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Agonists Suppress Adrenocortical Tumor Cell Proliferation and Induce Differentiation
Division of Endocrinology and Diabetes (M.J.B., I.S., F.B.), Department of Internal Medicine II, University Hospital Freiburg, D-79106 Freiburg, Germany; Division of Endocrinology (M.F., S.H.), Department of Internal Medicine, University Hospital Würzburg, D-97080 Würzburg, Germany; and Department of Internal Medicine (M.R.), University Hospital Innenstadt, Ludwig-Maximilians-University, 80336 Munich, Germany
Address all correspondence and requests for reprints to: Felix Beuschlein, M.D., Division of Endocrinology and Diabetes, Department of Internal Medicine II, Hugstetter Strasse 55, D-79106 Freiburg, Germany. E-mail: beuschlein{at}medizin.ukl.uni-freiburg.de.
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Abstract |
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ligands. Moreover, recent evidence has suggested that TZDs might have favorable effects in the treatment of a variety of tumors as differentiation-inducing agents. Adrenocortical carcinoma (ACC) is a rare tumor entity with poor prognosis due to its highly malignant phenotype and lack of effective treatment options. Objective: The purpose of this study was to investigate effects of TZDs on adrenocortical cancer cells.
Results: PPAR mRNA expression was detectable in all adrenocortical tumors including ACCs at similar levels. Furthermore, incubation of the adrenocortical tumor cell line NCI h295 with the PPAR agonist rosiglitazone led to a decrease in cell viability, a decrease of cellular proliferation, and an increase in apoptosis as well as steroidogenesis. On the molecular level, NCI h295 cells expressed higher levels of ACTH receptor (melanocortin receptor-2) mRNA upon treatment, whereas cyclin E mRNA was reduced, thus reflecting a shift toward an expression pattern found in less aggressive adrenocortical tumors in vivo. Accordingly, luciferase experiments confirmed an increased promoter activity for the melanocortin receptor-2 after stimulation with rosiglitazone. Coincubation with the specific PPAR antagonist GW9662 demonstrated the inhibition of TZD-induced increase in steroidogenesis, whereas growth suppression upon TZD treatment was not affected by GW9662.
Conclusions: Thus, both PPAR-dependent and PPAR-independent effects of TZD treatment are likely to contribute to the observed phenotypical effects on NCI h295 cells. Taken together, these data indicate that TZDs might have the potential to become an additional treatment option as differentiation-inducing agents in patients with ACC.
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Introduction |
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Peroxisome proliferator activated receptor (PPAR)- is a ligand-activated transcription factor and member of the nuclear hormone receptor superfamily that is involved in a variety of physiological processes (8). Upon activation by its ligand and heterodimerization with its obligate partner, the retinoid receptor, PPAR interacts with the peroxisome-proliferator response element (PPRE) in the promoter of its target genes (9). PPAR is expressed in a variety of tissues, predominantly in adipose tissue and large intestine epithelium but also in skeletal muscle, the retina, and lymphoid organs.
PPAR plays a pivotal role in adipocyte differentiation as well as glucose and lipid homeostasis (10, 11, 12). Accordingly, the thiazolidinediones (TZDs) rosiglitazone and pioglitazone, synthetic high-affinity ligands for PPAR, have been introduced into clinical practice to ameliorate insulin resistance in type 2 diabetes. The existence of approved PPAR agonists and the ability of PPAR-dependent pathways to induce cellular differentiation prompted research to explore whether stimulation of PPAR activity could curtail malignant cell growth. In fact, only recently several in vitro and in vivo studies demonstrated antitumor effects of PPAR agonists in breast cancer (13), liposarcoma (14), pituitary adenomas (15, 16), non-small-cell lung cancer (17), prostrate cancer (18), and thyroid carcinoma (19). Mechanistically, TZD-induced growth inhibition is mediated by induction of apoptosis or cell cycle arrest and differentiation, raising the possibility that in the appropriate context, the PPAR-signaling cascade could provide a new approach for pharmacological intervention in neoplastic disease (20).
In this study, we demonstrate that PPAR is abundantly expressed in human adrenal tumors including ACCs and normal adrenal tissue. Furthermore, treatment of the human adrenal cancer cell line NCI h295 with PPAR agonists results in growth inhibition, induction of apoptosis, and up-regulation of markers of adrenal differentiation, thus pointing toward the possibility of a new treatment option for patients with adrenocortical cancer.
