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-Mediated Growth Inhibition, Transactivation, and Differentiation Markers in Colon Cancer Cells
1 Departments of Biochemistry and Biophysics, 2 Veterinary Pathobiology, and 3 Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas; 4 Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, Texas; and 5 Division of Pathology and Laboratory Medicine, M. D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR)-mediated transactivation in HT-29, HCT-15, RKO, and SW480 colon cancer cell lines. Rosiglitazone also induces transactivation in these cell lines and inhibited growth of HT-29 cells, which express wild-type PPAR but not HCT-15 cells, which express mutant (K422Q) PPAR. In contrast, DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 inhibited growth of both HT-29 and HCT-15 cells with IC50 values ranging from 1 to 10 µmol/L. Rosiglitazone and diindolylmethane (DIM) analogues did not affect expression of cyclin D1, p21, or p27 protein levels or apoptosis in HCT-15 or HT-29 cells but induced keratin 18 in both cell lines. However, rosiglitazone induced caveolins 1 and 2 in HT-29 but not HCT-15 cells, whereas these differentiation markers were induced by DIM-C-pPhCF3 and DIM-C-pPhC6H5 in both cell lines. Because overexpression of caveolin 1 is known to suppress colon cancer cell and tumor growth, the growth inhibitory effects of rosiglitazone and the DIM compounds are associated with PPAR-dependent induction of caveolins. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has been extensively investigated and ligands for this receptor typically induce adipocyte differentiation and mediate tissue-specific anti-inflammatory responses. Synthetic thiazolidinediones (TZDs) such as rosiglitazone and pioglitazone are PPAR agonists that have been developed for treatment of insulin-resistant type 2 diabetes (1, 2, 3, 4)
.
Ikezoe et al. (5)
reported expression of wild-type PPAR mRNA in tumors from multiple tissues, including lung, breast, colon, prostate, osteosarcomas, acute myelogenous leukemia, adult T-cell leukemia, glioblastomas, B-cell acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, and myelodisplastic syndrome. In addition, PPAR mRNA was also expressed in 71 different cancer cell lines derived from hematopoietic and nonhematopoietic (lung, duodenal, prostate, breast, glioblastomas, and colon) tumors. Surprisingly, the reported PPAR mutations in colon cancer (6)
were not observed in the 10 colon cancer cell lines and 58 clinical samples examined in this study.
Studies in this laboratory have characterized a series of 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes [methylene or C-substituted diindolylmethanes (DIMs)] that activate PPAR-dependent transactivation and inhibit growth of MCF-7 breast cancer cells (7)
. Compounds containing p-CF3 (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and p-phenyl (DIM-C-pPhC6H5) were the most potent activators of PPAR and inhibitors of cell growth. Growth inhibition by C-substituted DIMs in MCF-7 cells was associated, in part, with proteasome-dependent down-regulation of cyclin D1.
A recent study reported that several colon cancer cells expressed either wild-type or mutant PPAR containing a point mutation at codon 422 (K422Q; ref. 8
). HCT-15 and other cells that express mutant PPAR were resistant to the growth inhibitory and differentiation-inducing effects of rosiglitazone, whereas these responses were induced by rosiglitazone in cells (e.g., HT-29) expressing wild-type PPAR. Results of this study show that PPAR-active C-substituted DIMs induce PPAR-dependent transactivation and differentiation-induced genes/proteins in both rosiglitazone-responsive (HT-29) and -nonresponsive (HCT-15) colon cancer cell lines. Caveolin 1 and caveolin 2 proteins were induced by rosiglitazone only in HT-29 cells, whereas C-substituted DIMs induced these proteins in both HT-29 and HCT-15 cells. The cell context-dependent growth inhibitory responses of rosiglitazone and PPAR-active C-substituted DIMs correlated with induction of caveolins, which exhibit tumor suppressor activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(sc-7273), Sp1 (sc-59), bcl-2, bax, poly(ADP-ribose) polymerase, cyclin D1 (sc-718), p21 (sc-756), p27 (sc-528), and caveolin 1 (sc-894) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Keratin-18 antibody was purchased from NeoMarkers (Fremont, CA). Caveolin 2 antibody was obtained from Transduction Laboratories (Lexington, KY). Reporter lysis buffer and luciferase reagent for luciferase studies were purchased from Promega (Madison, WI). ß-Galactosidase (ß-Gal) reagent was obtained from Tropix (Bedford, MA). Lipofectamine reagent and Plus reagent were supplied by Invitrogen (Carlsbad, CA). Western Lightning chemiluminescence reagent were from Perkin-Elmer Life Sciences (Boston, MA). The C-substituted DIMs were prepared in this laboratory by condensation of indole with p-substituted benzaldehydes, and compounds were >95% pure by gas chromatography-mass spectrometry.
