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Carcinogenesis Advance Access originally published online on August 27, 2007
Carcinogenesis 2007 28(11):2337-2346; doi:10.1093/carcin/bgm189
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

2-Cyano-lup-1-en-3-oxo-20-oic acid, a cyano derivative of betulinic acid, activates peroxisome proliferator-activated receptor {gamma} in colon and pancreatic cancer cells

Sudhakar Chintharlapalli1,{dagger}, Sabitha Papineni1,{dagger}, Shengxi Liu1, Indira Jutooru2, Gayathri Chadalapaka2, Sung-dae Cho1, Rajesh S. Murthy3, Youngjae You3 and Stephen Safe1,2,*

1 Institute of Biosciences and Technology, Texas A&M University Health Science Center,Houston, TX 77030-3303, USA
2 Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466, USA
3 Department of Chemistry, South Dakota State University, Brookings, SD 57007, USA

* To whom correspondence should be addressed. Tel: +979 845 5988; Fax: +979 862 4929; Email: ssafe{at}cvm.tamu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Betulinic acid (BA) is a phytochemical triterpenoid acid from bark extracts and is cytotoxic to cancer cells and tumors. We modified the A-ring of BA to give a 2-cyano-1-en-3-one moiety and the effects of the 2-cyano-lup-1-en-3-oxo-20-oic acid (CN-BA), 2-cyano derivative of BA, and its methyl ester (CN-BA-Me) were investigated in colon and pancreatic cancer cells. Both CN-BA and CN-BA-Me were highly cytotoxic to Panc-28 pancreatic and SW480 colon cancer cells. CN-BA and CN-BA-Me also induced differentiation in 3T3-L1 adipocytes, which exhibited a characteristic fat droplet accumulation induced by peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists. Based on these results, we investigated the activities of CN-BA and CN-BA-Me as PPAR{gamma} agonists using several receptor-mediated responses including activation of transfected PPAR{gamma}-responsive constructs, induction of p21 in Panc-28 cells and induction of caveolin-1 and Krüppel-like factor 4 in colon cancer cells. The results clearly demonstrated that both CN-BA and CN-BA-Me activated PPAR{gamma}-dependent responses in colon (caveolin-1) and pancreatic (p21) cancer cells, whereas induction of KLF4 by these compounds in colon cancer cells was PPAR{gamma} independent and also dependent on cell context. The PPAR{gamma} agonist activities of CN-BA and CN-BA-Me were structure-, response/gene- and cell context-dependent suggesting that these compounds are a novel class of selective PPAR{gamma} modulators with potential for clinical treatment of colon and pancreatic cancer.

Abbreviations: BA, betulinic acid; C-DIM, methylene-substituted diindolylmethanes; CDDO, 2-cyano-3,12-dioxo-18ß-oleana-1,19-diene-28-oic acid; ß-CDODA, 2-cyano-3,12-dioxo-18ß-olean-1,12-diene-30-oic acid; CN-BA, 2-cyano-lup-1-en-3-oxo-20-oic acid; CN-BA-Me, methyl ester of CN-BA; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; ß-GAL, ß-Galactosidase; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; TBST, Tris-buffered saline containing Tween-20


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Lup-20(29)-ene-3ß,28-diol (betulin) is a triterpene natural product found in extracts of many bushes and trees, and betulin can constitute up to 30% of the dry weight of bark from birch trees (1,2). Betulin has been used in folk medicine for treating skin diseases; however, betulinic acid (BA), which is both a natural product and chemical oxidation product of betulin, induces a broad range of pharmacological activities. BA and several derivatives exhibit anticancer activity, inhibit human immuno virus and other viruses through multiple pathways, are effective anti-bacterial and anti-malarial drugs and exhibit anti-inflammatory activity (1,2). The antitumorigenic activities of BA have been extensively investigated. Studies show that this compound inhibits tumor growth through multiple pathways and these responses are also cancer cell/tumor dependent (1–16). Pisha et al. (3) reported that BA selectively inhibited melanoma cancer cell and tumor growth and in in vivo studies this was accompanied by minimal toxic side effects at repeated doses of up to 500 mg/kg. Subsequent studies showed that BA was cytotoxic to many other cancer cell lines and this was associated with different activities (4–16). For example, BA induces apoptosis through decreased mitochondrial membrane potential, activation of mitogen-activated protein kinase and modulation of nuclear factor {kappa}B (4,5,14).

Structural modifications of BA and other lupane-derived triterpenoids differentially affect their pharmacologic activities (17–23). For example, modification of the C-20 exocyclic position of BA did not affect the cytotoxicity of these derivatives to a panel of prostate and colon cancer and melanoma cell lines (17). In contrast, A-ring modifications of BA containing a 1-ene-3-oxo moiety substituted at C-2 with electron-withdrawing groups were highly cytotoxic (19). These results were similar to ursane and oleanane triterpenoid acids where analogs containing electron-withdrawing substituents at C-2 within a 1-ene-3-one functionality were also highly cytotoxic to cancer cells compared with the parent acids (24–28). Typical among these synthetic derivatives were 2-cyano-3,12-dioxo-18ß-oleana-1,19-diene-28-oic acid (CDDO; synthesized from oleanolic acid) and 2-cyano-3,12-dioxo-18ß-olean-1,12-diene-30-oic acid (ß-CDODA; synthesized from glycyrrhetinic acid, a major constituent of licorice extracts). The high cytotoxicity of ß-CDODA, CDDO and related compounds was due, in part, to their peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist activity and ligands for this receptor are being developed as new anticancer drugs (29,30).