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Patients and Methods |
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Tissues of 32 patients with a variety of adrenal tumors were included in the study. The clinical data for these patients are shown in Table 1
. The clinical and pathological diagnosis was made according to established criteria. Adrenal tumor samples were collected during the German and Austrian Adrenal Network Multicenter Trial (21), whereas normal adrenal glands were obtained from brain-dead patients after organs had been removed for transplantation. The study protocol was approved by the local ethics committees of the Universities of Würzburg and Freiburg, and all of the patients consented to participate in the study. After removing adjacent fat tissue, the adrenal samples were snap frozen in liquid nitrogen and immediately stored at –80 C until analyzed.
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Total RNA was extracted from tissue samples using SV Total RNA isolation system (Promega, Madison, WI) according to the manufacturer’s protocol. Purity and yield of RNA was determined spectrophotometrically and integrity was confirmed by gel electrophoresis.
Reverse transcription was performed using the ImProm II RT system (Promega): after denaturation of 1 µg total RNA at 70 C for 5 min, reverse transcription was performed at 42 C for 60 min using Promega ImProm II reverse transcription system and oligo-dT20-primers according to the manufacturer’s protocol. Amplifications were then performed using the cDNA equivalent of 100 ng total RNA with Taq polymerase (Promega), 0.5 µM of each primer, 0.2 mM deoxynucleotide triphosphates, and 1.5 mM MgCl2. The sequences of the oligonucleotides used and annealing temperatures were as given in Table 2.
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) for 45 sec, and extension at 74 C for 45 sec, followed by a final extension step of 74 C for 10 min. Northern blot analysis
Twenty micrograms of total RNA from each tissue sample was electrophoresed on a 1% agarose gel containing 2% formaldehyde and blotted onto a positively charged nylon membrane (Hybond XL, Amersham, Aylesbury, UK). After labeling of the amplified cDNA probes with 50µCi 32P-
ATP using a random primed DNA labeling kit (Roche Applied Science, Mannheim, Germany), the membranes were hybridized using QuikHyb (Stratagene, Amsterdam, The Netherlands) hybridization solution. The blots were washed twice in 1x and 0.5x sodium chloride/sodium citrate buffer (each containing 0.1% sodium dodecyl sulfate) at 60 C, respectively, and exposed overnight at –80 C to BioMax film (Kodak, Rochester, NY) using an intensifying screen. The bio-imaging analyzer BAS-1500 (Fuji Photo Film Co., Tokyo, Japan) and MacBAS software version 2.4 (Fuji) was used to quantify the band intensities. After detection of the target RNA, the membrane was stripped and then reprobed.
The large number of tissue samples examined in this study required analysis of the samples on different blots. To ensure comparability of the relative mRNA expression of PPAR and melanocortin receptor-2 (MC2-R), one reference sample was carried out on all blots in parallel.
Cell culture
Rosiglitazone (supplied by GlaxoSmithKline, Munich, Germany), pioglitazone, and GW9662 (both purchased from Sigma, Taufkirchen, Germany) were dissolved in dimethylsulfoxide. The final concentration of dimethylsulfoxide in cell culture medium was adjusted to 0.1 and 0.2%, respectively. Synacthen (Tetrosactid, synthetic ACTH1–24 peptide, Novartis Pharma, Nuernberg, Germany) was dissolved in sterile PBS.
NCI h295 (CRL-10296; American Type Culture Collection, Manassas, VA) cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 5% fetal bovine serum (Invitrogen), antibiotics, 10–8 M hydrocortisone, 5 µg/ml insulin, 100 ng/ml transferrin, and 5.2 ng/ml SeO4 in a humidified atmosphere (95% air-5% CO2) before treatment with rosiglitazone or pioglitazone. Y1 cells (CCL-79; American Type Culture Collection) were cultured in Ham’s F10 medium (Invitrogen), supplemented with 2.5% fetal bovine serum (Invitrogen), 7.5% horse serum (Sigma), 50 U/ml penicillin, and 50 µg/ml streptomycin before treatment with rosiglitazone or pioglitazone.
