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Department of Pathophysiology, Endocrinology Unit (P.F., M.S., M.M., G.F.) and Gastroenterology Unit (E.C., M.T., C.G., S.M., A.G.), Center of Research Transfer and Higher Education, University of Florence, 50139 Florence, Italy
Address all correspondence and requests for reprints to: M. Mannelli, M.D., Department of Clinical Pathophysiology, Endocrine Unit, Viale Pieraccini, 6, 50139 Florence, Italy. E-mail: m.mannelli{at}dfc.unifi.it.
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Abstract |
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, which is a nuclear receptor acting as a transcription factor in several tissues. In the present study, we evaluated PPAR mRNA and protein expression in tissue samples of human adrenocortical carcinomas (ACCs), normal adrenal glands, and the human ACC cell line H295R. PPAR mRNA was expressed in six of eight ACC, two of three normal adrenal glands and the H295R cells. These results were confirmed by immunohistochemistry.
PPAR transcriptional activity in H295R cells, monitored by a reporter gene assay, was induced 2- to 3-fold by TZDs, such as rosiglitazone (RGZ) and pioglitazone, whereas in PPAR-transfected cells RGZ alone or RGZ plus 9-cis retinoic acid further increased reporter activity.
TZDs inhibited both the proliferation and invasiveness of H295R cells in a dose-dependent manner. Thymidine incorporation was reduced by about 60% by 20 µM of both TZDs. Cotreatment with the retinoic X receptor ligand 9-cis retinoic acid had an additive effect.
TZDs increased the number of cells in the G0/G1 phase and decreased them in the S phase. Western blot analysis showed that TZDs increased the expression of the cell cycle inhibitors p21 and p27 and reduced the expression of cyclin D1.
Twenty micromoles of RGZ and pioglitazone reduced H295R invasiveness through Matrigel by about 85%.
Zymography and ELISA tests showed that TZD inhibited metalloproteinase-2 secretion by H295R cells in a dose-dependent manner.
These data suggest that TZDs reduce the malignant potential of the H295R ACC cell line and, therefore, might potentially constitute a novel tool in the medical treatment of human ACCs.
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Introduction |
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Although several signaling pathways have been shown to be altered in these tumors (2, 3, 4, 5, 6, 7, 8), the sequence of events leading to proliferation and dedifferentiation is still unknown, making it difficult to target-specific antitumoral agents. Thus, due to the aggressiveness of ACC and the limited efficacy of the available medical treatment, any new drug that can be proven to be effective in counteracting adrenal carcinogenesis is warmly welcome.
Thiazolidinediones (TZDs) are a new class of antidiabetic drugs that attenuate the insulin resistance associated with obesity, hypertension, and impaired glucose tolerance in humans as well as several animal models of non-insulin-dependent diabetes mellitus (9). TZDs were found to be ligands for the peroxisome proliferator-activated receptor (PPAR)-, a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue but also in other tissues at much lower levels (10, 11). TZDs activate PPAR and promote association with the 9-cis retinoic X receptor (RXR) to form functional heterodimers that recognize its cognate response element at the level of the target genes (12, 13).
The PPAR/RXR signal is critical in a variety of biological processes, such as adipogenesis, glucose metabolism, and inflammation (14) and acts as an important cellular regulator by inhibiting growth and/or inducing differentiation and apoptosis of normal and tumor cells, including those of the breast (15) and colon (16). Treatment of breast and colon cancer cells with TZDs resulted in significant growth arrest both in culture and when implanted in nude mice (16). In addition, loss-of-function mutation of the PPAR gene has been found in some human colon and thyroid carcinomas (17, 18). As a consequence, PPAR has become a molecular target in anticancer drug development, and TZDs have been proposed as a therapy for PPAR-expressing tumors.
The aim of the present study was to evaluate the presence of PPAR in normal and pathological human adrenal tissue and evaluate the effects of PPAR agonists, such as TZDs, on cell growth and the invasiveness of the human ACC cell line H295R.
