Endocrinology, doi:10.1210/en.2005-0544
Endocrinology Vol. 147, No. 2 769-772
Copyright © 2006 by The Endocrine Society
Inhibition of 11ß-Hydroxysteroid Dehydrogenase Eliminates Impaired Glucocorticoid Suppression and Induces Apoptosis in Corticotroph Tumor Cells
Takeshi Nigawara,
Yasumasa Iwasaki,
Masato Asai,
Masanori Yoshida,
Machiko Kambayashi,
Hiroshi Sashinami,
Kozo Hashimoto and
Toshihiro Suda
Departments of Clinical Pathophysiology (T.N., Y.I., M.K.) and Medicine (M.A., M.Y.), Nagoya University Graduate School of Medicine and Hospital, Nagoya 466-8550, Japan; Departments of Endocrinology, Metabolism, and Infectious Diseases (T.N., T.S.) and Bacteriology (H.S.), Hirosaki University School of Medicine, Hirosaki 036-8562, Japan; and Department of Endocrinology, Metabolism, and Nephrology (Y.I., K.H.), Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku 783-8505, Japan
Address all correspondence and requests for reprints to: Yasumasa Iwasaki, M.D., Ph.D., Department of Medicine, Kochi University School of Medicine, 185-1 Kohasu, Oko-cho, Nankoku 783-8505. E-mail: iwasaki{at}med.kochi-u.ac.jp.
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Abstract
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Cushing’s disease is characterized by persistent ACTH secretion under hypercortisolemia. In an attempt to clarify the molecular mechanism, we examined the effect of 11ß-hydroxysteroid dehydrogenase (HSD) inhibition on glucocorticoid suppression of ACTH release using murine corticotroph tumor cells. We found that 11ß-HSD2, as well as -HSD1, was expressed in the cells and that its inhibition by carbenoxolone significantly improved the negative feedback effect of glucocorticoid. Carbenoxolone also enhanced apoptosis induced by cortisol. These effects are most likely attributable to inhibition of 11ß-HSD2 because only cortisol, a substrate of 11ß-HSD2, was present in these experimental conditions. We conclude that ectopic expression of 11ß-HSD2 is, at least in part, responsible for the impaired glucocorticoid suppression in corticotroph adenoma. Inhibition of 11ß-HSD2 may be applicable to the medical therapy for Cushing’s disease.
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Introduction
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CUSHING’S DISEASE IS caused by pituitary corticotroph adenoma with ACTH hypersecretion and the resultant hypercortisolemia. The cardinal characteristic of the disease is the impairment of the negative feedback effect by glucocorticoids; in the face of high blood cortisol levels, the tumor cells continue to secrete ACTH and proliferate, whereas normal corticotroph cells cease to secrete ACTH and degenerate (1, 2). The molecular mechanism of the impaired glucocorticoid suppression remains unclear; some studies have suspected that the glucocorticoid receptor is underexpressed or mutated, but none of these studies have reached a conclusive result (3, 4). Recently, Korbonits et al. (5) and Rabbitt et al. (6) reported an ectopic expression of the cortisol-inactivating enzyme 11ß-hydroxysteroid dehydrogenase (HSD) type 2 in human corticotroph tumor cells, which converts active cortisol to inactive cortisone. To clarify whether the ectopic expression of the enzyme is responsible for the glucocorticoid resistance, we examined the effect of 11ß-HSD inhibition on ACTH secretion and cell survival, employing AtT20 mouse corticotroph tumor cells as an in vitro model of human Cushing’s adenoma.
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Materials and Methods
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Cell culture
An AtT20 mouse corticotroph cell line transfected stably with the POMC-luciferase fusion gene (AtT20PL) was used (7). The cells were maintained in a T75 culture flask with DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin; Invitrogen) under a 5% CO2/95% atmosphere at 37 C. For each experiment, the cells were plated in 24-well plates with approximately 50% confluence. The culture medium was changed to DMEM supplemented with 0.5% fetal bovine serum treated with dextran-coated charcoal on the following day, and the cells were cultured for three to four more days, during which the culture medium was changed every other day (7).
