Tamoxifen-induced antitumorigenic/antiestrogenic action synergized by a selective aryl hydrocarbon receptor modulator.

Tamoxifen (TAM) is a highly effective selective estrogen receptor (ER) modulator used extensively for the treatment and prevention of breast cancer. However, prolonged treatment of women with TAM may be a risk factor for endometrial cancer, and research in our laboratory is focused on the development of selective aryl hydrocarbon receptor modulators that can be used in combination with TAM to improve its efficacy in the breast and inhibit TAM-induced endometrial effects. This study investigated the effects of the selective aryl hydrocarbon receptor modulators 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) alone and in combination with TAM in the carcinogen-induced mammary tumor model and in the ovariectomized uterotropic assay using female Sprague Dawley rats. The lowest effective dose of 6-MCDF that inhibited tumor growth was 50 microg/kg/day, and TAM was antitumorigenic at a dose of 100 microg/kg/day. In animals cotreated with TAM + 6-MCDF at doses of 100, 50, or 25 microg/kg/day of each compound, complete inhibition of mammary tumor growth was observed at all doses, and the results are consistent with a more than additive antitumorigenic response for the low dose group (25 + 25 microg/kg) and additive interactions at the 50 and 100 microg/kg doses. In a separate experiment, 6-MCDF (800 microg/kg) inhibited TAM-induced peroxidase activity and progesterone receptor binding in the ovariectomized rat uterus but did not affect TAM-induced bone growth in ovariectomized rats. This study also investigated the effects of TAM and 6-MCDF alone and in combination on ERalpha protein levels in MCF-7 human breast cancer cells as a model for studying interactions between these compounds. The results show that 6-MCDF decreased TAM-induced ERalpha levels in the absence or presence of 17beta-estradiol through proteasome activation, and these interactions may contribute to the observed combined antitumorigenic effects of these compounds.

Crosstalk between estrogen receptor alpha and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteasomes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that activates the aryl hydrocarbon receptor (AhR) and disrupts multiple endocrine signaling pathways. T47D human breast cancer cells express a functional estrogen receptor alpha (ERalpha) and AhR, and treatment of these cells with 17beta-estradiol (E2) or TCDD resulted in a rapid proteasome-dependent decrease in immunoreactive ERalpha and AhR proteins (>60-80%), respectively. E2 did not affect the AhR, whereas TCDD induced proteasome-dependent degradation of both the AhR and ERalpha in T47D and MCF-7 human breast cancer cells, and these responses were specifically blocked by proteasome inhibitors. Thus, TCDD-induced degradation of ERalpha may contribute to the antiestrogenic activity of AhR agonists and this pathway may be involved in AhR-mediated disruption of other endocrine responses.

Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins.

Treatment of HEC1A endometrial cancer cells with 10 nm 17beta-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from -2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor alpha (ERalpha) and reversed by the anti-estrogen ICI 182, 780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between -66 to -47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERalpha proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERalpha proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERalpha-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.

Estrogen and aryl hydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells.

Ishikawa endometrial cancer cells express the estrogen receptor (ER), and this study investigates aryl hydrocarbon receptor (AhR) expression and inhibitory AhR-ER crosstalk in this cell line. Treatment of Ishikawa cells with the AhR agonist [3H]2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) gave a radiolabeled nuclear complex that sedimented at 6.0 S in sucrose density gradients, and Western blot analysis confirmed that Ishikawa cells expressed human AhR and AhR nuclear translocator (Arnt) proteins. Treatment of Ishikawa cells with 10 nM TCDD induced a 9.7-fold increase in CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity and a 10.5-fold increase in chloramphenicol acetyltransferase (CAT) activity in cells transfected with pRNH11c containing an Ah-responsive human CYP1A1 gene promoter insert (-1142 to +2434). Inhibitory AhR-ER crosstalk was investigated in Ishikawa cells using E2-induced cell proliferation and transcriptional activation assays in cells transfected with E2-responsive constructs containing promoter inserts from the progesterone receptor and vitellogenin A2 genes. AhR agonists including TCDD, benzo[a]pyrene (BaP) and 6-methyl-1,3,8-trichlorodibenzofuran, inhibited 32-47% of the E2-induced responses. In contrast, neither estrogen nor progesterone inhibited EROD activity induced by TCDD in Ishikawa cells, whereas inhibitory ER-AhR crosstalk was observed in ECC-1 endometrial cells suggesting that these interactions were cell context-dependent.

Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements.

17beta-Estradiol (E2) induces expression of several genes via estrogen receptor (ER)-Sp1 protein interactions with GC-rich promoter elements in which Sp1 but not ER binds DNA. This study reports the ligand- and cell context-dependent ER(alpha)/Sp1 and ER(beta)/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ER(alpha) and ER(beta) proteins physically interact with Sp1 (coimmunoprecipitation) and preferentially bind to the C-terminal region of this protein in pull-down assays. E2- and antiestrogen-dependent transcriptional activation of ER(alpha)/Sp1 was observed in MCF-7, MDA-MB-231, and LnCaP cells, but not in HeLa cells. E2 did not affect or significantly decrease ER(beta)/Sp1 action, and antiestrogens had minimal effects in the same 4 cell lines. Exchange of activation function-1 (AF-1) domains of ER subtypes gave chimeric ER(alpha/beta) (AF-1alpha/AF-2beta) and ER(beta/alpha) (AF-1beta/AF-2alpha) proteins that resembled wild-type ER (alpha or beta) in terms of physical association with Sp1 protein. Transcriptional activation studies with chimeric ER(beta/alpha) and ER(alpha/beta) showed that only ER(alpha/beta) can activate transcription from an Sp1 element, not ER(beta/alpha). This indicates that the AF-1 domain from ER(alpha) is responsible for activation at an Sp1 element, independent of ER subtype context. In order to further characterize this observation, deletion constructs in the AF-1 domain of both ER(alpha) and ER(alpha/beta) were made, and transactivation studies indicated that the region between amino acids 79 and 117 of this domain is important for activation at an Sp1 element.

Mechanisms of inhibitory aryl hydrocarbon receptor-estrogen receptor crosstalk in human breast cancer cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that forms a functional heterodimeric complex with the AhR nuclear translocator (Arnt) protein. The environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a high affinity ligand for the AhR and has been extensively used to investigate AhR-mediated biochemical and toxic responses. TCDD modulates several endocrine pathways including inhibition of 17beta-estradiol-induced responses in the immature and ovariectomized rodent uterus and mammary gland and in human breast cancer cell lines. TCDD inhibits formation and growth of mammary tumors in carcinogen-induced rodent models and relatively nontoxic selective AhR modulators (SAhRMs) are being developed for treatment of breast cancer. The mechanisms of inhibitory AhR-estrogen receptor (ER) crosstalk have been investigated in MCF-7 breast cancer cells by analysis of promoter regions of genes induced by E2 and inhibited by TCDD. AhR-mediated inhibition of E2-induced cathepsin D, pS2, c-fos, and heat shock protein 27 gene expression involves direct interaction of the AhR complex with inhibitory pentanucleotide (GCGTG) dioxin responsive elements (iDREs) resulting in disruption of interactions between proteins binding DNA elements required for ER action and the basal transcription machinery. Mechanisms of inhibitory AhR-ER crosstalk indicate that functional iDREs are required for inhibition of some genes; however, results indicate that other interaction pathways are important including AhR-mediated proteasome-dependent degradation of the ER.

Estrogen and aryl hydrocarbon responsiveness of ECC-1 endometrial cancer cells.

ECC-1 endometrial cancer cells express estrogen receptor alpha (ER(alpha)), and 17beta-estradiol (E2) induces cell proliferation, cathepsin D mRNA levels, and reporter gene activity in cells transiently transfected with constructs derived from the human cathepsin D and creatine kinase B (pCD and pCKB, respectively) gene promoters. The comparative antiestrogenic activity of aryl hydrocarbon receptor (AhR) agonists and ER(alpha) antagonists were also determined in these endometrial cancer cells. A functional AhR was expressed in ECC-1 cells and AhR agonists including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibited E2-induced cell proliferation and transactivation. This was comparable to inhibitory AhR-ER crosstalk in breast cancer cell lines. The pure ER antagonist ICI 182,780 also exhibited antiestrogenic activity in ECC-1 cells; however, the results obtained for 4'-hydroxytamoxifen were response-specific. 4'-Hydroxytamoxifen alone did not induce ECC-1 cell proliferation but completely inhibited E2-induced cell proliferation. 4'-Hydroxytamoxifen primarily exhibited ER antagonist activities in transactivation assays and this contrasted to the predominant ER agonist responses observed in other endometrial cancer cell lines. The unique cellular context of ECC-1 cells was confirmed using pCKB and constructs expressing wild-type ER or ER variants expressing activation function 1 (AF1) or AF2 (ER-AF1 and ER-AF2, respectively). 4'-Hydroxytamoxifen did not induce reporter gene activity in cells cotransfected with pCKB and ER-AF1 or ER-AF2; however, in cotreatment studies (4'-hydroxytamoxifen plus E2), 4'-hydroxytamoxifen inhibited E2-induced transcriptional activation by ER-AF1 or ER-AF2. Thus, the primarily antiestrogenic activity observed for 4'-hydroxytamoxifen in ECC-1 cells may be related to the inability to activate gene expression through AF1-dependent pathways.

