Antiestrogen Binding Site Ligands Research Articles

Abstract 956: The liver-X-receptor-α is involved in the induction by Tamoxifen of breast cancer cell differentiation and death

Abstract Hormonotherapy with Tamoxifen (Tam) is one of the main strategies used world-wide for the treatment of estrogen receptor positive breast cancer. However, partial responses, tumor recurrence and acquired resistance to Tam may involve mechanisms unrelated to Estrogen Receptors (ER). Studies on non-ER related mechanisms of action of Tam led us to find that it induced breast cancer cell differentiation and death through the modulation of sterol metabolism and the stimulation of sterol oxidation (de Medina et al, Cell Death Differ, 2009), and that Tam inhibited cholesterol epoxide hydrolase (ChEH). ChEH catalyzes the trans hydration of cholesterol-5,6-epoxides (CE) and this activity is a property of the microsomal antiestrogen binding site (AEBS) (de Medina et al, PNAS, 2010). In the present study we report that Tam and AEBS ligands stimulated the oxidation of cholesterol into CE and induced the accumulation of CE in MCF-7 cells through the inhibition of ChEH at therapeutic concentrations. Since CE has been reported to be a modulator of Liver-X-Receptors (LXR), we investigated the impact of Tam and other AEBS ligands on LXR-dependent gene expression: Tam and AEBS ligands modulated the expression LXR-dependent genes in MCF-7 cells. This effect had a 24 to 48 hour lag time while the effect of direct LXR modulators was observable after 6 hours, suggesting an indirect action of AEBS ligands. The involvement of CE in the control of transcription by Tam and AEBS ligands was supported by the observation that vitamin E, which inhibited the formation and the accumulation of CE, totally blocked this transcriptional modulation. We found that knocking down LXR-α using interfering RNAs in MCF-7 cells blocked the transcriptional modulation of LXR-controlled genes induced by CE, Tam and AEBS ligands. Finally, we found that the knock down of LXR-α reproduced the vitamin E inhibition of the induction of differentiation and death of MCF-7 cells. Altogether, these data showed that LXR-α is implicated in the anticancer action of Tam and identified a new signaling pathway that could explain sensitivity and resistance to Tam. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 956. doi:1538-7445.AM2012-956

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.

Abstract 2918: Molecular identification of the cholesterol-5,6-epoxide hydrolase: A new target for selective estrogen receptor modulators and unsaturated fatty acids

Abstract The cholesterol-5,6-epoxide hydrolase (ChEH) is the last epoxyde hydrolase which coding gene remains to be identified. The ChEH catalyzes the hydration of 5,6α-epoxy-cholestanol (α-CE) and 5,6β-epoxy-cholestanol (β-CE), two autoxydation products of cholesterol, into cholestane-3β,5α,6β-triol (CT). We earlier reported that the antiestrogen binding site (AEBS) was a hetero-oligomeric complex made of two subunits: the 3β-hydroxysterol-Δ7-reductase (DHCR7) and 3β-hydroxysterol- Δ8- Δ7-isomerase (D8D7I), two enzymes involved in post-lanosterol cholesterol biosynthesis (Kedjouar et al, J Biol Chem, 2004). We have recently reported that AEBS ligands induced differentiation and apoptosis of human breast cancer cell lines through the intracellular accumulation of autoxydation products of sterols such as α-CE and β-CE (Payre et al, Mol Cancer Ther, 2008; de Medina et al, Cell Death &amp; Differ, 2009). Because the ChEH and AEBS displayed some biochemical similitudes, we have investigated, in the present study, the potential relationship that may exist between the ChEH and AEBS. We established that α-CE and β-CE (the substrates of the ChEH) and CT (the product of the catalytic activity of the ChEH), were high-affinity and competitive ligands of AEBS. AEBS ligands include selective estrogen receptors modulators (SERMs) such as tamoxifen and raloxifene, poly unsaturated fatty acids such as docosahexaenoic acid (DHA), oxysterols such as 7-hydroxycholesterol and 7-ketocholesterol, and diphenylmethane compounds such as N-Pyrrolidino-4-(phenylmethyphenoxyl)-ethanamine (PBPE) and tesmilifene. We established that all AEBS ligands were inhibitors of the ChEH at pharmacological concentrations. We demonstrated a positive correlation between the binding to AEBS and the inhibition of the ChEH catalytic activity by AEBS ligands (r2 = 0.95, n = 39, p &amp;lt; 0.0001) and showed that AEBS and the ChEH had the same pharmacological profile. Finally, the structural similarity between the ChEH and AEBS was established by co-expression or genetic invalidation in mammalian cells of the D8D7I and the DHCR7, which respectively allowed the reconstitution or the impairment of both AEBS and the ChEH activity. Collectively, these data demonstrate that AEBS and the ChEH are molecularly and pharmacologically similar. These results provide the first evidence of the molecular identification of the ChEH, which coding gene had never been reported. In conclusion, we have identified a mechanism by which AEBS ligands induce the accumulation of sterol autoxidation products that were previously shown to induce breast cancer cell differentiation and apoptosis. The ChEH constitutes a new target for the chemoprevention and the treatment of breast cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2918.

Antiulcerogenic and antisecretory effects of a novel diphenylmethane derivative and antiestrogen binding site ligand.

N,N-Diethyl-2-[4-(phenylmethyl)-phenoxy]-ethanamine hydrochloride (DPPE) is a para-diphenylmethane derivative that binds selectively and with high affinity to the microsomal antiestrogen binding site (AEBS). Recent studies with DPPE indicate that AEBS is closely related to a lower affinity non-H1, non-H2 histamine site that may be associated with calcium channels; the DPPE-AEBS site is different from that which verapamil binds, however, DPPE, but not verapamil, demonstrates antiproliferative effects in vitro and is antiestrogenic in vivo. We now show that DPPE profoundly inhibits restraint and cold stress and ethanol-induced gastric ulcer formation, accelerates ulcer healing, attenuates the stress-induced rise in plasma corticosterone level, and significantly reduces basal and H2 agonist (dimaprit)-stimulated and, to a lesser extent, bethanechol-stimulated gastric acid output in conscious rats. A nonulcerogenic but prostaglandin-depleting dose of indomethacin completely blocks the inhibitory effects of DPPE on stress ulcer formation. Conversely, verapamil only slightly attenuates dimaprit-stimulated gastric acid secretion and exacerbates ethanol-induced gastric ulcers; its anti-stress ulcer effects are only partially attenuated by indomethacin. These findings support the likelihood that the site of action of DPPE is different from that of verapamil, and that an effect on prostaglandins may, at least in part, contribute to its antiulcer and apparent cytoprotective effects.