RT-PCR
Total RNA was isolated from the AtT20PL cells using RNeasy RNA extraction kit (Qiagen, Valencia, CA), and 5 µg total RNA was used for the RT reaction with Moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen). A 12-wk-old male C57BL/6J mouse (CLEA Japan, Inc., Tokyo, Japan) was killed by cervical dislocation, and total RNA from the anterior pituitary gland was isolated and applied to RT by the same method as mentioned above. The method of the animal experiment was approved by the Ethical Committee of Hirosaki University. The cDNA samples obtained was then amplified by PCR with Taq DNA polymerase (Takara Shuzo, Tokyo, Japan). The PCR condition used for all the reactions was as follows: 96 C, 3 min, for initial denaturation; 95 C, 10 sec, 55 C, 30 sec, and 72 C 1 min for cycle amplification (30 cycles); and 72 C 6 min for final extension. The sequences of primer sets were: 11ß-HSD2 set A, forward 5'-CTAGAACTGCGTGACCTCTG-3'; reverse, 5'-TGGATGAAATACATGAGCCC-3'; set B, forward, 5'-GAGACAGCTAAGAAACTGGATG-3'; reverse, 5'-GCCAGGCTTGATAATGCTGAC-3'; 11ß-HSD1 set A, forward, 5'-GAGCCCATGTGGTATTGACTG-3', reverse, 5'-CTTTGATGATCTCCAGGGCG-3'; set B, forward, 5'-CAGACCAGAAATGCTCCAGG-3', and reverse, 5'-GGACACAGAGAGTGATGGACA-3'. All primer sets were designed to span at least one intron to avoid false-positive signals derived from genomic DNA.
Experiments
To examine the effect of 11ß-HSD inhibition on the suppression by cortisol of basal ACTH secretion and the 5'-promoter activity of POMC gene, AtT20PL cells were treated with cortisol (100 nM, 1 h for secretion study and 5 h for promoter study) and/or carbenoxolone (CBX; 1 µM for secretion study and 5 µM for promoter study, 24 h), and then the ACTH release in the culture media or the POMC promoter activity was estimated. For dose-response study, cells were treated with two different doses of CBX (1 and 5 µM) for 24 h and also with forskolin (10 µM) and cortisol (0.1–100 nM) for 5 h until the end of the experiment. In promoter activity experiments, the cells were harvested at the end of the designated time period and were subjected to luciferase assay (8). ACTH concentrations in the media were assayed with an immunoradiometric assay kit (Mitsubishi Kagaku Iatron, Tokyo, Japan).
To determine the effect of CBX and/or cortisol on cell viability, AtT20PL cells were treated with CBX (5 µM) and/or cortisol (100 nM) for 7 d, and the viability of the cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay (CellTiter96 Aqueous One Solution, Promega, Madison, WI). To determine the effect on cell growth, AtT20 cells (1 x 104) were seeded in 24-well plates and cultured with CBX (5 µM) and/or cortisol (100 nM) for 3 d. Media were then changed to those containing 160 kBq/well of 3H-thymidine (PerkinElmer, Boston, MA) 24 h before harvesting. Finally, cells were collected, washed, and measured for ß-ray activity in a liquid scintillation counter.
To examine whether apoptosis was involved in the decrease of AtT20 cells by CBX and cortisol, total DNA samples of the cells were extracted and were subjected to agarose gel electrophoresis. Apoptotic changes of the cells were also assessed by terminal deoxyribonucleotidyl transferase-mediated UTP nick-end labeling (TUNEL) kit (DeadEnd Fluorometric TUNEL system, Promega). Briefly, AtT20PL cells were treated with vehicle or with CBX (5 µM) and/or cortisol (100 nM) for 2 d. Then, using a confocal microscope, five fields (x200) near the center of each well were arbitrarily chosen, and the apoptotic cells in each field were counted and applied for statistical analysis (ANOVA).
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Results
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We first confirmed an expression of 11ß-HSD2 mRNA in AtT20PL cells by RT-PCR (Fig. 1A
). mRNA for 11ß-HSD1 was also amplified. In contrast, the expression of 11ß-HSD1, but not 11ß-HSD2, was observed in the normal mouse anterior pituitary gland.
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FIG. 1. Expression of 11ß-HSDs in AtT20PL cells and the effect of inhibition of the enzymes by CBX. A, Expression of 11ß-HSDs in AtT20PL cells and in the normal mouse anterior pituitary gland. RT-PCR analysis was carried out using two different sets of primers for each subtype of the enzyme. B, Effects of 11ß-HSDs inhibition by CBX on the suppression of the POMC gene 5'-promoter activity by cortisol. AtT20PL cells were treated with vehicle or cortisol and also with or without CBX (1 µM), and then the basal ACTH release and the 5'-promoter activity of POMC gene were estimated. C, Effect of CBX on the dose-dependent inhibition of cortisol on the forskolin-induced promoter activity. AtT20 PL cells were treated with vehicle or two different doses of CBX (1 and 5 µM) and also with vehicle or four different doses (100 pM to 100 nM) of cortisol under forskolin stimulation (10 µM). *, P < 0.05 vs. the value of the corresponding group treated with forskolin alone.
