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β-catenin signaling
European Journal of Pharmacology 791 (2016) 455–464
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
5-Acetyl goniothalamin suppresses proliferation of breast cancer cells via Wnt/β-catenin signaling Nittaya Boonmuen a, Natthakan Thongon a, Arthit Chairoungdua a, Kanoknetr Suksen a, Wilart Pompimon b, Patoomratana Tuchinda c, Vichai Reutrakul c,d, Pawinee Piyachaturawat a,e,n a
Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Lampang Rajabhat University, Lampang 52100, Thailand c Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand d Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand e Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 May 2016 Received in revised form 7 September 2016 Accepted 15 September 2016 Available online 16 September 2016
Styryl lactones are plant-derived compounds from genus Goniothalamus with promising anti-proliferation and anticancer properties. However, the exact mechanism and the target for their activities remained unclear. In the present study, we investigated the effect of 5-acetyl goniothalamin (5GTN) from Goniothalamus marcanii on Wnt/β-catenin signaling pathway which is a key regulator in controlling cell proliferation in breast cancer cells (MCF-7 and MDA-MB-231). 5GTN, a naturally occurring derivative of goniothalamin (GTN) mediated the toxicity to MCF-7 and MDA-MB-231 cells in a dose- and time- related manner, and was more potent than that of GTN. 5GTN strongly inhibited cell proliferation and markedly suppressed transcriptional activity induced by β-catenin in luciferase reporter gene assay. In consistent with this view, the expression of Wnt/β-catenin signaling target genes including c-Myc, cyclin D1 and Axin2 in MCF-7 and MDA-MB-231 cells were suppressed after treatment with 5GTN. It was concomitant with cell cycle arrest at G1 phase and cell apoptosis in MCF-7 cells. In addition, 5GTN enhanced glycogen synthase kinase (GSK-3β) activity and therefore reduced the expression of active form of β-catenin protein in MCF-7 and MDA-MB-231 cells. Taken together, 5GTN exhibited a promising anticancer effect against breast cancer cells through an inhibition of Wnt/β-catenin signaling. This pathway may be served as a potential chemotherapeutic target for breast cancer by 5GTN. & 2016 Elsevier B.V. All rights reserved.
Keywords: 5-Acetyl goniothalamin Anticancer Antiproliferation Breast cancer Styryl lactone Wnt signaling Chemical compounds studied in this article: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (PubChem CID:64965) Ellipticine (PubChem CID:3213) 5-bromo-2′-deoxyuridine (BrdU) (PubChem CID: 6035) Propidium iodide (PI) (PubChem CID: 104981) Doxorubicin (PubChem CID: 31703)
1. Introduction Wnt/β-catenin signaling pathway plays a central role in regulating cell proliferation in normal and various cancer cells (Amado et al., 2011). Mutation of Wnt/β-catenin pathway causing deregulation of the expression of Wnt/β-catenin target genes was demonstrated in mammary carcinoma and colorectal cancer (Holland et al., 2013). Approximately 40% of breast cancer cases showed increases in β-catenin (Incassati et al., 2010). The n Corresponding author at: Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Rd., Ratchatewee, Bangkok 10400, Thailand E-mail address: [email protected] (P. Piyachaturawat).
http://dx.doi.org/10.1016/j.ejphar.2016.09.024 0014-2999/& 2016 Elsevier B.V. All rights reserved.
dysregulation of Wnt/β-catenin signaling in cancer cells has been reported on various steps along the complex regulating pathway including increased β-catenin expression, inactivated negative regulators, and altered levels of Wnt ligands or other components of Wnt signaling (Incassati et al., 2010). Thus, the defect of Wnt signaling is one of the promising targets for chemoprevention and chemotherapy for cancers including breast cancer (Dakeng et al., 2012). In the absence of Wnt ligand, β-catenin, which is a key component of Wnt/β-catenin signaling pathway, is degraded via the proteasome as a result of the phosphorylation by the destruction complex proteins including the APC (adenomatous polyposis coli), axin, glycogen synthase kinase (GSK) 3-β, and casein kinase 1 (Amado et al., 2011). In the presence of Wnt ligand, on the other hand, binding of Wnt to a Frizzled receptor in the
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presence of the co-receptor, LRP5/6 (low density lipoprotein-related protein 5/6), subsequently reduces the activity of destruction complex to degrade β-catenin (Incassati et al., 2010). The stabilized cytoplasmic β-catenin is then translocated to the nucleus to bind to lymphoid enhancer-binding factor 1/T-cell factor (TCF/LEF) transcription factor, and stimulates the expression of TCF responsive target genes (Bao et al., 2012). The downstream transcriptional targets of β-catenin/TCF including cyclin D1 and c-Myc are important regulators of cell cycle process and proliferation particularly at the transition of G1/S and G2/M phases (Barcelos et al., 2014, Pooja et al., 2014). Naturally occurring compounds from plant have long been an attractive source of anticancer agents (Harvey, 2008). Of these, a styryl lactone goniothalamin (GTN), which is a low molecular weight phenolic compound from the genus Goniothalamus (Annonaceae), has been reported to exhibit a promising antiproliferative activity against a broad array of cancer cells including leukemia, kidney, pancreas, colon and breast cancer cells (Alabsi et al., 2013; Chen et al., 2005; Tantithanaporn et al., 2011). Its anticancer activities have been reported to be associated with induction of apoptosis, ROS production, DNA damage, and interruption of mitochondrial transmembrane potential in a variety of cancer cells (Alabsi et al., 2012; Inayat-Hussain et al., 2003; Yen et al., 2012). Although many anticancer activities of GTN have been proposed, the more specific signaling target to improve the efficiency and specificity of treatment has not been investigated. In an effort to search for more specific therapeutic target, the present study aimed to investigate the involvement of antiproliferative activity of a naturally occurring derivative of goniothalamin, 5-acetyl goniothalamin (5GTN) on Wnt/β-catenin signaling pathway, a critical pathway for regulating proliferation in cancer cells.
2. Materials and methods 2.1. Materials Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), antibiotic-antimycotic agents, trypsin, Lipofectamine 2000 and Trizol reagents were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), RIPA, and SuperSignal West Pico Chemiluminescent Substrate were from Thermoscientific (Cramlington, UK). SDS, 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and ellipticine were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dual-luciferase reporter assay was from Promega (Madison, WI, USA). SYBR kit was from Applied Biosystems (Carlsbad, CA, USA). Protease inhibitor cocktail and cell proliferation ELIZA, BrdU (colorimetric) were from Roche (Mannheim, Germany). Propidium iodide (PI)/RNase Staining Buffer and Annexin-V FITC apoptosis detection kit were obtained from BD Biosciences (San Jose, CA, USA). Doxorubicin was from Guanyu Bio-Tech Co. (Xijan, Republic of China). Phosphatase inhibitor cocktail and active-β-catenin (anti-ABC) clone 8E7 monoclonal antibody were from Millipore Corporation (Darmstadt, Germany). Βeta-catenin (H-102) and βactin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-GSK-3β (Ser9) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). GSK-3β antibody was from Abcam (Cambridge, MA, USA). The T-cell factor (TCF)reporter plasmids (TOPFLASH and the negative control FOPFLASH), β-catenin-FLAG and S33Y plasmids were generated as previously described (Bhukhai et al., 2012). Two styryl lactones; Goniothalamin (GTN) and 5-acetyl goniothalamin (5GTN) were isolated from Goniotalamus tapis and Goniothalamus marcanii, respectively as previously described (Ahmad et al., 1991). They were obtained as white crystals, m.p.
