digoxin and Adrenal-Cortex-Neoplasms

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells.

Topics: Adrenal Cortex Neoplasms; Aldosterone; Calcium Channel Blockers; Cell Line, Tumor; Cholesterol Side-Chain Cleavage Enzyme; Cytochrome P-450 CYP11B2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Digoxin; Enzyme Inhibitors; Humans; Hydrocortisone; Mineralocorticoid Receptor Antagonists; Ouabain; Phosphoproteins; RNA, Messenger; Steroid Hydroxylases

The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected. To exclude the possibility that DLI could be accounted for by cross-reaction with another known adrenal steroid, aminoglutethimide inhibition was accompanied by inhibition of 17 alpha-hydroxylase with SU-10603 and inhibition of 3 beta-hydroxysteroid dehydrogenase with cyanoketone. Once again, pregnenolone release was effectively inhibited, but no similar pattern of inhibition of DLI release was observed. Increasing the time of the incubation periods from 1 to 2 h did not change the pattern of secretion of pregnenolone or DLI. HPLC analysis of DLI released over prolonged culture periods into serum-supplemented nutrient medium showed high levels of DLI in a single major and several adjacent peaks. Analysis of the ability of extracts of Y-1-conditioned medium to compete with tritiated ouabain for binding to erythrocytes indicates that conditioned medium contained highly enriched levels of ouabain-like activity. On HPLC analysis, the distribution of this activity showed partial correlation with the distribution of DLI. These observations indicate that Y-1 cells produce and release significant quantities of a material with cardiac glycoside-like properties reflected in the cross-reactivity with antidigoxin antibodies and the ability to compete with ouabain for binding to erythrocytes. In substantiation of previous findings in chopped adrenal cultures, the cardiac glycoside-like activity does not appear to result from cholesterol side-chain cleavage or pregnenolone production, since inhibition of side-chain cleavage as well as subsequent 17 alpha-hydroxylation and 3 beta-dehydrogenation did not result in consistent inhibition of DLI release.

Topics: Adrenal Cortex Neoplasms; Aminoglutethimide; Animals; Blood Proteins; Cardenolides; Cell Line; Digoxin; Erythrocyte Membrane; Humans; Kinetics; Mice; Ouabain; Pregnenolone; Radioimmunoassay; Saponins