For Northern blot experiments, NCI h295 cells were cultured in 10-cm dishes and treated with 50 µM rosiglitazone for 0, 24, and 48 h in triplicates. Cells were harvested, RNA extracted, and Northern blotting carried out as described above. Quantification was performed by scanning of the individual Northern blots and calculation of the ratio of the density of the given gene product [MC2-R, steroid acute regulatory protein (StAR), side chain cleavage (SCC) enzyme] to the density of the housekeeping gene (glyceraldehydes-3-phosphate dehydrogenase).
Cell viability assay, cell proliferation assay, and caspase-3 and -7 assay
The cell viability assay is based on the transformation and colorimetric quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma).
In brief, cells were plated in 96-well plates at a density of 20,000 cells/well. After 24 h, cells were treated with various concentrations of rosiglitazone or pioglitazone. After 24, 48, or 96 h, respectively, MTT stock solution was added (final concentration 0.5 mg/ml), and cells were incubated at 37 C for 2 h. To stop the coloring reaction and dissolve the formed formazan crystals, a solubilization solution (10% sodium dodecyl sulfate, 0.01 M HCl) was added, and the mixture was incubated overnight at room temperature. The color intensity was measured at 555 nm using a multiplate ELISA reader.
Cell proliferation was measured using a colorimetric 5-bromo-2'-deoxyuridine cell proliferation ELISA (Roche Applied Science) according to the manufacturer’s protocol.
Caspase-3 and -7 activity was assayed using the Promega Caspase Glo 3/7 system. For the detection of DNA laddering, NCI h295 was cultured in 6-well plates until subconfluence, and rosiglitazone at a concentration of 50 µM was added for 3, 6, and 24 h. Cells were harvested and genomic DNA was extracted using Wizard Genomic DNA kit (Promega) according to the manufacturer’s protocol. Gel electrophoresis was carried out with 500 ng DNA using a 1.5% agarose gel stained with ethidium bromide.
Transfection experiments
Full-length constructs of the p450SCC and p450C17 promoter (kindly provided by Dr. Gary Hammer, University of Michigan, Ann Arbor, MI) as well as full-length and 5'-deletion constructs of the human MC2-R promoter (22) were used as luciferase reporter gene constructs and transiently transfected into NCI h295 and Y1 cells, respectively, using ExGen 500 (MBI Fermentas, St. Leon-Rot, Germany). The Renilla luciferase vector pRL (Promega) was cotransfected for normalization. Twenty-four hours after transfection, cells were stimulated with rosiglitazone 5 x 10–5 M or forskolin 10–5 M or left untreated, and activity was measured after 24 h of incubation using the dual-luciferase reporter assay system (Promega). All transfection experiments were performed at least in triplicate. Promoter analyses were performed using MatInspector software (Genomatix Software GmbH, Munich, Germany) (23).
Hormone assays in cell culture medium
NCI h295 cells were cultured in 24-well plates for 24 h with medium supplemented with rosiglitazone or vehicle. Cell culture medium was collected for cortisol measurement and cell viability was determined using the MTT assay.
For ACTH stimulation assays, cells were cultured for 24 h with medium supplemented with rosiglitazone or vehicle, medium was then replaced by fresh medium containing rosiglitazone or vehicle and synacthen, and medium was collected after further 24 h of incubation for measurement of cortisol. Cell viability was determined using the MTT assay as described above.
Cortisol in cell culture medium was determined using a commercially available cortisol immunoradiometric assay (competitive immunoassay, DPC Biermann, Bad Nauheim, Germany). Each experiment was done in triplicate. The intra- and interassay coefficients of variation were less than 8% and less than 12%, respectively.
Data analysis
All results are expressed as mean ± SEM. Statistical comparisons were analyzed by ANOVA and Fisher’s protective least significant difference test using Stat View 5 (SAS Institute Inc., Cary, NC). Statistical significance is defined as P < 0.05 and is indicated as an asterisk (1) in the figures.