Plasmids.
The Gal4 reporter containing 5x Gal4 response elements (pGal4) was kindly provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). Gal4DBD-PPAR construct (gPPAR) was a gift of Dr. Jennifer L. Oberfield (Glaxo Wellcome Research and Development, Research Triangle Park, NC). The PPAR-VP16 fusion plasmid (VP-PPAR) contained the DEF region of PPAR (amino acids 183–505) fused to the pVP16 expression vector and the GAL4-coactivator fusion plasmids pM-SRC1, pMSRC2, pMSRC3, pM-DRIP205, and pM-CARM-1 were kindly provided by Dr. Shigeaki Kato (University of Tokyo, Tokyo, Japan).
Transfection and Luciferase Assay.
Colon cancer cells were plated in 12-well plates at 1 x 105 cells/well in DMEM:Ham’s F-12 media supplemented with 2.5% charcoal-stripped FBS. After growth for 16 hours, various amounts of DNA, i.e., Gal4Luc (0.4 µg), ß-gal 0.04 µg), VP-PPAR (0.04 µg), pM SRC1 (0.04 µg), pMSRC2 (0.04 µg), pMSRC3 (0.04 µg), pMDRIP205 (0.04 µg), and pMCARM-1 (0.04 µg) were transfected by Lipofectamine or Lipofectamine Plus (Invitrogen) according to the manufacturer’s protocol. After 5 hours of transfection, the transfection mix was replaced with complete media containing either vehicle (DMSO) or the indicated ligand for 20 to 22 hours. Cells were then lysed with 100 mL of 1x reporter lysis buffer, and 30 µL of cell extract were used for luciferase and ß-gal assays. Lumicount was used to quantitate luciferase and ß-gal activities, and the luciferase activities were normalized to ß-gal activity.
Cell Proliferation Assay.
Cells were plated at a density of 2 x 104/well in 12-well plates and replaced the next day with DMEM:Ham’s F-12 media containing 2.5% charcoal-stripped FBS and either vehicle (DMSO) or the indicated ligand and concentration. Fresh media and compounds were added every 48 hours. Cells were counted at the indicated times using a Coulter Z1 cell counter. Each experiment was done in triplicate and results are expressed as means ± SE for each determination.
Fluorescence-Activated Cell Sorting Analysis.
HT-29 and HCT-15 cells were synchronized in serum-free media for 3 days and then treated with either the vehicle (DMSO) or the indicated compounds for 24 hours. Cells were trypsinized, centrifuged, and resuspended in staining solution containing 50 µg/mL propidium iodide, 4 mmol/L sodium citrate, 30 units/mL RNase, and 0.1% Triton X-100. After incubation at 37°C for 10 minutes, sodium chloride was added to give a final concentration of 0.15 mol/L. Cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA), using CellQuest (Becton Dickinson Immunocytometry Systems) acquisition software. Propidium iodide (PI) fluorescence was collected through a 585/42-nm bandpass filter, and list mode data were acquired on a minimum of 20,000 single cells defined by a dot plot of PI width versus PI area. Data analysis was performed in ModFit LT (Verity Software House, Topsham, ME) using PI width versus PI area to exclude cell aggregates.
Western Blot Analysis.
HT-29 and HCT-15 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. For the apoptosis experiments, cells were treated for 72 h with various compounds. Western blot analysis of whole cell lysates was determined as described previously (7
, 9)
. Band intensities were evaluated by scanning laser densitometry (Sharp Electronics Corporation, Mahwah, NJ) using Zero-D Scanalytics software (Scanalytics Corporation, Billerica, MA).
Statistical Analysis.
