Inhibition of β-catenin expression by embelin.

<p>(A) Whole cell lysates were prepared and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis using β-catenin, phospho-β-catenin, or GSK-3β antibodies was conducted. The numbers between the blots are the ratios of the intensity of bands after normalized with control. (B) Luciferase assay. TOPFlash or FOPFlash-transfected cells were treated with embelin. The luciferase activity was measured and the relative firefly luciferase activity, normalized by the renilla luciferase activity, is shown. (C) Cells were seeded on cover slip for 24 h and treated with 30 μM embelin up to 24 h. Then the cells were stained with β-catenin antibody and fluorescence was determined using confocal microscopy. (D) Expression of cyclin D1, c-Myc and MMP-7 was determined by using quantitative PCR and Western blot analysis. Values represent mean ± S.D. of three independent determinations.<b>*,</b>^<b>#</b><b>,</b>^<b>§</b>, significantly different from the untreated control (<i>p</i>< 0.05). The numbers between the blots are the ratios of the intensity of bands after normalized with control.</p

Embelin induces pro-apoptotic proteins and suppresses anti-apoptotic proteins in PC3 cells.

<p>(A), (C), (E) Translocation of pro-apoptotic protein (Bax), cytochrome <i>c</i>, AIF, and level of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1) were analyzed by western blotting. Cells were treated with embelin for the indicated time periods. After incubation, cells were harvested and the cytosolic and mitochondrial fractions were isolated. Extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis was conducted. GAPDH level was determined as loading controls for cytosol and total lysates. COX-4 level was determined as a loading control for mitochondria. Histone H1 level was determined as a loading control for nuclear fraction. C, cytosolic fraction, M, mitochondrial fraction, N, nuclear fraction. The numbers between the blots are the ratios of the intensity of bands after normalized with control. (B), (D), (F) Translocation of Bax (B), cytochrome <i>c</i> (D), and AIF (F). Cells were cultured on microscopic slides and treated with embelin for 24 h. After treatment, cells were fixed, permeabilized, and subsequently stained using specific antibody. Cells were then stained with the Texas-Red-labeled secondary antibody. Fluorescence was determined using confocal microscopy. G, Expression of VDAC1 and oligomerization of VDAC1. Embelin-treated cells were harvested and then incubated with sulfo-EGS (250 μM) for 25 min at 30°C. After proteins were resolved by SDS-PAGE (8%), VDAC1 proteins were measured using Western blot analysis. A 33 kDa band represents VDAC1 monomers while a band at 65 kDa represents the VDAC1 dimer. Mitochondrial fraction was isolated and resolved by SDS-PAGE (12%) and Western blot analysis was conducted. COX-4 level was determined as a loading control for mitochondria.</p

Inhibition of Akt and COX-2 expression by embelin in PC3 cells.

<p>(A) Cells were treated with 30 μM embelin for the indicated time periods, and whole cell lysates were prepared, and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis using phospho-Akt, total Akt, and COX-2 antibodies was conducted. The numbers between the blots are the ratios of the intensity of bands after normalized with control. (B) Cells were transfected with Myr-Akt plasmid and then treated with 30 μM of embelin for 24h. Cell viability was measured by CCK. Values represent mean ± S.D. of three independent determinations. <b>*,</b> significantly different from the untreated control cells (<i>p</i>< 0.01) and **, significantly different from the embelin only-treated cells (<i>p</i><0.001). Whole cell lysates were prepared and the levels of total Akt and phospho-Akt were determined by Western blot analysis. (C) AKT inhibitor IV treated cell lysates were prepared and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis using phosphor-Akt, total Akt, and GAPDH antibodies was conducted. Cells were treated with 0.312 μM of AKT inhibitor IV for 24 h. Cell viability was measured by CCK. Formazan formation was quantified by spectrophotometry at 450 nm. The percentage of cells surviving in each group relative to the control was calculated. (D) Cells were transfected with COX-2 luciferase vector and renilla luciferase vector for 48 h. Cells were subjected to the dual-luciferase assay. The relative firefly luciferase activity, normalized by the renilla luciferase activity, is shown. Values represent mean ± S.D. of three independent determinations.<b>*,</b> significantly different from the untreated control (<i>p</i>< 0.05).</p

Inhibition of cell migration and invasion by embelin in PC3 cells.

<p>(A) Cells were treated with 30 μM embelin in 6-well cell culture plate. A1-mm wide scratch was made across the cell layer using a sterile pipette tip. Each well was photographed at indicated time periods. Black lines on images portray the location of the cell front. The rate of cell migration was determined after estimation of the gap migrated at each time point. Values represent mean ± S.D. of three independent determinations.<b>*,</b> significantly different from the untreated control (<i>p</i>< 0.05). ^<b>#</b><b>,</b> significantly different from the untreated control (<i>p</i>< 0.05). (B) Cells were cultured in the upper chamber and incubated with embelin 30 μM for 24 h. The invading cells were fixed and stained, and cells that invaded the lower area of the membrane were photographed. Stained cells were destained with 30% acetic acid for 30 min and determined using microplate reader on 595 nm. The results were plotted and shown in the graph. Values represent mean ± S.D. of three independent determinations. (C) Cells were treated with embelin for 24 h, and conditioned media were harvested. After SDS-PAGE running gel including 0.1% gelatin, the gel was stained with coomassie blue solution and washed by destaining solution. The gel was photographed using ChemiDoc XRS.</p

GSK-3β inhibitor recovers embelin-mediated β-catenin downregulation.