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Results |
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mRNA independent of their endocrine activity and cellular differentiation
To identify adrenocortical tissues as a potential target for TZD treatment, a variety of adrenal tumors was screened for PPAR expression. All tumor and normal adrenal samples expressed PPAR mRNA and expression levels were similar in all types of tissue samples. Normal adrenal 100.0 ± 10.3%, ACC 98.7 ± 7.4%, cortisol-producing adenoma 99.1 ± 13.7%, aldosterone-producing adenoma 93.3 ± 15.3%, and nonfunctioning adenoma 125.5 ± 11.0% (mean percent of normal adrenal ± SEM, no significant differences). In contrast, ACTH receptor (MC2-R) expression significantly differed among the groups, with highest expression levels in aldosteronomas (201.6 ± 70.2%) and lowest in ACCs (17.8 ± 6.4%), which corresponds with earlier findings (24). Expression of MC2-R in cortisol-producing adenoma (107.8 ± 17.4%) and nonfunctioning adenoma (68.7 ± 16.1%) was comparable with normal adrenal glands (100.0 ± 13.0%). A representative Northern blot hybridized with a PPAR probe and a MC2-R probe is shown in Fig. 1A.
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As a model of ACC, human NCI h295 cells, which we demonstrated to express PPAR at levels comparable with that of adrenal tumor tissue (see Fig. 7A; data not shown) were treated with rosiglitazone (1 x 10–6 to 5 x 10–5 M) for up to 96 h.
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). Similarly, treatment for 48 h led to a significant decrease in cell viability at doses of rosiglitazone of 1 x 10–5 M (93.3 ± 1.2%, P = 0.005) and 5 x 10–5 M (67.9 ± 1.0%, P < 0.0001), compared with untreated cells (100.0 ± 1.3%, Fig. 2B). With an incubation time of 96 h, doses of rosiglitazone required to yield a significant response on cellular viability dropped to 1 x 10–6 (95.4 ± 1.1%, P = 0.02), whereas higher doses had more pronounced effects (5 x 10–6, 79.4 ± 1.0%, P < 0.0001; 1 x 10–5, 81.8 ± 0.9%, P < 0.0001; 5 x 10–5, 18.3 ± 0.6%, P < 0.0001) in comparison with untreated cells (100.0 ± 2.4%, Fig. 2C). The effects of pioglitazone treatment (1 x 10–6 to 5 x 10–5 M) on NCI h295 cells for 48 h were comparable with that of rosiglitazone (data not shown).
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Treatment with rosiglitazone inhibits cellular proliferation and increases apoptosis in NCI h295 cells
To further define the cause of the overall decrease in cell viability induced by TZD treatment, we assessed possible effects of rosiglitazone and pioglitazone on proliferation and apoptosis in NCI h295 cells.
After 24 h of incubation, rosiglitazone significantly suppressed cellular proliferation at a concentration of 1 x 10–6 M (89.9 ± 2.6%; P = 0.0007), whereas this effect was further increased in a dose-dependent manner (5 x 10–6, 84.8 ± 1.6%, P < 0.0001; 1 x 10–5, 81.6 ± 1.1%, P < 0.0001; 5 x 10–5, 71.9 ± 1.3%, P < 0.0001), compared with untreated cells (100.0 ± 2.1%, Fig. 3A). Longer treatment further substantiated the negative effects of rosiglitazone on cellular proliferation (48 h: 1 x 10–5, 69.1 ± 14.8%, P = 0.04; 5 x 10–5, 40.6 ± 6.7%, P = 0.0004; untreated, 100.0 ± 4.0%, Fig. 3B; 96 h: 1 x 10–5, 87.6 ± 2.4%, P = 0.004; 5 x 10–5, 28.9 ± 0.9%, P < 0.0001; untreated, 100.0 ± 3.2%, Fig. 3C). Pioglitazone treatment in doses as described above yielded comparable results (data not shown).
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). Caspase activation was further validated by the presence of DNA laddering in treated NCI h295 cells (Fig. 4B), suggesting a PPAR-dependent activation of the apoptosis pathway.
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treatment decreases proliferation and induces apoptosis in adrenocortical cells, effects that both lead to a decrease of cellular viability as assessed by the MTT test. Rosiglitazone leads to a decrease in expression of cyclin E but increased IGF-II mRNA levels
To further evaluate the effects of TZD treatment on the functional phenotype of NCI h295 cells, total RNA was extracted and subjected to Northern blot analysis after incubation with 5 x 10–5 M rosiglitazone for 0, 24, and 48 h, respectively.
After 48 h of treatment with 50 µM rosiglitazone, expression of cyclin E decreased from 100 ± 4.8% in controls to 77.5 ± 11.4%, whereas IGF-II mRNA increased from 100.0 ± 1.7% in untreated cells to 180.2 ± 16.3% (Fig. 5).
