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Mol Cancer Ther. 2008 Jul;7(7):2096-108. doi: 10.1158/1535-7163.MCT-07-2350.

Apigenin inhibits antiestrogen-resistant breast cancer cell growth through estrogen receptor-alpha-dependent and estrogen receptor-alpha-independent mechanisms.

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Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401, USA.

Abstract

Breast cancer resistance to the antiestrogens tamoxifen (OHT) and fulvestrant is accompanied by alterations in both estrogen-dependent and estrogen-independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either OHT or fulvestrant. We found that apigenin can function as both an estrogen and an antiestrogen in a dose-dependent manner. At low concentrations (1 mumol/L), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (>10 mumol/L), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either OHT or fulvestrant showed synergistic, growth-inhibitory effects on both antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or an antiestrogen, effects of the drug on estrogen receptor-alpha (ERalpha); transactivation activity, mobility, stability, and ERalpha-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERalpha and its coactivator amplified in breast cancer-1. However, higher doses (>10 mumol/L) of apigenin inhibited ERalpha mobility (as determined by fluorescence recovery after photobleaching assays), down-regulated ERalpha and amplified in breast cancer-1 expression levels, and inhibited multiple protein kinases, including p38, protein kinase A, mitogen-activated protein kinase, and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and a protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERalpha-dependent and ERalpha-independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.

PMID:: 18645020; [PubMed - indexed for MEDLINE]
PMCID:: PMC2559959

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Figure 1

A, Dose-dependent effects of apigenin on breast cancer MCF7 cell growth. To determine growth rates in the presence of apigenin (Api), cells were plated in 96-well dishes (2,000 per well) in basal medium for the indicated times and cell numbers were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-, diphenyltetrazolium bromide (MTT) assay. Relative cell growth rates (drug vs. vehicle) are shown in the presence of the indicated doses of apigenin, (*, P<0.05), as compared with DMSO-treated controls, Student's t test. B, Dose-dependent effects of apigenin on E2-induced breast cancer cell growth. MCF7 cells were treated with E2 or combinations of E2 with the indicated concentrations of apigenin and cell numbers determined by MTT assay after treatment. Points, mean (n = 6); bars, SE. **, P<0.01, 10 μM Api+E2 compared with 10 nM E2 (control), Student's t test. C, D, Fulvestrant and OHT inhibit apigenin-induced breast cancer cell growth. MCF7 cells were treated with combinations of apigenin and various concentrations of fulvestrant (ICI) or OHT. Cell numbers were determined by MTT assay after treatment. Points, mean (n = 6); bars, SE. *, P<0.05, **P<0.01, compared with DMSO treated controls, Student's t test. E, Dose-dependent effects of apigenin on MCF7, MCF7-F and MCF7-T cells and differential inhibitory effects of apigenin on growth of drug-sensitive and -resistant breast cancer cells. To determine growth rates in the presence of apigenin, MCF7, MCF7-T, and MCF7-F cells were plated in 96-well dishes (2,000 per well) in basal medium for the indicated times and cells treated with various doses of apigenin for 7 days. Cell numbers were then determined by MTT assay and relative cell growth rates (drug vs. vehicle) then determined. Points, mean (n = 6); bars, SE. *, P<0.05, **P<0.01, compared with DMSO treated controls, Student's t test. F, Differential expression of ERα, AIB1, PKA in drug-resistant breast cancer cell lines. ERα, AIB1 and PKA protein levels in MCF7, MCF7-F and MCF-T cells were determined by immunoblotting using specific antibodies. GAPDH was used as a loading control. Representative results of two independent experiments, each performed in duplicate, are shown.

Apigenin Inhibits Antiestrogen-resistant Breast Cancer Cell Growth through Estrogen Receptor-?-dependent and -independent Mechanisms

Mol Cancer Ther. 2008 July;7(7):2096-2108.

