ER-α36 mediates E2 and tamoxifen induced c-Myc expression.

<p>A, Western blot analysis of c-Myc expression in Hec1A/V and Hec1A/RNAi cells treated with 10 nM E2 or 2 µM Tam for 12 h. Levels of expression were normalized to the levels of β-actin, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells (−) vs. treatments. #, P<0.05 compared to Tam-treated Hec1A/V cells. B and C, Hec1A cells were treated for 12 h with 10 nM E2 or 2 µM Tam or together with 10 µM of MEK inhibitor U0126 or 50 µM PI3K inhibitor LY294002. Levels of c-Myc expression were normalized to the levels of β-actin, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells (−) vs. treatments. #, P<0.05 compared to E2- and Tam-treated Hec1A/V cells.</p

ER-α36 mediates tamoxifen induced activation of the MAPK/ERK in Hec1A cells.

<p>A and B, Hec1A cells were treated with 10 nM E2 or 2 µM Tam for the indicated time points. Levels of ERK1/2 phosphorylation were measured in protein extracts with Western blot analysis. Total ERK1/2 was used as loading control. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. C and D, ER-α36 expression in Hec1A/V and Hec1A/RNAi cells. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to Hec1A/V cells. E, Hec1A/V and Hec1A/RNAi cells treated with 10 nM E2 or 2 µM Tam were analyzed for the levels of ERK1/2 phosphorylation with Western blot. Total ERK1/2 was used as loading control, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells (−) vs. treatments. F, Lysates from Hec1A cells treated with DMSO (Lanes 1, 2 and 3), 10 nM E2 (Lanes 2 and 5), 2 µM Tam (Lanes 3 and 6) or pretreated with 10 µM U0126 (Lanes 4, 5 and 6) for 30 min were analyzed with Western blot analysis.</p

ER-α36 mediates E2 and tamoxifen induced activation of Akt.

<p>Hec1A cells were treated with 10 nM E2 (A) or 2 µM Tam (B) for the indicated time points and the lysates were immunoblotted with an antibody against phosphorylated Akt. Levels of phosphorylation were normalized with the total Akt protein, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. C, Western blot analysis of the Akt phosphorylation in Hec1A/V and Hec1A/RNAi ER36 cells treated with 10 nM E2 or 2 µM Tam for 10 min. Levels of phosphorylation were normalized with the total Akt protein, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. #, P<0.05 compared with E2- or Tam-treated Hec1A/V cells. D, Hec1A cells pretreated with 50 µM PI3K inhibitor LY294002 (LY, Lanes 4, 5 and 6) for 2 h and then treated with 10 nM E2 (Lanes 2 and 5) or 2 µM Tam (Lanes 3 and 6) for 10 min. E, Western blot analysis of Akt phosphorylation in MCF-7/ER36 cells treated with 2 µM Tam for the indicated time points. Expression was normalized to total Akt, and each bar represents mean value ± SEM (n = 3).*, P<0.05 compared for untreated cells. F, Lysates were prepared from MCF-7/ER36 cells treated with DMSO (Lanes 1 and 2), 2 µM of Tam (Lanes 2 and 4) or pretreated with 50 µM PI3K inhibitor LY294002 (LY, Lanes 3 and 4) for 2 h, and immunoblotted with antibodies against phosphorylated Akt or total Akt.</p

ER-α36 mediates E2 or tamoxifen induced activation of the MAPK/ERK in MCF-7 cells.

<p>A, Western blot analysis of ER-α36 expression in MCF-7/V and MCF-7/ER36 cells. Levels of expression were normalized to levels of β-actin, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to MCF-7/V cells. B and C, MCF-7/V cells treated with 10 nM E2 alone or with 2 µM Tam together for the indicated time points. Protein extracts were analyzed with Western blot analysis. Total ERK1/2 was used as loading control. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to control cells. D, MCF-7/ER36 cells treated with 2 µM Tam for different time points were analyzed for ERK1/2 phosphorylation with Western blot. Levels of expression were normalized to levels of the total ERK1/2, and each bar represents mean value ± SEM (n = 3).*, P<0.05 compared for untreated cells. E, Lysates were prepared from MCF-7/ER36 cells treated with DMSO (Lanes 1, 2 and 3), 10 nM E2 (Lanes 2 and 5), 2 µM of Tam (Lanes 3 and 6) or pretreated with 10 µM U0126 (Lanes 4, 5 and 6) for 30 min and immunoblotted with antibodies against pERK1/2 or total ERK1/2.</p

Icaritin inhibits Hec1A cells growth.