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Materials and Methods |
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Most of the chemicals and supplies were purchased from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose and Nytran were from Schleicher and Schuell, Inc. (Keene, NH). Agarose, trypsin, all restriction endonucleases, DNA-modifying enzymes, and tissue culture media were purchased from Life Technologies, Inc. (New Brunswick, NJ). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT). [Methyl-3H]thymidine ([3H]TdR) and D-threo-[dichloroacetyl-1,2-14C]chloramphenicol were purchased from NEN Life Science Products (Boston, MA). PPAR (E8), p21, p27, cyclin D1 (CD1), and ß-actin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rosiglitazone (RGZ) and pioglitazone (PGZ) were from Glaxo (Welwyn, UK) and Takeda Chemicals (Tokyo, Japan), respectively.
Tissue samples and cell cultures
A total of three normal human adrenals glands and eight adrenal carcinomas were used in this study. Normal adrenal glands were removed during an expanded nephrectomy due to renal carcinoma or from organ donors (age 32–72 yr). Approval for the use of human material was given by the university ethical committee. Informed consent was obtained from each patient. Adrenocortical fragments, collected immediately after surgery, were frozen in liquid nitrogen and stored at –80 C.
The human ACC cell line H295R was obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained at 37 C in a 95% air-5% CO2 fully humidified environment in a culture medium consisting of a 1:1 (vol/vol) mixture of DMEM/F-12 with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, enriched with a mixture of insulin/transferrin/selenium.
RNA extraction and RT-PCR
Total RNA was extracted from cultured cells and tissue samples using guanidinium-phenol-chloroform methods according to Chomczynski and Sacchi (19) with minor modifications (20). One microgram of the total RNA from tissue or tumor cells was reverse transcribed with the Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Paisley, UK) at 42 C for 60 min in a 20-µl mixture in the presence of random hexamers. Primers for human PPAR were 5'-TCTGGCCCACCAACTTTGGG-3' and 5'-CTTCACAAGCATGAACT-CCA-3'. Two microliters of a reverse-transcribed mixture was subjected to PCR in a 20-µl reaction solution [10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 0.01% gelatin, 20 mM deoxynucleotide triphosphate, 0.5 U of Taq polymerase (Life Technologies), and 0.25 pmol of primer]. Thirty-five cycles of reaction at 94 C for 50 sec, 60 C for 45 sec, and 72 C for 90 sec were carried out using a DNA thermal cycler (PerkinElmer Cetus, Norwalk, CT). The efficiency of the reverse transcription reaction was controlled in each sample by PCR amplification of human glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (5'-GCCAAAAGG-GTCATCATCTC-3', 5'-GTAGAGGCAGGGATGATGTTC-3').
Immunohistochemistry
Frozen sections of adrenal tissue (6 µm thick) were collected onto clean glass slides, dried overnight, and fixed in acetone and chloroform for 30 min. Cells plated onto sterile tissue culture chambers (Lab-tek, Naperville, IL) were washed with PBS, air dried for 15 min, and fixed in acetone. After brief air drying, the primary antibody (mouse antihuman PPAR monoclonal antibody) was diluted 1:20 in Tris-buffered solution (TBS) containing 0.1% BSA and applied for 30 min at room temperature in a humid chamber. Sections were washed twice in TBS for 5 min each and covered with a polyclonal rabbit antimouse antibody (Dakopatts; Dakocytomation Denmark A/S, Glostrup, Denmark) diluted 1:20 in the same buffer as described above. After 30 min incubation, the sections were rinsed twice in TBS for 5 min and incubated with alkaline phosphatase antialkaline phosphatase immune complex (Dakopatts) diluted 1:50 in TBS for 30 min. The chromogenic reaction was developed with new fuchsin and naphthol-as-biphosphate-phosphate for 30 min and finally counterstained with Mayer’s hemalum solution.