Although there are multiple structural differences between BA and oleanolic acid (the synthetic precursor of CDDO and CDODA) (Figure 1), we hypothesized that introduction of a 2-cyano group into the lupane skeleton of BA would generate a new class of PPAR{gamma} agonists. Previous studies showed that 2-cyano-lup-1-en-3-oxo-20-oic acid (CN-BA), the 2-cyano derivative of 20(29)-dihydro BA, was highly cytotoxic to cancer cells, and in this study, we compared the effects of BA, CN-BA and the corresponding methyl ester (CN-BA-Me) (Figure 1) in Panc-28 pancreatic and colon cancer cell lines. Results of growth inhibition studies showed that both CN-BA and CN-BA-Me were more cytotoxic than BA in pancreatic and colon cancer cells. CN-BA and CN-BA-Me but not BA induced PPAR{gamma}-dependent transactivation; however, the receptor-dependent induction of p21, caveolin1 and Krüppel-like factor 4 expression was cell context and gene dependent. These results demonstrate for the first time that CN-BA and CN-BA-Me are PPAR{gamma} agonists and their enhanced cytotoxicity compared with BA is due, at least in part, to activation of PPAR{gamma}. Moreover, the structure- and cell context-dependent activities of CN-BA and CN-BA-Me as PPAR{gamma} agonists suggest that these compounds are selective PPAR{gamma} modulators.


Figure 1
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  		Fig. 1. Structures of the triterpenoid acid BA, CN-BA/CN-BA-Me and ß-CDODA-Me. The ß-CDODA compounds were derived from glycyrrhetinic acid using a synthetic procedure analogous as outlined in this paper for preparation of CN-BA and CN-BA-Me from BA.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell lines and reagents
SW480, HT-29 and HCT-15 human colon cancer cells were kindly provided by Dr Stan Hamilton (M.D.Anderson Cancer Center, Houston, TX). Panc-28 human pancreatic cancer cells and 3T3-L1 pre-adipocytes were obtained from American Type Culture Collection (Manassas, VA). SW480, HT-29 and Panc-28 cells were maintained in Dulbecco's modified/Ham's F-12 (Sigma–Aldrich, St Louis, MO) with phenol red supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 5% fetal bovine serum and 10 ml/l 100x antibiotic anti-mycotic solution (Sigma). HCT-15 cells were maintained in RPMI 1640 (Sigma) supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 0.45% glucose, 0.24% N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 10% fetal bovine serum and 10 ml/l of 100x antibiotic anti-mycotic solution (Sigma). Cells were maintained at 37°C in the presence of 5% CO2. Reporter lysis buffer and luciferase reagent for luciferase studies were supplied by Promega (Madison, WI). ß-Galactosidase (ß-Gal) reagent was obtained from Tropix (Bedford, MA), and LipofectAMINE reagent was purchased from Invitrogen (Carlsbad, CA). The PPAR{gamma} antagonist N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide (T007) (31) was synthesized in this laboratory, and its identity and purity (>98%) was confirmed by gas chromatography–mass spectrometry.

Synthesis of CN-BA and CN-BA-Me
CN-BA and CN-BA-Me were prepared from betulin (Sigma–Aldrich) based on the previous methods (19). The synthesis from a key intermediate, methyl lup-2-eno[2,3-d]isoxazol-28-oate, is briefly described and only definite peaks in proton NMR are recorded. Methyl Lup-2-eno[2,3-d]isoxazol-28-oate. To a solution of methyl lupan-2-hydroxymethylene-3-oxo-28-oate (350 mg, 0.70 mmol) in ethanol (20 ml) and water (1 ml), hydroxylamine hydrochloride (488 mg, 7.02 mmol) was added. The reaction mixture was refluxed for 1 h, cooled to room temperature and concentrated under vacuum. Water was added to the reaction mixture and extracted with ethyl acetate (2x). The organic layer was then washed with brine (2x), separated, and the crude product was purified by a flash silica gel column using a solvent system of hexanes:ethyl acetate (95:5) to yield methyl lup-2-eno[2,3-d]isoxazol-28-oate as a white cream-colored solid. 1H NMR (400 MHz, CDCl3): {delta} 8.00 (1H, s), 3.68 (3H, s), 1.31, 1.21, 0.99, 0.98, 0.83 (each 3H, s), 0.89 (3H, d, J = 6.8 Hz), 0.78 (3H, d, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3): {delta} 177.6, 173.8, 151.1, 109.7, 57.8, 54.3, 52.0, 49.7, 49.6, 45.0, 43.4, 41.5, 39.7, 38.9, 38.1, 36.6, 35.6, 34.2, 32.8, 30.6, 30.5, 30.4, 29.5, 27.7, 23.8, 23.6, 22.2, 22.0, 19.6, 16.8, 16.5, 15.5, 15.4. Methyl 2-Cyano-lup-3-hydroxy-2-en-28-oate. To a solution of lup-2-eno[2,3-d]isoxazol-28-oate (250 mg, 0.50 mmol) in ether (30 ml) and methanol (15 ml) in an ice bath, 30% sodium methoxide in methanol (3071 mg, 56.87 mmol) was added drop wise. The reaction mixture was then stirred at room temperature for 2 h. After dilution with ether, the reaction mixture was washed with 5% hydrochloric acid (2x). The organic layer was separated and worked up by standard methods to yield crude methyl 2-cyano-lup-3-hydroxy-2-en-28-oate (240 mg, 96%) as a white solid, which was used for the next step without further purification. 1H NMR (400 MHz, CDCl3): {delta} 3.90 (1H, m), 3.67 (3H, s), 1.27, 1.17, 0.98, 0.95, 0.83 (each 3H, s), 0.89 (3H, d, J = 6.8 Hz), 0.79 (3H, d, J = 6.8 Hz). CN-BA-Me. A mixture of 2-cyano-lup-3-hydroxy-2-en-28-oate (230 mg, 0.46 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoguinone (117 mg, 0.52 mmol) in benzene (30 ml) was refluxed for 3 h. The reaction mixture was cooled in ice and filtered to remove reduced 2,3-dichloro-5,6-dicyano-1,4-benzoguinone. The filtrate was then concentrated under vacuum. The crude product was purified by a flash silica gel column using a solvent system of benzene:acetone (98:2) to yield CN-BA-Me (170 mg, 74%) as a pale brown solid. 1H NMR (400 MHz, CDCl3): {delta} 7.83 (1H, s), 3.66 (3H, s), 1.25, 1.18, 1.12, 1.00, 0.95 (each 3H, s), 0.87 (3H, d, J = 6.7 Hz), 0.75 (3H, d, J = 6.6 Hz). 13C NMR (100 MHz, CDCl3): {delta} 199.0, 177.4, 171.6, 115.8, 114.6, 57.6, 53.3, 52.0, 49.4, 45.7, 44.8, 44.4, 43.6, 42.6, 41.5, 38.8, 37.9, 34.2, 32.6, 30.4, 30.2, 28.5, 27.3, 23.7, 23.5, 22.1, 22.0, 19.6, 19.1, 17.2, 15.4, 15.2. ESI-HRMS Calcd for (C32H47NO3 + H): 494.3634. Found: 494.3685. Anal. (C32H47NO3) C, H. CN-BA. A mixture of CN-BA-Me (120 mg, 0.25 mmol) and lithiumiodide (720 mg) in dimethylformamide (2.4 ml) was refluxed for 2 h. The reaction mixture was cooled to room temperature and 5% hydrochloric acid was added. The reaction mixture was extracted with ethyl acetate (2x). The organic layer was then washed with water (2x) followed by washings with brine (2x). The organic layer was separated and worked up by standard methods. The crude product was purified by a flash silica gel column using a solvent system of hexanes:ethyl acetate (80:20) to yield CN-BA (93 mg, 80%) as a light yellow solid. 1H NMR (400 MHz, CDCl3): {delta} 10.29 (1H, broad s), 7.83 (1H, s), 1.27, 1.14, 1.04, 0.99, 0.99 (each 3H, s), 0.91 (3H, d, J = 6.8 Hz), 0.79 (3H, d, J = 6.7 Hz). 13C NMR (100 MHz, CDCl3): {delta} 199.0, 182.1, 171.5, 115.8, 114.8, 57.5, 53.3, 49.3, 45.8, 44.8, 44.4, 43.7, 42.7, 41.5, 39.0, 38.1, 34.3, 32.7, 32.7, 30.5, 30.3, 28.6, 27.4, 23.8, 23.5, 22.2, 22.1, 19.6, 19.2, 17.3, 15.4, 15.3. ESI-HRMS Calcd for (C31H45NO3 + H): 480.3478. Found: 480.3540. Anal. (C31H45NO3) C, H. CN-BA and CN-BA-Me were >97% pure by spectroscopic analysis.

