Identification and pharmacological characterization of cholesterol-5,6-epoxide hydrolase as a target for tamoxifen and AEBS ligands.
The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.
de Medina
Philippe
P
Institut National de la Santé et de la Recherche Médicale, U563, Equipe: Métabolisme, Oncogenése et Différenciation Cellulaire, 31052 Toulouse, France.
Paillasse
Michael R
MR
Segala
Gregory
G
Poirot
Marc
M
Silvente-Poirot
Sandrine
S
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D055162:Biocatalysis / Q000187:drug effects; D019556:COS Cells; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004965:Estrogen Antagonists / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008862:Microsomes, Liver / Q000378:metabolism; D015394:Molecular Structure; D011869:Radioligand Assay; D051381:Rats; D011960:Receptors, Estrogen / Q000378:metabolism; D013261:Sterols / Q000737:chemistry / Q000494:pharmacology*; D013629:Tamoxifen / Q000737:chemistry / Q000378:metabolism
D004965:Estrogen Antagonists; D008024:Ligands; D011960:Receptors, Estrogen; D013261:Sterols; D013629:Tamoxifen; D002784:Cholesterol; D004851:Epoxide Hydrolases