title: Selection and optimization of MCF-7 cell line for screening selective inhibitors of 11beta-hydroxysteroid dehydrogenase 2.
abstract: An 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) produces glucocorticoid (GC) from 11-keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11beta-HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non-selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11beta-HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11beta-HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone-derived cell lines. We demonstrated that MCF-7 cells had high expression for 11beta-HSD2, but not for 11beta-HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11beta-HSD2 was corroborated by constructing inhibitory response to an 11b-HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF-7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11beta-HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme.
authors.affiliation: Division of Electron Microscopic Research, Korea Basic Science Institute, 113 Gwahangno, Yuseong-gu, Daejeon, South Korea.
authors.forename: Chi Hyun
authors.initials: CH
authors.lastname: Kim
authors.forename: Young Sik
authors.initials: YS
authors.lastname: Cho
mesh_terms: D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2 / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; D006854:Hydrocortisone