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Rosiglitazone treatment increases MC2-R mRNA expression and steroidogenesis in NCI h295 cells
Expression levels of MC2-R mRNA as assessed by Northern blot substantially increased from 100 ± 6.4% of untreated cells to 340 ± 64% after 24 h of incubation and 393 ± 25% after 48 h of treatment. Endogenous StAR mRNA expression slightly increased to 159 ± 13% after 24 h and 125 ± 6% after 48 h of treatment with rosiglitazone, whereas SCC expression remained stable with 97 ± 7% after 24 h of incubation and decreased to 59 ± 1% after 48 h of incubation.
On a functional level, the observed increase in MC2-R expression was associated with a dose-dependent increase in cortisol secretion (5 x 10–5 M rosiglitazone for 24 h, 313 ± 10 ng/ml vs. untreated cells, 236 ± 13 ng/ml, P = 0.005). Taking into account the lower number of viable cells after rosiglitazone treatment, differences in cortisol secretion normalized by MTT assay within the same experiment yielded a significant difference in comparison with untreated cells (100.0 ± 8.2%) already at rosiglitazone doses of 5 x 10–6 M (134.5 ± 8.2%; P = 0.018) and above (1 x 10–5 M, 131.4 ± 7.6%, P = 0.028; 5 x 10–5 M, 170.7 ± 5.2%, P = 0.001; Fig. 6E). More pronounced differences in cortisol secretion upon rosiglitazone treatment were detectable when NCI h295 cells were stimulated with increasing doses of ACTH (0 nM ACTH, 100 ± 2.6% without rosiglitazone vs. 144 ± 4.7% with 50 µM rosiglitazone, P = 0.272; 1 nM ACTH, 92.6 ± 3.0 vs. 180.1 ± 11.5%, P = 0.040; 10 nM ACTH, 97.7 ± 3.6 vs. 203.0 ± 2.1%, P = 0.016; 100 nM ACTH, 120.1 ± 4.5 vs. 267.9 ± 62.4%, P = 0.002; Fig. 6F)
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To further substantiate the observed effects of rosiglitazone on endogenous MC2-R expression levels, MC2-R-, p450SCC-, and p450C17-luciferase constructs were transiently transfected into NCI h295 cells. In accordance with the results obtained by Northern blotting, rosiglitazone treatment led to a significant promoter activation of the transfected MC2-R construct (167.3 ± 9.0 vs. untreated 100 ± 3.6%, P = 0.008; forskolin 297.8 ± 22.0%, P < 0.0001). In contrast, p450SCC and p450C17 promoter activity was not significantly affected by rosiglitazone treatment (p450SCC: 82.4 ± 2.5 vs. 100 ± 4.2%, P = 0.6; p450C17: 94.2 ± 8.4 vs. 100.0 ± 6.2%, P = 0.9).
Promoter sequence analysis of the human MC2-R promoter revealed potential PPREs at position –712 to –692 bp, position –570 to –551 bp, position –479 to –459 bp, position –421 to –402 bp, position –116 to –96 bp, and position –46 to –26 bp with respect to the transcription start site. Accordingly, promoter activity of 5'-deletion constructs of the human MC2-R promoter showed continuous decrease of rosiglitazone-dependent promoter activation with decreasing promoter fragment length (rosiglitazone vs. untreated: 1 kb, 192.2 ± 9.0 vs. 100 ± 3.8%, P < 0.0001; 549 bp, 164.8 ± 6.5 vs. 100 ± 6.0%, P = 0.0004; 214 bp, 144.9 ± 12.8 vs. 100 ± 5.9%, P = 0.003; 64 bp, 134.5 ± 11.8 vs. 100 ± 13.8%, P = 0.46).
In PPAR-negative Y1 adrenocortical cancer cells, rosiglitazone does not increase MC2-R promoter activity
RT-PCR analysis demonstrated PPAR expression in NCI h295 adrenocortical cell line. In contrast, no PPAR mRNA could be amplified by RT-PCR from the murine adrenocortical cell line Y1 (Fig. 6C).
Transfection experiments with Y1 cells were performed as described above with full-length promoter constructsof MC2-R, p450SCC and C17-hydroxylase, respectively. Whereas incubation with forskolin significantly increased promoter activity of the MC2-R and p450SCC, incubation with rosiglitazone did not affect promoter activity of the MC2-R in Y1 cells (100.1 ± 10.9 vs. untreated 100.0 ± 7.3%).
