Aminoglutethimide suppresses adrenocorticotropin receptor expression in the NCI-h295 adrenocortical tumor cell line

Abstract

The adrenostatic compound aminoglutethimide (AG), a potent inhibitor of the P450 side chain cleavage enzyme, is used in the treatment of ACTH-dependent or adrenal Cushing's syndrome. Recently, AG has been shown to inhibit ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. To investigate whether ACTH-R down-regulation will also be induced in tumor cells, we studied the effect of AG on ACTH-R expression in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. The cells were incubated in triplicate with increasing doses of AG (3, 30, 300 µM) which suppressed steroid secretion dosedependently. After 48 h, cells were harvested, and total RNA was extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe. In parallel experiments, after preincubation with AG the cells were stimulated with ACTH (10 nM) for 10 min and the intracellular cAMP accumulation was determined by RIA. AG significantly suppressed the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 µM AG, 5 1%; 30 µM AG, 64 1%; 3 µM AG, 108 19% compared with control cells, 100 11%). The reduced ACTH-R mRNA expression was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of the ACTH-R protein. The adrenostatic compound metyrapone, an inhibitor of 11 -hydroxylase activity, also suppressed ACTH-R mRNA expression in a similar fashion. Stimulation of the protein kinase A pathway by simultaneous incubation of ACTH (10 nM) or forskolin (10 µM) together with AG was not able to overcome the steroid biosynthesis blockade, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression. Also, cortisol (12 µM) reversed the AGinduced ACTH-R mRNA expression. We conclude that AG induces profound ACTH-R down-regulation in the NCI-h295 cell line either by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability. This novel action of AG can be reversed by stimulation of the cAMP pathway and of the glucocorticoid-mediated signal transduction cascade. As the down-regulation occurs in vitro at concentrations which are reached during treatment with AG in humans it may contribute to its therapeutic activity in adrenal disease.

Steroidogenic acute regulatory protein mRNA expression in adrenal tumours

“…Fassnacht et al described a similar, but more profound effect on ACTH receptor mRNA expression (21). The expression of StAR mRNA was not changed by incubation with either aminoglutethimide or metyrapone in our experiments.…”

“…We have previously noted that the endocrine profile and the pattern of P450 enzyme expression of the NCI-h295 cell line changes during long-term culture conditions due to selection of different clones. With regard to P450scc expression, the NCI strain used in our experiments is known to respond well to stimulation by ACTH (21).…”

Altered Expression Patterns of Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in the Adrenal Cortex

“…After ACTH stimulation, H295R cells showed biphasic patterns of DHEA-S secretion. This biphasic reaction to ACTH has been reported previously (Jefcoate et al 1987;Le Roy et al 2000) and was explained by the biphasic upand downregulation of ACTH receptors in H295R cells (Fassnacht et al 1998(Fassnacht et al ,2000. This was reproduced also in a forskolin stimulation experiment (data not shown).…”

Very low-density lipoprotein (VLDL)-induced signals mediating aldosterone production

“…In subsequent studies, we also showed a similar effect in the HAC15 cell line (Tsai et al 2014), a clone of H295R, which shows good responses to AngII and ACTH . The ability of VLDL to enhance CYP11B2 expression is not additive with a near-maximal concentration of angiotensin II (10 nM) but VLDL enhances the stimulatory effect of the cAMP pathway agonist forskolin, even at a high dose (10 µM) that causes a large increase in cAMP levels and aldosterone production in H295R cells (Fassnacht et al 1998). In addition, VLDL's stimulation of steroidogenesis was not unique to aldosterone production.…”