Statistical differences between different groups were determined by ANOVA and Scheffe’s test for significance. The data are presented as mean ± SD for at least three separate determinations for each treatment.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Active C-Substituted DIMs.
and Table 1
. The results obtained in HT-29 and HCT-15 cells, which express wild-type and mutant (K422Q) PPAR (8)
, are illustrated in Fig. 1, A and B
. Rosiglitazone significantly inhibited growth of HT-29 cells at concentrations of 1, 5, and 10 µmol/L; however, the concentration required for IC50 was >10 µmol/L. In contrast, 1 µmol/L concentrations of DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 significantly (P < 0.05) inhibited growth of HT-29 cells, and IC50 values were <5 µmol/L for all three compounds. Rosiglitazone did not inhibit proliferation of HCT-15 cells (8)
; the PPAR-active C-substituted DIMs all significantly inhibited growth at 5 or 10 µmol/L concentrations, and IC50 values were <10 µmol/L. The results in Table 1
show that rosiglitazone treatment (1 to 10 µmol/L) minimally affected growth of SW480 or RKO cells, whereas IC50 values for the PPAR-active C-substituted DIMs varied from 1 to 5 or 5 to 10 µmol/L with DIM-C-pPhCF3 as the most potent compound in both cell lines.
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. The PPAR agonists decreased the percentage of HT-29 cells in S phase and increased the percentage in G0-G1. Rosiglitazone did not affect the percentage of HCT-15 cells in G0-G1, S, or G2-M phase of the cell cycle, and this was consistent with the failure of this compound to inhibit proliferation of this cell line (Fig. 1B)
. Surprisingly, 10 µmol/L DIM-C-pPhCF3 and DIM-C-pPhC6H5 also did not alter the percentage distribution of HCT-15 cells in G0-G1, S, or G2-M phase, although both compounds significantly inhibited growth of these cells (Fig. 1)
. Previous studies with HCT-15 cells showed that the PPAR agonist BRL-49653 did not inhibit growth of these cells or block G0-G1
S-phase progression (10)
.
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-Dependent Transactivation and Coactivator Interactions by Rosiglitazone, DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5. is expressed in SW480, RKO, HT-29, and HCT-15 cells (data not shown), and ligand-dependent activation of PPAR in HCT-15 and HT-29 colon cancer was determined in cells transfected with chimeric PPAR-GAL4 and a GAL4 response element plasmid (pGAL4) containing five tandem GAL4 response elements linked to a luciferase reporter gene (Fig. 3, A and B)
. Rosiglitazone (1 to 10 µmol/L) and 1 to 10 µmol/L DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 induced reporter gene activity in HCT-15 and HT-29 cells, and similar results were observed in RKO and SW480 cells (data not shown). The magnitude of the induction responses were <6-fold in the HCT-15 and HT-29 cells, and the relative potencies of the individual compounds were variable and dependent on cell context. DIM-C-pPhCH3 exhibited lower activity in the transactivation assays in the colon cancer cells as previously reported in breast cancer cell lines (7)
. Results in Fig. 3, C and D
, show that induction of luciferase activity by rosiglitazone and PPAR-active C-substituted DIMs in HT-29 and HCT-15 cells transfected with PPAR-GAL4/pGAL4 was inhibited by GW9662, a PPAR antagonist. Results summarized in Fig. 3E
show that 7.5 µmol/L GW9662 also significantly blocks the growth inhibitory effects of 2.5, 5.0, or 7.5 µmol/L DIM-C-pPhC6H5 in HCT-15 cells. In contrast, GW9662 alone was growth inhibitory in HT-29 cells. These data confirm the role of PPAR in mediating inhibition of HCT-15 cell growth by DIM-C-pPhC6H5.
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12,14-prostaglandin J2 (PGJ2) but not TZDs induced transactivation in a mammalian two-hybrid assay in COS-1 cells transfected with VP-PPAR and GAL4-coactivator chimeras (coactivators = SRC-1, SRC-2, SRC-3, and DRIP205/TBP/TRAP220). Therefore, we investigated ligand-dependent interactions of VP-PPAR with chimeric GAL4-coactivator in colon cancer cells transfected with the pGAL4-luc reporter gene construct (Fig. 4)
. PPAR-active C-substituted DIMs and rosiglitazone induced transactivation in HT-29 cells transfected with VP-PPAR and GAL4-PGC-1, and minimal interactions were observed using chimeric proteins containing SRC-1, SRC-2, SRC-3, DRIP205, or CARM-1. DIM-C-pPhCF3 also induced transactivation (< 2-fold) in cells transfected with GAL4-SRC-3, and DIM-C-pPhC6H5 did not significantly induce activity in cells transfected with GAL4-PGC-1. PPAR-active C-substituted DIMs induced transactivation in HCT-15 cells transfected with GAL4-PGC-1 and with one exception (DIM-C-pPhtBu/GAL4-SRC2), minimal interactions with other coactivators were observed. In SW480 and RKO colon cancer cells, a variable pattern of PPAR-coactivator interactions were observed and the PPAR-active C-substituted DIMs but not rosiglitazone-induced interactions with PGC-1 (data not shown).
