New mechanisms of adrenostatic compounds in a human adrenocortical cancer cell line
M. Fassnacht, S. Hahner, F. Beuschlein*, A. Klink*, M. Reincke* and B. Allolio
Schwerpunkt Endokrinologie, Department of Medicine, University of Wuerzburg, Germany; * Schwerpunkt Endokrinologie, Department of Medicine, University of Freiburg, Germany
| Abstract | Background Adrenostatic compounds are frequently used in the treatment of patients with Cushing's syndrome and act via direct inhibition of steroidogenic enzymes. However, additional mechanisms may be involved in the blockade of adrenal steroid secretion. We therefore investigated the effects of aminoglutethimide (AG), metyrapone (MTP) and etomidate (ETO) in the human NCI-h295 adrenocortical carcinoma cell line. Materials and methods Cells were incubated with increasing doses of the adrenostatic compounds. Steroid hormone secretion (cortisol, 17-OH-progesterone, DHEA-S) and cAMP synthesis were determined and Northern blot analysis and cell proliferation experi- ments were performed. Results ETO was the most potent adrenostatic compound inhibiting P450c11 hydroxy- lase at low concentrations (IC50 15 nM), and also blocking P450 side-chain cleavage (scc) enzyme (IC50 400 nM) at higher concentrations. The pattern of enzyme inhibition was similar for MTP with an IC50 of 3.5 p.M for P450c11 and 17 u.M for P450scc, while AG blocked P450scc with an IC50 of 10 u.M. AG significantly suppressed the baseline ACTH- R mRNA expression in a dose-dependent fashion (300 u.M AG: 5% ± 1%; 30 µM AG: 64% ± 1%; 3 µM AG: 108% ± 19% compared with control cells: 100% ± 11%) but increased glucocorticoid receptor mRNA. The reduced ACTH-R mRNA was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of ACTH-R protein. The simultaneous incubation of hydrocortisone together with AG reversed the inhibitory effect of AG on the ACTH-R expression. AG and ETO inhibited cell prolifera- tion in the NCI-h295 cells, but ETO was much more potent and showed antiproliferative effects at concentrations of 6 uM. The growth inhibition was not reversed by administra- tion of hydrocortisone. Conclusions Our data demonstrate that adrenostatic compounds not only act by suppres- sion of steroidogenic enzymes but can also influence both ACTH-R expression and cell proliferation in adrenal cells. As these effects occur in vitro at concentrations that are reached during treatment with theses drugs in patients, they are probably also of clinical relevance. Particularly the antiproliferative activity of ETO may be useful in Cushing's syndrome due to adrenocortical cancer. The interaction of steroidogenesis, ACTH-R and glucocorticoid receptor expression as well as cell proliferation provides a new concept of the intra-adrenal response to stress in humans. Keywords Adrenostatic compounds, Cushing's syndrome. Eur J Clin Invest 2000; 30 (Suppl. 3): 76-82 |
Schwerpunkt Endokrinologie, Department of Medicine, University of Wuerzburg, Germany (M. Fassnacht, S. Hahner, B. Allolio); Schwerpunkt Endokrinologie, Department of Medicine, University of Freiburg, Germany (F. Beuschlein, A. Klinik, M. Reinche).
Correspondence to: M. Fassnacht, MD, Schwerpunkt Endokrinologie, Medizinische Universitätsklinik Würzburg, 97080 Würzburg, Germany. Tel .: +49-931-2013122; fax: +49- 931-2012283; e-mail: M.Fassnacht@medizin.uni-wuerzburg.de
Introduction
Inhibitors of adrenal steroidogenesis (adrenostatic agents) play an important role in the management of patients with Cushing’s syndrome (CS). These compounds reduce cor- tisol secretion by blocking one or more of the steroido- genic enzymes (Table 1).
