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Introduction: It has been demonstrated that mTOR inhibitors have potent anti-proliferative effects in a human Medullary Thyroid Carcinoma (MTC) cell line. We here explore the possible role of roTOR inhibitors, Everolimus and BEZ235 (which also inhibits the PI3K pathway) on the effects of Insulin-like Growth Factor-1 (IGF-1) in human MTC primary cultures. Aim(s): To this purpose, 20 MTCs were dispersed in primary cultures, treated without or with 1uM Everolimus, 10nM-1uM BEZ235, and/or 50nM IGF-1. Materials and methods: Cell viability and apoptosis were evaluated after 48 h. We observed the phosphorylation of p70s6k by ELISA. Results: Everolimus and Bez 235 significantly reduced MTC cell viability, respectively, by 30% and 40 %, while IGF-1 enhanced cell viability, an effect completely blocked by mTOR inhibitors. Co-incubation with an IGF-1R blocking antibody enhanced the antiproliferative effects of mTOR inhibitors. Phosphorylation of p70S6K, a down-stream effector in the PI3K/Akt pathway of mTOR was as well-enhanced by IGF-1 and reduced by Everolimus and BEZ235 indicating that IGF-1 exerts its proliferative effects by inducing this pathway, which, in turn, can be effectively blocked by mTOR inhibitors. Conclusion: In conclusion, mTOR inhibitors reduced MTCs cell viability by inducing apoptosis, with a mechanism likely involving IGF-I signalling, suggesting that it might represent a possible medical treatment for persistent/recurrent MTCs. [ABSTRACT FROM AUTHOR]

Introduction: Bronchial carcinoids (BCs), originating from endocrine cells dispersed in the respiratory epithelium, can be divided into typical (TBC) and atypical (ABC). TBC are less aggressive, smaller, and much less likely to metastasize, while ABC are more aggressive and metastasize, roTOR has a central role in regulating cell growth, metabolism, and apoptosis. A differential roTOR activation in BCs has been previously demonstrated, suggesting that mTOR pathway might play a predictive role in patients eligible for mTOR-targeted therapies. Aim(s): To evaluate the effects of roTOR inhibitors on human BCs cell lines. Materials and methods: NCIH720 (ABC) and NCIH727 (TBC) cells were treated with Everolimus or with NVP-BEZ 235, a dual PI3K/mTOR inhibitor. Cell viability, caspase activity and cell cycle progression were evaluated after 72 h. Results: Everolimus inhibits cell viability in both cell lines with more pronounced effects on NCIH720, does not activate apoptosis but determines G1/G0 accumulation. NVPBEZ235 had similar effects to Everolimus in terms of cell viability in NCIH727, but was more potent in NCIH720 cells. Caspase activity was not induced by NVP-BEZ 235 in NCIH727, but was enhanced in NCIH720. Western blot and Q-PCR were performed to assess whether this differential effect is due to variations in roTOR expression in BCs cell lines, mTOR expression is 1.5-fold higher in NCIH720 compared to NCIH727. Conclusion: These data demonstrate that mTOR inhibitors affect BCs cell lines differently depending on mTOR expression. [ABSTRACT FROM AUTHOR]

The conjugation of drugs with nanoparticles represents an innovative approach for controlled and targeted administration of therapeutic agents. Nanoparticle-based systems have been tested for the inner ear therapy, increasing the drug diffusion and being detected in all parts of the cochlea when locally applied near the round window. In this study, glycerol monooleate liquid crystalline NanoParticles were conjugated with Dexamethasone (NPD), a hydrophobic drug already used for inner ear treatments but defective in solubility and bioavailability. NPD has been tested in vitro in the cell line OC-k3, a model of sensory cells of the inner ear, and the therapeutic efficacy has been evaluated against cisplatin, a chemotherapeutic compound known to induce ototoxicity. After comparing the physical chemical characteristics of NPD to the equivalent naïve nanoparticles, an initial investigation was carried out into the nanoparticle's uptake in OC-k3 cells, which takes place within a few hours of treatment without causing toxic damage up to a concentration of 50 µg/mL. The NPD delivered the dexamethasone inside the cells at a significantly increased rate compared to the equivalent free drug administration, increasing the half-life of the therapeutic compound within the cell. Concerning the co-treatment with cisplatin, the NPD significantly lowered the cisplatin cytotoxicity after 48 h of administration, preventing cell apoptosis. To confirm this result, also cell morphology, cell cycle and glucocorticoids receptor expression were investigated. In conclusion, the NPD system has thus preliminarily shown the potential to improve the therapeutic efficacy of treatments delivered in the inner ear and prevent drug-induced ototoxicity.