Cell proliferation assay
This assay is performed in 12-well tissue culture plates at the concentration of 2 x 104 cells per well, using Dulbecco’s modified Eagle’s medium (DMEM)/Ham's F-12 media containing 2.5% charcoal-stripped fetal bovine serum (FBS). The cells were counted on the initial day using Z1 cell counter (Beckman Coulter, Fullerton, CA) and then the cells were treated either with vehicle [dimethyl sulfoxide (DMSO)] or the indicated triterpenoid compounds, each sample in triplicate. Every 48 h, fresh medium was added along with the indicated compounds. The count of the cells was taken after 2, 4 and 6 days. The results are expressed as means ± standard errors for each set of triplicate.

Mammalian two-hybrid assay
The GAL4 reporter construct contains 5x GAL4 response elements (p GAL4), were kindly provided by Dr Marty Mayo (University of North California, Chapel Hill, NC). The GAL4-coactivator fusion plasmids pM-SRC1, pMSRC2, pMSRC3, pM-DRIP205, pM-CARM-1 and PPAR{gamma}-VP16 (Vp-PPAR{gamma}) containing the DEF region of the PPAR{gamma} (amino acids 183–505) fused to the pVP16 expression vector were kindly provided by Dr Shigeaki Kato (University of Tokyo, Tokyo, Japan). SW480 colon cancer cells were plated in 12-well tissue culture plates at 1 x 105 cells per well in DMEM/Ham's F-12 medium supplemented with 2.5% charcoal-stripped FBS. After allowing cells to adhere overnight, transient transfections were carried out with GAL4-Luc (0.4 µg), ß-GAL (0.04 µg), VP-PPAR{gamma} (0.04 µg), pM-SRC1 (0.04 µg), pM-PGC-1 (0.04 µg), pM-SMRT (0.04 µg), pM-TRAP220 (0.04 µg), pM-DRIP205 (0.04 µg) and pMCARM1 (0.04 µg) using LipofectAMINE2000 (Invitrogen) following the manufacturer's guidelines. After 6 h of transfection, cells were treated in triplicate either with vehicle (DMSO) or the indicated compound suspended in complete medium for 20–24 h. One hundred microliters per well of 1x Reporter Lysis Buffer (Promega) was used to lyse the cells and 30 µl of this lysate was used to perform the luciferase and ß-GAL assays using Lumicount (Perkin-Elmer Life and Analytical Sciences, Boston, MA). The luciferase activities obtained were normalized to the ß-gal activity.

Transfections
Cells were seeded on to the 12-well plates and 0.4 µg of GAL4-Luc, 0.04 µg of ß-GAL, 0.04 µg of GAL4DBD-PPAR{gamma}, 0.4 µg of p21-luc(FL) containing –2325 to +8 insert, 0.4 µg of p21-luc (–124) containing –124 to +8 insert and 0.4 µg of p21-LUC (–60) containing –60 to +8 insert were transfected using LipofectAMINE reagent (Invitrogen) following the manufacturer's protocol. Cells were treated either with vehicle or respective compounds suspended in complete medium after 6 h of transfection. Cell lysate is extracted after 20–22 h by adding 100 µl of 1x reporter lysis buffer per well and 30 µl of this extract is used to quantitate the luciferase activity using Lumicount (Perkin-Elmer Life and Analytical Sciences). Each experiment is done in triplicate and the results are normalized to the ß-GAL activity.