Transient transfection of culture cells
Cells were transfected at the density of 5 x 105 cells/60-mm dish with 2 µg peroxisome proliferator response element (adipocyte response element)-73-tk-luciferase reporter plasmid [containing three copes of the peroxisome proliferator response element from the adipocyte lipid binding protein gene ligated to a herpes simplex thymidine kinase promoter upstream from a luciferase gene] (21, 22), 0.15 µg of human PPAR expression plasmid, and 1 µg pSV2CAT (a vector containing Simian virus 40 early promoter and enhancer sequences that drives a chimeric chloramphenicol acetyl transferase gene) as an internal control by calcium phosphate precipitation. The total amount of DNA transfected was normalized with a carrier DNA (pcDNA3.1; Invitrogen Corp., Carlsbad, CA). Four hours later, the cells were exposed to PBS containing 15% glycerol for 3 min. The cells were rinsed twice with PBS, and fresh medium without serum and phenol red was added. Twenty-four hours after transfection, the cells were treated with TZDs. Twenty-four hours later, the cells were harvested, washed twice with PBS, and lysed in 900 µl lysis buffer (Promega, Madison, WI) containing 25 mM Tris (pH 7.8), 2 mM ethylenediaminetetraacetic acid, 20 mM dithiothreitol, 10% glycerol, and 1% Triton X-100. Fifty microliters of cell extract were incubated with luciferase assay reagent based on the original protocol by de Wet et al. (23). The number of relative light units with a 3-sec delay and a 30-sec incubation were measured using a Sirius1 luminometer (Berthold Detection System, Pforzheim, Germany). Chloramphenicol acetyl transferase activity was measured as previously described (24). The conversion of chloramphenicol to its acetylated products was quantified on an ß-scanner (Ambis System, San Diego, CA).
DNA synthesis assay
DNA synthesis was evaluated according to the amount of [3H]TdR incorporated into trichloroacetic acid-precipitated materials. The cells were seeded at the density of 5 x 104 cells/24-well plates and incubated in complete medium until they became 70% confluent. After 24-h incubation with phenol red and serum-free medium, the cells were treated with TZDs and/or 9-cis retinoic acid for 24 h and then pulsed for 4 h with 1.0 µCi/ml [3H]TdR (6.7 Ci/mmol). At the end of the pulsing period, ice-cold 10% trichloroacetic acid was added, and the dishes were kept on ice for 15 min. After another wash with trichloroacetic acid and one with methanol, the cells were solubilized in 0.2 N NaOH, and radioactivity was measured in the scintillation counter. Experiments were performed in triplicate and repeated at least three times.
Cell cycle analysis
The cells (4 x 105) were exposed to TZDs for 72 h in medium supplemented with 5% dialyzed FBS. Total cells were collected, washed, suspended in cold PBS, and stained in trypan blue. Both blue and nonblue cells were counted. The cells were adjusted to 1 x 106 viable cells/ml and fixed overnight in 2:1 ratio (vol/vol) of methanol/chloroform before staining with propidium iodide. Cell cycle status was analyzed with a flow cytometer (Becton Dickinson, Lincoln Park, NJ) and CellFIT cell-cycle analysis software (Becton Dickinson).
Protein extraction and Western blot
Whole-cell proteins were extracted from the H295R carcinoma cell line. The cells were cultured in the presence or absence of test agents and were homogenized in Laemmli buffer (25). Nuclear proteins were isolated from treated and untreated cells based on micropreparation methods (26). The nuclear extracts were suspended in 20 mM HEPES (pH 7.9), 40 mM NaCl, 1.5 MgCl2, 0.2 mM ethylenediaminetetraacetic acid, 25% glycerol, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged at 20,000 x g for 10 min at 4 C, and the supernatants were frozen in liquid nitrogen and stored at –80 C until use. Nuclear and whole-cell extracts (40 µg protein) were fractionated in 12% SDS-PAGE and electroblotted onto nitrocellulose filters. Proteins were detected by incubating the filters with the following primary antibodies: p21 (1:1000), p27 (1:500), CD1 (1:1000), and ß-actin (1:1000). Detection of the protein bands was performed using the Amersham enhanced chemiluminescence kit (Arlington Heights, IL).
Invasion assay
The ability of the cells to invade through a Matrigel-coated filter was measured in a Boyden chamber. Polyvinylpyrrolidone-free polycarbonate filters (pore size 8 µm) were coated with basement membrane Matrigel (200 µg/filter) (Collaborative Research Inc., Bedford, MA), as described in the standard protocol (27). The Matrigel was diluted to the desired final concentration with cold distilled water, applied to the filter, dried under the hood, and reconstituted with serum-free medium. The homogeneity of the coating was verified by protein staining. The coated filters were placed in Boyden chambers. Confluent cells were serum starved for 24 h and then washed, trypsinized, and suspended in serum-free medium with or without TZDs at the density of 1 x 105 and placed in the upper chambers. DMEM containing 1% FBS was placed in the lower compartments of the Boyden chambers. The chambers were incubated for 6 h in 5% CO2-95% air at 37 C. At the end of incubation, the cells of the upper surface were completely removed by wiping with a cotton swab. The filters were fixed in methanol and stained with hematoxylin and eosin. Cells from various areas of the lower surface were counted by a computerized video-image analysis system (Quantimet Q500MC, Leica Cambridge Ltd., Cambridge, UK). Each assay was performed at least three times in duplicate.