Periostin (POSTN), a member of the matricellular protein family, is a secreted adhesion-related protein produced in the periosteum and periodontal ligaments. Matricellular proteins are a nonstructural family of extracellular matrix (ECM) proteins that regulate a wide range of biological processes in both normal and pathological conditions. Recent studies have demonstrated the key roles of these ECM proteins in the tumor microenvironment. Furthermore, periostin is an essential regulator of bone and tooth formation and maintenance, as well as cardiac development. Also, periostin interacts with multiple cell-surface receptors, especially integrins, and triggers signals that promote tumor growth. According to recent studies, these signals are implicated in cancer cell survival, epithelial-mesenchymal transition (EMT), invasion, and metastasis. In this review, we will summarize the most current data regarding periostin, its structure and isoforms, expressions, functions, and regulation in normal and cancerous tissues. Emphasis is placed on its association with cancer progression, and also future potential for periostin-targeted therapeutic approaches will be explored., (© 2022. The Author(s).)

Temporal bone squamous cell carcinoma (TBSCC) is an uncommon malignancy with a poor prognosis in advanced cases. The dismal outcome of advanced TBSSC cases is largely due to the cancer's local aggressiveness and the complex anatomy of this region, as well as to persistent pitfalls in diagnosis and treatment. Molecular changes occur in malignancies before any morphological changes become visible, and are responsible for the disease's clinical behavior. The main purpose of this critical systematic review is to assess the level of knowledge on the molecular markers involved in the biology, behavior, and prognosis of TBSCC. A search (updated to March 2022) was run in PubMed, Scopus, and Web of Science electronic databases without publication date limits for studies investigating molecular markers in cohorts of patients with primary TBSCC. The search terms used were: "temporal bone" OR "external auditory canal" OR "ear", AND "cancer" OR "carcinoma" OR "malignancy". We preliminarily decided not to consider series with less than five cases. Twenty-four case series of TBSCC were found in which different analytical techniques had been used to study the role of several biomarkers. In conclusion, only very limited information on the prognostic role of molecular markers in TBSCC are currently available; prospective, multi-institutional, international prognostic studies should be planned to identify the molecular markers involved in the clinical behavior and prognosis of TBSCC. A further, more ambitious goal would be to find targets for therapeutic agents able to improve disease-specific survival in patients with advanced TBSCC.

The biocompatibility and the antioxidant activity of barium titanate (BaTiO 3 ) and lithium niobate (LiNbO 3 ) were investigated on a neuronal cell line, the PC12, to explore the possibility of using piezoelectric nanoparticles in the treatment of inner ear diseases, avoiding damage to neurons, the most delicate and sensitive human cells. The cytocompatibility of the compounds was verified by analysing cell viability, cell morphology, apoptotic markers, oxidative stress and neurite outgrowth. The results showed that BaTiO 3 and LiNbO 3 nanoparticles do not affect the viability, morphological features, cytochrome c distribution and production of reactive oxygen species (ROS) by PC12 cells, and stimulate neurite branching. These data suggest the biocompatibility of BaTiO 3 and LiNbO 3 nanoparticles, and that they could be suitable candidates to improve the efficiency of new implantable hearing devices without damaging the neuronal cells.