Aryl hydrocarbon (Ah) nonresponsiveness in estrogen receptor-negative MDA-MB-231 cells is associated with expression of a variant arnt protein.

Several studies have reported a correlation between expression of the estrogen receptor (ER) and aryl hydrocarbon (Ah) responsiveness in human breast cancer cell lines. MDA-MB-231 cells are ER-negative and Ah-nonresponsive; however, initial studies showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA levels (5.8-fold) and chloramphenicol acetyltransferase activity (2.6-fold) in high passage (Hp, >50 passages) cells transiently transfected with an Ah-responsive plasmid. In contrast, no induction responses were observed in low passage (Lp, <20 passages) cells. The Ah responsiveness of Hp compared to Lp MDA-MB-231 cells was associated with a >2-fold increased expression of the Ah receptor in Hp cells. Further analysis revealed that the apparent molecular weight of the Ah receptor mRNA transcript and immunoreactive protein were comparable in Lp MDA-MB-231 and Ah-responsive human HepG2 cells. In contrast, RT-PCR analysis of the Ah receptor nuclear translocator (Arnt) protein showed that HepG2 cells expressed the expected 2.6-kb transcript, whereas a 1.3-kb transcript was the major product in MDA-MB-231 cells. Western blot analysis confirmed that HepG2 cells primarily expressed a 97-kDa wild-type form of Arnt, whereas a dominant 36-kDa variant was expressed in MDA-MB-231 cells. Complete sequence analysis of the variant form of Arnt revealed a major deletion of the C-terminal region of the protein (aa 330 to 789). Like HepG2 cells, the wild-type 2.6-kb transcript was detected in ER-positive (Ah-responsive) MCF-7 cells, whereas the low-molecular-weight variant Arnt was dominant in ER-negative MDA-MB-231, MDA-MB-435, and Adriamycin-resistant MCF-7 cells. These results suggest that expression of this protein may be useful as a prognostic factor in breast cancer.

Inhibition of estrogen-induced activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-7 human breast cancer and other cell lines transfected with vitellogenin A2 gene promoter constructs.

The antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in several cell lines using transient transfection assays and constructs containing 5'-regulatory sequences from the estrogen (E2)-responsive vitellogenin (Vit) A2 gene linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. TCDD significantly inhibited CAT activity induced by E2 in MCF-7 human breast cancer cells transiently transfected with 5'-deletion plasmids containing the homologous promoter [(-821/+14)- and (-482/+14)-CAT] or the heterologous thymidine kinase (tk) promoter [(-821/-87)tk-, (-482/-87)tk-, (-397/-87)tk-, and (-331/-87)tk-CAT]. In parallel experiments using wild-type mouse Hepa 1c1c7 and human HeLa cells cotransfected with a human estrogen receptor expression plasmid, TCDD also inhibited E2-induced CAT activity. The role of the nuclear Ah receptor complex was confirmed by results of the following studies using MCF-7 or mouse Hepa 1c1c7 cells transiently transfected with E2-responsive Vit A2 gene 5'-promoter constructs: (i) for a series of Ah receptor ligands, there was a correlation between their antiestrogenic activity in MCF-7 cells and their rank order binding affinity for the Ah receptor; (ii) alpha-naphthoflavone, an Ah receptor antagonist, inhibited the antiestrogenic activity of TCDD in MCF-7 cells; and (iii) TCDD inhibited E2-induced CAT activity in Ah-responsive wild-type but not in Ah-nonresponsive class 2 mutant Hepa 1c1c7 cells. The antiestrogenic activity of TCDD was also observed in cells which transiently overexpressed the human estrogen receptor (ER), suggesting that the mechanism does not involve downregulation of the ER by TCDD.