80–82 °C and 90–93 °C, respectively. The GTN and 5GTN were assigned to the formula C13H12O2 and C15H14O4 by EIMS. The molecular structures were elucidated by spectroscopy technique such as NMR, IR, and UV. Their spectral data were similar to those previously reported (Ahmad et al., 1991). The chemical structures are shown in Fig. 1A. 2.2. Cell culture Human breast cancer (MCF-7), triple-negative breast cancer (MDA-MB-231) and human embryonic kidney (HEK-293) cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM and MEM, respectively. They were supplemented with 10% fetal bovine serum and 1% antibiotic (100 U/ml of penicillin and 100 mg/ml of streptomycin), and incubated at 37 °C in a 5% CO2 incubator with humidified atmosphere (Invitrogen, Carlsbad, CA, USA). 2.3. Cell viability and cell proliferation assay Cell viability was determined by using the colorimetric MTT assay. MCF-7 and MDA-MB-231 cells (3 103 cells/well) were seeded in 96-well plates and incubated for 24 h prior to treatment with GTN, 5GTN, ellipticine or doxorubicin at concentrations ranging from 0 to 50 mM for 24, 48, and 72 h. The compounds were dissolved in DMSO and the final concentration of DMSO in the medium was less than 0.05%. After treatment, the medium was removed and fresh medium containing 0.5 mg/ml MTT was added and incubated at 37 °C for 4 h. The dark blue formazan crystal product was dissolved with DMSO and measured at 540 nm using a Multiskan™ GO Microplate Spectrophotometer (Thermoscientific, Cramlington, UK). The result was calculated as % of cell viability. Cell proliferation was determined by a Cell Proliferation ELISA, BrdU colorimetric assay according to the manufacturer's instructions (Roche, Mannheim, Germany). Briefly, MCF-7 and MDA-MB231 cells were plated at 3 103 cells/well in 96-well plates and cultured for 24 h. After treatment with 5GTN or doxorubicin for 24 h, BrdU labeling solution was added and incubated for 2 h at 37 °C followed by fixation and DNA denaturation with FixDenat solution. BrdU-POD antibody was incubated for 90 min at room temperature. BrdU incorporation with newly synthesized DNA was detected by adding substrate solution and incubated at room temperature for developing optimal color. The optical density was measured by a Multiskan™ GO Microplate Spectrophotometer (Thermoscientific, Cramlington, UK). 2.4. Cell cycle analysis The proportion of cells at different phases of the cell cycle after treating with 5GTN was determined by flow cytometry. Briefly, MCF-7 cells (6 105 cells/well) were seeded in 6-well plates for 24 h and treated with 5GTN or doxorubicin for 24 h. Cells were then harvested, washed with PBS, and fixed in cold 70% ethanol overnight at 20 °C. Fixed cells were then washed and re-suspended in PI/RNase Staining Buffer in the dark for 30 min at 37 °C. Cells were monitored using a BD FACSCanto™ flow cytometer (BD Bioscience, San Jose, CA, USA) and DNA histograms of each sample were further analyzed with BD FACSDiva software. 2.5. Analyses of apoptosis (Annexin V-propidium iodide staining) Cell apoptosis was analyzed by annexin V-FITC and PI staining. MCF-7 cells were seeded in 6-well plates and incubated with 5GTN or doxorubicin for 24 h. Cells were washed twice with PBS and followed by gentle pipetting to detach cells. Cells were then co-
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Fig. 1. (A) Structures of goniothalamin (GTN) and 5-acetyl goniothalamin. (B) The cytotoxic effect on MCF-7 and MDA-MB-231 cells that were treated with GTN, 5GTN, doxorubicin, and ellipticine for 72 h. Cell viability was evaluated by MTT assay. IC50 value is the concentration that inhibits 50% of cell proliferation. (C) The anti-proliferative effect on MCF-7 and MDA-MB-231 cells that were evaluated by BrdU cell proliferation assay after treatment for 24 h. Data are means 7 S.E.M of three independent experiments in triplicate. **P o0.01; significantly different from the vehicle control.
incubated with annexin-V and PI for 15 min at room temperature in the dark followed by analysis in BD FACSCanto™ flow cytometer (BD Bioscience, San Jose, CA, USA) using FACSDiva software.
luciferase activity was normalized to Renilla luciferase activity as an internal control and expressed as the fold change compared with the cells transfected with the pcDNA3.1 empty vector alone.
2.6. Luciferase reporter gene assay
2.7. Reverse transcription and real-time quantitative PCR
To evaluate the effect on transcription activity, HEK293 cells were seeded at 2.5 103 cells/well in 96-well culture plates. After overnight culture, the cells were transiently transfected with 0.05 mg of TOPflash or FOPflash, 0.05 mg of Renilla luciferase reporter plasmid, which was used to evaluate the efficiency of transfection, and 0.1 mg of β-catenin-FLAG plasmid or S33Y using Lipofectamine 2000 according to the instructions of the manufacturer. Twenty four hours after the transfection, cells were treated with different concentrations of 5GTN for 24 h. Cells were then lysed and luciferase activities were measured using the dualluciferase reporter assay system according to the recommendations of the manufacturer (Promega, Madison, WI, USA). The firefly
The mRNA expression of cyclin D1, c-Myc and Axin2 were investigated by real-time quantitative PCR. Briefly, MCF-7 and MDAMB-231 cells (6 105 cells/well) were seeded in 6-well plates and treated with 5GTN for 24 h. Total RNAs were extracted using Trizol reagent (Invitrogen, USA) as described (Bhukhai et al., 2012). cDNA synthesis was conducted using the iScript TM Select cDNA synthesis kit according to the protocol of the manufacturer (BioRad, Hercules, CA, USA). Quantitative real-time PCRs of cyclin D1, c-Myc and Axin2 were performed on an equal amount of cDNA s using KAPA SYBR FAST quantitative PCR kit according to the instructions of the manufacturer (Applied Biosystems, Carlsbad, CA, USA) with the ABIPRISM-7500 sequence detection system and
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analysis software (Applied Biosystems, Carlsbad, CA, USA). The experiments were repeated in triplicate. The primers used were described as follows: Cyclin D1, F 5′-GATCAAGTGTGACCCGGACTG3′ and R 5′-CCTTGGGGTCCATGTTCTGC-3′;. c-Myc, F 5′-GCAGCGACTCTGAGGAGGAA-3′ and R 5′GGCCTCCAGCAGGCAGCACA-3′; Axin2, F 5′-CCTGGCTCCAGAAGATCACA-3′ and R 5′AGCATCCTCCGGTATGGAAT-3′; GAPDH, F 5′ATGCCCCCATGTTCGTCATG-3′ and R 5′-GCAGGAGGCATTGCTGATGA-3′. The target mRNA level was normalized to the GAPDH mRNA level.
positive control. The concentrations used in the cell proliferative assay were 5–15 mM, and 10 mM for 5GTN and doxorubicin, respectively. These concentrations were lower than their IC50 values at 24 h of MTT assay. As shown in Fig. 1C, 5GTN significantly decreased the proliferation of MCF-7 and MDA-MB-231 cells (Po0.01) in a dose related fashion whereas doxorubicin at 10 mM almost completely inhibited the cell proliferation. This result suggests that 5GTN profoundly inhibited the proliferation of MCF7 and MDA-MB-231 cells. In addition, 5GTN was also found to inhibit cell migration on MCF-7 and MDA-MB-231 cells by using wound healing assay (data not shown).
2.8. Western blot analysis The expressions of active-β-catenin, β-catenin, Phospho-GSK3β (Ser9), and GSK-3β proteins were investigated by Western blot analysis. Briefly, MCF-7 and MDA-MB-231 cells (6 105 cells/well) were seeded in 6-well plates and treated with 5GTN for 24 h. The treated cells were washed with PBS twice. Cells were harvested and lysed with modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1 mM NaF, 1 mM Na3VO4), 1 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail. After 20 min incubation on ice, cells were subjected to brief sonication and centrifuged at 13,000 g for 20 min at 4 °C. The supernatants were collected and concentrations of protein in the supernatants were determined by bicinchoninic acid biuret (BCA) assay. An equal amount of protein was used for Western blot analysis. Proteins were separated using 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dried milk, incubated with primary antibodies overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein was visualized using the SuperSignal Enhanced Chemiluminescence (ECL) Western blotting detection kit. The expression of β-actin was used as a loading control. 2.9. Statistical analyses All data were expressed as means 7the standard error of the mean (S.E.M). They were analyzed by one-way analysis of variance (ANOVA). Differences among the treatment groups were assessed by Dunnett post hoc test, using GraphPad Prism version 5.01 (GraphPad Software Inc, CA, and USA). Values of Po0.05 were considered to be significant.