Treatment with the PPAR antagonist GW9662 has no significant effect on rosiglitazone-induced growth inhibition but inhibits MC2-R up-regulation and steroidogenesis in NCI h295 cells
We used the specific and potent PPAR antagonist GW9662 to more directly assess PPAR-dependent mechanisms. Indeed, rosiglitazone-induced increase in MC2-R mRNA as assessed by Northern blot was antagonized by GW9662 treatment (Fig. 7A). Accordingly, cortisol secretion with and without treatment with rosiglitazone was significantly decreased when cells were incubated with 1 x 10–5 M GW9662 (1 x 10–5 M GW9662, 53.7 ± 3.6% vs. untreated cells 100 ± 1.4%; P = 0.0004; 1 x 10–5 M GW9662 + 5 x 10–5 M rosiglitazone, 86.1 ± 4.4% vs. 5 x 10–5 M rosiglitazone 115.8 ± 6.8%; P = 0.006; Fig. 7B).
However, rosiglitazone-induced decrease in cell viability could not be antagonized by GW9662 (0 M vs. 5 x 10–5 M rosiglitazone, P < 0.0001 for all doses of GW9662; Fig. 7C).
Taken together, these data indicate that the main effects on growth inhibition induced by rosiglitazone treatment are likely to be independent of PPAR activation, whereas induction of steroidogenesis seems to depend on PPAR-mediated pathways.
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Discussion |
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agonist therapy has only recently been introduced as a potential new treatment option for a variety of different malignancies resulting in suppression of tumor cell proliferation and induction of a more differentiated tumor phenotype (14, 17, 18). Although PPAR expression has already been demonstrated in the normal murine adrenal gland (25), no data have been available regarding its expression in human adrenal disease. As we show herein, PPAR mRNA expression is readily detectable in a variety of adrenal tumors, including ACC. Interestingly, PPAR expression levels seem to be independent of clinical parameters such as tumor size and hormonal profile or molecular markers of differentiation such as MC2-R expression. However, from a clinical standpoint, these results indicated that ACC could be a potential target for PPAR agonist therapy. In line with this notion, treatment of the human adrenocortical tumor cell line NCI h295 with the PPAR agonists rosiglitazone and pioglitazone resulted in a decrease of cellular viability in a time- and dose-dependent manner. This decrease in cell viability is associated with a suppression of cellular proliferation and an induction of apoptosis in these cells.
Although decreasing proliferation, rosiglitazone induced an increase in steroidogenesis in NCI h295 cells, a finding that was further substantiated by ACTH stimulation. On a transcriptional level, rosiglitazone treatment robustly increased MC2-R mRNA and StAR expression, although to a lesser extent. The finding on endogenous MC2-R expression was verified by luciferase assays using full-length MC2-R promoter constructs. Only recently MC2-R up-regulation upon TZD treatment has been demonstrated in murine preadipocytes and a PPRE has been identified in the promoter region of the murine MC2-R (26). Promoter analysis of the human MC2-R revealed several potential PPREs. Although we did not perform EMSA assays or site-directed mutagenesis at this stage to directly validate PPAR binding or functional activity of these predicted PPREs, luciferase assays with 5' deletion constructs suggested that the defined PPREs have functional significance. Furthermore, TZD treatment of Y1 cells that lack PPAR expression did not result in an increase in MC2-R expression, and rosiglitazone-induced effects in NCI h295 cells could be abolished by antagonist treatment. Taken together, these data provide good evidence that MC2-R expression can be increased by PPAR-dependent mechanisms. Moreover, the observed increase in steroidogenesis is likely to result from an increase in the responsiveness of NCI h295 cells to ACTH stimulation.