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Agonists.-active C-substituted DIMs on cyclin D1, p21, and p27 protein expression were investigated in HT-29 and HCT-15 after treatment with DMSO (control) and 5 or 7.5 µmol/L concentrations of these compounds for 24 hours. The results show that none of the treatments significantly affected levels of cyclin D1, p21, or p27 proteins (Fig. 5)
, and protein levels at other time points (6 and 12 hours) were also unchanged.
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agonists induce genes associated with differentiation in preadipocytes and cancer cell lines, and results in Fig. 6
show that keratin 18 is constitutively expressed in both HT-29 and HCT-15 cells. In the former cell line, 5 and 7.5 µmol/L rosiglitazone, DIM-C-pPhCF3, and DIM-C-pPhC6H5 significantly induced keratin 18 protein, and a maximal 2-fold induction response was observed for 7.5 µmol/L DIM-C-pPhC6H5. Rosiglitazone and PPAR-active C-substituted DIMs also induced a 2.5 to 3-fold increase in keratin 18 protein in HCT-15 cells, showing that induction of this differentiation marker did not correlate with expression of wild-type or mutant PPAR. A recent study showed that caveolins 1 and 2 were induced by TZDs in HT-29 cells (12)
. Rosiglitazone, DIM-C-pPhCF3, and DIM-C-pPhC6H5 also significantly induced caveolins 1 and 2 in this cell line, and protein levels were increased by 5 to 9-fold after treatment for 72 hours. Similar results were observed in HT-29 cells treated for only 48 hours with the PPAR agonists (data not shown). In contrast, DIM-C-pPhCF3 and DIM-C-pPhC6H5, but not rosiglitazone, induced caveolins 1 and 2 in HCT-15 cells after treatment for 72 hours (Fig. 7, C and E)
and 48 hours (data not shown). Induction of caveolins 1 and 2 by DIM-C-pPhCF3 and DIM-C-pPhC6H5 in HT-29 and HCT-15 cells was inhibited by cotreatment with the PPAR agonist GW9662; similar inhibition was observed for rosiglitazone in HT-29 cells (Fig. 7, E and F)
. Because caveolin 1 exhibits tumor suppressor and growth inhibitory activities, the results suggest that induction of caveolins may be important for the antiproliferative effects of PPAR-active C-substituted DIMs and rosiglitazone in colon cancer cells (Fig. 1)
.
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agonists also induce apoptosis in some cancer cell lines (13, 14, 15)
, and therefore, this response was also investigated in HT-29 and HCT-15 cells. PPAR-active compounds (10 µmol/L) did not induce poly(ADP-ribose) polymerase cleavage (a marker of apoptosis) or alter levels of bax or bcl-2 protein expression in HCT-15 or HT-29 cells (Fig. 8)
. In contrast, the proteasome inhibitor MG132 induced poly(ADP-ribose) polymerase cleavage in both cell lines but did not affect levels of bcl-2 or bax proteins. Thus, differences in the observed antiproliferative activities of rosiglitazone and PPAR C-substituted DIMs in HT-29 and HCT-15 cells (Fig. 1)
was not due to apoptosis or modulation of cell cycle proteins but correlated with their cell context-dependent differential induction of caveolins 1 and 2 but not keratin 18.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is overexpressed in multiple tumor types (5)
and studies in several laboratories have demonstrated that PGJ2 and TZDs inhibit growth of human cancer cell lines in vitro and also in xenograft models in vivo (7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
. Two reports (17
, 18)
showed that treatment of Min mice, which express a mutated inactive form of the adenomatous polyposis coli (APC) tumor suppressor gene, with troglitazone or BRL-49653 induced colon polyp formation. These results contrast with xenograft studies where PPAR agonists inhibit colon tumor growth (16)
. Moreover, a recent study reported that 100 or 200 ppm pioglitazone and bezafibrate in the diet of Apc-deficient mice reduced intestinal polyps and hyperlipidemia (19)
. Another study used PPAR+/–/Min mice and their crosses to show that inhibition of colon carcinogenesis in these mice by PPAR agonists is Apc dependent (20)
.
