Supplementary Material for: SILIBININ, AN HSP90 INHIBITOR, ON HUMAN ACTH-SECRETING ADENOMAS

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 µM silibinin with/without dexamethasone for up to 72 hours. 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 both in 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.

引言。糖皮质激素受体（glucocorticoid receptor）在调控促肾上腺皮质激素（corticotrophin, ACTH）分泌过程中发挥关键作用，其功能与热休克蛋白90（heat shock protein 90, HSP90）分子伴侣复合物密切相关。糖皮质激素反馈敏感性受损是人类促肾上腺皮质激素腺瘤（即库欣病）的标志性病理特征，该疾病目前可供选择的内科治疗手段十分有限。水飞蓟宾作为一种HSP90抑制剂，已在肿瘤性促肾上腺皮质激素细胞中开展相关研究，并有研究提出将其应用于库欣病的治疗。本研究旨在进一步探究水飞蓟宾对人类促肾上腺皮质激素腺瘤的体外作用效果。方法。本研究成功体外培养7株分泌ACTH的人垂体促肾上腺皮质激素腺瘤细胞，分别用10~50 μM的水飞蓟宾联合或不联合地塞米松处理，处理时长最长达72小时。检测培养基中ACTH的水平，并评估前阿片黑素细胞皮质激素（pro-opiomelanocortin, POMC）以及糖皮质激素受体（即NR3C1）的基因表达情况。结果。在不同的腺瘤样本中，水飞蓟宾均可降低自发性ACTH分泌，并在不同程度上恢复其对类固醇负反馈的敏感性。在对水飞蓟宾敏感的腺瘤样本中，对照组与地塞米松处理组的POMC表达水平均出现下调。值得注意的是，水飞蓟宾可降低NR3C1的组成型表达，并逆转地塞米松诱导的表达抑制。结论。本研究结果表明，水飞蓟宾可在单个人类促肾上腺皮质激素腺瘤中抑制ACTH的合成与分泌，并直接调控NR3C1的基因表达。上述研究结果揭示了该HSP90抑制剂对人类促肾上腺皮质激素腺瘤的潜在治疗价值，并为库欣病患者提供了一种创新的靶向治疗思路。