Western blot analysis
SW480, HT-29, HCT-15 and Panc-28 (3 x 105) colon cancer cells were seeded in 6-well tissue culture plates in DMEM/Ham's F-12 medium containing 2.5% charcoal-stripped FBS. Protein is extracted from the cells treated either with vehicle or indicated compounds suspended for 24 h except for caveolin-1 protein, which was done for 72 h. Samples were extracted in high salt buffer [50 mmol/l N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 500 mmol/l NaCl, 1.5 mmol/l MgCl2, 1 mmol/l ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 10% glycerol and 1% Triton X-100 (pH 7.5) and 5 µl/ml protease inhibitor cocktail (Sigma–Aldrich)]. Samples were incubated at 100°C for 2 min, separated on either 10 or 12% Sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The polyvinylidene difluoride membrane was blocked in 5% TBST-Blotto (10 mM Tris–HCl, 150 mM NaCl, pH 8.0, 0.05% Triton X-100 and 5% non-fat dry milk) for about 30 min and was then incubated in fresh 5% TBST-Blotto with 1:1000 for caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA), 1:1000 for p21(BD Pharmingen, Franklin Lakes, NJ) and 1:10 000 for ß-actin (Sigma) primary antibody overnight with gentle shaking at 4°C. After washing with Tris-buffered saline containing Tween-20 (TBST) for 10 min, the membrane was incubated with respective secondary antibody (1:5000) (Santa Cruz Biotechnology) in 5% TBST-Blotto for 3 h. The membrane is then washed with TBST for 10 min, incubated with chemiluminiscence reagent from Perkin-Elmer for 1 min and then exposed to Kodak X-OMAT AR autoradiography film (Eastman Kodak, Rochester, NY).

Differentiation and Oil red O staining
3T3-L1 pre-adipocytes were cultured on poly-lysine-coated coverslips with DMEM and 10% FBS at 5% CO2 in 24-well plates. At 2 days after confluence, cells were incubated with fresh media supplemented with 3-isobutyl-1-methylxanthine (0.5 mM), dexamethasone (1 µM), insulin (1.7 µM), and indicated compounds (0.25 µM). After 48 h, cells were changed to fresh media and treated with DMSO or indicated compounds for 5 days. Cells without any treatment for the entire 7 days were used as control. The cells were then fixed with 10% formalin. Fixed cells were washed with 60% isopropanol and stained with filtered 60% Oil red O in deionized water. After staining, cells were washed with water and photographed.

Semi-quantitative real-time polymerase chain reaction
SW480 and HT29 colon cancer cells were treated either with vehicle (DMSO) or indicated compounds and after 24 h total RNA was extracted using RNeasy Mini kit (Qiagen, Valencia, CA). RNA concentration was measured by UV 260:280 nm absorption ratio, and 2 µg RNA was used to synthesize cDNA using Reverse Transcription System (Promega). Polymerase chain reaction conditions were as follows: initial denaturation at 94°C (1 min) followed by 28 cycles of denaturation for 30 s at 94°C, annealing for 60 s at 55°C and extension at 72°C for 60 s and a final extension step at 72°C for 5 min. The mRNA levels were normalized using GAPDH as an internal housekeeping gene. Primers obtained from IDT (Coralville, IA) and used for amplification are as follows: KLF4 (sense 5'-CTA TGG CAG GGA GTC CGC TCC-3'; anti-sense 5'-ATG ACC GAC GGG CTG CCG TAC-3') and GAPDH (sense 5'-ACG GAT TTG GTC GTA TTG GGC G-3'; anti-sense 5'-CTC CTG GAA GAT GGT GAT GG-3'). Polymerase chain reaction products were electrophoresed on 1% agarose gels containing ethidium bromide and visualized under UV transillumination.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Figure 2 illustrates the effects of BA, CN-BA and CN-BA-Me on growth of SW480 and Panc-28 cells. All three compounds inhibit growth of both cell lines and IC50 values ranging from 1 to 5, 1 to 2.5 and 1 to 2.5 µM (Panc-28) and 1 to 5, 1.0 and 1 to 2.5 µM (SW480) were observed for BA, CN-BA and CN-BA-Me, respectively. CN-BA was the most cytotoxic compound in both cell lines and this confirms results of a previous report showing that 2-cyano derivatives of BA enhanced cytotoxicity (19). One of the hallmarks of PPAR{gamma} agonists is their induction of differentiation in 3T3-L1 adipocytes, which is characterized by accumulation of fat droplets, which can be detected by Oil red O staining. Results in Figure 1D show that both CN-BA and CN-BA-Me induce Oil red O staining in this assay, whereas BA does not induce this response (data not shown). These results suggest that these 2-cyano derivatives of BA exhibit activity associated with PPAR{gamma} agonists.


Figure 2
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  		Fig. 2. Cell proliferation and adipocyte differentiation assays. Panc-28 and SW480 cells were treated with different concentrations of BA (A), CN-BA (B) or CN-BA-Me (C) for 6 days and the number of cells were counted after treatment for 2, 4 or 6 days as described in Materials and methods. Results are expressed as means ± standard errors for three separate determinations for each treatment group. (D) Effects of CN-BA and CN-BA-Me on differentiation of 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 0.25 µM CN-BA, CN-BA-Me or DMSO. Induction of fat droplets by Oil red O staining was determined as described in Materials and methods. Induction of intense staining for fat droplets was observed in replicate (3) experiments.