Recent studies in this laboratory showed the C-substituted DIMs inhibited growth and G0-G1S progression of MCF-7 breast cancer cells and activated PPAR-dependent transactivation (7)
. The growth inhibitory properties of rosiglitazone and three PPAR-active C-substituted DIMs (DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5) were also determined in HT-29, HCT-15, SW480, and RKO colon cancer cell lines. At concentrations of 10 µmol/L, rosiglitazone significantly inhibited growth of HT-29 cells (IC50 > 10 µmol/L) but not the other cell lines, whereas the PPAR-active C-substituted DIMs inhibited growth of all four colon cancer cell lines with IC50 values between 1 and 10 µmol/L. The growth inhibitory effects of DIM-C-pPhC6H5 were inhibited by GW9662 in HCT-15 cells (Fig. 3E)
. A recent study showed that troglitazone inhibited growth and induced differentiation genes in HCT-15 cells (21)
. However, these responses were observed using 50 µmol/L troglitazone, suggesting that activation of some mutant (K422Q) PPAR-dependent responses in HCT-15 cells may be observed with higher concentrations of TZDs.
Rosiglitazone and the PPAR-active C-substituted DIM activated PPAR-GAL4/pGAL4 in all four cell lines (Fig. 3)
with variable potencies, and transactivation was inhibited after cotreatment with the PPAR antagonist GW9662. The pattern of coactivator-PPAR interactions in HT-29 and HCT-15 cells paralleled, in part, their responsiveness to the growth inhibitory effects of rosiglitazone and the PPAR-active C-substituted DIMs (Fig. 4)
. Both structural classes of PPAR agonists induced interactions between VP-PPAR and GAL4-PGC-1 in HT-29 cells, whereas only the C-substituted DIMs induced the same interaction in HCT-15 cells (Fig. 4)
. In contrast, the pattern of coactivator-PPAR interactions in SW480 and RKO cells was more complex, and minimal induction was observed for rosiglitazone in both cell lines (data not shown). The differences in rosiglitazone-induced transactivation in the mammalian two-hybrid assay in HT-29 and HCT-15 were observed using VP-PPAR (wild-type) in both cell lines. This suggests that differences in PGC-1-PPAR interactions in HT-29 and HCT-15 cells are not attributable to expression of the PPAR mutant (K422Q) in the latter cell line but related to differential expression in the two cell lines of other nuclear cofactors required for this response. Although PPAR-active C-substituted DIMs preferentially induce PPAR-PGC-1 interactions in HT-28 and HCT-15 cells, this was not uniformly observed for all ligands in these cells (Fig. 4)
, SW480 or RKO cells (data not shown). For example, DIM-C-pPhC6H5 inhibits HT-29 and HCT-15 cell growth (Fig. 1)
, induces caveolins 1/2 (Fig. 7)
, and both responses are inhibited by the PPAR agonist GW9662 (Figs. 3E
, 7E
, and 7F
). In contrast, 10 µmol/L DIM-C-pPhC6H5 does not induce transactivation in the mammalian two-hybrid assay in HT-29 cells using GAL4-PGC-1 and VP-PPAR (Fig. 4)
. Current studies are investigating expression of PGC-1 and other coactivators and their role in PPAR-dependent transactivation and growth inhibition in HT-29 and HCT-15 cells.
We further investigated the role of wild-type versus mutant (K422Q) PPAR expression in mediating ligand-dependent responses in HT-29 and HCT-15 cells by examining their effects on proteins critical for cell cycle progression and differentiation. In HT-29 cells, treatment with rosiglitazone or PPAR-active C-substituted DIMs inhibited G0-G1S-phase progression (Fig. 2A)
. These effects were not observed in HCT-15 cells (Fig. 2B)
and may be due to the high percentage of cells in G0-G1 with or without treatment (>74%). Despite differences in fluorescence-activated cell sorting analysis, neither rosiglitazone or the PPAR-active C-substituted DIMs significantly changed levels of the cell cycle regulatory proteins p27, p21, or cyclin D1 in HT-29 or HCT-15 cells (Fig. 5)
. Moreover, we did not observe PPAR agonist-dependent induction of apoptosis in either cell line (e.g., Fig. 8
), suggesting that the antiproliferative effects (Fig. 1)
are not dependent on modulation of cell cycle proteins or induction of apoptosis in HT-29 or HCT-15 cells.