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To examine the role of the enzymes, we then treated the cells with the specific 11ß-HSD inhibitor CBX. We found that CBX alone did not influence either basal ACTH release or POMC expression. On the other hand, in the presence of cortisol, simultaneous treatment with CBX significantly inhibited both ACTH secretion and POMC promoter activity (Fig. 1B
). In a dose-response study, only a high concentration of cortisol (10 nM or above) suppressed forskolin-induced POMC gene expression. In contrast, the minimal inhibitory concentration of cortisol was shifted to 1 nM and 100 pM in the presence of 1 and 5 µM of CBX, respectively, thus showing a dose-dependent enhancement of the glucocorticoid negative feedback inhibition. Although CBX inhibits both 11ß-HSD1 and -HSD2, the above effect was obtained in the presence of cortisol (a substrate for 11ß-HSD2), suggesting that the ectopic expression of 11ß-HSD2 is, at least partly, responsible for the functional glucocorticoid resistance.
We then examined the effect of inhibition of 11ß-HSDs on cell viability (Fig. 2A). In culture medium containing no cortisol, cells continued to proliferate regardless of the presence or absence of CBX. When cultured with cortisol alone (100 nM), cells appeared somewhat less thriving, but most of the cells survived. In distinct contrast, the majority of the cells cultured with both cortisol (100 nM) and CBX ceased to grow and eventually degenerated within a week. In addition, cells cultured with a high concentration of cortisol (1 µM) alone showed similar changes as CBX/cortisol (100 nM). The difference in cell viability among the groups, estimated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, was quantitatively significant (Fig. 2B). The same treatment did not affect cell proliferation itself, as determined by 3H-incorporation for the first 3 d, suggesting that the decrease in cell number observed (Fig. 2A) is not due to reduced growth rate (Fig. 2C).
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FIG. 2. The effect of 11ß-HSD inhibition on cell proliferation and viability. A, AtT20PL cells were treated with CBX (5 µM) and/or two different doses of cortisol (+, 100 nM; ++, 1 µM) for up to 7 d. Representative dual-phase microscopic views at d 7 (x200) are shown. B, Cells were treated with CBX (5 µM) and/or cortisol (100 nM) for 7 d, and the viability of the cells at d 7 was assessed by colorimetric assay (n = 4; CellTiter96 Aqueous One Solution, Promega). C, Cells were treated with CBX (5 µM) and/or cortisol (100 nM), and cell proliferation rate was assessed by 3H-thymidine incorporation study at d 3. *, P < 0.05 vs. control (CBX-/cortisol-).
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Finally, we examined the possible involvement of apoptotic changes. When AtT20 cells were treated with CBX (5 µM) and cortisol (100 nM) for 7 d, a clear laddering of DNA was observed (Fig. 3A). Furthermore, TUNEL study revealed that CBX enhanced the proapoptotic effect of cortisol (Fig. 3, B and C).
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FIG. 3. Apoptotic changes induced by 11ß-HSD inhibition. A, AtT20PL cells were treated with CBX (5 µM) plus cortisol (100 nM) for 7 d, and DNA laddering was analyzed by agarose gel electrophoresis. B and C, AtT20PL cells were treated with CBX (5 µM) plus cortisol (100 nM), and apoptotic changes were analyzed by fluorescent microscopic imaging (x200) of the TUNEL method (B) or by estimation of the number of TUNEL-positive cells per x200 visual field (n = 5) (C) on d 2. *, P < 0.05 vs. control (CBX-/cortisol-).
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Discussion
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Recent studies suggest that significant expression of 11ß-HSD2 is found in human corticotroph tumor cells (5, 6). Because 11ß-HSD2 converts active cortisol to inactive cortisone, the ectopic expression of the enzyme may account for the attenuated glucocorticoid suppressibility commonly observed in Cushing’s disease. Our data using murine corticotroph tumor cells expressing 11ß-HSD2 demonstrate for the first time that the inhibition of 11ß-HSDs by CBX enhances the glucocorticoid inhibition of both ACTH secretion and POMC expression. We also found that the inhibition results in cellular apoptosis, probably caused by the augmented action of cortisol (9). Thus, we assume that intracellular inactivation of cortisol to cortisone by 11ß-HSD2 is, at least in part, responsible for the glucocorticoid resistance observed in corticotroph tumor cells, in terms of both functional resistance and cell survival under hypercortisolemia.
