Embelin-Induced Apoptosis of Human Prostate Cancer Cells Is Mediated through Modulation of Akt and β-Catenin Signaling

Abstract

There is increasing evidence that embelin, an active component of Embelia ribes, induces apoptosis in human cancer cells, but the detailed mechanisms are still unclear. Here, we have investigated the effect of embelin on the growth of human prostate cancer cells. Embelin strongly inhibited cell growth especially in human prostate cancer cell lines, including PC3, DU145, LNCaP-LN3 and normal prostate epithelial cell, RWPE-1 compared to breast cancer (MDA-MB-231, MCF-7, and T47D), hepatoma (HepG2, Hep3B, and HuH-7), or choriocarcinoma (JEG-3). We observed that embelin induced apoptosis of PC3 cells in a time-dependent manner correlated with decreased expression of Bcl-2, Bcl-xL, and Mcl-1, increased translocation of Bax into mitochondria, and a reduction in the mitochondrial membrane potential. Furthermore, embelin induced voltage-dependent anion channel (VDAC) 1 expression and oligomerization, which may promote cytochrome c and AIF release. Because embelin was able to inhibit Akt activation and cyclooxygenase-2 expression, the effects on Wnt/ β-catenin signaling were determined. Embelin activated glycogen synthase kinase (GSK)-3β by preventing phosphorylation and suppressed β-catenin expression. Attenuation of β-catenin-mediated TCF transcriptional activity and gene transcription, such as cyclin D1, c-myc, and matrix metalloproteinase (MMP)-7, were shown in embelin-treated cells. The changes in β-catenin levels in response to embelin were blocked by lithium chloride, a GSK-3 inhibitor, indicating that embelin may decrease β-catenin expression via GSK-3β activation. Furthermore, exposure of PC3 cells to embelin resulted in a significant decrease in cell migration and invasion. In conclusion, these findings suggest that inhibition of Akt signaling and activation of GSK-3β partially contributes to the pro-apoptotic effect of embelin in prostate cancer cells.

Fig 1. Embelin inhibits the cell growth of human prostate cancer cell lines in a concentration- and time-dependent manner.

Cells were treated with embelin at various concentrations for the indicated times. 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. (A) PC3 cell. (B) DU145 cell. (C) LNCaP-LN3 cell. (D) RWPE-1 cell. (E) IC₅₀ values of embelin in various human cancer cell lines. The IC₅₀ values were analysed using GraphPad Prism software. Values represent mean ± S.D. of three independent determinations. *, significantly different from the untreated control cells (p< 0.05).

Fig 2. Embelin induces mitochondrial apoptosis in PC3 cells.

(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 Δψ _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. *, significantly different from the untreated control cells (p< 0.05).

Fig 3. Embelin induces pro-apoptotic proteins and suppresses anti-apoptotic proteins in PC3 cells.

(A), (C), (E) Translocation of pro-apoptotic protein (Bax), cytochrome c, 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 c (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.

Fig 4. Inhibition of Akt and COX-2 expression by embelin in PC3 cells.

(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. *, significantly different from the untreated control cells (p< 0.01) and **, significantly different from the embelin only-treated cells (p<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.*, significantly different from the untreated control (p< 0.05).

Fig 5. Inhibition of β-catenin expression by embelin.

(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.*, ^# , ^§, significantly different from the untreated control (p< 0.05). The numbers between the blots are the ratios of the intensity of bands after normalized with control.

Fig 6. GSK-3β inhibitor recovers embelin-mediated β-catenin downregulation.

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.

Fig 7. Inhibition of cell migration and invasion by embelin in PC3 cells.

(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.*, significantly different from the untreated control (p< 0.05). ^# , significantly different from the untreated control (p< 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.