3. Results 3.1. The cytotoxic and anti-proliferative effects of styryl lactones on breast cancer cells The cytotoxic effects of styryl lactones on MCF-7 and MDA-MB231 cells were evaluated by MTT assay. At 72 h of treatment, both GTN and 5GTN effectively inhibited cell growth in concentration and time related manners. As shown in Fig. 1B, the half maximal inhibitory concentrations (IC50) of GTN and 5GTN at 72 h on MCF-7 were 14.6 72.6 and 8.7 72.1 mM, respectively, whereas those of doxorubicin and ellipticine, used as positive controls, were 0.09 70.01 and 0.9 7 0.3 mM, respectively. Similarly, IC50 of GTN and 5GTN at 72 h on MDA-MB-231 were 3.8 70.3 and 1.7070.02 mM, respectively. It should be noted that the cytotoxic potency of 5GTN on both breast cancer cells was greater than that of GTN. Therefore, 5GNT was chosen for further investigations on the mechanism of action in more details. The anti-proliferative effects of 5GTN on MCF-7 and MDA-MB231 cells were assessed by BrdU cell proliferation assay at 24 h using doxorubicin, the current potent anticancer compound, as a
3.2. Effects of 5GTN on cell cycle arrest and apoptosis of breast cancer cells The proportion of DNA in MCF-7 cells at different stages of the cell cycle (sub G1, G1, S and G2) was analyzed by a flow cytometer after treatment with various concentrations of 5GTN for 24 h. As shown in Fig. 2A and B, 5GTN markedly increased the population of cells at the G1 phase in a dose related manner. Thus, 5GTN increased the percentage of cells at G1 phase from 36.0 71.6%, in control, to 74.2 70.5%, 79.57 0.9%, and 81.4 70.3%, by 5GTN at 5, 10, and 15 mM, respectively, whereas doxorubicin at 10 mM, a positive control, increased cell number at G1 phase to 85.2 71.4%. In contrast to the increased cells at G1 phase, the populations of cells at S and G2/M phases were substantially decreased, suggesting that 5GTN induced cell cycle arrest at G1 phase in MCF-7 cells. The effect of 5GTN on the apoptotic death process was further examined. The externalization of phosphatidylserine on the outer membrane of cells was double-stained with annexin V-FITC/PI and assessed by flow cytometry. After 24 h of treatment, 5GTN at 5, 10 and 15 mM induced late apoptosis of MCF-7 cells up to 8.6 7 0.3%, 18.8 70.3%, and 35.570.2%, respectively, compared to control (5.17 0.2%). Likewise, doxorubicin (10 mM) induced late apoptosis to approximately 21.570.6% (Fig. 3). The results suggest that the cytotoxic effect of 5GTN in MCF-7 cells was mediated by an induction of apoptotic cell death mechanism. 3.3. Inhibitory effects of 5GTN on TCF-dependent transcriptional activity and Wnt/β-catenin signaling pathway Wnt/β-catenin signaling cascade is a key pathway in controlling proliferation of cancer cells including breast cancer (Schlange et al., 2007). To determine the possible involvement of the antiproliferative effect of 5GTN with Wnt/β-catenin signaling pathway, a luciferase reporter gene assay in HEK 293 cells was employed by transient transfection with TOPflash or FOPflash luciferase constructs, β-catenin-FLAG, and Renilla plasmids into the cells. In the TOPflash-transfected cells, β-catenin expression showed an upregulation of the luciferase activity, indicating an up-regulation of the canonical Wnt/β-catenin activity. After treatment with 5GTN, a significant decrease (P o0.01) in TCF/β-catenin transcriptional activity in β-catenin-overexpressed HEK293 cells was noted (Fig. 4). In contrast, the inhibition of Wnt/β-catenin activity was not observed in FOPflash-transfected cells, the LEF mutant reporter plasmid. These data suggest that 5GTN essentially inhibited TCF/LEF-dependent activation of the canonical Wnt/β-catenin signaling activity. 3.4. Effects of 5GTN on target genes of Wnt/β-catenin signaling The effects of 5GTN on the expression of Wnt/β-catenin target genes were further investigated. As shown in Fig. 5, 5GTN treatment for 24 h markedly reduced the mRNA expressions of cyclin D1, c-Myc and Axin2 in both MCF-7 and MDA-MB-231 cells. These results further support the inhibitory effect of 5GTN on the β-
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Fig. 2. Analysis of cell cycle phases by flow cytometry. (A) Representative DNA content histogram showing distribution of MCF-7 cells at various phases of cell cycle after treatment with doxorubicin (10 mM) or 5GTN (5–15 mM) for 24 h. (B) Percentages of cell population in each cell-cycle phase are shown in each panel. Data are means 7S.E.M of three independent experiments. **Po 0.01; significantly different from the vehicle control.
catenin-induced transcriptional activity, which subsequently leads to the suppression of the Wnt target genes in breast cancer cells. 3.5. Modulating effect of 5GTN on the components of the canonical Wnt/β-catenin signaling in breast cancer cells Wnt/β-catenin signaling pathway is a complex protein interaction network. The transduction of Wnt signaling is administrated by several proteins. Therefore, proteins involved in the series of Wnt/β-catenin pathway were determined by western blotting. As shown in Fig. 6, treatment with 5GTN at 15 μM for
24 h markedly decreased the expression of β-catenin proteins in both total and the non-phosphorylated β-catenin in MCF-7 cells. On the other hand, the expression of total β-catenin protein in MDA-MB-231 cells was not changed. However, the expression of dephosphorylated or active form of β-catenin protein was significantly decreased after treatment with 10 μM of 5GTN. To further determine whether the inhibition of 5GTN on Wnt/ β-catenin signaling is mediated through GSK-3β, a negative regulator of β-catenin, we examined the level of total GSK-3β, as well as its phosphorylated form (Ser9), which represents the inactivated form of GSK-3β. Interestingly, treatment with 5GTN on
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Fig. 3. Representative FACS analyses showing 5GTN-induced apoptosis in MCF-7 cells. Cells were treated with 5GTN or doxorubicin (positive control) at the indicated concentrations for 24 h. After co-stained with annexin V and propidium iodine, cells were then subjected to fluorescence activating cell sorting on a BD FACS Canto machine. Lower left, lower right, upper right, and upper left quadrant indicate live (FITC /PI ) cells, early apoptotic (FITC þ/PI ) cells, late apoptotic (FITC þ /PIþ ) cells and necrotic (FITC /PI þ) cells, respectively.
β-catenin plasmid. The luciferase activity-induced by β-catenin S33Y mutant was much greater than that of β-catenin wild type (Fig. 7C). After 5GTN treatment, the luciferase activity-induced by only β-catenin wild type was significantly decreased, whereas the luciferase activity-induced by β-catenin S33Y mutant was not altered. Taken together, these results suggest that 5GTN inhibited Wnt/β-catenin signaling through the activation of GSK-3β activity, leading to the reduction of active β-catenin protein expression. 4. Discussion
Fig. 4. TCF reporter activity was decreased in 5GTN-treated cells. HEK293 cells were transiently transfected with either TOPflash or FOPflash, β-catenin-FLAG, and Renilla luciferase vectors. Cells were then treated with 5GTN for 24 h. Luciferase assay was determined and results are expressed as relative luciferase units compared with the cell transfected with β-catenin-FLAG vector with vehicle control. Values are means 7S.E.M of three independent experiments. *P o 0.05 and **Po 0.01; significantly different from the vehicle control.
MCF-7 and MDA-MB-231 cells for 24 h did not alter total GSK-3β level, but significantly decreased the phosphorylated GSK-3β (P o0.01) (Fig. 7A and B). The inhibitory effect of 5GTN on Wnt/βcatenin signaling through GSK-3β was confirmed by luciferase reporter assay using β-catenin S33Y mutant, a GSK-3β insensitive
In the present study, we have demonstrated the promising anticancer effect of a styryl lactone 5GTN, a naturally occurring compound, against breast cancer cell lines (MCF-7 and MDA-MB231), and showed that this effect was mediated through an inhibition of Wnt/β-catenin signaling pathway. Interestingly, 5GTN suppressed TCF/LEF transcriptional activity, which is responsible for transcription of the molecular targets of Wnt/β-catenin signaling namely, cyclin D1, c-Myc, and Axin2 in both MCF-7 and MDA-MB-231 cells. In addition, the expressions of both total and active β-catenin, the key protein of Wnt signaling pathway which is highly expressed in breast cancer cells, were markedly decreased by 5GTN. The decreased β-catenin protein by 5GTN was accompanied by increased the GSK-3β activity demonstrated by suppression of the GSK-3β (Ser9) phosphorylation. Therefore, 5GTN, at least, exerted the inhibitory action at the transcriptional levels, and modulated the regulation of β-catenin degradation for suppressing cell proliferation. To our knowledge, this is the first report on the anticancer activity of 5GTN through inhibition of
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Fig. 5. Effect of 5GTN on the target genes of Wnt signaling. (A) MCF-7 and (B) MDAMB-231 cells were treated with 5GTN for 24 h. Total RNAs were extracted for quantitative PCR of the Wnt target genes: cyclin D1, c-Myc, and Axin2. Data are means 7 S.E.M of three independent experiments. *P o 0.05 and **Po 0.01; significantly decrease from the vehicle control; †P o 0.05 and ††P o 0.01; significantly increase from the vehicle control.