Indolo[3,2-b]carbazole: a dietary-derived factor that exhibits both antiestrogenic and estrogenic activity.

BACKGROUND: Indole-3-carbinol (I3C) and related compounds have been identified in vegetables of the Brassica genus. I3C and its acid-derived condensation product, indolo[3,2-b]carbazole (ICZ), bind to the aryl hydrocarbon (Ah) receptor and induce CYP1A1/1A2 gene expression in both in vivo and in vitro models. I3C also inhibits mammary tumor development in rodent models. PURPOSE: The major focus of this study was to investigate the induction of CYP1A1-dependent activity and antiestrogenic effects of ICZ in the MCF-7 human breast cancer cell line and determine if induction of CYP1A1 is required for observed antiestrogenic responses. METHODS: The induction of CYP1A1 in MCF-7 cells was determined by measuring time- and concentration-dependent changes in ethoxyresorufin O-deethylase (EROD) activity in response to ICZ treatment. The effects of ICZ on occupied nuclear estrogen receptor (ER) levels and inhibition of estrogen (17 beta-estradiol [E2])-induced cell proliferation, [3H]thymidine uptake, secretion of the 52-kd protein, and nuclear progesterone receptor (PR) levels were also measured. Chloramphenicol acetyl transferase (CAT) activity was assayed in MCF-7 cells transiently transfected with an estrogen-responsive vit-CAT plasmid. Competitive binding to rat cytosolic ER was also examined. RESULTS: ICZ (> or = 10 nM) induced CYP1A1 in MCF-7 human breast cancer cells. This compound also elicited a diverse spectrum of antiestrogenic responses, including inhibition of E2-induced cell proliferation, [3H]thymidine uptake, occupied nuclear PR binding, and CAT activity in cells transfected with the estrogen-responsive vit-CAT plasmid. In nuclear extracts from ICZ-treated cells, there was a decrease in ER levels and binding to an estrogen-responsive element in a gel shift assay. I3C also decreased nuclear ER binding in MCF-7 cells. ICZ bound with low affinity to the ER and exhibited weak estrogen-like activity. CONCLUSIONS: Like other Ah receptor agonists, ICZ is antiestrogenic in human breast cancer cells, and this activity is consistent with the inhibitory activity of I3C on mammary tumor formation in rodents. ICZ-induced antiestrogenic responses can be observed at times or concentrations in which EROD activity is unchanged, indicating an interaction between the Ah receptor and ER-mediated endocrine pathways that is independent of P450-induced hormone metabolism. ICZ also is a weak estrogen in MCF-7 cells and binds to the ER. IMPLICATIONS: The current focus on the role of dietary and environmental estrogens in human disease should take into account the possible contra-active effects of Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), ICZ, I3C, and related compounds that exhibit antiestrogenic activity.

Benzo[a]pyrene-resistant MCF-7 human breast cancer cells. A unique aryl hydrocarbon-nonresponsive clone.

Wild-type MCF-7 human breast cancer cells were cultured for 3 months in 1 microM benzo[a]pyrene (BaP), and resistant clones were screened for inducibility of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One of the BaP-resistant (BaPR) clones exhibited unique genotypic expression which distinguished it from both wild-type and drug-resistant (AdrR) variant MCF-7 cells. Glutathione levels, glutathione S-transferase activities, estrogen receptor levels, estrogen responsiveness, and expression of the multidrug-resistant MDR1 and MRP mRNA levels were similar in the wild-type and BaPR cells, whereas these parameters were reported to be altered in AdrR cells. In contrast, TCDD induced CYP1A1 gene expression and inhibited selected estrogen-induced responses in wild-type but not BaPR MCF-7 cells. Treatment of wild-type and BaPR cells with [3H]TCDD resulted in formation of the radiolabeled aryl hydrocarbon (Ah) 6 S nuclear receptor complex in both cell lines. The loss of Ah responsiveness in the BaPR variant cells correlated with the failure of the nuclear or transformed cytosolic Ah receptor complex to bind genomic dioxin-responsive elements as determined in gel retardation assays.