Potential signaling pathways induced by lignans.

<p>Potential signaling pathways induced by lignans.</p

Differential regulation of cell cycle by lignans.

<p>MCF-7 cells were treated with vehicle (<b>A</b>) or 10 nM E₂ (<b>C</b>), or 10 μM each of lignans (<b>E</b>, <b>G</b>, <b>I</b>, <b>K</b>, and <b>M</b>) for the indicated times. Cell extracts were subjected to Western blot analysis for cyclin D1, CDK4, cyclin E and β-actin (control). The results of three independent experiments are summarized along with the statistical evaluation in panels <b>D</b> (for E₂), and <b>F</b>, <b>H</b>, <b>J</b>, <b>L</b> and <b>N</b> (for lignans). Statistical significance of data compared with the negative control (lane 1) is shown as * (<i>p</i> < 0.05).</p

Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors - Fig 1

<p><b>Chemical structure (A) and cell-proliferation assay (B) for lignans.</b> (<b>A</b>) The phenylpropane backbone is shadowed. (<b>B</b>) MCF-7 cells were treated with vehicle (dimethylsulfoxide, DMSO), E₂ (10 nM) or different concentrations of chemicals as indicated: 1, 1 nM; 2, 10 nM; 3, 100 nM; 4, 1 μM; 5, 10 μM; and 6, 100 μM (on the left), and 10 μM EL (on the right). After incubation for 72 h, cell proliferation was examined by sulforhodamine B (SRB) assay. The rates of cell proliferation in response to E₂ or lignans to that of a control (DMSO) are shown in the graph. *: <i>p</i> < 0.05; vs. control (C), #: <i>p</i> < 0.05; vs. E₂, or §: <i>p</i> < 0.05; vs. EL. ICI: ICI 182,780, an ER antagonist.</p

Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors

<div><p>Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (<i>R</i> values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E₂). Significant correlations were observed among lignans (<i>R</i> values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level.</p></div

Secretion of MCP-1 in response to stimulation with lignans.

<p>MCF-7 cells were treated with E₂ (10 nM) or each of the lignans (10 μM) for 72 h in the presence or absence of ICI 182,780 (ICI). Then, the supernatants were collected and ELISA was performed according to the manufacturer's instructions. The data represent the mean ± SD of three independent experiments. *: <i>p</i> < 0.05; vs. control (C), or #: <i>p</i> < 0.05; vs. ICI 182,780 (-).</p

Western-blot analysis of Erk1/2 and Akt signaling pathways induced by lignans.

<p>Active/total Erk1/2 and Akt were analyzed by Western blotting. MCF-7 cells were treated with 10 nM E₂ (<b>A</b>) or 10 μM each of lignans (<b>C</b>, <b>E</b>, <b>G</b>, <b>I</b>, and <b>K</b>) in the presence or absence of inhibitors, ICI 182,780 (ICI) or LY294002 (LY), for the indicated times (minutes), and cell extracts were subjected to Western blot analysis for phosphorylated (P-) or total (T-) proteins as indicated. The results of three independent experiments are summarized along with the statistical evaluation in panels <b>B</b> (for E₂), and <b>D</b>, <b>F</b>, <b>H</b>, <b>J</b>, and <b>L</b> (for lignans). Statistical significance of data compared with the negative (lane 1) or positive (lanes 3) controls is shown as * (<i>p</i> < 0.05; vs. lane 1) or § (<i>p</i> < 0.05; vs. lane 3).</p

Estrogenic gene expression profiles of lignans revealed by DNA microarray assay.

<p>Correlation of gene expression profiles was examined between individual pairs of E₂ and lignans. The gene expression profiles for these chemicals were compared using a set of 150 estrogen-responsive genes in scatter-plot graphs. The vertical and horizontal axes indicate log₂ values of the signal intensities. <i>R</i>- and <i>p</i>-values were calculated for each graph on the basis of linear regression between two profiles.</p

(A and B) HeLa cells with stable knockdown of Kank (Kank KD) show increased activation of RhoA, whereas this activation is significantly blocked by cell-permeable C3 transferase

