Lack of CYP1A1 expression is involved in unresponsiveness of the human hepatoma cell line SK-HEP-1 to dioxin

The aryl hydrocarbon receptor (AhR) mediates a wide variety of toxic effects due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The human hepatoma cell line SK-HEP-1 expresses AhR and ARNT. However, TCDD failed to induce CYP1A1 and XRE-dependent reporter genes in these cells. Although CYP1A1 was not induced by TCDD exposure, both CYP1B1 and AhR repressor (AhRR) were constitutively expressed. The AhR antagonist alpha-naphthoflavone altered the basal level of XRE-dependent reporter gene expression dose-dependently. As our results suggested the activation of AhR signals by putative endogenous ligands, we established SK-HEP-1-derived cell lines that stably expressed CYP1A1. The inducibility of XRE-dependent reporter genes and CYP1B1 by TCDD was restored in these cells. Our findings demonstrated the presence of endogenous ligands in SK-HEP-1 cells due to the absence of the metabolizing enzyme CYP1A1, but not CYP1B1, which allowed the constitutive expression of AhR target genes.     

3',4'-dimethoxyflavone as an aryl hydrocarbon receptor antagonist in human breast cancer cells.

Treatment of MCF-7 and T47D human breast cancer cells with 3', 4'-dimethoxyflavone (3',4'-DMF) alone did not induce CYP1A1-dependent ethoxyresorufin O:-deethylase (EROD) activity or reporter gene activity in cells transfected with an aryl hydrocarbon (Ah)-responsive construct (pRNH11c). In contrast, 1 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced up to a 50- to 80-fold increase in EROD and reporter gene activity in MCF-7 and T47D cells. In cells cotreated with 1 nM TCDD plus 0.1-10 microM 3',4'-DMF, there was a concentration-dependent decrease in the TCDD-induced responses, with 100% inhibition observed at the 10 microM concentration. Gel mobility shift assays using rat liver cytosol and breast cancer cell nuclear extracts showed that 3',4'-DMF alone did not transform the AhR to its nuclear binding form, but inhibited TCDD-induced AhR transformation in rat liver cytosol and blocked TCDD-induced formation of the nuclear AhR complex in MCF-7 and T47D cells. TCDD also inhibited estrogen-induced transactivation in MCF-7 cells, and this response was also blocked by 3',4'-DMF, confirming the AhR antagonist activity of this compound in breast cancer cells.     

Aryl hydrocarbon receptor gene silencing with small inhibitory RNA differentially modulates Ah-responsiveness in MCF-7 and HepG2 cancer cells

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNAs for the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) mRNAs in MCF-7 breast cancer cells resulted in a 60 to 80% decrease in levels of AhR and ARNT proteins in whole-cell extracts and decreased binding of nuclear extracts to 32P-labeled dioxin-responsive element. siRNA for the AhR also decreased 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 protein, CYP1A1-dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17beta-estradiol (E2) induces proliferation of MCF-7 cells through enhanced G0/G1 --> S phase progression, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell-cycle progress were partially blocked in MCF-7 cells transfected with siRNA for AhR. The results also indicated that siRNA-dependent decreases in AhR protein in MCF-7 cells were accompanied by increased G0/G1 --> S phase progression, suggesting a growth-inhibitory role for the "endogenous" AhR. Surprisingly, TCDD alone induced G0/G1 --> S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 --> S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (cdk2), and cdk4. In the absence of ligand, the AhR exhibits growth-inhibitory (MCF-7) and growth-promoting (HepG2) activity that is cell context-dependent.     

Structure-dependent Ah receptor agonist activities of chlorinated biphenylenes.

Polychlorinated biphenylenes (PCBP) have been identified as combustion by-products that bind the aryl hydrocarbon receptor (AhR) and exhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like activity. This study investigates the Ah-responsiveness of 2,3,6,7-tetrachlorobiphenylene (2,3,6,7-CBP), 2,3,6-CBP, 2,3-CBP and 2-CBP in breast cancer cells. MCF-7 or ZR-75 cells were treated with different concentrations (1-100 nM) of the compounds alone to determine their activity as inducers of CYP1A1 protein expression or luciferase activity in cells transfected with a construct (pDRE(3)) containing three tandem dioxin responsive elements (DREs) linked to a luciferase reporter gene. In both assays, the order of potency was 2,3,6,7-CBP>2,3,6-CBP>2,3-CBP approximately 2-CBP, and 2,3,6,7-CBP and TCDD were equipotent. Similar results were also observed in an antiestrogenic assay in MCF-7 cells, confirming the high AhR agonist activity of 2,3,6,7-CBP in breast cancer cells.     