In recent decades, interest has increased in the role of reactive oxygen species (ROS) in health and disease. The ROS are key causative factors in several hearing loss pathologies including ototoxicity, noise trauma, cochlear ageing and ischemic injury. In order to investigate ROS effects on inner ear cells and counteract them, we developed an in vitro model of oxidative stress by exposing the inner ear cell line OC-k3 to hydrogen peroxide (H 2 O 2 ) at concentrations able to affect in vivo cellular components but allowing cell survival. The treatment with high concentrations (20 and 30 μM) resulted in reduction of cell viability, activation of apoptosis/necrosis and alteration of morphology, cell cycle progression and antioxidant defences. The ROS effects in inner ear cells are difficult to assess in vivo. Organocultures may provide preservation of tissue architecture but involve ethical issues and can be used only for a limited time. An in vitro model that could be commercially available and easy to handle is necessary to investigate inner ear oxidative stress and the ways to counteract it. The OC-k3 line is a suitable in vitro model to study ROS effects on inner ear cells because the observed cell alterations and damages were similar to those reported in studies investigating ROS effects of ototoxic drugs, noise trauma and cochlear ageing., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A cochlear implant (CI) is an electronic device that enables hearing recovery in patients with severe to profound hearing loss. Although CIs are a successful treatment for profound hearing impairment, their effectivity may be improved by reducing damages associated with insertion of electrodes in the cochlea, thus preserving residual hearing ability. Inner ear trauma leads to inflammatory reactions altering cochlear homeostasis and reducing post-operative audiological performances and electroacoustic stimulation. Strategies to preserve residual hearing ability led to the development of medicated devices to minimize CI-induced cochlear injury. Dexamethasone-eluting electrodes recently showed positive outcomes. In previous studies by our research group, intratympanic release of dexamethasone for 14 days was able to preserve residual hearing from CI insertion trauma in a Guinea pig model. Long-term effects of dexamethasone-eluting electrodes were therefore evaluated in the same animal model. Seven Guinea pigs were bilaterally implanted with medicated rods and four were implanted with non-eluting ones. Hearing threshold audiograms were acquired prior to implantation and up to 60 days by recording compound action potentials. For each sample, we examined the amount of bone and fibrous connective tissue grown within the scala tympani in the basal turn of the cochlea, the cochleostomy healing, the neuronal density, and the correlation between electrophysiological parameters and histological results. Detection of tumor necrosis factor alpha, interleukin-6, and foreign body giant cells showed that long-term electrode implantation was not associated with an ongoing inflammation. Growth of bone and fibrous connective tissue around rods induced by CI was reduced in the scala tympani by dexamethasone release. For cochleostomy sealing, dexamethasone-treated animals showed less bone tissue growth than negative. Dexamethasone did not affect cell density in the spiral ganglion. Overall, these results support the use of dexamethasone as anti-inflammatory additive for eluting electrodes able to protect the cochlea from CI insertion trauma., (Copyright © 2020 Simoni, Gentilin, Candito, Borile, Romanato, Chicca, Nordio, Aspidistria, Martini, Cazzador and Astolfi.)

