Methylation status of Vitamin D receptor gene promoter in benign and malignant adrenal tumors

We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5' regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesi

Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

PELP1 acts as an estrogen receptor (ER) coactivator that exerts an essential role in the ER's functions. ER coregulators have a critical role in the progression and response to hormonal treatment of estrogen-dependent tumors. We previously demonstrated that, in adrenocortical carcinoma (ACC), ER\u3b1 is upregulated and that estradiol activates the IGF-II/IGF1R signaling pathways defining the role of this functional cross-talk in H295R ACC cell proliferation. The aim of this study was to determine if PELP1 is expressed in ACC and may play a role in promoting the interaction between ER\u3b1 and IGF1R allowing the activation of pathways important for ACC cell growth. The expression of PELP1 was detected by Western blot analysis in ACC tissues and in H295R cells. H295R cell proliferation decrease was assessed by A3-(4,5-Dimethylthiaoly)-2,5-diphenyltetrazolium bromide (MTT) assay and [3H] thymidine incorporation. PELP1 is expressed in ACC tissues and in H295R cells. Moreover, treatment of H295R with E2 or IGF-II induced a multiprotein complex formation consisting of PELP1, IGF1R, ER\u3b1, and Src that is involved in ERK1/2 rapid activation. PELP1/ER/IGF1R/c-Src complex identification as part of E2- and IGF-II-dependent signaling in ACC suggests PELP1 is a novel and more efficient potential target to reduce ACC growth

Statins reduce intratumor cholesterol affecting adrenocortical cancer growth

Mitotane causes hypercholesterolemia in ACC patients. We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to ER\u3b1 action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production and ER\u3b1 activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ER\u3b1 function. A mitochondrial target of ER\u3b1, the respiratory complex IV (COX IV) was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. In vivo experiments confirmed the ability of simvastatin to reduce tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ER\u3b1, COX IV expression and activity and increase TUNEL positive cells. Collectively these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production, inhibits mitochondrial respiratory chain inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth

Primary aldosteronism and metabolic syndrome.

Hypertension is frequently associated with interrelated risk factors of metabolic origin, including abdominal obesity, dyslipidemia, and alterations in glucose homeostasis, all promoting the pathogenesis of arteriosclerosis. Clustering of these risk factors, defined as metabolic syndrome, is associated with an overall high cardiovascular risk profile. This article reviews current knowledge regarding the prevalence and characteristics of the metabolic syndrome in primary aldosteronism, and discusses a possible pathophysiological link between aldosterone and its individual components other than hypertension. An abnormal glucose metabolism due to insulin resistance appears to be linked to aldosterone overproduction, and seems the major contributor to metabolic dysfunction in primary aldosteronism. Impairment of insulin action may be also due to concurrent environmental factors (hypokalemia?), and/or it might occur in compartments other than fat tissue (liver? skeletal muscle?). Higher rates of cardiovascular events reported in primary aldosteronism could be due in part to the increased prevalence of the metabolic syndrome in this disorder. Regression of glucometabolic complications after the cure of aldosterone excess should be confirmed by larger studies, and the influence on the natural history of primary aldosteronism by using agents potentially able to correct metabolic abnormalities should be further explore

Insulin signaling in adipose tissue of patients with primary aldosteronism

OBJECTIVE: We studied phosphorylation of insulin-receptors substrate downstream molecules: 1) in the ex-vivo visceral adipose tissue (VAT) of patients with aldosterone-producing adenoma (APA) (no.=7) and non-functioning adenoma (NFA) (no.=7) undergoing laparoscopic adrenalectomy; 2) in aldosterone-treated sc adipocytes of subjects (no.=5) who requested abdominoplasty. PATIENTS AND METHODS: Western blotting was used to detect phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in VAT from APA and NFA patients, and in subcutaneous adipocytes pre-treated with different aldosterone concentrations. Phosphorylation of Akt and ERK1/2 was similar in VAT of patients with APA and NFA. Pre-treatment in adipocytes with both physiological (1 nM) and pharmacological (10 μM) doses of aldosterone did not affect basal or insulin-induced phosphorylation of Akt and ERK1/2. CONCLUSIONS: Our data give further evidence that insulin signaling in human VAT is not affected by primary aldosterone overproduction

Isolated R171Q amino acid change in MEN1 gene: polymorphism or mutation?