 
The PPAR{gamma} agonist activity of BA and related compounds was determined in SW480 cells transfected with PPAR{gamma}-GAL4/pGAL4 and a PPRE3-luc construct (Figure 3A). The results show that 2.5–10 µM CN-BA and CN-BA-Me induced transactivation, whereas BA was inactive in this assay. The PPAR{gamma} agonist activities were also determined in SW480 cells using the same constructs but treated with CN-BA and CN-BA-Me alone or in combination with the PPAR{gamma} antagonist T007, and in all cases, the induced activities were inhibited by T007 (Figure 3B). A similar approach was used in Panc-28 cells transfected with PPAR{gamma}-GAL4/pGAL4 and PPRE3-luc (Figure 3C), and CN-BA induced luciferase activity that was inhibited in cells co-treated with CN-BA plus T007. Not surprisingly, BA was inactive in these assays; however, results obtained for CN-BA-Me were highly inconsistent in Panc-28 cells compared with the colon cancer cell line (Figure 3A and B). CN-BA-Me exhibited minimal induction in cells transfected with PPAR{gamma}-GAL4/pGAL4 and no induction was observed in Panc-28 cells transfected with PPRE3-luc (data not shown). These results were observed in replicate experiments suggesting that there were structure-dependent differences (CN-BA versus CN-BA-Me) for activation of the PPAR{gamma}-GAL4/pGAL4 or a PPRE3-luc constructs in Panc-28 (but not SW480) cells.


Figure 3
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  		Fig. 3. Activation of PPAR{gamma} in SW480 and Panc-28 cells by BA, CN-BA and CN-BA-Me. SW480 cells (A and B) were transfected with PPAR{gamma}-GAL4/pGAL4 or PPRE3-luc treated with DMSO (control) or different concentrations of the compounds, and luciferase activity determined as described in Materials and methods. (C) Activation of PPAR{gamma} in Panc-28 cells. Cells were transfected with PPAR{gamma}-GAL4/pGAL4 or PPRE3-luc treated with DMSO or different concentrations of CN-BA and CN-BA-Me alone or in combination with 10 µM T007, and luciferase activity determined as described in Materials and methods. Results in (A–C) are expressed as means ± standard errors for three replicate determinations for each treatment group, and significant (P < 0.05) induction by the BA derivatives (*) and inhibition after co-treatment with T007 (**) are indicated. For studies in Panc-28 cells, we only observed induction of luciferase activity using CN-BA and not with CN-BA-Me or BA over several sets of experiments. (D) Mammalian two-hybrid assay in SW480 cells transfected with VP-PPAR{gamma} and GAL4-coactivator chimeras. SW480 cells were transfected with VP-PPAR{gamma}, coactivator-GAL4/pGAL4, treated with different concentrations of CN-BA or CN-BA-Me and 5 µM ß-CDODA-Me, and luciferase activity was determined as described in Materials and methods. Results are expressed as means ± standard errors for three replicate determinations for each treatment group, and significant (P < 0.05) induction is indicated by an asterisk.

 
PPAR{gamma} agonists are structurally diverse and induce tissue-specific receptor-dependent responses that are typical of selective PPAR{gamma} modulators (29,30). Similar results have been observed for agonists that bind and activate other nuclear receptors and this structure-dependent effect is due, in part, to tissue-specific expression of coactivators and other nuclear proteins that exhibit ligand structure-dependent interactions with receptors. Results in Figure 3D summarize the effects of CN-BA and CN-BA-Me on induction of luciferase activity in SW480 cells transfected VP-PPAR{gamma} and GAL4 coactivator and GAL4-SMRT (a co-repressor) expression plasmids. We used ß-CDODA-Me, a triterpenoid methyl ester derivative, which also contains a 2-cyano-1-en-3-one function (Figure 1) and activates PPAR{gamma} in colon cancer cells (28), as a comparative reference compound for the mammalian two-hybrid assay. The results show that CN-BA, CN-BA-Me and ß-CDODA-Me significantly induced luciferase activity in SW480 cells transfected with VP-PPAR{gamma} and GAL4-PGC-1 and GAL4-SRC-1, but not GAL4-AIB1, GAL4-TIFII, GAL4-TRAP220 and GAL4-SMRT. In contrast, only CN-BA-Me also activated GAL4-CARM1 indicating differences between CN-BA and CN-BA-Me in the mammalian two-hybrid assay, suggesting that even among these two acid–ester analogs, some tissue-specific selective PPAR{gamma} modulator activity might be expected. The data are consistent with the differences observed for CN-BA and CN-BA-Me in activation of transfected constructs in Panc-28 cells (Figure 3C).

