Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation

Abstract

Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1(omega5) was purified from geranium (Pelargonium x hortorum) and shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERalpha-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1(omega5) inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1(omega5) inhibited estradiol (E(2))-induced estrogen response element (ERE) reporter activity and transcription of the endogenous E(2) target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1(omega5) did not compete with E(2) for ERalpha or ERbeta binding, nor did AnAc 24:1(omega5) reduce ERalpha or ERbeta steady-state protein levels in MCF-7 cells; rather, AnAc 24:1(omega5) inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1(omega5) interaction with ERalpha ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1(omega5) interaction with the ERalpha DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1(omega5) inhibited E(2)-ERalpha interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1(omega5) inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses.

Fig. 1

AnAc 24:1^ω5 inhibits the proliferation of human breast cancer cells. MCF-10A normal, immortalized, breast cells and MCF-7, LCC9, LY2, and MDA-MB-231 breast cancer cells were grown in the presence of 10 nM E₂, 100 nM 4-OHT, or 50 µM AnAc 24:1^ω5, alone or in combination, as indicated, for 48 h prior to examining BrdU incorporation as described in Materials and Methods. Values are the mean ± SEM of 3–5 independent experiments in which each treatment within that experiment was performed in quadruplicate. Treatments that were significantly different (P<0.05) from EtOH control are designated (a) and treatments in combination with E₂ that were significantly different (P<0.05) from E₂ alone are designated (b).

Fig. 2

AnAc 24:1^ω5 inhibits cell cycle progression and stimulates apoptosis. FACS analysis (A) was used to determine the distribution of cells in G1-, S-, and G2/M- phases of the cell cycle in MCF-7, LY2 and MCF-10A breast cell lines. Cells were treated with EtOH, 10 nM E₂, 100 nM 4-OHT or 20 µM AnAc 24:1^ω5 alone or in combination as indicated and described in Materials and Methods. For apoptosis assays (B), MDA-MB-231 and MCF-7 cells were incubated with the indicated concentrations of AnAc 24:1^ω5 or, as positive controls, 100 nM 4-OHT for MCF-7 and 1 µM doxorubicin for MDA-MB-231 for 48 h. Apoptosis was evaluated by an Elisa kit that measures histone-associated DNA fragments in mono- and oligonucleosomes as an index of relative apoptosis as described in Materials and Methods. Values are the mean of quadruplicate determinations ± SEM. * Significantly different from the EtOH control, p < 0.05.

Fig. 3

AnAc 24:1^ω5 inhibits E₂-induced target gene transcription. To measure endogenous gene transcription, MCF-7, LCC9, LY2, MCF-10A, and MDA-MB-231 cells were serum-starved for 48 h and then treated for 6 h with EtOH, 10nM E₂, and 10, 20, or 40 µM AnAc 24:1^ω5 alone or in combination as indicated and as described in Materials and Methods. RNA levels of the target genes CCND1 (cyclin D1, A), CATD (cathepsin D, B) and TFF1 (pS2, C) were analyzed by real-time QRT-PCR as described in Materials and Methods. For MCF-10A and MDA-MB-231, CCND1 basal expression was ~25- and 114- fold higher than MCF-7 cells, respectively. For LY2, MCF-10A, and MDA-MB-231, CTSD basal expression was ~2-, 172-, and 138- fold higher than MCF-7 cells, respectively. No TFF1 expression was detected in cell lines other than MCF-7 (C). The effect of AnAc 24:1^ω5 on each ER subtype (D) was examined in HEK-293 cells that were co-transfected with ERα (top) or ERβ (middle) in addition to an ERE-luciferase reporter and pRL-TK as described in Materials and Methods. MCF-7 cells (bottom) were transfected with the same ERE-luciferase reporter and pRL-TK as described in Materials and Methods. Twenty-four hours after transfection, the cells were treated with ethanol (EtOH), 10 nM E₂ or the indicated concentrations of AnAc 24:1^ω5 alone (solid lines, open squares) or in combination with 10 nM E₂ (dashed lines, filled circles). Dual luciferase activity was assayed as described in Materials and Methods. Data are displayed as relative luciferase activity (fold difference) in which the EtOH activity was set to 1. For all panels, data are the mean ± SEM from 3 separate experiments. Values that were significantly different (P<0.05) from EtOH control are designated (a) and values from combined treatments that were significantly different (P<0.05) compared to E₂ alone are designated (b).

Fig. 4

AnAc 24:1^ω5 does not compete with [³H]E₂ for binding ERα or ERβ, but inhibits ERE binding. Ligand binding assays (A and B) utilized baculovirus expressed human ERα or ERβ incubated with [³H] E₂ and the indicated concentrations of E₂ or AnAc 24:1^ω5. [³H]E₂ specific binding was determined by HAP assay as described in Materials and Methods. Values are the average ± SEM of triplicates. EMSA assays utilized baculovirus-expressed ERα (C) and ERβ (D) incubated with [³²P]-labeled EREc38 in the absence (no ligand) or presence of E₂ (with ligand), plus increasing concentrations of AnAc 24:1^ω5 as indicated. EMSA was performed as described in Materials and Methods. An antibody against either ERα (G20 in C) or FLAG (used to detect FLAG-ERβ in D) was added to the indicated reaction mixtures to confirm the specificity of the retarded ER-ERE complex. SS = supershift of the ER-ERE with the indicated ERα or FLAG antibodies.

Fig. 5

AnAc 24:1^ω5 inhibits E₂-ERα occupancy of the pS2 (TFF1) gene promoter in MCF-7 cells and does not accelerate ERα or ERβ protein degradation. MCF-7 cells were treated with EtOH, 10 nM E₂, 10 µM AnAc 24:1^ω5, or 10 nM E₂ plus 10 µM AnAc 24:1^ω5 for 20 min and ChIP assays were performed as described in Materials and Methods. QRT-PCR (A) was performed for ERα occupancy on the pS2 ERE in ChIP samples and calculation of relative promoter enrichment was described in Materials and Methods. Values are the average ± std of two separate experiments. PCR products of pS2 reactions (B) from the input or indicated ChIP assay samples were separated on a 1.5% agarose gel and visualized by EtBr staining. To examine ERα (C) and ERβ (D) protein stability, MCF-7 cells were treated with 10 µM AnAc 24:1^ω5 or EtOH for the indicated times. WCE were separated by SDS PAGE and immunoblotted for ERα or ERβ using two different subtype-specific antibodies for each ER subtype. Blots were stripped and reprobed with β-actin for normalization and the bar graphs show the average of two replicate experiments ± std. No statistical differences were determined. Arrows (D) indicate the expected 50 kDa band of ERβ.

Fig. 6

Surflex-dock depiction of PDB structure of AnAc 24:1^ω5 docking with the ERα DBD. In each panel, the structure on left shows original PDB structure and the one on the right illustrates AnAc 24:1^ω5 bound to the DNA binding domain (DBD) as side view (A) and top view (B), i.e., looking through ERα DBD protein to the DNA.