Zymography
Gelatinolytic activity was determined in the supernatants of TZD-treated and untreated ACC cell lines. For this purpose, cells were cultured for 24 h in serum-free medium, washed twice, and finally treated with TZDs for another 24 h. The supernatants were collected, concentrated using centrifugal filter devices (Centricon) (Millipore Corp., Bedford, MA), and the protein content determined using BCA protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (20 µg) were mixed with sodium dodecyl sulfate sample buffer without reducing agents and incubated for 20 min at 37 C. For gelatinolytic activity, the assay samples were separated on polyacrylamide gels containing 1 mg/ml gelatin. After electrophoresis, the gels were stained for 1 h in a 45% methanol/10% acetic acid mixture containing Coomassie brilliant blue G250 and destained. Zymograms were photographed after 10 h of incubation at 37 C when clear bands of lysis were visible in the cloudy background. Zymographic analyses were performed in at least three independent experiments.
ELISA
Matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium were measured by Biotrak. Human MMP-2 and Biotrak human MMP-9 were measured with ELISA kits (Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands), respectively, following the manufacturer’s instructions. MMP-2 can be measured in a range of 1.5–24 ng/ml, and the sensitivity of the assay is 0.37 ng/ml. MMP-9 can be measured in the range of 1–32 ng/ml, and the sensitivity of the assay is 0.6 ng/ml. All experiments were performed in duplicate and repeated three times.
Statistical analysis
Data were expressed as mean ± SD. Statistical correlation of data was checked for significance by the ANOVA and paired Student’s t test. The corresponding probability is given.
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Results |
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expression and activity in human normal and pathological adrenal tissues and human H295R adrenal carcinoma cell lines
The expression of PPAR was detected by RT-PCR and immunohistochemistry in human normal and pathological adrenal tissues and the H295R cell line. PPAR mRNA was detected in six of eight adrenal carcinomas (Fig. 1A
) and two of three normal adrenal glands as well as the H295R cell line (Fig. 1B). In the normal adrenal gland, a weak immunoreactivity for PPAR was detected in a few nuclei (Fig. 1C), whereas in ACCs the nuclear expression of the receptor was present in a large number of cells (Fig. 1D). PPAR immunostaining also had a typical nuclear and perinuclear granular distribution in the H295R cells (Fig. 1E). The clinical features of patients harboring ACCs and tumor characteristics are reported in Table 1. The PPAR transactivation in the H295R cancer cell line was monitored by the activity of transfected adipocyte response element-73-tk-luciferase reporter cells (Fig. 2). Both TZDs (RGZ and PGZ) induced reporter activity about 2- to 3-fold over the control. Similarly, 9-cis retinoic acid, the agonist of the PPAR heterodimeric partner RXR, induced reporter activity about 3-fold over the control. Cotreatment with 9-cis retinoic acid and RGZ showed an additive stimulatory effect by inducing reporter activity approximately 2-fold over the single receptor ligand treatments. Transfection with 0.15 µg of human PPAR expression plasmid stimulated reporter activity more than 7-fold over the control, and treatment of PPAR-transfected cells with RGZ or 9-cis retinoic acid caused an additional 3- to 5-fold increase in the reporter activity (P < 0.01; Fig. 2).
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The effects of TZDs on adrenal carcinoma cell [3H]TdR incorporation are reported in Fig. 3. Incubation with both RGZ (Fig. 3A) and PGZ (Fig. 3B) resulted in dose-dependent growth inhibition of H295R cells. The potency of the two TZDs was similar with the maximal effect (about 50% inhibition) obtained at 20 µM with both TZDs, even if RGZ effects start being statistically significant at the dose of 5 µM (P < 0.05 vs. control). H295R treatment with different concentrations of 9-cis retinoic acid was able to reduce DNA synthesis in a dose-dependent manner (Fig. 3C). In addition, the simultaneous incubation of the cells with RGZ (5 and 10 µM) and 9-cis retinoic acid (10 µM) resulted in an additive inhibitory effect of [3H]TdR incorporation, which was statistically significant vs. RGZ and 9-cis retinoic acid alone (P < 0.05; Fig. 3D).