Wnt signaling pathway. This pathway may serve as a potential therapeutic intervention for the anticancer 5GTN. Several anticancer activities of GTN have previously been reported including the production of intracellular ROS causing DNA damage and apoptosis, which is mediated through the loss of mitochondrial transmembrane potential, increases in pro-apoptotic proteins, and activation of caspases 3, 7 and 9 activities (Inayat-Hussain et al., 2003; Kuo et al., 2011; Semprebon et al., 2015). The anticancer effect of GTN has also been demonstrated in mice (Vendramini-Costa et al., 2010) and rats (Mosaddik et al., 1999) with little cytotoxicity on normal cells (Alabsi et al., 2012). However, the effect as well as its mechanism of action of 5GTN has not been reported. Since 5GTN is a naturally occurring compound from our local plant and exhibits greater potency than its parent GTN, it
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is of our interest due to its availability. The present study investigated its effects and mechanism, particularly the new target entity in the cells that has not yet been reported. We focused on the Wnt/β-catenin signaling pathway, which is known to relate with cancer cell proliferation and aggressiveness. The aberrant expressions of integral components in Wnt/β-catenin signaling such as β-catenin and its destruction complex has a clinical impact on carcinogenesis and progression of breast cancer (Prasad et al., 2009). The primary breast cancers were reported to have the elevated levels of nuclear and/or cytoplasmic β-catenin, which is a key regulator of the Wnt signaling pathway (Incassati et al., 2010). Moreover, lacking of the secreted inhibitors of Wnt signaling such as secreted Frizzle-related protein 1 (sFRP1), Wnt inhibitory factor, and DKK have frequently been found in breast tumors (Incassati et al., 2010). In the present study, 5GTN has a profound inhibitory effect on the Wnt/β-catenin signaling pathway in breast cancer cells. After 5GTN treatment on MDA-MB-231 cells, the expression of active β-catenin protein was reduced without an alteration of total β-catenin protein expression. This finding was different from those in MCF-7 cells in which 5GTN reduced both active and total β-catenin proteins. Such discrepancy of the results may be related to the differences of the regulation of intracellular signaling and susceptibility of different cancer cells to the compound (Bayerlová et al., 2015, Schlange et al., 2007). The control of cell cycle progression is an important target to prevent cancer growth and the mutation of cell cycle regulators at G1/S transition is commonly reported in most cancers (Molinari, 2000). Our results showed that 5GTN induced cell cycle arrest at G1 phase and also decreased cells at S phase. This profound effect on cell cycle arrest may be related to its inhibitory effect on Wnt/ β-catenin mediated transcription activity, suppressing regulators of the cell cycle and eventually promoting cell apoptosis. This notion is supported by the reduction of β-catenin/TCF transcriptional activity and its target genes (cyclin D1, c-Myc, and Axin2). Surprisingly, the effects of 5GTN on the expression of growth promoting genes (cyclin D1and c-Myc) at low dose and high dose were different. It is not clear on how the expressions of cyclin D1 and c-Myc were increased after exposure to low doses of 5GTN in MCF-7 and MDA-MB-231 cells. However, this similar non-monotonic or biphasic effect has often been reported in chemotherapeutic agents including natural plant-derived compounds; such as resveratrol, epigallocatechin, and flavonoids (Bao et al., 2015; Calabrese, 2013). The biphasic dose response is known as the hormetic effect of cells which is characterized by showing stimulatory effects at low doses and inhibitory effects at higher doses (Bao et al., 2015). It was suggested as an adaptive response induced by a moderate level of biological factors (Bao et al., 2015). The molecular mechanism for this paradoxical effect is largely unknown. It has been suggested that one single chemical may act via two receptor subtypes that mediate opposing stimulatory and inhibitory pathways with different agonist affinity and receptor capacity (Bao et al., 2015; Calabrese, 2013). Although, 5GTN did not increase the proliferation of cells with the up-regulation of cyclin D1 and c-Myc in the present study, these findings raise the safety concern about using long term even at low doses of chemotherapeutic agents in our study using 5GTN and also by other studies (Bao et al., 2015). The potent anticancer activity of GTN has been reported to be derived from the presence of α, β-unsaturated δ-lactone, which is a key component to act as a Michael acceptor in biological systems. Michael acceptor is able to conjugate with nucleophilic group of biomolecules such as cysteine and glutathione (Bruder et al., 2013). In the present study, 5GTN had higher cytotoxic potency on breast cancer cells than GTN. This finding was consistent with the previous report showing the potent cytotoxicity of semisynthetic 5GTN of cheliensisin A against a panel of cancer cell lines
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Fig. 6. Effect of 5GTN on the expression of β-catenin protein. (A) MCF-7 and MDA-MB-231 cells were treated with 5GTN for 24 h and then Western blotting was performed for dephosphorylated β-catenin and total β-catenin proteins. Expression of β-actin was used as protein loading control. (B) Bar graphs represent the mean normalized densitometry values of dephosphorylated β-catenin, and total β-catenin. Data are means 7 S.E.M of three independent experiments. *Po 0.05 and **Po 0.01; significantly different from the vehicle control.
(Deng et al., 2011). The reason for the greater potency by substitution of acetyl group to GTN is not clear. In an earlier study on the synthesis of goniothalamin analogs, the replacement of the oxygen atom by nitrogen of aza-goniothalamin was found to decrease the cytotoxicity of GTN, which was suggested to relate to the unsaturated system of being less attacked by nucleophiles (Barcelos et al., 2014). However, the introduction of acyl group at the nitrogen atom could restore its cytotoxicity to the level of GTN suggesting that the acyl moiety could return the electron acceptor properties to the range of α, β-unsaturated δ-lactone of GTN (Barcelos et al., 2014). By analogy, it is likely that the substitution of acetyl group in our 5GTN would promote the electrophile property of α, β-unsaturated δ-lactone unity of GTN. Recently, GTN has been reported to strongly inhibit the exportin (CRM1), the export receptor, which involves in the transports of many nuclear export proteins (Wach et al., 2010). From the X-ray crystallography study, α, β-unsaturated ester of the styryl lactone GTN covalently binds to hydrophobic binding grove of exportin and blocks the exportin-dependent nuclear export that may account for the GTN-mediated cytotoxicity (Gademann, 2011). GSK-3β is considered as a multifunctional serine/threonine kinase in the cytoplasm, which is a negative regulator of β-catenin degradation in Wnt pathway (Caspi et al., 2008). However, it was also reported to play an important role in the nucleus by forming nuclear complexes of GSK-3β with β-catenin and results in the reduction of TCF-mediated transcription (Caspi et al., 2008). In the present study, there is a possibility that 5GTN, which contains
similar α, β-unsaturated δ-lactone to GTN, might inhibit the exportin-mediated GSK-3β export from the nucleus. However, 5GTN did not alter total GSK-3β, but only phosphorylated GSK-3β was decreased. It is likely that 5GTN targeted on the destruction of βcatenin protein by increasing the functional activity of GSK-3β, may not relate to its expression. In addition, this effect of 5GTN on GSK-3β activity was also confirmed by the lacking of the effect in S33Y, which is a mutated β-catenin insensitive to GSK-3β-mediated phosphorylation. Hence, the activation of GSK-3β might lead to the decrease of β-catenin, which subsequently down regulated the β-catenin/TCF transcriptional activity and the expression of target genes involving with cell proliferation, cell cycle, and apoptosis. However, further experiments are required to clarify the nuclear action of 5GTN in Wnt/β-catenin signaling pathway. Although 5GTN essentially displayed Wnt inhibitory activity in the present study, the styryl lactone structure and Michael acceptor capacity of goniothalamin have been reported to have a wide range of biological activities including anti-inflammation and genotoxicity (Seyed et al., 2014). Those alternative mechanism of actions of styryl lactone should not be excluded from its anticancer activity. In summary, Wnt/β-catenin signaling pathway is the molecular target for anticancer activity of 5GTN in breast cancer cells. 5GTN inhibited the β-catenin-mediated the expression of cancer cell proliferation related target genes and therefore induced apoptosis. This pathway may serve as a promising chemotherapeutic intervention pathway for this anticancer compound.
