title: Mitotane exhibits dual effects on steroidogenic enzymes gene transcription under basal and cAMP-stimulating microenvironments in NCI-H295 cells.
abstract: Adrenocortical carcinoma (ACC) is an extremely rare and aggressive endocrine malignancy with a poor prognosis. The most common symptom of ACC is hypercortisolism (Cushing's syndrome), which has the highest mortality. Mitotane is used as a steroidogenesis inhibitor for Cushing's syndrome or as a chemical adrenalectomy drug for ACC. Mitotane induces adrenal cortex necrosis, mitochondrial membrane impairment, and irreversible binding to CYP proteins. In this study, we explored the molecular effect of mitotane on steroidogenesis in human adrenocortical cancer NCI-H295 cells. Mitotane (10-40μM) inhibited basal and cAMP-induced cortisol secretion but did not cause cell death. Mitotane exhibited an inhibitory effect on the basal expression of StAR and P450scc protein. Furthermore, 40μM of mitotane significantly diminished StAR, CYP11A1 and CYP21 mRNA expression. HSD3B2 and CYP17 seem to be insensitive to mitotane. The stimulatory effects of mitotane on CYP11B1 were more remarkable than its inhibitory effects. In contrast, the activation of cAMP signaling strongly elevated the expression of all these genes. Mitotane (40μM) almost completely neutralized this positive effect and returned 8-Br-cAMP-induced StAR, CYP11A1, CYP17 and CYP21 mRNA to control levels. After cAMP activation, mitotane did not change the levels of CYP11B1 mRNA. The present study demonstrates that mitotane can inhibit cortisol biosynthesis due to a non-specific interference with the gene transcription of steroidogenic enzymes under both basal and 8-Br-cAMP-activated conditions in NCI-H295 cells. We also identified that StAR and CYP11A1 key enzymes that participate in the rate-limiting step of steroidogenesis, were more sensitive to mitotane. In addition, the biphasic effect of mitotane on CYP11B1 was also elucidated.
authors.affiliation: Department and Institute of Physiology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan, ROC. rosename@pchome.com.tw
authors.forename: Chia-Wen
authors.initials: CW
authors.lastname: Lin
authors.forename: Yen-Hwa
authors.initials: YH
authors.lastname: Chang
authors.forename: Hsiao-Fung
authors.initials: HF
authors.lastname: Pu
mesh_terms: D000306:Adrenal Cortex Neoplasms / Q000201:enzymology* / Q000235:genetics; D018268:Adrenocortical Carcinoma / Q000201:enzymology* / Q000235:genetics; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006854:Hydrocortisone / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D008939:Mitotane / Q000494:pharmacology*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D059016:Tumor Microenvironment / Q000187:drug effects / Q000502:physiology*
chemical_list: D018931:Antineoplastic Agents, Hormonal; D004791:Enzyme Inhibitors; D008939:Mitotane; D000242:Cyclic AMP; D013250:Steroid Hydroxylases; D006854:Hydrocortisone