Daily Intraperitoneal Administration of Rosiglitazone Does Not Improve Lung Function or Alveolarization in Preterm Rabbits Exposed to Hyperoxia.

Thiazolidinediones (TZDs) are potent PPARγ agonists that have been shown to attenuate alveolar simplification after prolonged hyperoxia in term rodent models of bronchopulmonary dysplasia. However, the pulmonary outcomes of postnatal TZDs have not been investigated in preterm animal models. Here, we first investigated the PPARγ selectivity, epithelial permeability, and lung tissue binding of three types of TZDs in vitro (rosiglitazone (RGZ), pioglitazone, and DRF-2546), followed by an in vivo study in preterm rabbits exposed to hyperoxia (95% oxygen) to investigate the pharmacokinetics and the pulmonary outcomes of daily RGZ administration. In addition, blood lipids and a comparative lung proteomics analysis were also performed on Day 7. All TZDs showed high epithelial permeability through Caco-2 monolayers and high plasma and lung tissue binding; however, RGZ showed the highest affinity for PPARγ. The pharmacokinetic profiling of RGZ (1 mg/kg) revealed an equivalent biodistribution after either intratracheal or intraperitoneal administration, with detectable levels in lungs and plasma after 24 h. However, daily RGZ doses of 1 mg/kg did not improve lung function in preterm rabbits exposed to hyperoxia, and daily 10 mg/kg doses were even associated with a significant lung function worsening, which could be partially explained by the upregulation of lung inflammation and lipid metabolism pathways revealed by the proteomic analysis. Notably, daily postnatal RGZ produced an aberrant modulation of serum lipids, particularly in rabbit pups treated with the 10 mg/kg dose. In conclusion, daily postnatal RGZ did not improve lung function and caused dyslipidemia in preterm rabbits exposed to hyperoxia.

PI3K/Akt/mTOR pathway involvement in regulating growth hormone secretion in a rat pituitary adenoma cell line.

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.

Expression and Function of the Costimulatory Receptor SLAMF1 Is Altered in Lymphocytes From Patients With Autoimmune Thyroiditis.

Context: Signaling lymphocytic activation molecule family 1 (SLAMF1) is a costimulatory receptor expressed by most immune cells. Its role in autoimmune thyroid disease (AITD) is not well known. Objective: To analyze the expression and function of the costimulatory receptor SLAMF1 in lymphocytes of patients with AITD. Design: Cross-sectional, prospective, single-center study. Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid. Patients: Twenty-eight patients with AITD (17 with Graves disease and 11 with Hashimoto thyroiditis) and 21 controls. Intervention: Multiparametric flow cytometry and immunofluorescence techniques to analyze the expression of SLAMF1 in peripheral blood (n = 28) and thyroid tissue (n = 5) mononuclear cells. Assay of inhibition of cellular proliferation to study the function of SLAMF1 in CD4+CD25+ T regulatory (Treg) cells. Main Outcome Measure: Expression levels and the function of SLAMF1 in lymphocytes in AITD patients and controls. Results: Expression of SLAMF1 was significantly increased in peripheral blood CD4+, T helper 17, and CD19+ B cells from AITD patients. Immunofluorescence microscopy detected the presence of SLAMF1+ lymphocytes in thyroid inflammatory cell infiltrate. Functional studies showed that SLAMF1 engagement in Treg cells increased their suppressive function in healthy controls but not in AITD patients. Conclusions: The altered expression of SLAMF1, as well as its defective function observed in patients with AITD, may have a relevant role in the defective immune-regulatory function observed in this condition.

mTOR inhibitors response and mTOR pathway in pancreatic neuroendocrine tumors.

Medical therapy of pancreatic neuroendocrine tumors (P-NET) may take advantage of Everolimus treatment. However, the extent of therapeutic response cannot be predicted. This study was aimed to identify the possible predictive markers of response to Everolimus in P-NET. We found that Everolimus reduced the cell viability and induced apoptosis in primary cultures of 6 P-NET (P-NET-R), where the proliferative and antiapoptotic effects of IGF1 were blocked by Everolimus. On the contrary, 14 P-NET primary cultures (P-NET-NR) were resistant to Everolimus and IGF1, suggesting an involvement of PI3K/AKT/mTOR pathway in the mechanism of resistance. The response to Everolimus in vitro was associated with an active AKT/mTOR pathway and seemed to be associated with a greater clinical aggressiveness. In addition, a patient sensitive to Everolimus in vitro was sensitive to this drug in vivo also and showed a positive p-AKT immunohistochemistry (IHC) at tissue level. Similarly, a patient resistant to Everolimus treatment after surgery was not sensitive to the drug in vitro and had a negative p-AKT IHC staining. Therefore, present data confirm that P-NET primary cultures may be considered a model for testing medical treatment efficacy and that IHC characterization of p-AKT might help in identifying human P-NET who can benefit from Everolimus treatment. These data encourage conducting a prospective multicenter study involving different groups of P-NET patients treated with Everolimus.

Protein Kinase C Delta restrains growth in ACTH-secreting pituitary adenoma cells.

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.

Inhibition of epithelial growth factor receptor can play an important role in reducing cell growth and survival in adrenocortical tumors.

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.

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 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 enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells.

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.