Previous studies in this laboratory have shown that PPAR{gamma} agonists induce p21 and p27 and decrease cyclin D1 expression in Panc-28 cells, and only the former response is receptor dependent (32). Results in Figure 4C show that both CN-BA and CN-BA-Me induce p21 protein expression in Panc-28 cells, and this is also accompanied by induction of p27 and down-regulation of cyclin D1 (data not shown) as reported previously for a series of PPAR{gamma}-active methylene-substituted diindolylmethanes (C-DIM) analogs in this cell line (32). Co-treatment of Panc-28 cells with 5 µM CN-BA and CN-BA-Me plus the 10 µM T007 significantly inhibited induction of p21, confirming that induction of p21 was PPAR{gamma} dependent (Figure 4A). In contrast, induction of p21 by BA was not inhibited after co-treatment with T007 and this was consistent with results of transactivation studies showing that BA does not activate PPAR{gamma} in Panc-28 or SW480 (Figure 3). Figure 4B shows that BA, CN-BA and CN-BA-Me induce transactivation in Panc-28 cells transfected with p21-luc(Fl), which contains the –2325 to +8 region of the p21 promoter. In cells co-treated with BA and related compounds plus the PPAR{gamma} antagonist T007, the induction of luciferase activity by CN-BA and CN-BA-Me was inhibited, whereas BA-induced activity was unaffected by T007. The results complement the immunoblot data confirming that induction of p21 by CN-BA/CN-BA-Me was PPAR{gamma} dependent, whereas induction of p21 by BA was PPAR{gamma} independent. We further investigated induction of luciferase activity in Panc-28 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 (1–6) and the results of the transfection studies suggest that these GC-rich sites are necessary for CN-BA- and CN-BA-Me-induced transactivation. Deletion analysis of the p21 promoter indicated that loss of inducibility [i.e. p21-luc(60)] was associated with loss of GC-rich sites 3 and 4, whereas CN-BA significantly induced activity but only at the 7.5 µM concentration, suggesting sites 3 and 4 were also important for this compound but induction could also be observed using constructs containing only GC-rich sites 5 and 6. Previous studies show that PPAR{gamma}-dependent activation of p21 by other PPAR{gamma} agonists is also dependent on GC-rich sites 3 and 4 and involves PPAR{gamma}/Sp-dependent activation of p21. The ligand-dependent recruitment of PPAR{gamma} to the p21 promoter by CN-BA and CN-BA-Me was further investigated in a ChIP assay in Panc-28 cells treated with the BA, CN-BA and CN-BA-Me for 1 or 2 h. The results (Figure 4D) show that both CN-BA and CN-BA-Me recruited PPAR{gamma} to the proximal GC-rich region of the p21 promoter and this was also accompanied by enhanced binding of Sp1. In contrast, BA did not induce PPAR{gamma} interactions with the p21 promoter in the ChIP assay. This is consistent with receptor-independent activation of p21 by BA and the mechanism of this response is currently being investigated. As a control for this experiment, transcription factor TFIIB bound to the proximal region of the GAPDH gene promoter but not to exon 1 of the CNAP1 gene.


Figure 4
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  		Fig. 4. Induction of p21 by BA, CN-BA and CN-BA-Me in Panc-28 cells. (A) Induction of p21 protein. Panc-28 cells were treated with the different compounds as indicated for 24 h, and whole-cell lysates were obtained and analysed by immunoblots as described in Materials and methods. Induction of p21-luc (B) and p21 deletion constructs (C) in Panc-28 cells. Cells were transfected with the various constructs, treated with DMSO, BA, CN-BA, CN-BA-Me alone or in combination with T007, and luciferase activity determined as described in Materials and methods. Results of all transactivation studies in this figure are presented as means ± standard errors for at least three separate determinations for each treatment group. Significant (P < 0.05) induction compared with solvent (DMSO) control (*) and inhibition by co-treatment with T007 (**) are indicated. (D) Chromatin immunoprecipitation assays. Primers designed for the proximal region of the p21 promoter (i) were used for a ChIP assay in Panc-28 cells and (ii) treated with DMSO, 5 µM BA, 5 µM CN-BA and 5 µM CN-BA-Me for 1 or 2 h. Analysis of interactions of Sp1 and PPAR{gamma} with the p21 promoter were carried out in the ChIP assay as described in Materials and methods. The ChIP assay was also used to examine binding of TFIIB to the GAPDH promoter (positive control) (iii) and to exon 1 of CNAP1 (negative control) as described in Materials and methods.

 
PPAR{gamma} agonists such as CDDO, ß-CDODA and related esters and PPAR{gamma}-active C-DIMs also induce receptor-dependent expression of caveolin-1 in colon cancer cells (28,33–35). Figure 5A shows that CN-BA and CN-BA-Me but not BA induce caveolin-1 in HT-29 cells and similar results were observed in HCT-15 cells (Figure 5B). In contrast, BA, CN-BA and CN-BA-Me did not induce caveolin-1 expression in SW480 cells, and the latter two compounds decreased expression of this protein (Figure 5C). Co-treatment of HT-29 and HCT-15 cells with CN-BA/CN-BA-Me plus the PPAR{gamma} antagonist T007 resulted in inhibition of the induced caveolin-1 response, confirming that induction was PPAR{gamma} dependent (Figure 5D). Thus, receptor-dependent activation of caveolin-1 by CN-BA and CN-BA-Me was dependent on cell context and this correlated with results of previous studies with CDDO and the 18{alpha} and 18ß isomers of CDODA-Me where CDDO and {alpha}-CDODA-Me but not ß-CDODA-Me induced caveolin-1 in HT-29 and SW480 cells, whereas like CN-BA/CN-BA-Me, ß-CDODA induced caveolin-1 in HT-29 but not in SW480 cells.


Figure 5
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  		Fig. 5. Induction of caveolin-1 expression in colon cancer cells. HT-29 (A), HCT-15 (B) and SW480 (C) cells were treated with DMSO, different concentrations of BA, CN-BA or CN-BA-Me for 72 h. Caveolin-1 expression was determined by western blot analysis as described in Materials and methods. Similar results were observed in replicate experiments. (D) Effects of T007 on induction of caveolin-1. HCT-15 or HT-29 cells were treated with DMSO or different concentrations of CN-BA and CN-BA-Me alone or in combination with 5 µM T007 and caveolin-1 expression was determined by western blot analysis as described in Materials and methods.

 
Previous studies showed that {alpha}- and ß-CDODA-Me induced the tumor suppressor gene KLF-4 in HT-29 and SW480 colon cancer cells (28), and the results in Figure 6 summarize the effects of CN-BA and CN-BA-Me on KLF4 expression in HT-29 and SW480 cells. In the former cell line, CN-BA and CN-BA-Me induced KLF4 mRNA levels and similar results were observed for ß-CDODA-Me, which was used as a positive control for this cell line. However, in HT-29 cells co-treated with CN-BA, CN-BA-Me and ß-CDODA-Me plus the PPAR{gamma} antagonist T007, induction of KLF4 was significantly decreased only for ß-CDODA-Me. In contrast, CN-BA and CN-BA-Me did not induce KLF4 expression in SW480 cells, whereas ß-CDODA-Me treatment enhanced KLF4 mRNA as described previously (28). The differences between CN-BA/CN-BA-Me and ß-CDODA-Me as inducers of KLF4 mRNA levels in colon cancer cells clearly distinguished between two classes of structurally related PPAR{gamma} agonists derived from triterpenoid acids and confirm that CN-BA/CN-BA-Me are a novel class of PPAR{gamma} antagonists. In addition, we also confirmed that BA/CN-BA induced apoptosis in SW480 and Panc-28 cells, and results in Figure 6C show that both compounds induced caspase-dependent PARP cleavage in these cell lines.


