endothelin-1 and Adrenocortical-Carcinoma

The human adrenocortical carcinoma-derived SW-13 cell line is currently used to study the interrelationships occurring between cytokines and growth factors and endothelins (ET) and adrenomedullin (AM). SW-13 cells express either ET-1 and AM or growth factors, and several cytokines stimulate ET-1 and AM release from SW-13 cells. However, neither the morphology and steroid-hormone secretion of SW-13 cells nor the expression of ET and AM receptors and the effects of ET and AM on SW-13 cell growth have been investigated. Electron microscopy showed that SW-13 cells were deprived of the typical organelles involved in steroid-hormone synthesis (i.e. mitochondrial with tubular cristae, smooth endoplasmic reticulum and lipid droplets), their prominent ultrastructural features being rough endoplasmic reticulum cisternae, free ribosomes and mitochondria with laminar cristae. Accordingly, steroid-hormone secretion was very low: no cortisol was produced and only very small amounts of aldosterone and its precursors were released. No appreciable secretory response to physiological concentrations of ACTH was observed. Reverse transcription-polymerase chain reaction showed the expression of pro ET-1 and proAM genes, as well as detected the mRNAs of only the ET- and AM-receptor subtypes, which are currently thought to mediate the growth-promoting action of these peptides: i.e. the ETA and AM2 receptors. In keeping with these observations, both ET-1 and AM markedly stimulated the growth of SW-13 cells, by enhancing the proliferation and lowering the apoptosis rate. Taken together, our findings allow us to conclude that SW-13 cannot be used for investigating the mechanisms involved in the regulation of steroid-hormone secretion, but are a suitable and useful model to study the role of endogenous ET and AM systems in the autocrine-paracrine control of human adrenocortical-cell growth.

Topics: Adrenocortical Carcinoma; Adrenocorticotropic Hormone; Adrenomedullin; Apoptosis; Cell Line, Tumor; Cell Proliferation; DNA, Complementary; Endothelin-1; Humans; Microscopy, Electron, Transmission; Peptides

Adrenocortical carcinoma is a rare neoplasm with poor prognosis. Endothelin-1 (ET-1) has been implicated in carcinogenesis, but has never been studied in this neoplasm. A 76-year-old woman with Cushing's syndrome due adrenocortical carcinoma was operated on and the tumour removed was studied by immunohistochemistry for ET-1. Patient history illustrates the poor prognosis of this cancer that became metastatic after one year. Immunohistochemical studies disclosed a strong expression of ET-1 by adrenocortical carcinoma cells. As shown in other cancers, ET-1 expression by adrenocortical carcinoma may suggest a pathogenic role of ET-1 in tumorigenesis that possibly could be countered by ET-1 receptor antagonists. These agents could open new therapeutic perspectives to treat a carcinoma known to have a poor prognosis.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Aged; Cushing Syndrome; Endothelin-1; Female; Humans; Immunohistochemistry

Aldosterone has recently been implicated in the pathogenesis of heart failure. The purpose of the present study was to determine the effect of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors that are also involved in heart failure, on aldosterone secretion by human adrenocortical carcinoma NCIH295R cells grown in 96-well plates. Ang II stimulated the production of aldosterone dose-dependently in serum-free medium, and the presence of serum drastically decreased aldosterone secretion. In contrast, ET-1-stimulated aldosterone production absolutely required serum. Under optimal conditions, ET-1 was more effective than Ang II as an aldosterone secretagogue. In a suboptimal condition of 2.5% serum, ET-1 and Ang II at 1 microM produced 63 and 76 pmol aldosterone/mg protein, respectively, while 230 pmol aldosterone/mg protein was generated upon coincubation with ET-1 and Ang II. The effect of ET-1 was inhibited dose-dependently by the selective ETA receptor antagonist BQ-123 with an IC50 of 23 nM, but the selective ETB receptor antagonist RES-701 had no effect up to 10 microM. These results suggest that ET-1 and Ang II stimulated aldosterone secretion synergistically in NCIH295R cells and that the effect of ET-1 was mediated via the ETA receptor subtype.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Aldosterone; Angiotensin II; Cell Line, Tumor; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-2; Endothelin-3; Humans; Peptides, Cyclic; Receptor, Endothelin A; Receptor, Endothelin B; Serum; Up-Regulation

The production and secretion of peptides by adrenocortical tumors have not been well studied. We therefore studied the production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1 in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive-adrenomedullin and immunoreactive-endothelin were detected in the culture medium of SW-13 cells (27.7 +/- 1.6 fmol/10 (5) cells/24 h and 11.0 +/- 0.8 fmol/10 (5) cells/24 h, respectively, mean +/- SEM, n = 6). Northern blot analysis showed the expression of adrenomedullin mRNA and endothelin-1 mRNA in SW-13 cells. On the other hand, no significant amount of calcitonin gene-related peptide, corticotropin-releasing hormone, neuropeptide Y, or urocortin was secreted by SW-13 cells. Treatment with ACTH (10(-9)-10(-7) mol/l), angiotensin II (10(-9)-10(-7) mol/l), or dexamethasone (10(-8)-10(-6) mol/l) for 24 h had no significant effects on immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium of SW-13. Treatment with tumor necrosis factor (TNF)-alpha (20 ng/ml) increased significantly both immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium. Interferon-gamma (100 U/ml) increased the immunoreactive-endothelin levels, but not immunoreactive-adrenomedullin levels, whereas interleukin-1 (IL-1)beta (10 ng/ml) increased immunoreactive-adrenomedullin levels, but not immunoreactive-endothelin levels. These findings indicate that SW-13 human adrenocortical carcinoma cells produce and secrete two vasoactive peptides, adrenomedullin, and endothelin-1 and that the secretion of these two peptides is modulated differently by cytokines.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Adrenomedullin; Endothelin-1; Humans; Interferon-gamma; Interleukin-1; Peptides; Radioimmunoassay; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vasoconstrictor Agents; Vasodilator Agents

The role played by endothelin (ET-1) and its receptor subtypes A and B (ET(A) and ET(B)) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ET(A), and ET(B) complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ET(B) agonist IRL-1620; this effect was not inhibited by either the ET(A) antagonist BQ-123 or the ET(B) antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ET(B), but not ET(A), activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active ET-1 biosynthetic pathway and are provided with ET(A) and ET(B) receptors; 2) ET-1 regulates in an autocrine/paracrine fashion the secretion of aldosterone by NCI-H295 cells by enhancing both AS transcription and raising the intracellular Ca2+ concentration; and 3) the former effect of ET-1 probably involves the activation of both receptor subtypes, whereas calcium response is exclusively mediated by the ET(B) receptor.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Aspartic Acid Endopeptidases; Autoradiography; Base Sequence; Calcium; Cytochrome P-450 CYP11B2; DNA, Complementary; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; Metalloendopeptidases; Oligopeptides; Piperidines; Polymerase Chain Reaction; Protein Precursors; Receptors, Endothelin; Tumor Cells, Cultured