<p>Embelin- treated cells were co-treated with LiCl 20 mM for 24 h. (A) Whole cell lysate were prepared, and extracted proteins were resolved by SDS-PAGE (10%) and Western blot analysis was conducted. GAPDH level was determined as a loading control. The numbers between the blots are the ratios of the intensity of bands after normalized with control. (B) TOPFlash or FOPFlash-transfected cells were treated with embelin. The luciferase activity was measured and the relative firefly luciferase activity, normalized by the renilla luciferase activity, is shown. (C) Cells were seeded on cover slip for 24 h and treated with 30 μM embelin for 24 h. Cells were stained with β-catenin antibody and fluorescence was determined using confocal microscopy.</p

Embelin induces mitochondrial apoptosis in PC3 cells.

<p>(A) Cells were incubated for the indicated time periods with embelin (30 μM), and were stained with annexin V-FITC and PI. Apoptosis was measured by flow cytometry. The cell populations were discriminated in each quadrant as viable cells in the lower left (annexin V negative/PI negative), early apoptotic cells in the upper left (annexin V positive/PI negative), late apoptotic cells in the upper right (annexin V positive/PI positive), and necrotic cells in the lower right quadrant (annexin V negative/PI positive). (B) Induction of DNA fragmentation in PC3 cells by embelin. Cells were incubated for the indicated time periods with embelin. Chromosomal DNA was isolated and DNA fragmentation was determined. M, DNA marker; P, paclitaxel (1 μM). (C) After PC3 cells were incubated with embelin for time periods, cells were labeled with 2 M JC-1 for 30 min and Δ<i>ψ</i>_m was determined by confocal microscopy. The ratio of JC-1 aggregate (red) to monomer (green) intensity was calculated with Image J software. Values represent mean ± S.D. of three independent determinations. <b>*,</b> significantly different from the untreated control cells (<i>p</i>< 0.05).</p

CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation

<div><p>Cytochrome P450 1B1 (CYP1B1) is a major E₂ hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of <i>CTNNB1</i>, <i>ZEB2</i>, <i>SNAI1</i>, and <i>TWIST1</i>. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE₂), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction.</p></div

CYP1B1 enhances cell proliferation.

<p>(A) Relative cell viability determined by CCK assay subsequent to induction of CYP1B1 in MCF-7 cells following CYP1B1 overexpression. Data are representative of experiments in triplicate. (*<i>p</i>≤0.05) (B-D) PCNA was measured by western blot and confocal microscopy following (B) CYP1B1 overexpression, and (C) CYP1B1 knockdown. (D) Confocal microscopic analysis of MCF-7 cells treated with 5 μM DMBA and 10 μM TMS for 48 h. Cells were pre-treated with TMS for 1 h prior to DMBA.</p

CYP1B1 upregulates Sp1 expression.

<p>(A-D) Sp1 mRNA levels were measured in MCF-7 cells by (A) RT-PCR following CYP1B1 overexpression, (B) qRT-PCR following CYP1B1 overexpression, (C) RT-PCR following CYP1B1 knockdown, (D) qRT-PCR following CYP1B1 knockdown. Data are representative of experiments in triplicate. (*<i>p</i>≤0.05) (E-H) Sp1 protein levels were measured by western blot (E) in MCF-10A cells following CYP1B1 overexpression, (F) in MCF-7 cells following adenoviral CYP1B1 overexpression, (G) in MCF-7 cells following CYP1B1 knockdown, and (H) in MCF-7 and MCF-10A cells following TMS treatment (0, 1, 5, and 10 μM) for 48 h. (I-K) Using confocal microscopic analysis, Sp1 levels were determined following (I) adenoviral CYP1B1 overexpression in MCF-7 cells and (J-K) treatment with 5 μM DMBA in the presence of 10 μM TMS for 48 h in (J) MCF-7 and (K) MCF-10A cells. Cells were pre-treated with TMS for 1 h prior to DMBA.</p

CYP1B1 activates Wnt/β-catenin signaling by inducing β-catenin expression and nuclear localization.

<p>(A-B) mRNA expression of β-catenin and Wnt/β-catenin signaling target genes in MCF-7 cells following CYP1B1 overexpression was determined by (A) RT-PCR, (B) qPCR, and (C-D) mRNA expression of β-catenin and Wnt/β-catenin signaling target genes in MCF-7 cells following CYP1B1 knockdown was determined by (C) RT-PCR, and (D) qPCR. (E) mRNA expression of β-catenin and Wnt/β-catenin signaling target genes in MCF-10A cells following CYP1B1 knockdown was determined by qPCR. (F) β-catenin/TCF/LEF promoter activity was determined using dual-luciferase assay following CYP1B1 overexpression in MCF-7 cells. Data are representative of experiments in triplicate. (*<i>p</i>≤0.05) Wnt/β-catenin signaling proteins were measured following (G) CYP1B1 overexpression in MCF-7 and MCF-10A cells, (H) adenoviral CYP1B1 overexpression in MCF-7 cells, (I) CYP1B1 knockdown in MCF-7 cells, and (J) treatment with TMS (0, 1, 5, and 10 μM) for 48 h in MCF-7 and MCF-10A cells. (K) Confocal microscopic analyses of β-catenin following treatment with 5 μM DMBA in the presence of 10 μM TMS for 48 h in MCF-7 cells and (L) CYP1B1 overexpression in MCF-10A cells. (M-N) Confocal microscopic analyses in adenoviral CYP1B1 overexpressed MCF-7 cells for (M) CYP1B1, and (N) β-catenin. (O) β-catenin proteins in nucleus or cytosol were measured following treatment with 5 μM DMBA in the presence of 10 μM TMS for 48 h in MCF-7 cells.</p