At first glance, these data on increased steroidogenesis are contradicting with results obtained from patients with polycystic ovary syndrome and patients with hypercortisolism treated with PPAR agonists. TZD treatment has been shown to decrease ovarian sex steroid production (27) and directly inhibit P450c17 enzyme activity (28). Moreover, rosiglitazone has been evaluated as an alternative treatment option in patients with central Cushing’s syndrome. In this context, PPAR agonists result in a decrease of corticotroph proliferation and decrease in ACTH secretion (15, 16). However, these in vitro and clinical data as well as the data presented herein are clearly dependent on the experimental context. Direct inhibition of P450c17 activity in yeast microsomes does not preclude the increase in steroidogenesis in a whole-cell system through transcriptional regulation. In addition, because theca cells lack MC2-R expression, rosiglitazone is unlikely to affect ovarian steroidogenesis equivalent to adrenal steroidogenesis. Recent follow-up data from patients with Cushing’s disease on treatment with TZDs indicate favorable clinical outcome in a subgroup of patients, whereas in others the treatment fails to improve hormonal hypersecretion (29). Although effects of PPAR agonists on adrenal steroidogenesis in hyperplastic adrenal glands in the context of Cushing’s disease might well vary from that in an adrenocortical tumor cell line, our in vitro data give evidence that the observed failure to normalize cortisol hypersecretion might in part be due to the induction of a higher steroidogenic activity of the adrenal cortex.
In addition to its role in the regulation of adrenal steroidogenesis, there is an increasing body of evidence suggesting that ACTH is also implicated in adrenal differentiation. In a series of 20 cases with benign and malignant adrenocortical tumors, we have demonstrated an association between loss of heterozygosity (LOH) of the MC2-R gene and an advanced tumor stage and a more rapid course of disease than in carcinoma patients without LOH (30). These data give indirect evidence that allelic loss of the MC2-R gene in adrenocortical tumors can result in loss of differentiation, a characteristic feature of human tumorigenesis that is associated with clonal expansion of a malignant cell clone. Thus, the PPAR-dependent increase in MC2-R expression is in line with the concept that in addition to its effects on proliferation and apoptosis, TZDs can act as inducers of a more differentiated adrenocortical phenotype.
Maternal LOH of the 11p15 region together with duplication of the paternal allele resulting in overexpression of the IGF-II gene and loss of p57KIP2 gene expression are associated with a malignant phenotype in sporadic adrenocortical tumors (31). Whereas IGF-II leads to proliferation of adrenocortical cells, abrogation of p57KIP2 gene expression results in increased expression levels of G1 cyclins, namely cyclin E (32), which ultimately participates in disruption of normal cell cycle control in adrenocortical tumors. Accordingly, IGF-II overexpression and increased cyclin E levels have been evaluated as independent predictors of poor clinical outcome (32, 33, 34). On a molecular level, PPAR-dependent decrease in cellular proliferation is associated with a decrease of cyclin E expression levels, indicating similar effects of PPAR signaling on adrenal cell cycle regulation as observed in other tumor entities (16, 35).
IGF-II is one of the most potent growth factors for the adrenal cortex both during development (36) and in the context of tumorigenesis (37). Accordingly, proliferation of NCI h295 cells has been demonstrated to be dependent on auto- or paracrine effects of IGF-II secretion (38). Surprisingly, despite the clear growth-suppressive effects of TZD treatment on adrenocortical cells, IGF-II levels significantly increased in a time-dependent manner. In fact, if pathophysiological relevant, these findings have to be interpreted as TZD-induced phenotypical changes downstream of IGF-II action or as TZD-induced effects independent and dominant of known effects of IGF-II. In each case, although the less aggressive cellular phenotype indicates that activation of the PPAR-dependent pathways might directly or indirectly overcome autocrine growth-promoting effects of IGF-II, this finding merits caution and needs to be evaluated in more detail in the future.
To further define effects of TZD treatment on NCI h295 cells as PPAR-dependent or PPAR-independent mechanisms, we used the potent and selective PPAR antagonist GW9662 (39). In line with the potential PPREs in the promoter region of the MC2-R gene, we demonstrate that GW9662 treatment decreases rosiglitazone-induced MC2-R expression in NCI h295 cells. Accordingly, both baseline and rosiglitazone-induced steroidogenesis was significantly blocked after incubation with GW9662. Thus, these findings indicate that TZD-induced differentiation of human adrenocortical cancer cells, defined as a shift toward a more steroidogenic phenotype, is mediated through activation of PPAR-dependent pathways. In contrast, GW9662 treatment does significantly increase cellular viability in rosiglitazone-treated and untreated cells. As such, these findings indicate that the main growth-inhibiting effects of TZDs are mediated by other, PPAR-independent pathways.