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but a weak activator of PPAR (21), had a negligible effect on H295R cell proliferation (not shown). TZD treatment alters cell cycle progression in adrenal cancer cells
Preliminary experiments evaluating trypan blue exclusion and lactate dehydrogenase leakage from H295R cells into the culture medium showed that TZDs induced growth inhibition rather than cytotoxicity because the number of vitally stained cells was higher than 90% in all experiments at any given time point and concentration used. Based on this observation, we assessed the effect of RGZ and PGZ on cell cycle progression. Little change in cell cycle distribution was observed at 12 h with 20 µM TZDs in H295R cells (data not shown). Both RGZ (20 µM) and PGZ (20 µM) increased the proportion of cells in the G0/G1 phase at 24 h and the growth arrest persisted at later time points. The increased number of cells in the G0/G1 phase was mirrored by a proportional decrease in cells in the S phase (Fig. 4A).
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) and in a significant reduction of CD1. The expression of ß-actin, used as an internal control, did not change under any experimental condition. TZDs inhibit H295R cancer cells invasiveness involving MMP-2
The effect of TZDs on the ability of cells to invade through reconstituted basement membranes was analyzed using Matrigel-coated invasion chambers, as described in Materials and Methods. Incubation with RGZ inhibited matrix invasive-ness in a dose-dependent manner (Fig. 5). A similar effect was also demonstrated using PGZ (data not shown).
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, a 72-kDa band corresponding to MMP-2 and two faint bands at 94 and 88 kDa corresponding to latent and activated MMP-9, respectively, were found in the conditioned medium of the human adrenal carcinoma H295R cell line. A significant reduction in the 72-kDa gelatinolytic band was demonstrated after 24-h treatment with RGZ or PGZ (20 µM). On the contrary, TZD treatment had a negligible effect on the MMP-9 forms (Fig. 6A).
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, both RGZ and PGZ inhibited MMP-2 secretion in a dose-dependent manner. On the contrary, TZD treatment did not influence MMP-9 secretion (data not shown). |
Discussion |
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Medical treatment has not changed substantially in the last decades and still relies mainly on the use of the adrenolytic agent mitotane (1), alone or in association with other chemotherapeutical agents (30).
On the whole, approximately 50% of the patients affected with ACCs will not survive more than 2 yr from diagnosis, and 5-yr mortality is approximately 80% (31).
Ignorance of the processes leading to ACC formation has hindered the targeting of new effective drugs. Nevertheless, rapidly expanding research on the mechanisms that modulate gene expression has started offering new tools for medical therapy of cancer. Among these, the use of PPAR agonists in the treatment of some types of cancer has been suggested by their proven effects on cell growth (inhibition) and differentiation (stimulation). In fact, PPAR agonists have been shown to reduce growth and invasiveness in several types of tumors (10, 11, 16).
In this paper, we demonstrate that human ACC expresses PPAR and that TZDs are able to reduce cell growth and invasiveness of the ACC cell line H295R.
Levels of PPAR expression seemed higher in the ACCs than the normal adrenal glands, although they were variable and were measured semiquantitatively. It is not known whether PPAR expression is associated with a better prognosis. Our series is too limited to reach any conclusion, although a lack of correlation is suggested. Indeed, in the two patients whose tumors did not express PPAR (Table 1), we observed one death after 3 yr (patient 1) and a recurrence (patient 3), whereas among the other six patients affected by PPAR-positive tumors, four died (patients 4, 6, 7, and 8) and two remain disease free (patients 2 and 5).
We also demonstrated that PPAR is active at the transcriptional level in H295R cells, in which both the inhibitory effects on growth and invasiveness were dose dependent and present at concentrations similar to those achieved in vivo by TZD administration in diabetic patients (32).
The additive effect exerted on cell growth inhibition by TZD and 9-cis retinoic acid is in line with the well-known synergistic action exerted by PPAR and RXR agonists (33).