In this study, CBX clearly enhanced the glucocorticoid suppression. More precisely, CBX alone did not have any effect but showed a significant inhibitory effect on both basal ACTH secretion and POMC 5'-promoter activity in the presence of cortisol. Furthermore, CBX clearly enhanced the dose-dependent inhibition of cortisol on forskolin-induced POMC gene expression. Although CBX inhibits both 11ß-HSD1 and -HSD2, the effect of CBX is probably caused by the inhibition of 11ß-HSD2 because only cortisol, a substrate for the enzyme, was present in the culture system.
Glucocorticoid hormone is known to exert a potent inhibitory effect on normal pituitary corticotroph cells, and chronic administration of large doses of glucocorticoid elicits cellular atrophy and/or degeneration in vivo (10). In this sense, 11ß-HSD2 appears to contribute to the survival of the corticotroph tumor cells under hypercortisolemia. Indeed, our data show that CBX plus cortisol results in an unequivocal decrease of the AtT20PL cell population. Interestingly, a supraphysiological concentration of cortisol (1 µM) mimicked the effect of CBX plus cortisol (100 nM), probably because the high concentration of cortisol overwhelmed the inactivating capacity of 11ß-HSD2.
In this study, we used cortisol instead of corticosterone. Because corticosterone is the physiological glucocorticoid in the mouse and has higher mineralocorticoid activity, the use of corticosterone might have produced different results. However, because the affinity of corticosterone to 11ß-HSD2 is reported to be similar to or even higher than that of cortisol (11), we would expect the results of 11ß-HSD2 inhibition by CBX to be even more pronounced.
Rabbitt et al. (6) reported, using the primary culture of human nonfunctioning or somatotroph tumor cells, that glucocorticoid treatment in the presence of 11ß-HSD2 inhibitor glycyrrhetinic acid exerted an antiproliferative effect. In our study using murine corticotroph tumor cells, however, the apparent decrease in cell number may not entirely be caused by a growth inhibitory effect of glucocorticoid because 3H-thymidine incorporation was not affected by the treatment during the early period of cultivation. Instead, we found that inhibition of 11ß-HSD2 induced cellular apoptosis, probably because of the increased concentration and action of intracellular cortisol. It is well known that glucocorticoid hormones are major inducers of apoptosis in some cell types (10) and are associated with growth inhibition of tumor cells (12, 13).
Our functional study using murine corticotroph tumor cells, as well as the previous studies using specimens of human pituitary adenoma (5, 6), may have a clinical implication for the medical therapy of Cushing’s disease. The current treatment of choice in Cushing’s corticotroph adenoma is surgical resection via the transsphenoidal approach. However, nonsurgical measures are needed for patients whose tumors are difficult to localize, or those with recurrence, both of which are frequently encountered (14, 15). Although irradiation is one of the options, it does not exhibit a prompt effect and sometimes causes eventual panhypopituitarism. Administration of steroidogenesis inhibitors (metyrapone and mitotane) is also effective in some patients although not well-tolerated because of their profound side effects. Cyproheptadine and bromocriptine, which have been tried in the past, have shown only limited benefit. In this context, the inhibitor of 11ß-HSD2 may be a promising candidate for the medical therapy of Cushing’s disease. Licorice derivatives such as CBX and glycyrrhetinic acid are commonly used as food additives worldwide, and their general safety is recognized. We think that their possible side effects (hypertension and hypokalemia) can at least partly be avoided by the concomitant use of specific mineralocorticoid receptor antagonists such as spironolactone or eplerenone, theoretically without affecting the expected therapeutic effect at the pituitary tumor. The development of a specific drug delivery system to the tumor should also be undertaken.
A novel, potent pharmacological agent against Cushing’s disease would be of significant use as an adjuvant therapy to surgical resection or as the primary therapy in inoperable cases. Further accumulation of evidence is awaited before clinical application.
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Acknowledgments
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We thank Ms. Tatsuyo Miura for excellent technical assistance.
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Footnotes
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First Published Online October 27, 2005
Abbreviations: CBX, Carbenoxolone; HSD, hydroxysteroid dehydrogenase; TUNEL, terminal deoxyribonucleotidyl transferase-mediated UTP nick-end labeling.
Received May 5, 2005.
Accepted for publication October 17, 2005.
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Abstract
Introduction