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Fig. 7. Effect of 5GTN on the expression of GSK-3β protein. (A) MCF-7 and MDA-MB-231 cells were treated with 5GTN for 24 h and then Western blotting was performed for phosphorylated GSK-3β and total GSK-3β proteins. Expression of β-actin was used as protein loading control. (B) Bar graphs represent the mean normalized densitometry values of phosphorylated GSK-3β and total GSK-3β ratio. (C) GSK-3β-dependent inhibitory effect of 5GTN on Wnt/β-catenin signaling. HEK293 cells were transiently transfected with either TOPflash, β-catenin-FLAG or constitutively active β-catenin mutant S33Y, which is insensitive to GSK-3β-mediated phosphorylation, and Renilla luciferase vectors. Cells were then treated with 5GTN for 24 h. Luciferase assay was determined and results are expressed as relative luciferase units compared with the cell transfected with pcDNA3.1-transfected cells. Values are means 7 S.E.M of three independent experiments. *Po 0.05 and **Po 0.01; significantly different from the vehicle control.
Acknowledgment This research project is supported by the Thailand Research Fund (TRF)-Mahidol University through the Royal Golden Jubilee Ph.D. Program (PHD/0170/2552 to PP and NB), the research grants from Mahidol University, and the TRF through the International Research Network (IRN-58W0004), and the plant compounds from the Center of Excellence for Innovation in Chemistry (PERCHCIC). We are grateful to Prof. Chumpol Pholpramool for his critical
reading of the manuscript.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
5-Acetyl goniothalamin suppresses proliferation of breast cancer cells via Wnt/β-catenin signaling Nittaya Boonmuen a, Natthakan Thongon a, Arthit Chairoungdua a, Kanoknetr Suksen a, Wilart Pompimon b, Patoomratana Tuchinda c, Vichai Reutrakul c,d, Pawinee Piyachaturawat a,e,n a
Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Lampang Rajabhat University, Lampang 52100, Thailand c Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand d Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand e Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 May 2016 Received in revised form 7 September 2016 Accepted 15 September 2016 Available online 16 September 2016
Styryl lactones are plant-derived compounds from genus Goniothalamus with promising anti-proliferation and anticancer properties. However, the exact mechanism and the target for their activities remained unclear. In the present study, we investigated the effect of 5-acetyl goniothalamin (5GTN) from Goniothalamus marcanii on Wnt/β-catenin signaling pathway which is a key regulator in controlling cell proliferation in breast cancer cells (MCF-7 and MDA-MB-231). 5GTN, a naturally occurring derivative of goniothalamin (GTN) mediated the toxicity to MCF-7 and MDA-MB-231 cells in a dose- and time- related manner, and was more potent than that of GTN. 5GTN strongly inhibited cell proliferation and markedly suppressed transcriptional activity induced by β-catenin in luciferase reporter gene assay. In consistent with this view, the expression of Wnt/β-catenin signaling target genes including c-Myc, cyclin D1 and Axin2 in MCF-7 and MDA-MB-231 cells were suppressed after treatment with 5GTN. It was concomitant with cell cycle arrest at G1 phase and cell apoptosis in MCF-7 cells. In addition, 5GTN enhanced glycogen synthase kinase (GSK-3β) activity and therefore reduced the expression of active form of β-catenin protein in MCF-7 and MDA-MB-231 cells. Taken together, 5GTN exhibited a promising anticancer effect against breast cancer cells through an inhibition of Wnt/β-catenin signaling. This pathway may be served as a potential chemotherapeutic target for breast cancer by 5GTN. & 2016 Elsevier B.V. All rights reserved.
Keywords: 5-Acetyl goniothalamin Anticancer Antiproliferation Breast cancer Styryl lactone Wnt signaling Chemical compounds studied in this article: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (PubChem CID:64965) Ellipticine (PubChem CID:3213) 5-bromo-2′-deoxyuridine (BrdU) (PubChem CID: 6035) Propidium iodide (PI) (PubChem CID: 104981) Doxorubicin (PubChem CID: 31703)
1. Introduction Wnt/β-catenin signaling pathway plays a central role in regulating cell proliferation in normal and various cancer cells (Amado et al., 2011). Mutation of Wnt/β-catenin pathway causing deregulation of the expression of Wnt/β-catenin target genes was demonstrated in mammary carcinoma and colorectal cancer (Holland et al., 2013). Approximately 40% of breast cancer cases showed increases in β-catenin (Incassati et al., 2010). The n Corresponding author at: Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Rd., Ratchatewee, Bangkok 10400, Thailand E-mail address: [email protected] (P. Piyachaturawat).
http://dx.doi.org/10.1016/j.ejphar.2016.09.024 0014-2999/& 2016 Elsevier B.V. All rights reserved.
dysregulation of Wnt/β-catenin signaling in cancer cells has been reported on various steps along the complex regulating pathway including increased β-catenin expression, inactivated negative regulators, and altered levels of Wnt ligands or other components of Wnt signaling (Incassati et al., 2010). Thus, the defect of Wnt signaling is one of the promising targets for chemoprevention and chemotherapy for cancers including breast cancer (Dakeng et al., 2012). In the absence of Wnt ligand, β-catenin, which is a key component of Wnt/β-catenin signaling pathway, is degraded via the proteasome as a result of the phosphorylation by the destruction complex proteins including the APC (adenomatous polyposis coli), axin, glycogen synthase kinase (GSK) 3-β, and casein kinase 1 (Amado et al., 2011). In the presence of Wnt ligand, on the other hand, binding of Wnt to a Frizzled receptor in the
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presence of the co-receptor, LRP5/6 (low density lipoprotein-related protein 5/6), subsequently reduces the activity of destruction complex to degrade β-catenin (Incassati et al., 2010). The stabilized cytoplasmic β-catenin is then translocated to the nucleus to bind to lymphoid enhancer-binding factor 1/T-cell factor (TCF/LEF) transcription factor, and stimulates the expression of TCF responsive target genes (Bao et al., 2012). The downstream transcriptional targets of β-catenin/TCF including cyclin D1 and c-Myc are important regulators of cell cycle process and proliferation particularly at the transition of G1/S and G2/M phases (Barcelos et al., 2014, Pooja et al., 2014). Naturally occurring compounds from plant have long been an attractive source of anticancer agents (Harvey, 2008). Of these, a styryl lactone goniothalamin (GTN), which is a low molecular weight phenolic compound from the genus Goniothalamus (Annonaceae), has been reported to exhibit a promising antiproliferative activity against a broad array of cancer cells including leukemia, kidney, pancreas, colon and breast cancer cells (Alabsi et al., 2013; Chen et al., 2005; Tantithanaporn et al., 2011). Its anticancer activities have been reported to be associated with induction of apoptosis, ROS production, DNA damage, and interruption of mitochondrial transmembrane potential in a variety of cancer cells (Alabsi et al., 2012; Inayat-Hussain et al., 2003; Yen et al., 2012). Although many anticancer activities of GTN have been proposed, the more specific signaling target to improve the efficiency and specificity of treatment has not been investigated. In an effort to search for more specific therapeutic target, the present study aimed to investigate the involvement of antiproliferative activity of a naturally occurring derivative of goniothalamin, 5-acetyl goniothalamin (5GTN) on Wnt/β-catenin signaling pathway, a critical pathway for regulating proliferation in cancer cells.