Figure 6
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  		Fig. 6. Induction of KLF4 gene expression apoptosis by BA and related compounds. Induction of KLF4 in HT-29 (A) and SW480 (B) cells. Cells were treated with different concentrations of BA derivatives, ß-CDODA-Me or T007 alone or in combination, and KLF4 mRNA levels were determined by real-time polymerase chain reaction as described in Materials and methods. Each experiment was replicated (>3x) and T007 did not inhibit KLF4 mRNA induction by BA, CN-BA and CN-BA-Me, whereas 60–80% of the response induced by ß-CDODA-Me was inhibited by T007. KLF4 mRNA levels were not induced in SW480 cells by BA derivatives. (C) Induction of apoptosis. Cells were treated for 24 h with BA and related compounds, and whole-cell lysates were examined by western blot analysis as described in Materials and methods.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
PPAR{gamma} is over-expressed in tumors from multiple tissues and cell lines (36) and PPAR{gamma} agonists are being developed as mechanism-based drugs for cancer chemotherapy. PPAR{gamma} agonists typically inhibit cancer cell growth and this is associated with induction of p21 and/or p27 and down-regulation of cyclin D1 and cells treated with these compounds also exhibit morphological and biochemical features of apoptosis. The mechanisms of the growth inhibitory/pro-apoptotic responses induced by different structural classes of PPAR{gamma} agonists are cell context and gene dependent, and induction of both receptor-dependent and independent responses are observed. For example, the thiazolidinedione troglitazone induces non-steroidal inflammatory drug-activated gene-1 (NAG-1) in HCT-116 colon cancer cells through receptor-independent activation of kinase pathways, whereas induction of NAG-1 by 15-deoxy-{Delta}12,14-prostaglandin J2 in HCT-116 cells is PPAR{gamma} dependent and inhibited by PPAR{gamma} antagonist (37–39). PPAR{gamma}-active C-DIMs induce caveolin-1 expression in HT-29 and HCT-15 colon cancer cells, whereas rosiglitazone induced caveolin-1 only in the former cell lines (33). The induction responses by both compounds were inhibited by PPAR{gamma} antagonists and cell context-dependent differences of C-DIMs and rosiglitazone in HCT-15 cells were associated with expression of mutant PPAR{gamma} (K422Q) in this cell line (34) and the mutant receptor was insensitive to rosiglitazone.

CDDO and its related methyl ester and imidazole derivatives are PPAR{gamma} agonists (27,35) and are potent anticancer drugs currently undergoing clinical trials. These triterpenoid acid derivatives of oleanolic acid, a phytochemical used in traditional medicine induce multiple receptor-independent and some receptor-dependent responses including the receptor-dependent induction of caveolin-1 in colon cancer cells (35). Although oleanolic acid is only weakly cytotoxic to cancer cells, the introduction of the 2-cyano-substituted 1-en-3-oxo moiety into the A-ring of oleanolic acid greatly enhanced the cytotoxicity of the resulting 2-cyano derivatives including CDDO that also has an enone system in the C-ring (24–26) and similar results were observed for the corresponding 2-cyano derivatives of glycyrrhetinic acid, namely {alpha}-CDODA and ß-CDODA-Me (28). Moreover, in studies with glycyrrhetinic acid analogs, it was shown that the 2-cyano group was required for PPAR{gamma} agonist activity.

The major structural differences between BA and oleanolic and glycyrrhetinic acids are their 5- and 6-member E-rings, respectively (Figure 1), and the position of substituents in this ring. However, despite these structural differences, introduction of the 2-cyano-1-ene-3-oxo system into the A-ring of BA gave CN-BA, which activated PPAR{gamma} in both SW480 and Panc-28 cell lines (Figures 2 and 3) and enhanced the cytotoxicity of these compounds compared with BA (Figure 1). Surprisingly, CN-BA-Me activated PPAR{gamma}-GAL4/pGAL4 and PPRE-luc in SW480 but was much less effective in activating these constructs in Panc-28 cells, and these cell context-dependent differences in CN-BA and CN-BA-Me suggest that these compounds may be selective PPAR{gamma} agonists or modulators.

Selective receptor modulators exhibit tissue-selective receptor agonist activities and differences between diverse structural classes of these compounds can be discerned in mammalian two-hybrid assays using VP-PPAR and GAL4-coactivator chimeras (28,33,35). This assay has some relevance for identifying selective receptor modulators since differences between selective receptor modulators may be due, in part, to their interaction with coactivator proteins. Induction of luciferase activity in colon cancer cells transfected with VP-PPAR{gamma}/GAL4-coactivator constructs is dependent on the coactivator and structure of the PPAR{gamma} agonist (28,33,35). C-DIM PPAR{gamma} agonists induce transactivation in cells transfected with GAL4-PGC-1 (33), whereas CDDO and CDDO-Me are active in cells transfected with GAL4-chimeras containing SRC-1, SRC-2 (TIFII), SRC-3 (A1B1), TRAP220, PGC-1 and CARM-1 (35). ß-CDODA and {alpha}-CDODA-Me activate GAL4-chimeras containing PGC-1 and SRC-1 and PGC-1 and SRC-2, respectively (28). CN-BA activates GAL4-chimeras containing PGC-1 and SRC-1 in SW480 cells and resembles ß-CDODA-Me. CN-BA-Me activates PGC-1, SRC-1 and CARM-2 (Figure 3D), whereas these compounds did not activate GAL4-chimeras containing SRC-2, TRAP220 or SMRT (data not shown). The unique pattern for CN-BA-Me in the mammalian two-hybrid assay highlights differences that are dependent only on methylation of the 20-carboxyl group in the E-ring, and these results are consistent with differences between CN-BA and CN-BA-Me in their activation of transfected PPAR{gamma}-responsive constructs in Panc-28 cells (Figure 3C).