Sensorineural hearing loss due to aging, noise exposure, trauma or drug ototoxicity is irreversible because cochlear hair cells and neurons cannot regenerate. Recently, therapeutic strategies involving nanoparticles have been developed as innovative drug delivery systems. Thermodynamically stable liquid crystalline nanoparticles based on the polar lipid glycerol monooleate (GMO NP, cubosomes), nontoxic and able to encapsulate both hydrophilic and hydrophobic compounds, were produced and tested for biocompatibility in an immortalized Organ of Corti derived cell line (OC-k3), through cell viability and cytomorphological assays, and Western blot expression profiles of apoptotic markers. Overall, the GMO NP were biocompatible in OC-k3 at the doses and time tested, supporting previous data obtained in a neuronal cell line (PC12). The results encourage further tests on GMO NP-mediated drug release with improved target specificity and could be useful to develop innovative therapies against sensorineural hearing loss., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cis-diamminedichloridoplatinum (II) (cisplatin) is a chemotherapeutic drug currently prescribed for the treatment of many types of human cancer, but its use is associated with numerous adverse effects, one of which is ototoxicity. Cisplatin-induced hearing loss is mainly attributed to oxidative stress, but recent data suggest that inflammation could be the trigger event leading to inner ear cell death through endoplasmic reticulum (ER) stress, autophagy, necroptosis, and then intrinsic apoptosis. In this review, we look at the molecular targets of cisplatin, and the intracellular pathways underlying its ototoxicity. Special attention is devoted to signal transduction processes involving cisplatin that may promote the development of new strategies to prevent cisplatin-induced hearing loss, an adverse event with severe social impacts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 h onwards by decamethylcyclopentasiloxane. The number of neurites per cell was not affected by hexadimethylsiloxane, but was significantly reduced from 24 h onwards by octamethyltrisiloxane and decamethylcyclopentasiloxane. Neurite length was reduced by hexadimethylsiloxane only at 24 h, and by octamethyltrisiloxane and decamethylcyclopentasiloxane at all exposure intervals. In controls exposed to silicone or glass rods cell viability was reduced only after 24 h, 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: Insulin-like growth factor 1 (IGF1) controls growth hormone (GH) secretion via a negative feed-back loop that may disclose novel mechanisms possibly useful to control GH hyper-secretion. Our aim was to understand whether PI3K/Akt/mTOR pathway is involved in IGF1 negative feedback on GH secretion., Methods: Cell viability, GH secretion, Akt, and Erk 1/2 phosphorylation levels in the rat GH3 cell line were assessed under treatment with IGF1 and/or everolimus, an mTOR inhitior., Results: We found that IGF1 improves rat GH3 somatotroph cell viability via the PI3K/Akt/mTOR pathway and confirmed that IGF1 exerts a negative feedback on GH secretion by a transcriptional mechanism. We demonstrated that the negative IGF1 loop on GH secretion requires Akt activation that seems to play a pivotal role in the control of GH secretion. Furthermore, Akt activation is independent of PI3K and probably mediated by mTORC2. In addition, we found that Erk 1/2 is not involved in GH3 cell viability regulation, but may have a role in controlling GH secretion, independently of IGF1., Conclusion: Our data confirm that mTOR inhibitors may be useful to reduce pituitary adenoma cell viability, while Erk 1/2 pathway may be considered as a useful therapeutic target to control GH secretion. Our results open the field for further studies searching for effective drugs to control GH hyper-secretion.

Growth Hormone may influence neoplastic development of endometrial epithelium towards endometrial adenocarcinoma, which is one of the most occurring tumors in acromegalic patients. Since chemoresistance often develops in advanced endometrial adenocarcinoma, we investigated whether Growth Hormone might influence the development of chemoresistance to drugs routinely employed in endometrial adenocarcinoma treatment, such as Doxorubicin, Cisplatin, and Paclitaxel. Growth Hormone and Growth Hormone receptor expression was assessed by immunofluorescence in two endometrial adenocarcinoma cell lines, AN3 CA and HEC-1-A cells. Growth Hormone effects were assessed investigating cell viability, caspase3/7 activation, ERK1/2, and protein kinase C delta protein expression. AN3 CA and HEC-1-A cells display Growth Hormone and Growth Hormone receptor. Growth Hormone does not influence cell viability in both cells lines, but significantly reduces caspase 3/7 activation in AN3 CA cells, an effect blocked by a Growth Hormone receptor antagonist. Growth Hormone rescues AN3 CA cells from the inhibitory effects of Doxorubicin and Cisplatin on cell viability, while it has no effect on Paclitaxel. Growth Hormone does not influence the pro-apoptotic effects of Doxorubicin, but is capable of rescuing AN3 CA cells from the pro-apoptotic effects of Cisplatin. On the other hand, Growth Hormone did not influence the effects of Doxorubicin and Paclitaxel on HEC-1A cell viability. The protective action of Growth Hormone towards the effects of Doxorubicin may be mediated by ERK1/2 activation, while the pro-apoptotic effects of Cisplatin may be mediated by protein kinase C delta inhibition. All together our results indicate that Growth Hormone may differentially contribute to endometrial adenocarcinoma chemoresistance. This may provide new insights on novel therapies against endometrial adenocarcinoma chemoresistant aggressive tumors.