2. Materials and methods 2.1. Materials Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), antibiotic-antimycotic agents, trypsin, Lipofectamine 2000 and Trizol reagents were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), RIPA, and SuperSignal West Pico Chemiluminescent Substrate were from Thermoscientific (Cramlington, UK). SDS, 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and ellipticine were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dual-luciferase reporter assay was from Promega (Madison, WI, USA). SYBR kit was from Applied Biosystems (Carlsbad, CA, USA). Protease inhibitor cocktail and cell proliferation ELIZA, BrdU (colorimetric) were from Roche (Mannheim, Germany). Propidium iodide (PI)/RNase Staining Buffer and Annexin-V FITC apoptosis detection kit were obtained from BD Biosciences (San Jose, CA, USA). Doxorubicin was from Guanyu Bio-Tech Co. (Xijan, Republic of China). Phosphatase inhibitor cocktail and active-β-catenin (anti-ABC) clone 8E7 monoclonal antibody were from Millipore Corporation (Darmstadt, Germany). Βeta-catenin (H-102) and βactin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-GSK-3β (Ser9) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). GSK-3β antibody was from Abcam (Cambridge, MA, USA). The T-cell factor (TCF)reporter plasmids (TOPFLASH and the negative control FOPFLASH), β-catenin-FLAG and S33Y plasmids were generated as previously described (Bhukhai et al., 2012). Two styryl lactones; Goniothalamin (GTN) and 5-acetyl goniothalamin (5GTN) were isolated from Goniotalamus tapis and Goniothalamus marcanii, respectively as previously described (Ahmad et al., 1991). They were obtained as white crystals, m.p.
80–82 °C and 90–93 °C, respectively. The GTN and 5GTN were assigned to the formula C13H12O2 and C15H14O4 by EIMS. The molecular structures were elucidated by spectroscopy technique such as NMR, IR, and UV. Their spectral data were similar to those previously reported (Ahmad et al., 1991). The chemical structures are shown in Fig. 1A. 2.2. Cell culture Human breast cancer (MCF-7), triple-negative breast cancer (MDA-MB-231) and human embryonic kidney (HEK-293) cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM and MEM, respectively. They were supplemented with 10% fetal bovine serum and 1% antibiotic (100 U/ml of penicillin and 100 mg/ml of streptomycin), and incubated at 37 °C in a 5% CO2 incubator with humidified atmosphere (Invitrogen, Carlsbad, CA, USA). 2.3. Cell viability and cell proliferation assay Cell viability was determined by using the colorimetric MTT assay. MCF-7 and MDA-MB-231 cells (3 103 cells/well) were seeded in 96-well plates and incubated for 24 h prior to treatment with GTN, 5GTN, ellipticine or doxorubicin at concentrations ranging from 0 to 50 mM for 24, 48, and 72 h. The compounds were dissolved in DMSO and the final concentration of DMSO in the medium was less than 0.05%. After treatment, the medium was removed and fresh medium containing 0.5 mg/ml MTT was added and incubated at 37 °C for 4 h. The dark blue formazan crystal product was dissolved with DMSO and measured at 540 nm using a Multiskan™ GO Microplate Spectrophotometer (Thermoscientific, Cramlington, UK). The result was calculated as % of cell viability. Cell proliferation was determined by a Cell Proliferation ELISA, BrdU colorimetric assay according to the manufacturer's instructions (Roche, Mannheim, Germany). Briefly, MCF-7 and MDA-MB231 cells were plated at 3 103 cells/well in 96-well plates and cultured for 24 h. After treatment with 5GTN or doxorubicin for 24 h, BrdU labeling solution was added and incubated for 2 h at 37 °C followed by fixation and DNA denaturation with FixDenat solution. BrdU-POD antibody was incubated for 90 min at room temperature. BrdU incorporation with newly synthesized DNA was detected by adding substrate solution and incubated at room temperature for developing optimal color. The optical density was measured by a Multiskan™ GO Microplate Spectrophotometer (Thermoscientific, Cramlington, UK). 2.4. Cell cycle analysis The proportion of cells at different phases of the cell cycle after treating with 5GTN was determined by flow cytometry. Briefly, MCF-7 cells (6 105 cells/well) were seeded in 6-well plates for 24 h and treated with 5GTN or doxorubicin for 24 h. Cells were then harvested, washed with PBS, and fixed in cold 70% ethanol overnight at 20 °C. Fixed cells were then washed and re-suspended in PI/RNase Staining Buffer in the dark for 30 min at 37 °C. Cells were monitored using a BD FACSCanto™ flow cytometer (BD Bioscience, San Jose, CA, USA) and DNA histograms of each sample were further analyzed with BD FACSDiva software. 2.5. Analyses of apoptosis (Annexin V-propidium iodide staining) Cell apoptosis was analyzed by annexin V-FITC and PI staining. MCF-7 cells were seeded in 6-well plates and incubated with 5GTN or doxorubicin for 24 h. Cells were washed twice with PBS and followed by gentle pipetting to detach cells. Cells were then co-
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Fig. 1. (A) Structures of goniothalamin (GTN) and 5-acetyl goniothalamin. (B) The cytotoxic effect on MCF-7 and MDA-MB-231 cells that were treated with GTN, 5GTN, doxorubicin, and ellipticine for 72 h. Cell viability was evaluated by MTT assay. IC50 value is the concentration that inhibits 50% of cell proliferation. (C) The anti-proliferative effect on MCF-7 and MDA-MB-231 cells that were evaluated by BrdU cell proliferation assay after treatment for 24 h. Data are means 7 S.E.M of three independent experiments in triplicate. **P o0.01; significantly different from the vehicle control.
incubated with annexin-V and PI for 15 min at room temperature in the dark followed by analysis in BD FACSCanto™ flow cytometer (BD Bioscience, San Jose, CA, USA) using FACSDiva software.
luciferase activity was normalized to Renilla luciferase activity as an internal control and expressed as the fold change compared with the cells transfected with the pcDNA3.1 empty vector alone.
2.6. Luciferase reporter gene assay
2.7. Reverse transcription and real-time quantitative PCR
To evaluate the effect on transcription activity, HEK293 cells were seeded at 2.5 103 cells/well in 96-well culture plates. After overnight culture, the cells were transiently transfected with 0.05 mg of TOPflash or FOPflash, 0.05 mg of Renilla luciferase reporter plasmid, which was used to evaluate the efficiency of transfection, and 0.1 mg of β-catenin-FLAG plasmid or S33Y using Lipofectamine 2000 according to the instructions of the manufacturer. Twenty four hours after the transfection, cells were treated with different concentrations of 5GTN for 24 h. Cells were then lysed and luciferase activities were measured using the dualluciferase reporter assay system according to the recommendations of the manufacturer (Promega, Madison, WI, USA). The firefly
The mRNA expression of cyclin D1, c-Myc and Axin2 were investigated by real-time quantitative PCR. Briefly, MCF-7 and MDAMB-231 cells (6 105 cells/well) were seeded in 6-well plates and treated with 5GTN for 24 h. Total RNAs were extracted using Trizol reagent (Invitrogen, USA) as described (Bhukhai et al., 2012). cDNA synthesis was conducted using the iScript TM Select cDNA synthesis kit according to the protocol of the manufacturer (BioRad, Hercules, CA, USA). Quantitative real-time PCRs of cyclin D1, c-Myc and Axin2 were performed on an equal amount of cDNA s using KAPA SYBR FAST quantitative PCR kit according to the instructions of the manufacturer (Applied Biosystems, Carlsbad, CA, USA) with the ABIPRISM-7500 sequence detection system and
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analysis software (Applied Biosystems, Carlsbad, CA, USA). The experiments were repeated in triplicate. The primers used were described as follows: Cyclin D1, F 5′-GATCAAGTGTGACCCGGACTG3′ and R 5′-CCTTGGGGTCCATGTTCTGC-3′;. c-Myc, F 5′-GCAGCGACTCTGAGGAGGAA-3′ and R 5′GGCCTCCAGCAGGCAGCACA-3′; Axin2, F 5′-CCTGGCTCCAGAAGATCACA-3′ and R 5′AGCATCCTCCGGTATGGAAT-3′; GAPDH, F 5′ATGCCCCCATGTTCGTCATG-3′ and R 5′-GCAGGAGGCATTGCTGATGA-3′. The target mRNA level was normalized to the GAPDH mRNA level.
positive control. The concentrations used in the cell proliferative assay were 5–15 mM, and 10 mM for 5GTN and doxorubicin, respectively. These concentrations were lower than their IC50 values at 24 h of MTT assay. As shown in Fig. 1C, 5GTN significantly decreased the proliferation of MCF-7 and MDA-MB-231 cells (Po0.01) in a dose related fashion whereas doxorubicin at 10 mM almost completely inhibited the cell proliferation. This result suggests that 5GTN profoundly inhibited the proliferation of MCF7 and MDA-MB-231 cells. In addition, 5GTN was also found to inhibit cell migration on MCF-7 and MDA-MB-231 cells by using wound healing assay (data not shown).