The PPAR{gamma} agonist activities of CN-BA and CN-BA-Me and their role as selective receptor modulators were further investigated using four receptor-mediated responses, namely (i) the induced differentiation of 3T3-L1 adipocytes, (ii) induction of the cyclin-dependent kinase inhibitor p21 in Panc-28 cells and the induction of (iii) caveolin-1 and (iv) KLF4 in colon cancer cells. Both CN-BA and CN-BA-Me induced differentiation of 3T3-L1 adipocytes and this was characterized by accumulation of fat droplets, which are visualized by Oil red O staining (Figure 2D). Previous studies showed that PPAR{gamma}-active C-DIMs induced p21 expression in Panc-28 cells and this response was associated with interactions of PPAR{gamma} with the proximal GC-rich region of the p21 promoter (32). Similar results were obtained for both CN-BA and CN-BA-Me that induced p21 expression in Panc-28 cells and reporter gene activity in cells transfected with p21-luc(F1), and both responses were inhibited after co-treatment with PPAR{gamma} antagonist T007 (Figure 4A). Deletion analysis of the p21 promoter suggested that for CN-BA-Me, GC-rich sites 5 and 6 were required for activation of p21, whereas CN-BA also induced activity with p21-luc(60) that only contained GC-rich sites 5 and 6. Nevertheless, sites 3 and 4 appear to play an important role for both CN-BA-Me and CN-BA, and these same sites were also required for PPAR{gamma}-dependent activation of p21 by C-DIMs (32). It has also been reported previously that progesterone receptor and androgen receptor agonists induce p21 through receptor-Sp protein interactions with GC-rich sites 3 and 4 and site 3, respectively (40,41), suggesting that these GC-rich sites in the p21 promoter are important targets for nuclear receptors. We also showed that BA induced PPAR{gamma}-independent activation of p21, and the differences between BA versus CN-BA/CN-BA-Me are evident not only after treatment with the PPAR{gamma} antagonist T007 (Figure 4A and B) but also in the recruitment of PPAR{gamma} to the p21 promoter by CN-BA/CN-BA-Me but not BA in a ChIP assay in Panc-28 cells (Figure 4D).

In a recent study with the 18{alpha} and 18ß isomers of CDODA-Me, we showed that both compounds induced caveolin-1 in HT-29 and HCT-15 cells, whereas only {alpha}-CDODA-Me induced caveolin-1 in SW480 cells (28). These results suggested that in SW480 cells, the stereochemistry at C-18 of CDODA that influences the confirmation of the E-ring also differentially affected PPAR{gamma}-dependent activation of caveolin-1. CN-BA and CN-BA-Me but not BA induced caveolin-1 in HT-29 and HCT-15 cells and co-treatment with the PPAR{gamma} antagonist T007 inhibited the induction response (Figure 6). In contrast, CN-BA and CN-BA-Me do not induce caveolin-1 in SW480 cells as observed previously for 18ß isomer of CDODA; however, the stereochemistry at C-18 for the BA derivatives is {alpha}, suggesting that the cell context-dependent activation of caveolin-1 by the cyano-substituted triterpenoid acids is dependent not only on the stereochemistry at C-18 but also on the structure of the E-ring. The presence of the 5-membered E-ring with carboxy and isopropyl substituents (Figure 1) resulted in loss of PPAR{gamma}-dependent induction of caveolin-1 in SW480 cells by CN-BA and CN-BA-Me and this cell context-dependent response was consistent with the activity of these compounds as selective receptor modulators. The cytotoxicity of BA and CN-BA derivatives (Figure 2) was due not only to growth inhibition but also to induction of apoptosis (Figure 6C), which was not inhibited by PPAR{gamma} antagonists (data not shown), suggesting a receptor-independent pro-apoptotic pathway which is currently being investigated.

The CDODA-Me compound induced the tumor suppressor gene KLF4 in SW480 and HT-29 colon cancer cells, and this response was inhibited by T007 (28). In contrast, CN-BA/CN-BA-Me did not induce KLF4 mRNA in SW480 cells and induction of this gene in HT-29 cells was receptor independent. These data, coupled with the effects of the cyano-substituted compounds on transactivation in the mammalian two-hybrid and reporter gene assays, adipocyte differentiation, p21 and caveolin-1 expression, demonstrate that CN-BA and CN-BA-Me represent a novel class of selective PPAR{gamma} agonists in colon and pancreatic cancer cells. The concentration-dependent differences in the activation of p21 and PPAR{gamma}-GAL4/PPRE3-luc (≥2.5 µM) and induction of Oil red O staining and caveolin-1 (≤0.5 µM) may be due, in part, to relatively short (24 h) and longer (72–120 h) treatment times, respectively. However, differences in gene responsiveness may also be due to other nuclear proteins and competition by receptor complexes bound to response elements on different gene promoters for common nuclear cofactors. The activities of CN-BA and CN-BA-Me coupled with their cytotoxicity (Figure 2) suggest that the receptor-dependent and independent responses induced by these compounds will be advantageous for further development of these compounds for clinical applications in the treatment of colon and pancreatic cancer.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (ES09106, CA108718 and CA112337 [GenBank] ); the M.D.Anderson Cancer Center Pancreatic Cancer Spore (P20-CA10193) and the Texas Agricultural Experiment Station.


   Footnotes
 
{dagger} Both authors contributed equally to this work. Back


   Acknowledgments
 
Conflict of interest statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received June 8, 2007; revised August 13, 2007; accepted August 13, 2007.


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