Context: Metastatic disease is responsible for the majority of endocrine cancer deaths. New therapeutic targets are urgently needed to improve patient survival rates., Objective: The proto-oncogene PTTG1-binding factor (PBF/PTTG1IP) is overexpressed in multiple endocrine cancers and circumstantially associated with tumor aggressiveness. This study aimed to understand the role of PBF in tumor cell invasion and identify possible routes to inhibit its action. Design, Setting, Patients, and Interventions: Thyroid, breast, and colorectal cells were transfected with PBF and cultured for in vitro analysis. PBF and cortactin (CTTN) expression was determined in differentiated thyroid cancer and The Cancer Genome Atlas RNA-seq data., Primary Outcome Measure: Pro-invasive effects of PBF were evaluated by 2D Boyden chamber, 3D organotypic, and proximity ligation assays., Results: Our study identified that PBF and CTTN physically interact and co-localize, and that this occurs at the cell periphery, particularly at the leading edge of migrating cancer cells. Critically, PBF induces potent cellular invasion and migration in thyroid and breast cancer cells, which is entirely abrogated in the absence of CTTN. Importantly, we found that CTTN is over-expressed in differentiated thyroid cancer, particularly in patients with regional lymph node metastasis, which significantly correlates with elevated PBF expression. Mutation of PBF (Y174A) or pharmacological intervention modulates the PBF: CTTN interaction and attenuates the invasive properties of cancer cells., Conclusion: Our results demonstrate a unique role for PBF in regulating CTTN function to promote endocrine cell invasion and migration, as well as identify a new targetable interaction to block tumor cell movement.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Information regarding the safety of herbal drugs is often not reported. We describe the case of a 65-year-old woman referred to us for a iatrogenic hypercortisolism, who denied any previous steroid consumption. She reported only a chronic application of a phytocosmetic cream, containing ethanol extract of the Cardiospermum halicacabum (CH) plant. Adrenal insufficiency occurred after the cream application was stopped. CH is used in traditional and Western medicine for its documented anti-inflammatory properties. Once the presence of synthetic glucocorticoids was ruled out in the phytocosmetic product, we investigated whether and how its chronic application could have caused the iatrogenic hypercortisolism., Methods: Liquid chromatography high-resolution mass spectrometry (LC-HRMS) was performed to exclude the presence of known glucocorticoids in the cream. ELISA assay and Western blot analysis were employed to assess ACTH secretion and the glucocorticoid receptor expression respectively in murine ACTH-secreting pituitary adenoma cells AtT-20/D16v-F2, treated with dexamethasone, CH tincture, and mifepristone alone or in combination. To detect specific interaction of CH extract with the glucocorticoid receptor, we performed a dual-luciferase reporter assay in HEK293 cells., Results: In AtT-20/D16v-F2 cells, CH extract showed to significantly reduce basal and CRH-induced ACTH secretion and the glucocorticoid receptor expression, similarly to dexamethasone; these effects were counteracted by mifepristone. In HEK293 cells, dexamethasone significantly induced luciferase activity after 24- and 36-hour treatment and CH tincture only after 36 hours; these effects were antagonized by mifepristone., Conclusions: CH extract displays a glucocorticoid-like activity, by means of a direct binding to the glucocorticoid receptor.

Protein Kinase C Delta (PRKCD) has been highlighted among disrupted pathways in corticotroph adenomas. PRKCD is expressed at low level in human corticotroph adenomas and controls cell cycle in vitro. Therefore, PRKCD may play an important role in the development/progression of corticotroph adenomas, warranting further studies to understand the role of PRKCD and related pathways in restraining pituitary cell growth. We evaluated PRKCD role in influencing cell behavior in terms of cell viability, hormone expression and protein expression profile, by silencing PRKCD in AtT-20/D16v-F2 cells. PRKCD silencing increases cell viability, enhances hormone expression and induces morphological changes associated with deregulation of adhesion molecules. PRKCD silencing is associated with an increase in Epithelial Growth Factor Receptor (EGFR) expression, a marker of tumor aggressive behavior, and sensitivity to anti-EGFR molecules. PRKCD might restrain corticotroph adenoma cells from acquiring an aggressive behavior, candidating PRKCD as a possible molecular target for the treatment of corticotroph adenomas., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Medical treatment of adrenocortical carcinoma (ACC) is still far from optimal, since even molecular targeted therapy failed to demonstrate striking results. Clinical trials enrolling ACC patients with high tissue vascular endothelial growth factor receptor (VEGFR) expression levels showed controversial results after treatment with Sunitinib, possibly due to variability in the expression of drug targets, which include epidermal growth factor receptor (EGFR). To better clarify this issue, we evaluated whether VEGFR may play a crucial role in ACC responsiveness to Sunitinib and whether EGFR may represent an alternative target in ACC medical treatment, by employing two ACC cell lines, the NCI-H295 and SW13 cells lines, and adrenocortical tissues primary cultures. Our data show that VEGF/VEGFR system may not be crucial in modulating ACC proliferation and responsiveness to Sunitinib. In addition, by cell viability, proliferation and caspase activation assays we found that Sunitinib inhibits adrenocortical cell viability acting, at least in part, through EGFR, that, in turn, is crucial for EGF proliferative effect on adrenocortical cells. The latter depends, at least in part, on ERK 1/2 activation. An EGFR selective inhibitor was highly effective in reducing cell viability in an adrenocortical tumor primary culture and in the SW13 cells, which express high EGFR levels. Our results suggest that EGFR inhibitors could represent effective therapeutic tools in ACC patients whose tumors express high EGFR levels, that, in turn, may be considered a predictive factor of response. Accurate molecular tumor profiling is crucial to predict drug efficacy and to tailor ACC patients therapeutic approach., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