Aminoglutethimide (AG) was used as an anticonvulsant for several years before its adrenostatic activity was
| Aminoglutethimide | P450scc enzyme |
|---|---|
| Metyrapone | P450c11 hydroxylase |
| P450scc enzyme | |
| Etomidate | P450c11 hydroxylase |
| P450scc enzyme | |
| Trilostane | 3-ß-hydroxysteroid-dehydrogenase |
| Ketoconazole | P450c17 enzyme |
| P450c11 hydroxylase | |
| P450scc enzyme |
discovered. AG inhibits both P450 side-chain cleavage (scc) enzyme and aromatase [1]. A number of studies have demonstrated the usefulness of AG (0-5-2 g day 1) in the treatment of patients with CS [2-5]. If AG monother- apy is insufficient to induce remission of hypercortisolism, it can be successfully combined with metyrapone [6].
Metyrapone (MTP) also inhibits a variety of P450 enzymes also, primarily 11ß-hydroxylase [7-9]. MTP is as effective as AG in the normalization of serum cortisol in CS.
The hypnotic imidazole derivative etomidate (ETO) is both in vivo and in vitro the most potent adrenostatic drug available [10-12]. ETO interacts with the cytochrome P450 enzymes 11ß-hydroxylase (P450c11) and side-chain cleavage enzyme (P450scc) [13-17]. A single hypnotic induction dose leads to adrenal inhibition for several hours [18,19]. However, used as low dose therapy it is well tolerated without hypnotic side-effects and rapidly decreases serum cortisol in patients with CS [20-22].
Until now there has been no evidence that these adrenostatic drugs act by any other mechanisms beside impairment of enzyme activity. With the establishment of the human adrenocortical cell line NCI-h295 in 1990 [23] a good model has become available to study adrenal function in more detail [24-27]. Therefore, the aim of our study was to investigate whether adrenostatic agents influ- ence adrenal function beyond the know inhibition of steroidogenic enzymes.
Materials and methods
Cell culture
The NCI-h295 tumour cell line was maintained in RPMI 1640 medium supplemented with transferrin, insulin, selenium and 2% fetal calf serum (TIS medium). The cells were incubated for 48 h with increasing con- centrations of the adrenostatic compounds AG (60 nM-300 µM), MTP (60nM-300µM), ETO (0-06 nM-60 MM) and with ACTH (10nM), forskolin (10 µM), hydrocortisone (12µM), respectively. At the end of the incubation period steroid hormones (cortisol, 17-OH-progesterone, DHEA-S) were determinated in cell supernant by established radioimmunoassay (RIA) and
cells were prepared for RNA extraction. To measure the cAMP response to ACTH stimulation cells were grown in 24-well plates (1.5 million cells per well) in TIS medium. Cells were pretreated with and without AG (3-300 M) for 48 h and incubated with and without ACTH (10 nm) for 10 min. Intracellular cAMP accumulation was deter- mined by RIA.
For cell proliferation experiments cells were seeded in 24 well plates and incubated with increasing concentra- tions of AG and ETO. After 2, 4, 6 and 8 days of culture, the cells were examined by trypan blue staining for cell viability and counted with a coulter counter. The number of cells is given as mean ± SEM.
RNA extraction and Northern analysis
Total RNA was extracted using the guanidinium isothio- cyante method and blotted after electrophoresis and hybridised with a human ACTH-R cDNA probe [28] and a human glucocorticoid receptor cDNA probe [29]. The blots were stripped and re-hybridized with a mouse ß-actin cDNA probe for standardization. Autoradio- graphic images were digitalized with a video camera and computer-based image analysis system using the IMAGE program (NIMH, National Institute of Health, Bethesda, USA). The mean mRNA expression ± SEM was expressed as a percentage of the untreated control cells (= 100%) after normalization for ß-actin expression.
Results
Inhibition of steroidogenic enzymes
Treatment of NCI-h295 cells for 48 h with the adreno- static compounds led to a dose-dependent decrease of cortisol and DHEA-S secretion. The release of 17-OH- progesterone was increased in cells treated with low concentrations of ETO (0.6-60 nM) and MTP (60 nM-6 µM), indicating inhibition of 11ß-hydroxylase (P450c11). In contrast higher concentrations of ETO and MTP led to a decrease of 17-OH-progesterone due to inhibition of P450scc enzyme. With AG a dose-dependent decrease of all steroids was observed. ETO was the most potent adrenostatic compound inhibiting the P450c11 hydroxylase with an IC50 of 15 nM and the P450scc enzyme with an IC50 of 400 nM. The pattern of enzyme inhibition was similar for MTP with an IC50 of 3.5 MM for P450c11 and 17 MM for P450scc, while AG blocks P450scc with an IC50 of 17 u.M (Fig. 1).