<p>(A) The chemical structure of icaritin. (B) Effects of icaritin on the growth inhibition of Hec1A cells. Cells were maintained in phenol red-free media with 2.5% charcoal-stripped fetal serum for 24 h, and then treated with the indicated concentration of icaritin. Cells were harvested at different time points as the indicated and proliferation potential was assessed by MTT assay. Results of eight independent experiments were averaged and mean ± SEM. *, <i>P</i><0.05 compared to control cells.</p

Icaritin induced Hec1A cells apoptosis.

<p>(A) Visualization of apoptotic cells by the TUNEL assay on Hec1A cells. (B) DNA fragmentation was evaluated using a Cell Death Detection ELISA kit. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to control cells. (C) The apoptotic status was determined by Annexin V/PI staining method. Percentages of negative (viable) cells, annexin V-positive (early apoptotic) cells, PI-positive (necrotic) cells, or annexin V and PI double-positive (late apoptotic) cells were shown (mean of three independent experiments) by a flow cytometry analysis.</p

Effects of icaritin on MAPK pathways.

<p>Hec1A cells were treated with the indicated icaritin concentration or the indicated interval, total cellular extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated forms of ERK1/2 (A), JNK (B) and p38 (C). Membranes were reprobed with antibodies against total ERK1/2, JNK and p38 for normalization.</p

Effects of icaritin on cell cycle regulators in Hec1A cells.

<p>(A) Hec1A cells were treated with the indicated concentration of icaritin for 24 h. The levels of cyclin D1 and cdk4 were determined by western blot. Protein levels of β-actin were also measured as controls. (B) Hec1A cells were treated with the indicated concentration of icaritin for 24 h. The levels of p21 and p27 were determined by western blot. Protein levels of β-actin were also measured as controls.</p

Icaritin induced Hec1A cells apoptosis was mediated through ERK1/2 activation.

<p>(A) Hec1A cells were treated with icaritin in the presence or absence of 10 µM U0126, 10 µM SP600125, or 40 µM SB203580 for 24 h, Cell growth was determined by MTT. The bars represent the mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared for the icaritin-treated group. (B) Hec1A cells were treated with icaritin in the presence or absence of 10 µM U0126, 10 µM SP600125, or 40 µM SB203580 for 24 h, and DNA fragmentation was determined. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to icaritin-treated cells. (C) Hec1A cells were treated with icaritin in the presence or absence of 10 µM U0126, 10 µM SP600125, or 40 µM SB203580 for 24 h, and caspase-3 activity was determined by caspase-Glo assay. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to icaritin-treated cells. (D) Hec1A cells were pretreated or not pretreated with 10 µM U0126 then added with DMSO (vehicle) or 10 µM icaritin for 12 h, 24 h respectively. Protein extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated ERK1/2. Protein levels of ERK1/2 were also measured as controls. (E) Hec1A cells were pretreated or not pretreated with 10 µM U0126 then added with DMSO (vehicle) or 10 µM icaritin for 12 h, 24 h respectively. Protein extracts were prepared and the cleavage of PARP was analyzed with Western bolt. Protein levels of β-actin were also measured as controls. (F) Hec1A Cells were pretreated with or without z-VAD-fmk (10 µM) were further incubated with 10 µM icaritin for 24 h. Protein extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated ERK1/2. Protein levels of ERK1/2 were also measured as controls.</p

Induction of caspases activities by icaritin.

<p>(A) Hec1A cells were treated with the indicated icaritin concentration or the indicated time points, total cellular extracts were prepared and subjected to Western blot assay to measure levels of cleaved-caspase-3 and cleaved-caspase-9. Protein levels of β-actin were also measured as controls. (B) Hec1A cells were treated with the indicated icaritin concentration or the indicated time points, total cellular extracts were prepared and subjected to Western blot assay using antibodies against PARP. Protein levels of β-actin were also measured as controls. (C) Hec1A cells were fixed and labeled for cytochrome c (red) and DNA (blue). (D) Hec1A cells were pretreated with 10 µM of z-VAD-fmk for 1 h, followed with 10 µM icaritin for 24 h, and DNA fragmentation and caspase-3 activity were determined. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to control cells. (E) Hec1A cells were pretreated with 10 µM of z-VAD-fmk for 1 h, followed with 10 µM icaritin for 24 h. Protein extracts were prepared and subjected to Western blot assay using antibody against PARP. Protein levels of β-actin were also measured as controls.</p