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 μM 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 ~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.

Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocortical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotoxicity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at concentrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater efficacy of combination therapy (mitotane plus chemotherapeutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.

Mitotane represents the mainstay medical treatment for metastatic, inoperable or recurrent adrenocortical carcinoma. Besides the well-known adverse events, mitotane therapy is associated also with endocrinological effects, including sexual and reproductive dysfunction. The majority of male patients undergoing adjuvant mitotane therapy show a picture of hypogonadism, characterized by low free testosterone and high sex hormone binding globulin levels and unmodified LH concentrations. Since mitotane has been shown to have direct pituitary effects, we investigated whether mitotane may influence both cell viability and function of gonadotroph cells in the settings of two pituitary cell lines. We found that mitotane reduces cell viability, induces apoptosis, modifies cell cycle phase distribution and secretion of gonadotroph cells. The present data strengthen previous evidence showing a direct mitotane effect at pituitary level and represent a possible explanation of the lack of LH increase following decrease in free testosterone in patients undergoing adjuvant mitotane therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Magmas is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. Magmas is overexpressed in the majority of human pituitary adenomas and in a mouse ACTH-secreting pituitary adenoma cell line. Here we report that Magmas is highly expressed in two out of four rat pituitary adenoma cell lines and its expression levels inversely correlate to the extent of cellular response to staurosporine in terms of apoptosis activation and cell viability. Magmas over-expression in rat GH/PRL-secreting pituitary adenoma GH4C1 cells leads to an increase in cell viability and to a reduction in staurosporine-induced apoptosis and DNA fragmentation, in parallel with the increase in Magmas protein expression. These results indicate that Magmas plays a pivotal role in response to pro-apoptotic stimuli and confirm and extend the finding that Magmas protects pituitary cells from staurosporine-induced apoptosis, suggesting its possible involvement in pituitary adenoma development.

Medical therapy for Cushing's disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushing's syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

Bronchial carcinoids (BCs) are rare neuroendocrine tumors that are still orphans of medical treatment. Human BC primary cultures may display resistance to everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), in terms of cell viability reduction. Our aim was to assess whether the novel dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235 is effective in everolimus-resistant human BC tissues and cell lines. In addition, we searched for possible markers of the efficacy of mTOR inhibitors that may help in identifying the patients who may benefit from treatment with mTOR inhibitors, sparing them from ineffective therapy. We found that NVP-BEZ235 is twice as potent as everolimus in reducing cell viability and activating apoptosis in human BC tissues that display sensitivity to mTOR inhibitors, but is not effective in everolimus-resistant BC tissues and cell lines that bypass cyclin D1 downregulation and escape G0/G1 blockade. Rebound AKT activation was not observed in response to treatment with either mTOR inhibitor in the 'resistant' BC cells. In addition to total mTOR levels, putative markers of the sensitivity of BCs to mTOR inhibitors are represented by AKT, p70S6K (RPS6KB2), and ERK1/2 (MAPK3/1) protein levels. Finally, we validated these markers in an independent BC group. These data indicate that the dual PI3K/mTOR inhibitor NVP-BEZ235 is more potent than everolimus in reducing the proliferation of human BC cells. 'Resistant' cells display lower levels of mTOR, p70S6K, AKT, and ERK1/2, indicating that these proteins may be useful as predictive markers of resistance to mTOR and PI3K/mTOR inhibitors in human BCs.