Possible aryl hydrocarbon receptor-independent pathway of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced antiproliferative response in human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ligand with high affinity for the aryl hydrocarbon receptor (AhR). It suppresses 17β-estradiol (E2)-induced cell proliferation in human breast cancer cells. Although it has been theorized that the AhR is involved in TCDD-induced antiestrogenic activity and antiproliferation in human breast cancer cells, some evidence suggests that these activities of chlorinated aromatic compounds also occur by AhR-independent pathways. Here, we investigated the possibility of TCDD-induced antiproliferative responses in human breast cancer cells through AhR-independent pathways. Compared with that in vehicle-treated controls, DNA synthesis was significantly suppressed in MCF-7 cells and ZR75-1 cells treated with TCDD at a very low concentration (0.01 nM), whereas that in human ovarian carcinoma OVCAR3 cells, human cervical carcinoma HeLa cells and human choriocarcinoma JEG-3 cells was unaffected, even by exposure to 10 nM TCDD. The suppression induced by TCDD was not associated with the estrogen receptor α-signaling pathway. Another AhR agonist, 3,3',4,4',5-pentachlorobiphenyl, had no effect on DNA synthesis in MCF-7 cells at concentrations high enough to induce the transactivation function of the AhR. Furthermore, in MCF-7 cells, knockdown of the AhR by RNA interference had no effect on TCDD-induced antiproliferation. These findings suggest that the principal pathways of TCDD-induced antiproliferation in breast cancer cells are not AhR dependent.     

Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin

The aryl hydrocarbon receptor (AhR), when activated by exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates expression of several phase I and phase II enzymes and is also involved in the regulation of cell proliferation. Several studies suggest that endogenous AhR ligand(s) may exist. One putative endogenous ligand is indirubin, which was recently identified in human urine and bovine serum. We determined the effect of indirubin in MCF-7 breast cancer cells on induction of the activities of cytochromes P450 (CYP) 1A1 and 1B1, as measured by estradiol and ethoxyresorufin metabolism, and on induction of the CYP1A1 and CYP1B1 mRNAs. With 4-hr exposure, the effects of indirubin and TCDD at 10nM on CYP activity were comparable, but the effects of indirubin, unlike those of TCDD, were transitory. Indirubin-induced ethoxyresorufin-O-deethylase activity was maximal by 6-9 hr post-exposure and had disappeared by 24 hr, whereas TCDD-induced activities remained elevated for at least 72 hr. The effects of indirubin on CYP mRNA induction were maximal at 3 hr. Indirubin was metabolized by microsomes containing cDNA-expressed human CYP1A1 or CYP1B1. The potency of indirubin was comparable to that of TCDD in a CYP1B1-promoter-driven luciferase assay, when MCF-7 cells were co-exposed to the AhR ligands together with the CYP inhibitor, ellipticine. Thus, if indirubin is an endogenous AhR ligand, then AhR-mediated signaling by indirubin is likely to be transient and tightly controlled by the ability of indirubin to induce CYP1A1 and CYP1B1, and hence its own metabolism.     

The aryl hydrocarbon receptor antagonist, 3'methoxy-4'nitroflavone,attenuates 2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent regulation of growth factor signaling and apoptosis in the MCF-10A cell line

Previous studies have demonstrated that 2,3,7,8 tetracholorodibenzo-p-dioxin (TCDD) mimics epidermal growth factor receptor (EGFR) signaling in the MCF-10A human mammary epithelial cell line and protects cells from EGF withdrawal-induced apoptosis. These effects appear to be due to the ability of TCDD to increase the expression of transforming growth factor-alpha (TGFalpha), a known EGFR ligand. Because TCDD's effects occurred at concentrations as low as 1 nM, a role for the aryl hydrocarbon receptor (AhR) was hypothesized. In the present study, 3'methoxy-4'nitroflavone (MNF), a known AhR antagonist, was used to analyze AhR signaling in this cell line. MNF suppressed TCDD-dependent dioxin response element (DRE)-driven luciferase activity at concentrations as low as 10 nM. In addition, MNF attenuated TCDD's ability to inhibit apoptosis and to activate Akt and Erk1,2, two EGFR-dependent signaling molecules. Finally, the TCDD-dependent increase in TGFalpha mRNA was also suppressed by MNF. MNF's effects on TCDD action in the MCF-10A cell line occurred at concentrations ranging from 1 nM for Akt phosphorylation and TGFalpha expression to 100 nM for inhibition of apoptosis. Attenuation of TCDD-dependent luciferase activity occurred at concentrations as low as 10 nM, which suggests that TCDD inhibits apoptosis in human mammary epithelial cells by multiple mechanisms.