Studio dell’azione del Mitotane, farmaco adrenolitico, sulla funzionalità ipofisaria

Mitotane (o,p_-DDD), a derivative of the insecticide dichlorodiphenyltrichloroethane, has been widely used for treatment of adrenocortical carcinoma (ACC). ACC is a rare neoplasm characterized by a dismal prognosis, with less than 50 % of patients surviving 5 yr after diagnosis. Because the majority of patients undergoing radical resection relapse, adjuvant therapy has been considered as an option. Indeed, it has been shown that adjuvant mitotane treatment has beneficial effects on outcome in ACC patients. Over the years, mitotane therapy has been thoroughly debated because of its toxicity that limited patient compliance and possibly drug efficacy, due to the previously employed high doses without blood concentration monitoring. The important advancement represented by mitotane monitoring allowed to demonstrate that mitotane activity and toxicity is correlated with the achieved blood drug concentrations. However, the undesired effects of mitotane also manifest at concentrations considered to have a therapeutic impact (14–20 mg/liter, corresponding to 44–62 microM), representing a major hurdle to treat adjuvantly patients who are free of disease after radical surgery. Mitotane treatment is associated with several adverse events. Recently, the effects of mitotane on the endocrine system have been thoroughly investigated in patients treated adjunctively after complete ACC removal, where perturbation in thyroid function is characterized by a decrease in FT4 levels over time, inversely correlated with mitotane concentration. Free T3 (FT3) and TSH levels do not change significantly, mimicking central hypothyroidism. Previously, mitotane has been demonstrated to increase T4-binding globulin (TBG) and to compete with T4 for TBG binding sites, but these phenomena do not explain the changes in thyroid function pattern observed during adjuvant therapy. It has been suggested that mitotane might impair pituitary TSH secretion, interfere with FT3, FT4, and/or TSH assays, or affect deiodase activity, thus changing the FT4/FT3 ratio. However, no evidence is currently available to support these hypotheses. Additionally, reduced ACTH levels were observed in patients treated adiuvantly with mitotane. The aim of our study was to investigate whether mitotane may interfere with thyroid hormone and TSH assays in human serum or directly affect TSH secretion in a mouse model of thyrotrope cells. Additionally, the aim of this study was to evaluate mitotane effects on adrenocorticotrope function and ACTH secretion in a mouse model of adrenocorticotrope cells, in order to explain the perturbation of ACTH levels in patients treated adiuvantly with mitotane. Our results demonstrate that mitotane does not interfere with thyroid hormone laboratory tests but directly reduces both secretory activity and cell viability on pituitary TSH secreting mouse cells. These data represent a possible explanation of the biochemical picture consistent with central hypothyroidism in patients undergoing mitotane therapy and open new perspectives on the direct pituitary effects of this drug. Moreover, mitotane inhibits adrenocorticotrope cell viability, inducing apoptotic mechanisms, and reduces ACTH secretion, both basally and after CRH stimulation. These results suggest that mitotane profoundly affect pituitary function, probably without cell specificity

IGF-I influences everolimus activity in medullary thyroid carcinoma

Context: Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. It has been previously demonstrated that insulin-like growth factor I (IGF-I) protects MTC from the effects of antiproliferative drugs. Everolimus, an mTOR inhibitor, has shown potent antiproliferative effects in a human MTC cell line, TT, and in two human MTC primary cultures. Objective: To verify whether IGF-I may influence the effects of everolimus in a group of human MTC primary cultures. Design: We collected 18 MTCs that were dispersed in primary cultures, treated without or with 10 nM-1 mu M everolimus and/or 50 nM IGF-I. Cell viability was evaluated after 48 h, and calcitonin (CT) secretion was assessed after a 6 h incubation. IGF-I receptor downstream signaling protein expression profile was also investigated. Results: Everolimus significantly reduced cell viability in eight MTC [by similar to 20%; P < 0.01 vs. control; everolimus-responders (E-R) MTCs], while cell viability did not change in 10 MTCs [everolimus-non-responders (E-NR) MTCs]. In E-R MTCs, IGF-I blocked the antiproliferative effects of everolimus that did not affect CT secretion, but blocked the stimulatory effects of IGF-I on this parameter. IGF-I receptor downstream signaling proteins were expressed at higher levels in E-NR MTC as compared to E-R MTCs. Conclusion: IGF-I protects a subset of MTC primary cultures from the antiproliferative effects of everolimus and stimulates CT secretion by an mTOR mediated pathway that, in turn, may represent a therapeutic target in the treatment of aggressive MTCs

Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors

Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures and in the human pancreatic endocrine cancer cell line, BON1. To this aim six human PNN dispersed in primary cultures and BON1 cells were treated without or with 1–10 μM Enzastaurin and/or 100 nM IGF1 in the presence or absence of serum. Cell viability and apoptosis were evaluated after 48–72 h; Chromogranin A (CgA) and/or insulin secretion was assessed after 6 h of incubation. PKC expression was investigated by immunofluorescence and western blot. We found that Enzastaurin significantly reduced human PNN primary culture cell viability, as well as CgA and insulin secretion. Moreover, in the BON1 cell line Enzastaurin inhibited cell proliferation at 5 and 10 μM by inducing caspase-mediated apoptosis, and reduced phosphorylation of glycogen synthetase kinase 3β (GSK3β) and of Akt, both downstream targets of PKC pathway and pharmacodynamic markers for Enzastaurin. In addition, Enzastaurin blocked the stimulatory effect of IGF1 on cell proliferation, and reduced CgA expression and secretion in BON1 cells. Two different PKC isoforms are expressed at different levels and have partially different subcellular localization in BON1 cells. In conclusion, Enzastaurin reduces cell proliferation by inducing apoptosis, with a mechanism likely involving GSK3β signaling, and inhibits secretory activity in PNNin vitromodels, suggesting that Enzastaurin might represent a possible medical treatment of human PNN

Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment

Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized protooncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake

Strategies to use microRNAs as therapeutic targets

Abstract MicroRNAs (miRNAs) are non-coding RNAs generated from endogenous hairpin-shaped transcripts that powerfully regulate gene expression at post-transcriptional level. Each miRNA is capable to regulate the expression levels of hundreds of transcripts and each mRNA may have more than one miRNA recognition sequence. There is emerging evidence that deregulation of miRNA expression leads to the alteration of pivotal physiological functions contributing to the development of diseases and neoplasms, including pituitary adenoma. This review is aimed at providing the up-to-date knowledge concerning deregulated miRNAs of pituitary tumors and their functions. In order to take stock, pituitary tumors have been sub-divided in different classes on the basis of tumor features (histotype, dimension, aggressiveness). The overview takes full consideration of the recent advances in miRNAs role as potential therapeutics and biomarkers

Polydimethylsiloxanes biocompatibility in PC12 neuronal cell line

Cochlear implants, the only way to recover from severe/profound hearing loss, may cause adverse effects, among which reactions to silicone materials coating implant electrodes, leading to apoptosis and necrosis of spiral ganglion cells. Our aim was to evaluate whether three polydimethylsiloxane (PDMS) compounds (hexadimethylsiloxane, octamethyltrisiloxane, decamethylcyclopentasiloxane) used in silicone rods could exert toxic effects on an in vitro neuronal cell model (PC12). Cell viability, morphology and mRNA expression levels of apoptotic markers were evaluated on PC12 cells at different PDMS dilutions up to 6 days of exposure. The results showed that at the highest concentrations tested cell viability was reduced by hexadimethylsiloxane and octamethyltrisiloxane at all times of exposure, but only from 72\u2009h onwards by decamethylcyclopentasiloxane. The number of neurites per cell was not affected by hexadimethylsiloxane, but was significantly reduced from 24\u2009h onwards by octamethyltrisiloxane and decamethylcyclopentasiloxane. Neurite length was reduced by hexadimethylsiloxane only at 24\u2009h, and by octamethyltrisiloxane and decamethylcyclopentasiloxane at all exposure intervals. In controls exposed to silicone or glass rods cell viability was reduced only after 24\u2009h, but neurite number and length was never reduced at any exposure interval. Biomolecular investigations showed that apoptotic markers did not change in any experimental condition, suggesting that PDMS are biocompatible. The reduction of cell viability and neurite number and length caused by exposure to these compounds was probably caused by a PDMS surface film formed over the cell medium, preventing air exchange, and not by the release of cytotoxic molecules