2.8. Western blot analysis The expressions of active-β-catenin, β-catenin, Phospho-GSK3β (Ser9), and GSK-3β proteins were investigated by Western blot analysis. Briefly, MCF-7 and MDA-MB-231 cells (6 105 cells/well) were seeded in 6-well plates and treated with 5GTN for 24 h. The treated cells were washed with PBS twice. Cells were harvested and lysed with modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1 mM NaF, 1 mM Na3VO4), 1 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail. After 20 min incubation on ice, cells were subjected to brief sonication and centrifuged at 13,000 g for 20 min at 4 °C. The supernatants were collected and concentrations of protein in the supernatants were determined by bicinchoninic acid biuret (BCA) assay. An equal amount of protein was used for Western blot analysis. Proteins were separated using 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dried milk, incubated with primary antibodies overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein was visualized using the SuperSignal Enhanced Chemiluminescence (ECL) Western blotting detection kit. The expression of β-actin was used as a loading control. 2.9. Statistical analyses All data were expressed as means 7the standard error of the mean (S.E.M). They were analyzed by one-way analysis of variance (ANOVA). Differences among the treatment groups were assessed by Dunnett post hoc test, using GraphPad Prism version 5.01 (GraphPad Software Inc, CA, and USA). Values of Po0.05 were considered to be significant.
3. Results 3.1. The cytotoxic and anti-proliferative effects of styryl lactones on breast cancer cells The cytotoxic effects of styryl lactones on MCF-7 and MDA-MB231 cells were evaluated by MTT assay. At 72 h of treatment, both GTN and 5GTN effectively inhibited cell growth in concentration and time related manners. As shown in Fig. 1B, the half maximal inhibitory concentrations (IC50) of GTN and 5GTN at 72 h on MCF-7 were 14.6 72.6 and 8.7 72.1 mM, respectively, whereas those of doxorubicin and ellipticine, used as positive controls, were 0.09 70.01 and 0.9 7 0.3 mM, respectively. Similarly, IC50 of GTN and 5GTN at 72 h on MDA-MB-231 were 3.8 70.3 and 1.7070.02 mM, respectively. It should be noted that the cytotoxic potency of 5GTN on both breast cancer cells was greater than that of GTN. Therefore, 5GNT was chosen for further investigations on the mechanism of action in more details. The anti-proliferative effects of 5GTN on MCF-7 and MDA-MB231 cells were assessed by BrdU cell proliferation assay at 24 h using doxorubicin, the current potent anticancer compound, as a
3.2. Effects of 5GTN on cell cycle arrest and apoptosis of breast cancer cells The proportion of DNA in MCF-7 cells at different stages of the cell cycle (sub G1, G1, S and G2) was analyzed by a flow cytometer after treatment with various concentrations of 5GTN for 24 h. As shown in Fig. 2A and B, 5GTN markedly increased the population of cells at the G1 phase in a dose related manner. Thus, 5GTN increased the percentage of cells at G1 phase from 36.0 71.6%, in control, to 74.2 70.5%, 79.57 0.9%, and 81.4 70.3%, by 5GTN at 5, 10, and 15 mM, respectively, whereas doxorubicin at 10 mM, a positive control, increased cell number at G1 phase to 85.2 71.4%. In contrast to the increased cells at G1 phase, the populations of cells at S and G2/M phases were substantially decreased, suggesting that 5GTN induced cell cycle arrest at G1 phase in MCF-7 cells. The effect of 5GTN on the apoptotic death process was further examined. The externalization of phosphatidylserine on the outer membrane of cells was double-stained with annexin V-FITC/PI and assessed by flow cytometry. After 24 h of treatment, 5GTN at 5, 10 and 15 mM induced late apoptosis of MCF-7 cells up to 8.6 7 0.3%, 18.8 70.3%, and 35.570.2%, respectively, compared to control (5.17 0.2%). Likewise, doxorubicin (10 mM) induced late apoptosis to approximately 21.570.6% (Fig. 3). The results suggest that the cytotoxic effect of 5GTN in MCF-7 cells was mediated by an induction of apoptotic cell death mechanism. 3.3. Inhibitory effects of 5GTN on TCF-dependent transcriptional activity and Wnt/β-catenin signaling pathway Wnt/β-catenin signaling cascade is a key pathway in controlling proliferation of cancer cells including breast cancer (Schlange et al., 2007). To determine the possible involvement of the antiproliferative effect of 5GTN with Wnt/β-catenin signaling pathway, a luciferase reporter gene assay in HEK 293 cells was employed by transient transfection with TOPflash or FOPflash luciferase constructs, β-catenin-FLAG, and Renilla plasmids into the cells. In the TOPflash-transfected cells, β-catenin expression showed an upregulation of the luciferase activity, indicating an up-regulation of the canonical Wnt/β-catenin activity. After treatment with 5GTN, a significant decrease (P o0.01) in TCF/β-catenin transcriptional activity in β-catenin-overexpressed HEK293 cells was noted (Fig. 4). In contrast, the inhibition of Wnt/β-catenin activity was not observed in FOPflash-transfected cells, the LEF mutant reporter plasmid. These data suggest that 5GTN essentially inhibited TCF/LEF-dependent activation of the canonical Wnt/β-catenin signaling activity. 3.4. Effects of 5GTN on target genes of Wnt/β-catenin signaling The effects of 5GTN on the expression of Wnt/β-catenin target genes were further investigated. As shown in Fig. 5, 5GTN treatment for 24 h markedly reduced the mRNA expressions of cyclin D1, c-Myc and Axin2 in both MCF-7 and MDA-MB-231 cells. These results further support the inhibitory effect of 5GTN on the β-
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Fig. 2. Analysis of cell cycle phases by flow cytometry. (A) Representative DNA content histogram showing distribution of MCF-7 cells at various phases of cell cycle after treatment with doxorubicin (10 mM) or 5GTN (5–15 mM) for 24 h. (B) Percentages of cell population in each cell-cycle phase are shown in each panel. Data are means 7S.E.M of three independent experiments. **Po 0.01; significantly different from the vehicle control.
catenin-induced transcriptional activity, which subsequently leads to the suppression of the Wnt target genes in breast cancer cells. 3.5. Modulating effect of 5GTN on the components of the canonical Wnt/β-catenin signaling in breast cancer cells Wnt/β-catenin signaling pathway is a complex protein interaction network. The transduction of Wnt signaling is administrated by several proteins. Therefore, proteins involved in the series of Wnt/β-catenin pathway were determined by western blotting. As shown in Fig. 6, treatment with 5GTN at 15 μM for
24 h markedly decreased the expression of β-catenin proteins in both total and the non-phosphorylated β-catenin in MCF-7 cells. On the other hand, the expression of total β-catenin protein in MDA-MB-231 cells was not changed. However, the expression of dephosphorylated or active form of β-catenin protein was significantly decreased after treatment with 10 μM of 5GTN. To further determine whether the inhibition of 5GTN on Wnt/ β-catenin signaling is mediated through GSK-3β, a negative regulator of β-catenin, we examined the level of total GSK-3β, as well as its phosphorylated form (Ser9), which represents the inactivated form of GSK-3β. Interestingly, treatment with 5GTN on
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Fig. 3. Representative FACS analyses showing 5GTN-induced apoptosis in MCF-7 cells. Cells were treated with 5GTN or doxorubicin (positive control) at the indicated concentrations for 24 h. After co-stained with annexin V and propidium iodine, cells were then subjected to fluorescence activating cell sorting on a BD FACS Canto machine. Lower left, lower right, upper right, and upper left quadrant indicate live (FITC /PI ) cells, early apoptotic (FITC þ/PI ) cells, late apoptotic (FITC þ /PIþ ) cells and necrotic (FITC /PI þ) cells, respectively.
