Antonio Stigliano

AME Position Statement on Adrenal Incidentaloma.

OBJECTIVE To assess currently available evidence on adrenal incidentaloma and provide recommendations for clinical practice. DESIGN A panel of experts (appointed by the Italian Association of Clinical Endocrinologists (AME)) appraised the methodological quality of the relevant studies, summarized their results, and discussed the evidence reports to find consensus. RADIOLOGICAL ASSESSMENT Unenhanced computed tomography (CT) is recommended as the initial test with the use of an attenuation value of ≤10 Hounsfield units (HU) to differentiate between adenomas and non-adenomas. For tumors with a higher baseline attenuation value, we suggest considering delayed contrast-enhanced CT studies. Positron emission tomography (PET) or PET/CT should be considered when CT is inconclusive, whereas fine needle aspiration biopsy may be used only in selected cases suspicious of metastases (after biochemical exclusion of pheochromocytoma). HORMONAL ASSESSMENT: Pheochromocytoma and excessive overt cortisol should be ruled out in all patients, whereas primary aldosteronism has to be considered in hypertensive and/or hypokalemic patients. The 1 mg overnight dexamethasone suppression test is the test recommended for screening of subclinical Cushings syndrome (SCS) with a threshold at 138 nmol/l for considering this condition. A value of 50 nmol/l virtually excludes SCS with an area of uncertainty between 50 and 138 nmol/l. MANAGEMENT Surgery is recommended for masses with suspicious radiological aspects and masses causing overt catecholamine or steroid excess. Data are insufficient to make firm recommendations for or against surgery in patients with SCS. However, adrenalectomy may be considered when an adequate medical therapy does not reach the treatment goals of associated diseases potentially linked to hypercortisolism.

3,5,3′-triiodothyronine (T3) is a survival factor for pancreatic β-cells undergoing apoptosis

3,5,3′‐triiodothyronine (T3) is essential for the growth and the regulation of metabolic functions, moreover, the growth‐stimulatory effect of T3 has largely been demonstrated and the pathways via which T3 promotes cell growth have been recently investigated. Type 1 diabetes (T1D) is due to the destruction of β‐cells, which occurs even through apoptosis. Aim of our study was to analyze whether T3 could have an antiapoptotic effect on cultured β‐cells undergoing apoptosis. We have demonstrated that T3 promotes cell proliferation in islet β‐cell lines (rRINm5F and hCM) provoking an increment in cell number (up to 55%: rRINm5F and 45%: hCM), cell viability, and BrdU incorporation, and regulating the cell cycle‐related molecules (cyc A, D1, E, and p27kip1). T3 inhibited the apoptotic process induced by streptozocin, S‐Nitroso‐N‐Acetylpenicylamine (SNAP), and H2O2 via regulation of the pro‐ and anti‐apoptotic factors Bcl‐2, Bcl‐XL, Bad, Bax, and Caspase 3. The T3 protective effect was PI‐3 K‐, but not MAPK‐ or PKA‐mediated, involving pAktThr308. Thus, T3 could be considered a survival factor protecting islet β‐cells from apoptosis. J. Cell. Physiol. 206: 309–321, 2006.

HPLC–mass spectrometry method for quantitative detection of neuroendocrine tumor markers: Vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid

BACKGROUND The urinary excretion of vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) can be increased in the presence of neuroblastic and carcinoid tumors. The former is characterized by defective catecholamine metabolism which results in high urinary levels of VMA and HVA. The latter shows an altered metabolism of tryptophan and an increased synthesis of serotonin, producing high 5-HIAA urinary concentrations. METHODS We describe an HPLC-tandem mass spectrometric method for the simultaneous quantification of VMA, HVA and 5-HIAA in human urine. The chromatographic separation is performed on a reversed-phase C18 column. Instrumental analysis is performed on a Q-Trap 2000 triple quadrupole/ion trap mass spectrometer. RESULTS The method is fast and does not require sample pre-treatment. Multiple calibration curve exhibited consistent linearity and reproducibility. Linear responses were observed in the concentration range 0-50 mg/l for each analyte. Limits of detection were 0.001 mg/l for VMA, 0.015 mg/l for 5-HIAA and 0.050 mg/l for HVA with a signal-to-noise ratio of 3. Limits of quantification were 0.005 mg/l for VMA, 0.050 mg/l for 5-HIAA and 0.1 mg/l for HVA with a signal-to-noise ratio of 10. CONCLUSIONS This method can be proposed as a tool for neuroendocrine tumor markers detection.

Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

Thiazolidinediones, specific peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.

Low correspondence between K-ras mutations in pancreatic cancer tissue and detection of K-ras mutations in circulating DNA.

Objective: K-ras is the most frequently mutated gene in pancreatic cancer; reported rates range from 70% to 90%. The aim of this study was to evaluate the correspondence between K-ras mutations in pancreatic cancer tissue and in circulating DNA and the value of K-ras mutations as serological marker. Methods: The research was conducted in 30 patients with pancreatic cancer in whom both plasma and neoplastic tissues were available. Such research was extended to circulating DNA isolated from 40 patients with chronic pancreatitis. Mutations in codon 12 were examined by mutant allele-specific amplification method and by direct sequencing. Serum values of routinely used tumor markers such as carbohydrate antigen (Ca) 19.9, carcinoembryonic antigen, Ca 50, and Ca 242 have been tested in all the patients enrolled in this study. Results: K-ras mutations were detected in 70% of neoplastic tissue samples, but no mutated DNA resulted in circulating DNA samples. The 60% of patients with tissue K-ras mutation showed elevation of some tumor markers among Ca 19.9, carcinoembryonic antigen, Ca 50, and Ca 242. As a whole, these last showed low sensitivity (20%-56.67%) and specificity (56.67%-77.5%) when compared with chronic pancreatitis. Conclusion: Over the years, there has been no change in the direction of an earlier diagnosis by serological markers, and also, these data indicate that K-ras mutation in serum is an unsatisfactory method for the detection in patients with pancreatic cancer as well as in patients with high risk of progression toward neoplastic pancreatic disease.

Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrified. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.

Increased metastatic lymph node 64 and CYP17 expression are associated with high stage prostate cancer.

The metastatic lymph node 64 (MLN64), which is localized in the human chromosome 17, encodes a protein with strong homology with steroidogenic acute regulatory protein. Its overexpression in human breast carcinomas and MLNs led to the hypothesis that this protein could be involved in intraneoplastic steroidogenesis. In the present study, we investigated the expression of MLN64 in prostate cancer, another hormone-dependent tumor, and compared its expression with that of CYP17, the gene encoding for the key enzyme of androgen synthesis. We investigated by RT-PCR the expression of MLN64 and CYP17 in 60 prostatic tumors and compared their expression with the stage of disease and the appearance of relapses in a follow-up of 24 months. We found MLN64 and CYP17 expressed in all samples examined, with significantly higher expression in neoplastic tissues with respect to normal tissues (NTs). Moreover, only in neoplastic but not in NTs, a positive linear correlation was found between MLN64 and CYP17 gene expression. MLN64 and CYP17 expression seems to correlate with high stage, high Gleason score and short relapse-free time. These data, for the first time, demonstrate the presence of MLN64 and CYP17 expression in both normal and neoplastic prostatic tissues. The biological role of MLN64 in human prostate and, particularly, in neoplastic tissue is still unclear. Our findings concerning MLN64 and CYP17 gene expression and their significant positive correlation in human prostate cancer may suggest their possible role in intraneoplastic autonomous steroidogenesis.

The TRβ1 Is Essential in Mediating T3 Action on Akt Pathway in Human Pancreatic Insulinoma Cells

Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor β1 has been hypothesized. The serine‐threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic β‐cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non‐genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRβ1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor β1. We showed that the thyroid receptor β1 mediates the T3 regulation of the cdk4·cyc D1·p21CIP1·p27KIP1 complex formation and activity. In addition TRβ1 is essential for the T3 upregulation of the Akt targets β‐catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the β1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway. J. Cell. Biochem. 106: 835–848, 2009.

Targeting Estrogen Receptor-α Reduces Adrenocortical Cancer (ACC) Cell Growth in Vitro and in Vivo: Potential Therapeutic Role of Selective Estrogen Receptor Modulators (SERMs) for ACC Treatment

CONTEXT Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis and no effective treatment. ACC is characterized by an increased production of IGF-II and by estrogen receptor (ER)-α up-regulation. OBJECTIVE The objective of this study was to define the role played by ERα in 17β-estradiol (E2)- and IGF-II-dependent ACC growth and evaluate whether selective estrogen receptor modulators are effective in controlling ACC growth in vivo. EXPERIMENTAL DESIGN The human adrenocortical cell line H295R was used as an in vitro model and to generate xenograft tumors in athymic nude mice. RESULTS In H295R cells IGF-II controlled expression of steroidogenic factor-1 that, in turn, increased aromatase transcription and, consequently, estrogen production, inducing cell proliferation. ERα silencing significantly blocked E2- and IGF-II-dependent cell proliferation. This effect was dependent on the regulation of cyclin D1 expression by ERα, activated in response to both E2 and IGF-II. In fact, IGF-II induced ERα activation by phosphorylating serine 118 and 167. Furthermore, we demonstrated that ERα mediated E2-induced nongenomic signaling that stimulated IGF-I receptor (IGF1R), ERK1/2, and AKT phosphorylation, resulting in a ligand-independent activation of the IGF1R-induced pathway. In addition, E2 potentiated this pathway by up-regulating IGF1R expression as a consequence of increased cAMP-responsive element binding protein activation and binding to IGF1R promoter. The estrogen antagonist, hydroxytamoxifen, the active metabolite of tamoxifen, reduced IGF1R protein levels and both E2- and IGF-II-induced cell proliferation. Moreover, H295R xenograft growth was strongly reduced by tamoxifen. CONCLUSION These findings establish a critical role for ERα in E2- and IGF-II-dependent ACC proliferation and provide a rationale for targeting ERα to control the proliferation of ACC.

Proteomic analysis of peripheral T lymphocytes, suitable circulating biosensors of strictly related diseases

T lymphocytes and/or their subpopulations from peripheral blood may represent molecular sensors to be used for the evaluation of gene expression modification in physiological and pathological conditions, providing a unique and easily available biological model for integrated studies of gene expression in humans. In this study, a proteomic approach was applied to evaluate the association between changes in T cell protein expression patterns and specific diseased conditions. In particular, two hyperandrogenic syndromes were studied, sharing many clinical and biochemical signs: polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH). Comparison of proteomic maps of T lymphocytes derived from patients affected by PCOS or CAH with those derived from healthy subjects showed that 14 proteins are expressed differentially in both PCOS and CAH, 15 exclusively in PCOS and 35 exclusively in CAH. Seventeen of these proteins have been identified by mass spectrometry analysis. Furthermore, proteomic data mining by hierarchical clustering was performed, highlighting T lymphocytes competence as a living biosensor system.