Mechanism of Action of α-Naphthoflavone as an Ah Receptor Antagonist in MCF-7 Human Breast Cancer Cells

doi:10.1006/taap.1993.1101

Toxicology and Applied Pharmacology

Volume 120, Issue 2, June 1993, Pages 179-185

https://doi.org/10.1006/taap.1993.1101 Get rights and content

Abstract

α-Naphthotlavone (αNF) and 6-methyl-1,3,8-trichlorodiben-zofuran (MCDF) inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 gene expression in MCF-7 human breast cancer cells and also decreased the accumulation of the nuclear [³H]TCDD-aryl hydrocarbon (Ah) receptor complex. Nuclear extracts from cells treated with 10⁻⁶ M αNF and incubated with a dioxin responsive element (DRE, 26-mer) did not form a retarded band in a gel mobility shift assay. In contrast, incubation of nuclear extracts from cells treated with 10⁻⁶ M MCDF and DRE gave a retarded band and this is consistent with the antiestrogenic and Ah receptor agonist activity of MCDF in human breast cancer cells. αNF was further investigated as an Ah receptor antagonist by determining the inhibition by αNF of TCDD-induced antiestrogenicity in MCF-7 cells. TCDD (10⁻⁹ M) inhibited the 17β-estradiol-induced proliferation of MCF-7 cells and the secretion of the 52-kDa protein. In cotreatment studies, αNF (10⁻⁸ to 10⁻⁶ M) caused a concentration-dependent decrease in the antiestrogenic responses elicited by TCDD. In addition, αNF inhibited the TCDD-induced down-regulation of nuclear estrogen receptor levels in MCF-7 cells. αNF (10−6 M) alone was inactive as an estrogen or antiestrogen and in cotreatment studies did not affect 17β-estradiol-induced responses in MCF-7 cells. Tamoxifen (10⁻⁷ M), an antiestrogen which acts through the estrogen receptor, also inhibited 17β-estradiol-induced cell proliferation and aNF did not affect the tamoxifen-mediated antiproliferative response. Thus, aNF antagonized TCDD-induced CYP1A1 gene expression in MCF-7 cells and also acted as an anti-antiestrogen for TCDDmediated antiestrogemcity in these cells. These results were consistent with the low levels of DRE binding observed with nuclear extracts from cells treated with 10⁻⁹ M TCDD plus αNF (10⁻⁸ to 10⁻⁶ M) or 10⁻⁶ M αNF alone. Thus, αNF appears to act as an Ah receptor antagonist in MCF-7 cells by decreasing the levels of transcriptionally active nuclear Ah receptor complexes.

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AHR signaling limits inflammation by damping proinflammatory responses in KCs, particularly the production of chemokines and cytokines recruiting and further activating immune cells, such as IL-17–producing cells into the site of inflammation (Di Meglio et al., 2014a). Increased metabolic activity in R26Cyp1a1 mice faithfully phenocopied Ahr deficiency, leading to exacerbated skin inflammation, which in turn was attenuated by the inhibition of CYP1A1 enzymatic activity by α-NF, a potent CYP1 inhibitor (Wincent et al., 2012), also described in early studies as a partial AHR antagonist (Merchant et al., 1993). Interestingly, inhibition of physiological CYP1A1 activity in wild-type mice did not influence Aldara-induced skin inflammation (data not shown), suggesting that the metabolic turnover of AHR physiological ligands is carefully optimized under normal conditions, in keeping with the known harmful effects of electrophilic compounds produced during xenobiotics bioactivation.
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This makes the ZET a suitable in vitro model to be added to the testing battery that we are currently developing for PDT testing of PS UVCBs. α-Naphtoflavone (ANF) is a well-known and commonly used AhR inhibitor in both in vivo and in vitro studies (Gasiewicz and Rucci, 1991; Merchant et al., 1993; Timme-Laragy et al., 2007). However, the present study selected 6,2’,4’-trimethoxyflavone (TMF), and not ANF, to study the role of AhR in mediating the observed effects in both the EST and AhR CALUX assay because of the relatively higher selectivity of TMF as an AhR inhibitor.
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Regular ArticleMechanism of Action of α-Naphthoflavone as an Ah Receptor Antagonist in MCF-7 Human Breast Cancer Cells

Abstract

Regular Article
Mechanism of Action of α-Naphthoflavone as an Ah Receptor Antagonist in MCF-7 Human Breast Cancer Cells