We suggest that the isolated R171Q amino acid change might be regarded as a point mutation with low penetrance MEN1 phenotype, rather than a harmless polymorphism. Therefore, MEN1 patients carrying this genetic alteration, as well as clinically unaffected carriers, should undergo a careful endocrine investigation and a close clinical and biochemical follow-up

Retention of heterozygosity at chromosome 7p22 and 11q13 in aldosterone-producing tumours of patients with familial hyperaldosteronism not remediable by glucocorticoids

The mechanisms involved in aldosterone overproduction and adrenal cell proliferation of FH-II remain unknown. The genetic basis of this disorder may involve heritable alterations of more than one gene and/or reflect an unrestrained stimulation of aldosterone synthesis and cell growth factors

Retention of heterozygosity at chromosome 7p22 and 11q13 in aldosterone-producing tumors of patients with familial hyperaldosteronism not remediable by glucocorticoids

The mechanisms involved in aldosterone overproduction and adrenal cell proliferation of FH-II remain unknown. The genetic basis of this disorder may involve heritable alterations of more than one gene and/or reflect an unrestrained stimulation of aldosterone synthesis and cell growth factors

Genetic forms of primary aldosteronism

Numerous recent reports suggest that primary aldosteronism (PAL) is much more common than previously thought, accounting for 5-10% of hypertensive patients with most being normokalaemic. A recent Framingham study analysis has revealed the aldosterone/renin ratio, a marker of autonomous aldosterone production, to be an independent predictor of blood pressure progression and hypertension development. The description of two familial forms and Framingham results showing significant heritability of the aldosterone/renin ratio suggests a genetic basis for PAL. One rare, glucocorticoid-remediable, familial form (familial hyperaldosteronism type I [FH-I]), is caused by an adrenocorticotropic hormone-regulated, hybrid CYP11B1/CYP11B2 gene mutation and is associated with a wide spectrum of phenotypic expression from normotension to severe hypertension, which may cause early death from stroke, but is readily controlled by giving low-dose glucocorticoids. Identification of the underlying mutation has permitted development of genetic tests, greatly facilitating diagnosis. Familial hyperaldosteronism type II (FH-II), which is not glucocorticoid-remediable and not associated with the hybrid gene mutation, is at least five times more common than FH-I. Linkage studies have implicated a locus at chromosome 7p22 in three of five families with FH-II so far studied, and candidate genes within the linked locus are currently being closely examined. Since FH-II is clinically indistinguishable from apparently non-familial PAL, mutations causing FH-II are likely to be operative in the wider PAL population. As has occurred with FH-I, the search for its genetic basis brings with it the hope of new, more streamlined genetic methods of detection and a better understanding of its pathophysiology

Effects of Taxol on the human NCI-H295 adrenocortical carcinoma cell line.

Abstract We investigated the effects of taxol, an antimicrotubule agent active in different cancers, on the human NCI-H295 steroid-secreting adrenocortical carcinoma cell line. Cells were incubated for 48, 72 or 96 h with taxol 10(-10)-10(-4) M. Cell viability was evaluated by MTT assay with IC50 calculation. Apoptosis was investigated by measuring DNA fragmentation with ELISA assay after cell exposure to taxol at IC50 for 24 h. For secretion studies, aldosterone, cortisol, testosterone and dehydroepiandrosterone-sulphate (DHEA-S) were measured by RIA in the conditioned medium after 96 h exposure to taxol 10(-10)-10(-6) M, and expressed as percentage of steroid production by control cells. By MTT, taxol induced a dose-dependent inhibition of cell proliferation, with ICs50 at 72-96 h corresponding to blood levels achieved in vivo in patients with other types of cancer. Nuclear fragmentation, morphologically confirmed at electron microscopy, showed a 4-fold increase after exposure to taxol. With 10(-6) M taxol, aldosterone decreased to 48%, cortisol to 61%, testosterone to 76% and DHEA-S to 89% of steroid production by control cells. Taxol is an effective cytotoxic and antiproliferative agent in a human adrenocortical steroid-secreting carcinoma cell line. Apoptosis induced by the drug is involved in neoplastic cell death