Influence on ACTH receptor and glucocorticoid receptor expression
Incubation of the cells for 48 h with AG significantly reduced ACTH-R mRNA expression in a dose-dependent manner but increased glucocorticoid receptor mRNA. In
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contrast, forskolin increased the accumulation of ACTH- R mRNA and decreased glucocorticoid receptor mRNA (Fig. 2a,b). Similar suppression of ACTH-R mRNA was induced by incubation with MTP.
To investigate the functional significance of reduced ACTH-R mRNA levels, we evaluated the cAMP response to ACTH in NCI-h295 cells. ACTH-stimulated cAMP accumulation was significantly reduced after pretreatment with AG in doses ≥ 30 p.M (300 MM AG: 786 ± 29; 30 MM AG: 860 ± 76 vs. control cells: 1130 ± 118 fmol well-1 10 min-1; P < 0.01) indicating reduced functional ACTH-R expression (Fig. 2c).
Simultaneous incubation of hydrocortisone (12 p.M) together with AG showed a significant up-regulation of the ACTH-R mRNA compared with cells incubated with AG only, indicating that glucocorticoids at higher concen- trations increase ACTH-R expression (Fig. 3).
A)
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30μM AG
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Stimulation of the protein kinase A pathway by incuba- tion of ACTH or forskolin increased ACTH-R expres- sion. The simultaneous incubation of these compounds with AG was not able to overcome the steroid biosynthesis inhibition, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression (Fig. 3).
Effects of cell proliferation
AG and ETO inhibited cell proliferation in a dose- dependent fashion. Treatment with AG reduced the cell growth at high concentrations ≥ 300 p.M, whereas ETO exhibited a significant antiproliferative activity at concen- trations of ≥ 6 p.M (day 8: 0.6 MM ETO 88% ± 4%,
ACTH-R mRNA expression in %
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6 MM ETO 78% ± 5%, 60 µM ETO 61% ± 9% com- pared with control cells: 100% ± 8%; P < 0-01) (Fig. 4).
In contrast to the suppression of ACTH-R expression growth inhibition induced by AG and ETO was not sig- nificantly reversed by administration of hydrocortisone to cell medium (Fig. 4).
Discussion
The major finding of our experiments in adrenal cells is the observation that different adrenostatic compounds not only block steroidogenic enzymes but also inhibit ACTH receptor expression and cell proliferation. Thus these drugs have pleiotropic effects in adrenal tumour cells.
In this study we were able to show that the incubation of the NCI-h295 cell line with adrenostatic compounds suppresses the ACTH-R mRNA abundance in a dose- dependent fashion. Decreased ACTH-R mRNA levels are paralleled by a profound reduction of the cAMP response to ACTH, demonstrating reduced expression of func- tional ACTH-R in this system. The actions of AG on ACTH-R mRNA expression in the NCI-h295 cell line can be reversed by addition of hydrocortisone, suggesting that the glucocorticoid signal transduction cascade is involved in regulation of the ACTH-R expression. Similar results were shown by Picard-Hagen et al. in ovine adrenocortical cells [30], indicating a general effect in adrenal cells and not only in a cancer cell line. Therefore, adrenostatic compounds may modulate ACTH-R expres- sion also in vivo. Suppression of ACTH-R protein may be desirable in patients with adrenal pathology. For example, in patients with adrenocortical carcinoma, ACTH may be one of the factors stimulating cell proliferation via the protein kinase A pathway or other second messengers. Down-regulation of ACTH-R expression in this situation could desensitize the tumour cells to the stimulatory action of ACTH.