PPAR agonists also induce genes/proteins associated with differentiation in colon cancer cells (8
, 12
, 19
, 22
, 23)
. For example, troglitazone induced villin and intestinal alkaline phosphatase mRNA levels in several colon cancer cell lines (23)
, and TZDs and PGJ2 induced caveolin 1 and caveolin 2 protein expression in HT-29 cells (12)
. Keratin 18 and 20 proteins and CEACAM6 mRNA levels were minimally expressed in HCT-15 cells but were not affected by treatment with rosiglitazone; however, after transfection of HCT-15 cells with wild-type PPAR, rosiglitazone induced these responses (8)
. A direct comparison of PPAR agonist-induced expression of differentiation markers in HCT-15 versus HT-29 cells has not been reported previously. The results in Fig. 6
show that rosiglitazone, DIM-C-pPhCF3, and DIM-C-pPhC6H5 significantly induced keratin 18 in both HT-29 and HCT-15 cells, indicating that keratin 18 protein induction was independent of PPAR agonist structure and expression of wild-type or mutant (K422Q) PPAR. In contrast, induction of caveolin 1 and caveolin 2 in HT-29 and HCT-15 was dependent on both ligand structure and cell context (Fig. 7)
. Rosiglitazone induced caveolins 1 and 2 in HT-29 but not in HCT-15 cells, whereas PPAR-active C-substituted DIMs induced caveolins 1 and 2 in both HT-29 and HCT-15 cells, and these responses were inhibited after cotreatment with GW9662. This pattern of induced caveolin expression parallels the growth inhibitory effects of rosiglitazone and PPAR-active C-substituted DIMs in HT-29 and HCT-15 cells (Fig. 1)
, suggesting that induced caveolin 1 and caveolin 2 expression may contribute to growth inhibition of colon cancer cells. This observation is consistent with a study by Bender et al. (24)
, showing that overexpression of caveolin 1 in HT-29 or DLD (HCT-15) cells significantly decreased their tumorigenicity in athymic nude mouse xenograft models.
In summary, this study demonstrates the PPAR-active C-substituted DIMs induce PPAR-dependent transactivation in colon cancer cells expressing wild-type (HT-29) or mutant (K422Q) (HCT-15) PPAR. Moreover, these compounds also inhibit growth of HT-29 and HCT-15 cells, whereas equivalent concentrations of rosiglitazone are active only in the former cell line. The enhanced growth inhibitory activity of PPAR-active C-substituted DIMs versus rosiglitazone in HCT-15 cells correlated with the induction of caveolins 1 and 2 by the former compounds and not by rosiglitazone (Fig. 7)
. Current studies are focused on the mechanisms of the growth inhibitory effects of this new class of PPAR agonists in other colon cancer cell lines and the role of caveolins and other genes/proteins in mediating PPAR-dependent antitumorigenic activities.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, Veterinary Research Building 409, College Station, TX 77843-4466. Phone: (979) 845-5988; Fax: (979) 862-4929; E-mail: ssafe{at}cvm.tamu.edu
Received 2/ 5/04. Revised 6/ 1/04. Accepted 6/ 4/04.