Nevertheless, the mechanisms through which TZDs exert their antiproliferative and antiinvasive effects on H295R cells cannot be ascertained in the present study. Indeed, in an attempt to elucidate whether the effects of TZDs on H295R cells were PPAR dependent, we used the specific PPAR inhibitor GW9662, but the concentrations necessary to inhibit transcriptional activity turned out to be toxic for the cells (Ferruzzi, P., personal observation).
The mechanisms through which TZDs have been shown to exert antitumoral effects in other human cancers, such as colon, breast, pancreas, and prostatic cancer, have yet to be completely clarified. They vary from one cell type to another and have been found to sometimes be independent of PPAR presence.
Troglitazone, another PPAR agonist, such as the natural activator 15d-PGJ2, has been shown to cause a growth arrest in cells that do not express PPAR, through a depletion of intracellular calcium deposits, inhibition of RNA-dependent protein kinase, and phosphorylation of the eukaryotic initiation factor 2 (34). Moreover, troglitazone is able to inhibit the proliferation of leucemic cell KU812 through a PPAR-independent mechanism (35). In contrast, RGZ inhibits DNA synthesis of colon (36), glioma (37), and pancreatic cancer cells (38) through a mechanism that is strictly dependent on PPAR expression.
In addition, the inhibition exerted by TZDs on cell invasiveness may be PPAR independent. In pancreas tumor cells, TZDs inhibit invasiveness independently of PPAR expression with a mechanism involving inhibition of gelatinolytic and fibrinolytic activity via repression of the MMP-2 secretion and increased expression of the PAI-1 inhibitor, respectively (38).
In line with these observations, our data seem to suggest that TZD inhibit invasiveness in H295R cells, at least in part through a decrease in MMP-2 activity. Indeed, although MMP-2 secretion by H295R cells seems lower than in other cancer cell lines (39), the results of the ELISA showed a clear dose-dependent inhibitory effect exerted by both RGZ and PGZ.
Additional antitumoral effects can be exerted by PPAR ligands in vivo through the inhibition of angiogenesis. In fact, PPAR is highly expressed in tumor endothelium and is activated by TZD in cultured endothelial cells (40). Furthermore, in human and bovine endothelial cells, TZDs inhibit vascular endothelial growth factor-induced migration and MMP activity (41, 42).
Therefore, in view of the multiple PPAR-dependent and -independent effects exerted by TZDs, it is not surprising that a strict correlation between PPAR expression and antitumoral effects of PPAR agonists does not exist in vivo.
The different mechanisms of action mediating the various effects of TZDs may also explain the differences observed in the doses causing inhibition of growth and invasiveness. Nevertheless, the elucidation of the mechanisms leading to the inhibition of growth and invasiveness by TZD in H295R cells was beyond the aim of the present paper and deserves additional study with the appropriate experimental designs.
The target of our study was, indeed, to evaluate whether TZDs might also exert antitumoral effects on human H295R cells. The demonstration that they inhibit growth and invasiveness in a human ACC cell line is a first step that broadens the prospect of research on medical therapy for ACC.
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Footnotes |
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First Published Online December 7, 2004
Abbreviations: ACC, Adrenocortical carcinoma; CD1, cyclin D1; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; [3H]TdR, [methyl-3H]thymidine; MMP, matrix metalloproteinase; PGZ, pioglitazone; PPAR, peroxisome proliferator-activated receptor; RGZ, rosiglitazone; RXR, retinoic X receptor; TBS, Tris-buffered solution; TZD, thiazolidinedione.
Received May 24, 2004.
Accepted November 23, 2004.
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References |
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ligands are potent inhibitors of angiogenesis in vitro and in vivo. J Biol Chem 274:9116–9121This article has been cited by other articles:
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S. Han and J. Roman Rosiglitazone suppresses human lung carcinoma cell growth through PPAR{gamma}-dependent and PPAR{gamma}-independent signal pathways. Mol. Cancer Ther., February 1, 2006; 5(2): 430 - 437. [Abstract] [Full Text] [PDF]
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, Mock transfected cells; 
, PPAR
, PGZ 20 µM. B, Expression of proteins related to the cell cycle (CD1, p21, and p27) in cells treated with TZDs. A representative of three independent experiments yielding similar results is shown.
