Silibinin, an HSP90 Inhibitor, on Human ACTH-Secreting Adenomas

Abstract

Introduction: The glucocorticoid receptor is pivotal to control corticotrophin (ACTH) secretion, and its function is closely linked to the heat shock protein 90 (HSP90) chaperone complex. Impaired sensitivity to glucocorticoid feedback is a hallmark of human corticotroph adenomas, i.e., Cushing's disease, a disorder with few medical treatment options. Silibinin, a HSP90 inhibitor, has been studied in tumoral corticotroph cells and its use proposed in Cushing's disease. Aim of the present study was to further investigate the effect of silibinin on human corticotroph adenomas in vitro.

Methods: Seven human ACTH-secreting pituitary adenomas were established in culture and treated with 10-50 µ<sc>m</sc> silibinin with/without dexamethasone for up to 72 h. ACTH medium levels were measured, and POMC and glucocorticoid receptor, i.e., NR3C1, gene expression assessed.

Results: Silibinin reduced spontaneous ACTH secretion and restored sensitivity to steroid negative feedback to a different extent in individual adenomas. POMC expression was decreased in both control and dexamethasone-treated wells in specimens sensitive to silibinin. Interestingly, silibinin reduced constitutive NR3C1 expression and reversed the dexamethasone-induced inhibition.

Conclusions: Our findings indicate that silibinin can inhibit ACTH synthesis and secretion in individual human corticotroph adenomas and directly affects NR3C1 gene expression. These results reveal promising effects of this HSP90 inhibitor on human corticotroph adenomas and support an innovative target treatment for patients with Cushing's disease.

Keywords: Cushing’s disease; Glucocorticoid feedback; Heat shock protein 90; Silibinin.

Fig. 1

ACTH concentrations in ACTH-secreting pituitary adenoma specimens during incubation with silibinin. Black bars represent control wells (C), white bars wells incubated with silibinin at 10, 20, and 50 µm. Bars represent mean ± SEM of triplicate wells; *indicates p < 0.05 silibinin versus control wells.

Fig. 2

ACTH concentrations in ACTH-secreting pituitary adenoma specimens during incubation with dexamethasone and silibinin. Black bars represent control wells (C), shaded bars indicate effect of 10 nm dexamethasone alone (D), and white bars indicate wells incubated with dexamethasone and 10, 20, and 50 µm silibinin. Bars represent mean ± SEM of triplicate wells; *indicates p < 0.05 dexamethasone versus control wells; ^# indicates p < 0.05 silibinin+ dexamethasone versus dexamethasone-treated wells.

Fig. 3

POMC quantification in ACTH-secreting pituitary adenoma specimens after 72 h incubation with 10 µm silibinin (a) and silibinin + dexamethasone ((b) shaded bars: 10 nm dexamethasone; white bars: dexamethasone and 10 µm silibinin). Dashed line indicates POMC levels in control wells. Bars represent mean ± SEM of triplicate wells; *indicates p < 0.05 versus control wells; ^# indicates p < 0.05 silibinin + dexamethasone versus dexamethasone-treated wells.

Fig. 4

NR3C1 quantification in ACTH-secreting pituitary adenoma specimens after 72 h incubation with 10 µm silibinin (a) and silibinin + dexamethasone ((b) shaded bars: 10 nm dexamethasone; white bars: dexamethasone and 10 µm silibinin). Dashed line indicates NR3C1 levels in control wells. Bars represent mean ± SEM of triplicate wells; * indicates p < 0.05 versus control wells; ^# indicates p < 0.05 silibinin+ dexamethasone versus dexamethasone-treated wells.