cells. Int J Cancer 92: 733-737.
Mueller E, Smith M, Sarraf P, Kroll T, Aiyer A, Kaufman DS, Oh W, Demetri G, Figg WD, Zhou XP, et al. (2000) Effects of ligand activation of peroxisome proliferator-activated receptor in human prostate cancer. Proc Natl Acad Sci U S A 97: 10990-10995.[Abstract/Free Full Text]
Qin C, Morrow D, Stewart J, Spencer K, Porter W, Smith R III, Phillips T, Abdelrahim M, Samudio I, and Safe S (2004) A new class of peroxisome proliferator-activated receptor (PPAR) agonists that inhibit growth of breast cancer cells: 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes. Mol Cancer Ther 3: 247-259.[Abstract/Free Full Text]
Rieusset J, Touri F, Michalik L, Escher P, Desvergne B, Niesor E, and Wahli W (2002) A new selective peroxisome proliferator-activated receptor antagonist with antiobesity and antidiabetic activity. Mol Endocrinol 16: 2628-2644.[Abstract/Free Full Text]
Saez E, Olson P, and Evans RM (2003) Genetic deficiency in Pparg does not alter development of experimental prostate cancer. Nat Med 9: 1265-1266.
Samudio I, Konopleva M, Hail N Jr, Shi YX, McQueen T, Hsu T, Evans R, Honda T, Gribble GW, Sporn M, et al. (2005) 2-Cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im) directly targets mitochondrial glutathione to induce apoptosis in pancreatic cancer. J Biol Chem 280: 36273-36282.[Abstract/Free Full Text]
Schopfer FJ, Lin Y, Baker PR, Cui T, Garcia-Barrio M, Zhang J, Chen K, Chen YE, and Freeman BA (2005) Nitrolinoleic acid: an endogenous peroxisome proliferator-activated receptor ligand. Proc Natl Acad Sci U S A 102: 2340-2345.[Abstract/Free Full Text]
Segawa Y, Yoshimura R, Hase T, Nakatani T, Wada S, Kawahito Y, Kishimoto T, and Sano H (2002) Expression of peroxisome proliferator-activated receptor (PPAR) in human prostate cancer. Prostate 51: 108-116.
Yang CC, Ku CY, Wei S, Shiau CW, Chen CS, Pinzone JJ, Ringel MD, and Chen CS (2006) Peroxisome proliferator-activated receptor -independent repression of prostate-specific antigen expression by thiazolidinediones in prostate cancer cells. Mol Pharmacol 69: 1564-1570.[Abstract/Free Full Text]
Yang CC, Wang YC, Wei S, Lin LF, Chen CS, Lee CC, Lin CC, and Chen CS (2007) Peroxisome proliferator-activated receptor gamma-independent suppression of androgen receptor expression by troglitazone mechanism and pharmacologic exploitation. Cancer Res 67: 3229-3238.[Abstract/Free Full Text]
作者单位：Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas (S.P., S.S.); and Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas (S.C., S.S.)