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S. Chintharlapalli, S. Papineni, S. Liu, I. Jutooru, G. Chadalapaka, S.-d. Cho, R. S. Murthy, Y. You, and S. Safe 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 Carcinogenesis, November 1, 2007; 28(11): 2337 - 2346. [Abstract] [Full Text] [PDF]
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 J.-R. Weng, C.-H. Tsai, S. K. Kulp, D. Wang, C.-H. Lin, H.-C. Yang, Y. Ma, A. Sargeant, C.-F. Chiu, M.-H. Tsai, et al. A Potent Indole-3-Carbinol Derived Antitumor Agent with Pleiotropic Effects on Multiple Signaling Pathways in Prostate Cancer Cells Cancer Res., August 15, 2007; 67(16): 7815 - 7824. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, I. Jutooru, A. McAlees, and S. Safe Structure-dependent activity of glycyrrhetinic acid derivatives as peroxisome proliferator-activated receptor {gamma} agonists in colon cancer cells Mol. Cancer Ther., May 1, 2007; 6(5): 1588 - 1598. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, and S. Safe 1,1-Bis(3'-Indolyl)-1-(p-substitutedphenyl)methanes Inhibit Growth, Induce Apoptosis, and Decrease the Androgen Receptor in LNCaP Prostate Cancer Cells through Peroxisome Proliferator-Activated Receptor {gamma}-Independent Pathways Mol. Pharmacol., February 1, 2007; 71(2): 558 - 569. [Abstract] [Full Text] [PDF]
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S. D. Cho, K. Yoon, S. Chintharlapalli, M. Abdelrahim, P. Lei, S. Hamilton, S. Khan, S. K. Ramaiah, and S. Safe Nur77 Agonists Induce Proapoptotic Genes and Responses in Colon Cancer Cells through Nuclear Receptor-Dependent and Nuclear Receptor-Independent Pathways Cancer Res., January 15, 2007; 67(2): 674 - 683. [Abstract] [Full Text] [PDF]
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P. Lei, M. Abdelrahim, and S. Safe 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit ovarian cancer cell growth through peroxisome proliferator-activated receptor-dependent and independent pathways. Mol. Cancer Ther., September 1, 2006; 5(9): 2324 - 2336. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, and S. Safe 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through PPAR{gamma}-dependent and PPAR{gamma}-independent pathways Mol. Cancer Ther., May 1, 2006; 5(5): 1362 - 1370. [Abstract] [Full Text] [PDF]
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M. Abdelrahim, K. Newman, K. Vanderlaag, I. Samudio, and S. Safe 3,3'-Diindolylmethane (DIM) and its derivatives induce apoptosis in pancreatic cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5 Carcinogenesis, April 1, 2006; 27(4): 717 - 728. [Abstract] [Full Text] [PDF]
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J. A. Savino III, J. F. Evans, D. Rabinowitz, K. J. Auborn, and T. H. Carter Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane. Mol. Cancer Ther., March 1, 2006; 5(3): 556 - 563. [Abstract] [Full Text] [PDF]
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M. Konopleva, W. Zhang, Y.-X. Shi, T. McQueen, T. Tsao, M. Abdelrahim, M. F. Munsell, M. Johansen, D. Yu, T. Madden, et al. Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells. Mol. Cancer Ther., February 1, 2006; 5(2): 317 - 328. [Abstract] [Full Text] [PDF]
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W. Kassouf, S. Chintharlapalli, M. Abdelrahim, G. Nelkin, S. Safe, and A. M. Kamat Inhibition of Bladder Tumor Growth by 1,1-Bis(3'-Indolyl)-1-(p-Substitutedphenyl)Methanes: A New Class of Peroxisome Proliferator-Activated Receptor {gamma} Agonists Cancer Res., January 1, 2006; 66(1): 412 - 418. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, S. J. Baek, S. Liu, and S. Safe 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 Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 Mol. Pharmacol., December 1, 2005; 68(6): 1782 - 1792. [Abstract] [Full Text] [PDF]
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 S. Chintharlapalli, R. Burghardt, S. Papineni, S. Ramaiah, K. Yoon, and S. Safe Activation of Nur77 by Selected 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes Induces Apoptosis through Nuclear Pathways J. Biol. Chem., July 1, 2005; 280(26): 24903 - 24914. [Abstract] [Full Text] [PDF]
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S. Chintharlapalli, S. Papineni, M. Konopleva, M. Andreef, I. Samudio, and S. Safe 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 {gamma}-Dependent and -Independent Pathways Mol. Pharmacol., July 1, 2005; 68(1): 119 - 128. [Abstract] [Full Text] [PDF]
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R. Contractor, I. J. Samudio, Z. Estrov, D. Harris, J. A. McCubrey, S. H. Safe, M. Andreeff, and M. Konopleva A Novel Ring-Substituted Diindolylmethane,1,1-Bis[3'-(5-Methoxyindolyl)]-1-(p-t-Butylphenyl) Methane, Inhibits Extracellular Signal-Regulated Kinase Activation and Induces Apoptosis in Acute Myelogenous Leukemia Cancer Res., April 1, 2005; 65(7): 2890 - 2898. [Abstract] [Full Text] [PDF]
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