An increasing body of evidence from the recent literature indicates modulation of cellular growth upon TZD treatment independent of PPAR (40, 41, 42, 43). Possible PPAR-independent mechanisms include induction of cellular acidosis through inhibition of Na+/H+ exchanger (40), inhibition of translational initiation through calcium store depletion (44), and release of apoptotic factors from the mitochondria through production of reactive oxygen species (45). Our results indicate that TZD treatment has diverse effects on adrenocortical cancer cells including both PPAR-dependent and PPAR-independent mechanisms that together result in a more differentiated and less aggressive tumor phenotype. Similar results have been reported for pancreas carcinoma in which tumor growth has been defined as a PPAR-dependent mechanism, whereas tumor invasiveness has been reported to be regulated independent of PPAR-mediated pathways (43).
Obviously in vitro results do not necessarily translate into clinical practice; thus, before considering the use of PPAR agonists as an additional treatment option for patients with adrenal carcinoma, questions regarding dosage and treatment effectivity and toxicity remain to be answered. TZDs such as rosiglitazone are in widespread clinical use for the treatment of diabetes type 2. Unlike the now-withdrawn first generation of TZDs, rosiglitazone appears to have little propensity for damage to hepatic cells (46). Plasma levels measured after standard rosiglitazone treatment (8 mg) are in the range of 1 µM (47). However, higher doses also are well tolerated and result in similar percentage of adverse events than placebo in double-blind clinical trials (48, 49). In addition, in preclinical studies animals received up to 150 mg/kg·d without obvious toxicity (15, 16). Because our results indicate significant effects on adrenocortical cell viability and proliferation in a dosage range similar to that reached during treatment for type 2 diabetes, these findings are encouraging to proceed with further studies on more physiological animal models of adrenal tumorigenesis.
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Acknowledgments |
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In addition, we are indebted to Dr. Gary Hammer (University of Michigan, Ann Arbor, MI) for kindly providing the SCC and C17 promoter constructs as well as Kai Dallmeier, Dominik Schulte, and Dörte Ortmann for technical advice and assistance with the in vitro experiments.
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Footnotes |
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First Published Online May 10, 2005
1 See Acknowledgments for a listing of the German and Austrian Adrenal Network members. 
Abbreviations: ACC, Adrenocortical carcinoma; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; LOH, loss of heterozygosity; MC2-R, melanocortin receptor-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome-proliferator response element; SCC, side-chain cleavage enzymes; StAR, steroid acute regulatory protein; TZD, thiazolidinedione.
Received July 1, 2004.
Accepted April 22, 2005.
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: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 276:37731–37734 in the control of lipid metabolism. J Lipid Res 43:177–186: implications for cardiovascular disease. Hypertension 43:297–305 and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci USA 95:8806–8811 and the retinoid X receptor. Proc Natl Acad Sci USA 94:237–241 receptor is a novel therapeutic target for ACTH-secreting pituitary adenomas. Nat Med 8:1281–1287[CrossRef][Medline]
receptor ligands: novel therapy for pituitary adenomas. J Clin Invest 111:1381–1388[CrossRef][Medline]
activation inhibits tumor progression in non-small-cell lung cancer. Oncogene 23:100–108[CrossRef][Medline]
in human prostate cancer. Proc Natl Acad Sci USA 97:10990–10995 activation induces cell cycle arrest via the p53-independent pathway in human anaplastic thyroid cancer cells. Jpn J Cancer Res 93:1358–1365[CrossRef][Medline]
ligand troglitazone in patients with liposarcoma. Proc Natl Acad Sci USA 96:3951–3956 ligand rosiglitazone in Cushing’s disease. Eur J Endocrinol 151:173–178[Abstract]
reduces the growth rate of pancreatic cancer cells through the reduction of cyclin D1. Life Sci 70:1565–1575[CrossRef][Medline]
inhibits adipocyte differentiation. J Biol Chem 275:1873–1877-independent mechanism in breast cancer-derived cell lines. Clin Cancer Res 10:7022–7030-dependent and -independent growth inhibition of gastrointestinal tumour cells. Genes Cells 9:1113–1123}, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPAR{} agonist rosiglitazone, independently of PPAR{} activation. Br J Pharmacol 8:8
independent mechanisms. Gut 53:1688–1697 and mediated by inhibition of translation initiation. Cancer Res 61:6213–6218. J Biol Chem 279:8976–8985 and its ligand-dependent growth inhibition in human brain tumor cell lines. Jpn J Cancer Res 93:660–666[CrossRef][Medline]
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