Stable Kank KD or control cells were treated with 1 μg/ml of cell-permeable C3 transferase and analyzed by GST-RBD pull-down assays. The levels of active RhoA were quantified and the relative levels compared with the control (lane 1) are shown in B. The results shown are the means ± SD of triplicate experiments. *, P < 0.01 compared with the control; ***, P < 0.01 compared with the control and the stable Kank KD. (C) Cell migration induced by 10 μg/ml insulin or Kank KD is inhibited by 2.5 μg/ml of cell-permeable C3 transferase and 10 μM Y-27632. The cells were treated as indicated and the cell migration assay was performed as described in . The results shown are the means ± SD of triplicate experiments. *, P < 0.01 compared with the control; ***, P < 0.01 compared as indicated.<p><b>Copyright information:</b></p><p>Taken from "Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K–Akt signaling"</p><p></p><p>The Journal of Cell Biology 2008;181(3):537-549.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364698.</p><p></p

(A) The amino acid sequences of candidate 14-3-3–binding (149–156 and 162–169) and Akt-phosphorylation (162–167) motifs

The consensus sequences of the 14-3-3–binding motif and the Akt-phosphorylation motif are also shown. (B) Binding of 14-3-3 to wild-type or mutant Kank. Either Ser154 or Ser167, or both, in Kank were replaced by Ala. HEK293T cells expressing wild-type Kank (WT, lanes 2 and 3) or Kank containing mutations (S154A, S167A, and S154A + S167A; lanes 4, 5, and 6, respectively) were subjected to immunoprecipitation followed by Western blotting using the indicated antibodies. (C) The interaction between Kank and 14-3-3 is direct and requires Ser167. GST-fused 14-3-3 isoforms expressed in bacteria were pulled down using the in vitro translated coiled-coil domain (Coil; amino acids 90–360) of Kank or its mutant containing S167A (Coil). Coprecipitates were subjected to Western blotting and detected by staining with streptavidin-HRP. (D) Ser167 of Kank is phosphorylated by Akt in vitro. GST-14b (amino acids 161–171 of Kank, lane 3) and GST-14bM (amino acids 161–171 of Kank containing a mutation of S167A, lane 4), GST, a negative control (lane 1), and GST-GSK3β, a positive control (lane 2), which were all expressed in bacteria, were subjected to phosphorylation by Akt. The samples before (−Akt) or after (+Akt) treatment with Akt along with Coomassie staining are shown. (E) Phosphorylation of the 14-3-3–binding motif by Akt enhances the association of Kank with 14-3-3. MBP-Coil with (+) or without (−) phosphorylation was subjected to pull-down assays with GST 14-3-3 isoforms γ, ε, η, or θ or control GST and the bound MBP-tagged protein was detected using an anti-MBP antibody.<p><b>Copyright information:</b></p><p>Taken from "Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K–Akt signaling"</p><p></p><p>The Journal of Cell Biology 2008;181(3):537-549.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364698.</p><p></p

(A and B) Tetracycline-inducible TREx-293 cells stably expressing Kank (A) or Kank (B) were subjected to cell migration assays using TransWell

TREx-293 cells expressing tetracycline-inducible FLAG-Kank or FLAG-Kank with (lanes 3 and 4) or without (lanes 1 and 2) insulin (10 μg/ml) stimulation were used for cell migration assays. The ratios of migration compared with the control were calculated. The results shown are the means ± SD of triplicate experiments. Tet, tetracycline induction. *, P < 0.01 compared with the control ; **, P < 0.05 compared with the control. (C) The expression of FLAG-Kank and FLAG-Kank is induced in tetracycline-inducible TREx-293 cells. The expression of FLAG-Kank (lanes 1–4) and FLAG-Kank (lanes 5–8) with or without the treatment with 10 μg/ml insulin and/or tetracycline was examined by Western blotting. (D) Knockdown of Kank enhances cell migration. The esiRNA of Kank (Kank KD, lanes 2 and 4) and control esiRNA (lanes 1 and 3) were prepared as described in Materials and methods and transfected into HEK293 cells. Cell migration assays were performed using these transfected cells in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 10 μg/ml insulin. The relative migration was calculated as described in A. *, P < 0.01 compared with the control; ***, P < 0.01 compared with the control and P < 0.05 compared with Kank KD. (E) The expression of endogenous Kank is decreased by the esiRNA of Kank. The expression levels of endogenous Kank were determined by Western blotting. The cells were transfected and stimulated as described in D.<p><b>Copyright information:</b></p><p>Taken from "Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K–Akt signaling"</p><p></p><p>The Journal of Cell Biology 2008;181(3):537-549.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364698.</p><p></p