It is noteworthy that it is impossible to explain the effects of the adrenostatic drugs on the ACTH-R only by
the influence of the PKA-system or the glucocorticoid transduction pathway. Therefore, we postulate the exis- tence of a third pathway in the regulation of the ACTH- R. Possible candidates for such a ‘third player’ are SF-1, DAX-1 or other still unknown transcription factors or steroid metabolites.
Interestingly the adrenostatic compounds also demon- strate an antiproliferative activity in the NCI-h295 cell line (Fig. 4). As this inhibition was not reversed by incu- bation with 5 M hydrocortisone (a concentration pro- duced by untreated control cells after 10 days) the antiproliferative effect is obviously not mediated via decreased cortisol secretion. Moreover, we were able to
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show that hydrocortisone alone does not significantly influence cell proliferation. Since the antiproliferative effect is most pronounced in ETO-treated cells, this drug may be particularly useful in the treatment of patients with CS due to adrenocortical cancer.
In this context it is important to note that all drugs used in our experiments were effective within the ‘thera- peutic range’ for humans. In patients receiving 1000 mg AG or 1000 mg MTP, respectively, plasma levels of either compound reach 70 p.M [31-33]. However, higher daily doses for these drugs have been recommended for CS [22,34,35]. Clinical studies with ETO verified that plasma levels up to 6 uM are nonhypnotic and well tolerated [21]. These data clearly indicate that our findings may also have clinical significance.
ACTH up-regulates its own receptor mRNA and ACTH binding sites in human, ovine and bovine adreno- cortical cells up to 20-fold [25,36-41]. This effect is mediated through the cAMP signal transduction cascade since it can be mimicked by treatment with forskolin and CAMP.
ACTH-R expression in neoplastic adrenal tissue seems to be regulated by additional factors other than plasma ACTH, since high ACTH-R mRNA levels have been found in adrenal adenomas of patients with adrenal CS and suppressed plasma ACTH [41-43]. Apparently, glu- cocorticoids are important factors in ACTH-R regulation. Dexamethasone enhances the ACTH-induced cAMP and cortisol response and increases the ACTH-R mRNA levels in adrenal cells [30,44-47]. In our experiments, the physiological glucocorticoid hydrocortisone induced a sig- nificant ACTH-R mRNA up-regulation counteracting the effect of adrenostatic drugs. However, in vivo, glucocorti- coids on their own are not able to maintain ACTH-R up- regulation. For example, down-regulation of ACTH-R
mRNA has been shown in rats treated with dexametha- sone i.p. suppressing plasma ACTH levels [41]. Therefore, in vivo ACTH is of major importance in ACTH-R up-regulation and glucocorticoids may merely enhance the effect of ACTH.
Based on these findings, we postulate the following model of intra-adrenal response to ACTH in humans: During stress the body is able to activate different systems to rapidly sensitize the adrenal cortex to ACTH. Such a self-intensifying system is useful, because steroid pro- ducing cells are not able to store intracellular hormones, yet secretion of large amounts of cortisol during stress is necessary for survival. Probably the main actor is ACTH itself. As mentioned earlier ACTH increases the expression of its own receptor. This up-regulation of ACTH-R by its own ligand is a unique phenomenon in G-protein coupled receptors. The stimulatory effect on ACTH-R expression is amplified by an increased steroidogenesis. High local cortisol concentration causes further up-regulation of the ACTH-R. This concept is fur- ther supported by recent analysis to the ACTH-R promo- tor region. Naville et al. have identified several putative cAMP response elements [48] and our group has recently found a glucocorticoid response element in the human ACTH-R promotor region (A. Klink unpublished obser- vation). To interrupt this positive short loop feedback sys- tem glucocorticoids act mainly via the long-loop negative feedback on the pituitary ACTH secretion (Fig. 5).
In summary, our data demonstrate that adrenostatic compounds influence both ACTH-R expression and cell proliferation in the NCI-h295 cell line and our findings lead to a new concept of the intra-adrenal response to stress. These effects represent an hitherto unknown action of the steroidogenic inhibitors and may be clinically rel- evant in patients with adrenal neoplasms.
-
ACTH
+
negative feedback
ACTH receptor
+
+
CAMP
+
steroidogenesis
+
proliferation
cortisol
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