The functional aftermath of microRNA (miRNA) dysregulation in ACTH-secreting pituitary adenomas has not been demonstrated. miRNAs represent diagnostic and prognostic biomarkers as well as putative therapeutic targets; their investigation may shed light on the mechanisms that underpin pituitary adenoma development and progression. Drugs interacting with such pathways may help in achieving disease control also in the settings of ACTH-secreting pituitary adenomas. We investigated the expression of 10 miRNAs among those that were found as most dysregulated in human pituitary adenoma tissues in the settings of a murine ACTH-secreting pituitary adenoma cell line, AtT20/D16v-F2. The selected miRNAs to be submitted to further investigation in AtT20/D16v-F2 cells represent an expression panel including 5 up-regulated and 5 down-regulated miRNAs. Among these, we selected the most dysregulated mouse miRNA and searched for miRNA targets and their biological function. We found that AtT20/D16v-F2 cells have a specific miRNA expression profile and that miR-26a is the most dysregulated miRNA. The latter is overexpressed in human pituitary adenomas and can control viable cell number in the in vitro model without involving caspase 3/7-mediated apoptosis. We demonstrated that protein kinase Cδ (PRKCD) is a direct target of miR-26a and that miR26a inhibition delays the cell cycle in G1 phase. This effect involves down-regulation of cyclin E and cyclin A expression via PRKCD modulation. miR-26a and related pathways, such as PRKCD, play an important role in cell cycle control of ACTH pituitary cells, opening new therapeutic possibilities for the treatment of persistent/recurrent Cushing's disease.

Context: GH and IGF-I play a role in breast cancer (BC) development. We previously demonstrated that GH protects the estrogen receptor (ER) positive BC-derived MCF7 cell line toward the cytotoxic effects of doxorubicin (D), independently of IGF-I. This issue may be important in ER negative BC cells that are more aggressive and more likely to develop chemoresistance., Aim of the Study: The aim of this study was to evaluate whether GH may impact chemoresistance phenotype of ER-negative BC-derived MDA-MB-231 cell line and investigate the possible mechanisms implicated in the protective action of GH toward the cytotoxic effects of D in both ER-positive and ER-negative BC-derived cell lines., Results: GH protects ER-negative MDA-MB-231 cells from the cytotoxic effects of D and GH receptor antagonist pegvisomant reduces GH-induced DNA synthesis also in these cells. In both MDA-MB-231 and MCF7 cells, GH does not revert D-induced G2/M accumulation but significantly reduces basal and D-induced apoptosis, an effect blocked by pegvisomant. Glutathione S-transferase activity is not implicated in the protective effects of GH, whereas D-induced apoptosis depends on c-Jun N terminal kinase (JNK) activation. GH reduces both basal and D-stimulated JNK transcriptional activity and phosphorylation., Conclusions: In human BC cell lines, GH directly promotes resistance to apoptosis induced by chemotherapeutic drugs independently of ER expression by modulating JNK, further broadening the concept that GH excess may hamper cytotoxic BC treatment. These findings support the hypothesis that blocking GH receptor may be viewed as a potential new therapeutic approach to overcome chemoresistance, especially in ER-negative BC.

We investigate the role of protein kinase C (PKC) in the control of medullary thyroid carcinoma (MTC) cell proliferation by a PKC inhibitor, Enzastaurin, in human MTC primary cultures and in the TT cell line. We found that PKC inhibition reduces cell proliferation by inducing caspase-mediated apoptosis and blocks the stimulatory effect of IGF-I on calcitonin secretion. Enzastaurin reduces PKCβII (Thr500) phosphorylation, indicating a direct involvement of this isoform as well as the phosphorylated levels of Akt (Ser 473) and glycogen synthase kinase (Ser9), PKC pathway downstream targets and pharmacodynamic markers for PKC inhibition. PKCβII and PKCδ enzyme isoforms expression and localization were investigated. These data indicate that in vitro PKC is involved in the control of human MTC proliferation and survival by modulating apoptosis, with a mechanism that implicates PKCβII inhibition and translocation in different subcellular compartments. Targeting PKC may represent a useful therapeutic approach for controlling MTC proliferation.