Figure 4

A, Apigenin can induce ERα degradation in breast cancer cell lines. Breast cancer MCF7, MCF7-T, MCF7-F cells were treated with 10 nM E2, 100 nM ICI (fulvestrant), 100 nM OHT, or 10 μM apigenin for 24 h. Whole cell lysates were then prepared, subjected to SDS-PAGE, and blotted. ERα levels were measured and analyzed using Western blotting and a LICOR imaging system (described under “Materials and Methods”). Upper panel, Western blot image. Lower panel, quantitative analysis of Western blot of ERα protein from upper panel, normalized to GAPDH using LICOR Odyssey software. B, Apigenin induced ERα degradation is dose-dependent. MCF7 cells were treated with the indicated doses of apigenin for 24 h and ERα levels analyzed using Western blotting and LICOR imaging system (upper panel, Western blot image; lower panel, quantitative analysis of Western blot normalized to GAPDH). C, Apigenin can induce co-activator AIB1 degradation. Breast cancer MCF7 cells were treated with 10 nM E2, 100 nM ICI, 100 nM OHT, or 10 μM apigenin for 24 h. Whole cell lysates were prepared, subjected to SDS-PAGE, and blotted. AIB1 levels were measured and analyzed using Western blotting and LICOR imaging (upper panel, Western blot image; lower panel, quantitative analysis of Western blot of AIB1 protein from upper panel, normalized to GAPDH). For all experiments, representative results of two independent experiments, with each performed in duplicate, are shown. D, Apigenin induces ERα and AIB1 degradation in breast cancer cell lines through the proteasome. Breast cancer MCF7 cells were treated with 10 μM apigenin and MG132 for 8 h as indicated. ERα levels were analyzed by Western blotting and a LICOR imaging system. E, De novo protein synthesis is not required for apigenin-induced ERα and AIB1 degradation. MCF7 cells were treated with apigenin and CHX for 8 h, as indicated. ERα and AIB1 levels were analyzed using Western blotting and LICOR imaging system. F, AIB1 mRNA is down-regulated by apigenin. MCF7 cells were treated by 10 μM apigenin for 8 h. ERα mRNA and AIB1 mRNA were quantified by RT-qPCR. **P<0.01, compared with DMSO-treated controls, Student's t test.

Apigenin Inhibits Antiestrogen-resistant Breast Cancer Cell Growth through Estrogen Receptor-?-dependent and -independent Mechanisms

Mol Cancer Ther. 2008 July;7(7):2096-2108.

Figure 5

A, Apigenin can induce an ERα-AIB1 interaction. Protein interactions were examined by yeast 2-hybrid system. The yeast strain PJ69–4A was transformed with pAD-GAL4-rAIB1 and pBD-GAL4-ERαAF2 and grown in liquid yeast culture with the appropriate ligand. The strength of the interaction of AIB1 with the receptor was determined by measuring β-gal activity from three independent transformants. β- galactosidase activity represents the level of interaction between AIB1 and ERα in the absence or presence or of Apigenin (10^-6 M) or E2 (10^-9 M). **P<0.01, compared with DMSO-treated controls, Student's t test. B, Apigenin-induced ERα interaction with AIB1 in MCF7 cells. Co-immunoprecipitation was used to examine the ERα-AIB1 interaction in MCF7 cells. ERα was precipitated from cell lysates using an anti-ERα antibody in the presence or absence of ligand (1 μM E2, ICI and apigenin; NH, no hormone). The presence of AIB1 in the pull-down complex was examined by immunoblotting using an AIB1-specific antibody. To assess the amount of precipitated ERα in the complex, the same membrane was then re-probed with an ERα antibody. Normal rabbit IgG was used as a negative control. Representative results of two independent experiments, each performed in duplicate, are shown. C, AIB1 can enhance apigenin-induced ERα transcription activity in the yeast system. ERα transcription activity was examined in a yeast estrogen reporter assay. Yeast cells (RS188N) were transformed with expression vectors for ERα and an estrogen-responsive reporter construct (ERE-β-Gal), in the absence or presence of the expression vector for AIB1. Cells were treated with or without apigenin. Values are means ± SD, n=4. *, P<0.05, **P<0.01, compared with DMSO-treated controls by Student's t test. D, AIB1 can enhance apigenin-induced ERα transcription activity in HeLa cells. ERα transcriptional activity was determined by measuring luciferase activity in HeLa cells transfected with/without pcDNA3-AIB1, ERE2pS2-Luc, pSG5-ERα and pCMV-β-Gal. Cells were treated with 10 nM E2 or 1 μM apigenin for 24 h. Luciferase activity was corrected for transfection efficiency by expressing it as ratio of luciferase to β-galactosidase activities. Values are means ± SD, n=4. **P<0.01, with AIB1 compared with the vector control by Student's t test.

Apigenin Inhibits Antiestrogen-resistant Breast Cancer Cell Growth through Estrogen Receptor-?-dependent and -independent Mechanisms

Mol Cancer Ther. 2008 July;7(7):2096-2108.

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Apigenin inhibits antiestrogen-resistant breast cancer cell growth through estrogen receptor-alpha-dependent and estrogen receptor-alpha-independent mechanisms.

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