Supplementary Material for: A New Generation Somatostatin-Dopamine Analogue Exerts Potent Antitumoral Actions on Pituitary Neuroendocrine Tumor Cells

Background: Pituitary neuroendocrine tumors (PitNETs) represent approximately 15% of all intracranial tumors and usually are associated with severe comorbidities. Unfortunately, a relevant number of patients do not respond to currently available pharmacological treatments, that is, somatostatin analogs (SSAs) or dopamine-agonists (DA). Thus, novel, chimeric somatostatin/dopamine compounds (dopastatins) that could improve medical treatment of PitNETs have been designed. Objective: This study aims to determine the direct therapeutic effects of a new-generation dopastatin, BIM-065, on primary cell cultures from different PitNETs subtypes. Methods: Thirty-one PitNET-derived cell cultures (9 corticotropinomas, 9 somatotropinomas, 11 nonfunctioning pituitary adenomas [NFPAs], and 2 prolactinomas), were treated with BIM-065, and key functional endpoints were assessed (cell viability, apoptosis, hormone secretion, expression levels of key genes, free cytosolic [Ca2+]i dynamics, etc.). AtT-20 cell line was used to evaluate signaling pathways in response to BIM-065. Results: This chimeric compound decreased cell viability in all corticotropinomas and somatotropinomas tested, but not in NFPAs. BIM-065 reduced ACTH, GH, chromogranin-A and PRL secretion, and increased apoptosis in corticotropinomas, somatotropinomas, and NFPAs. These effects were possibly mediated through modulation of pivotal signaling cascades like [Ca2+]i kinetic and Akt- or ERK1/2-phosphorylation. Conclusions: Our results unveil a robust antitumoral effect in vitro of the novel chimeric compound BIM-065 on the main PitNET subtypes, inform on the mechanisms involved, and suggest that BIM-065 could be an efficacious therapeutic option to be considered in the treatment of PitNETs.

背景：垂体神经内分泌肿瘤（Pituitary neuroendocrine tumors, PitNETs）约占所有颅内肿瘤的15%，通常伴随严重并发症。遗憾的是，相当一部分患者对现有药物治疗方案无应答，即生长抑素类似物（somatostatin analogs, SSAs）或多巴胺激动剂（dopamine-agonists, DAs）。为此，科研人员已设计出可改善垂体神经内分泌肿瘤内科治疗效果的新型嵌合生长抑素/多巴胺化合物——多帕他汀（dopastatins）。

目的：本研究旨在探究新一代多帕他汀类化合物BIM-065对不同亚型垂体神经内分泌肿瘤原代细胞培养物的直接治疗作用。

方法：本研究采用31株垂体神经内分泌肿瘤来源的细胞培养物（9株促肾上腺皮质激素腺瘤、9株生长激素腺瘤、11株无功能垂体腺瘤[nonfunctioning pituitary adenomas, NFPAs]及2株催乳素腺瘤），以BIM-065进行干预，并检测多项关键功能终点，包括细胞活力、细胞凋亡、激素分泌、关键基因表达水平、游离胞质钙离子[Ca2+]i动态变化等。同时利用AtT-20细胞系评估BIM-065干预后的信号通路变化。

结果：该嵌合化合物可降低所有受试促肾上腺皮质激素腺瘤与生长激素腺瘤的细胞活力，但对无功能垂体腺瘤无此作用。BIM-065可抑制促肾上腺皮质激素（ACTH）、生长激素（GH）、嗜铬粒蛋白A及催乳素（PRL）的分泌，并促进促肾上腺皮质激素腺瘤、生长激素腺瘤与无功能垂体腺瘤的细胞凋亡。上述效应可能通过调控关键信号级联反应实现，例如游离胞质钙离子动力学变化以及蛋白激酶B（Akt）或细胞外调节蛋白激酶1/2（ERK1/2）的磷酸化水平。

结论：本研究结果证实，新型嵌合化合物BIM-065在体外对主要亚型的垂体神经内分泌肿瘤具有显著的抗肿瘤作用，并阐明了其潜在作用机制，提示BIM-065有望成为垂体神经内分泌肿瘤治疗的有效候选方案。