β-catenin plasmid. The luciferase activity-induced by β-catenin S33Y mutant was much greater than that of β-catenin wild type (Fig. 7C). After 5GTN treatment, the luciferase activity-induced by only β-catenin wild type was significantly decreased, whereas the luciferase activity-induced by β-catenin S33Y mutant was not altered. Taken together, these results suggest that 5GTN inhibited Wnt/β-catenin signaling through the activation of GSK-3β activity, leading to the reduction of active β-catenin protein expression. 4. Discussion
Fig. 4. TCF reporter activity was decreased in 5GTN-treated cells. HEK293 cells were transiently transfected with either TOPflash or FOPflash, β-catenin-FLAG, and Renilla luciferase vectors. Cells were then treated with 5GTN for 24 h. Luciferase assay was determined and results are expressed as relative luciferase units compared with the cell transfected with β-catenin-FLAG vector with vehicle control. Values are means 7S.E.M of three independent experiments. *P o 0.05 and **Po 0.01; significantly different from the vehicle control.
MCF-7 and MDA-MB-231 cells for 24 h did not alter total GSK-3β level, but significantly decreased the phosphorylated GSK-3β (P o0.01) (Fig. 7A and B). The inhibitory effect of 5GTN on Wnt/βcatenin signaling through GSK-3β was confirmed by luciferase reporter assay using β-catenin S33Y mutant, a GSK-3β insensitive
In the present study, we have demonstrated the promising anticancer effect of a styryl lactone 5GTN, a naturally occurring compound, against breast cancer cell lines (MCF-7 and MDA-MB231), and showed that this effect was mediated through an inhibition of Wnt/β-catenin signaling pathway. Interestingly, 5GTN suppressed TCF/LEF transcriptional activity, which is responsible for transcription of the molecular targets of Wnt/β-catenin signaling namely, cyclin D1, c-Myc, and Axin2 in both MCF-7 and MDA-MB-231 cells. In addition, the expressions of both total and active β-catenin, the key protein of Wnt signaling pathway which is highly expressed in breast cancer cells, were markedly decreased by 5GTN. The decreased β-catenin protein by 5GTN was accompanied by increased the GSK-3β activity demonstrated by suppression of the GSK-3β (Ser9) phosphorylation. Therefore, 5GTN, at least, exerted the inhibitory action at the transcriptional levels, and modulated the regulation of β-catenin degradation for suppressing cell proliferation. To our knowledge, this is the first report on the anticancer activity of 5GTN through inhibition of
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Fig. 5. Effect of 5GTN on the target genes of Wnt signaling. (A) MCF-7 and (B) MDAMB-231 cells were treated with 5GTN for 24 h. Total RNAs were extracted for quantitative PCR of the Wnt target genes: cyclin D1, c-Myc, and Axin2. Data are means 7 S.E.M of three independent experiments. *P o 0.05 and **Po 0.01; significantly decrease from the vehicle control; †P o 0.05 and ††P o 0.01; significantly increase from the vehicle control.
Wnt signaling pathway. This pathway may serve as a potential therapeutic intervention for the anticancer 5GTN. Several anticancer activities of GTN have previously been reported including the production of intracellular ROS causing DNA damage and apoptosis, which is mediated through the loss of mitochondrial transmembrane potential, increases in pro-apoptotic proteins, and activation of caspases 3, 7 and 9 activities (Inayat-Hussain et al., 2003; Kuo et al., 2011; Semprebon et al., 2015). The anticancer effect of GTN has also been demonstrated in mice (Vendramini-Costa et al., 2010) and rats (Mosaddik et al., 1999) with little cytotoxicity on normal cells (Alabsi et al., 2012). However, the effect as well as its mechanism of action of 5GTN has not been reported. Since 5GTN is a naturally occurring compound from our local plant and exhibits greater potency than its parent GTN, it
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is of our interest due to its availability. The present study investigated its effects and mechanism, particularly the new target entity in the cells that has not yet been reported. We focused on the Wnt/β-catenin signaling pathway, which is known to relate with cancer cell proliferation and aggressiveness. The aberrant expressions of integral components in Wnt/β-catenin signaling such as β-catenin and its destruction complex has a clinical impact on carcinogenesis and progression of breast cancer (Prasad et al., 2009). The primary breast cancers were reported to have the elevated levels of nuclear and/or cytoplasmic β-catenin, which is a key regulator of the Wnt signaling pathway (Incassati et al., 2010). Moreover, lacking of the secreted inhibitors of Wnt signaling such as secreted Frizzle-related protein 1 (sFRP1), Wnt inhibitory factor, and DKK have frequently been found in breast tumors (Incassati et al., 2010). In the present study, 5GTN has a profound inhibitory effect on the Wnt/β-catenin signaling pathway in breast cancer cells. After 5GTN treatment on MDA-MB-231 cells, the expression of active β-catenin protein was reduced without an alteration of total β-catenin protein expression. This finding was different from those in MCF-7 cells in which 5GTN reduced both active and total β-catenin proteins. Such discrepancy of the results may be related to the differences of the regulation of intracellular signaling and susceptibility of different cancer cells to the compound (Bayerlová et al., 2015, Schlange et al., 2007). The control of cell cycle progression is an important target to prevent cancer growth and the mutation of cell cycle regulators at G1/S transition is commonly reported in most cancers (Molinari, 2000). Our results showed that 5GTN induced cell cycle arrest at G1 phase and also decreased cells at S phase. This profound effect on cell cycle arrest may be related to its inhibitory effect on Wnt/ β-catenin mediated transcription activity, suppressing regulators of the cell cycle and eventually promoting cell apoptosis. This notion is supported by the reduction of β-catenin/TCF transcriptional activity and its target genes (cyclin D1, c-Myc, and Axin2). Surprisingly, the effects of 5GTN on the expression of growth promoting genes (cyclin D1and c-Myc) at low dose and high dose were different. It is not clear on how the expressions of cyclin D1 and c-Myc were increased after exposure to low doses of 5GTN in MCF-7 and MDA-MB-231 cells. However, this similar non-monotonic or biphasic effect has often been reported in chemotherapeutic agents including natural plant-derived compounds; such as resveratrol, epigallocatechin, and flavonoids (Bao et al., 2015; Calabrese, 2013). The biphasic dose response is known as the hormetic effect of cells which is characterized by showing stimulatory effects at low doses and inhibitory effects at higher doses (Bao et al., 2015). It was suggested as an adaptive response induced by a moderate level of biological factors (Bao et al., 2015). The molecular mechanism for this paradoxical effect is largely unknown. It has been suggested that one single chemical may act via two receptor subtypes that mediate opposing stimulatory and inhibitory pathways with different agonist affinity and receptor capacity (Bao et al., 2015; Calabrese, 2013). Although, 5GTN did not increase the proliferation of cells with the up-regulation of cyclin D1 and c-Myc in the present study, these findings raise the safety concern about using long term even at low doses of chemotherapeutic agents in our study using 5GTN and also by other studies (Bao et al., 2015). The potent anticancer activity of GTN has been reported to be derived from the presence of α, β-unsaturated δ-lactone, which is a key component to act as a Michael acceptor in biological systems. Michael acceptor is able to conjugate with nucleophilic group of biomolecules such as cysteine and glutathione (Bruder et al., 2013). In the present study, 5GTN had higher cytotoxic potency on breast cancer cells than GTN. This finding was consistent with the previous report showing the potent cytotoxicity of semisynthetic 5GTN of cheliensisin A against a panel of cancer cell lines
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Fig. 6. Effect of 5GTN on the expression of β-catenin protein. (A) MCF-7 and MDA-MB-231 cells were treated with 5GTN for 24 h and then Western blotting was performed for dephosphorylated β-catenin and total β-catenin proteins. Expression of β-actin was used as protein loading control. (B) Bar graphs represent the mean normalized densitometry values of dephosphorylated β-catenin, and total β-catenin. Data are means 7 S.E.M of three independent experiments. *Po 0.05 and **Po 0.01; significantly different from the vehicle control.
(Deng et al., 2011). The reason for the greater potency by substitution of acetyl group to GTN is not clear. In an earlier study on the synthesis of goniothalamin analogs, the replacement of the oxygen atom by nitrogen of aza-goniothalamin was found to decrease the cytotoxicity of GTN, which was suggested to relate to the unsaturated system of being less attacked by nucleophiles (Barcelos et al., 2014). However, the introduction of acyl group at the nitrogen atom could restore its cytotoxicity to the level of GTN suggesting that the acyl moiety could return the electron acceptor properties to the range of α, β-unsaturated δ-lactone of GTN (Barcelos et al., 2014). By analogy, it is likely that the substitution of acetyl group in our 5GTN would promote the electrophile property of α, β-unsaturated δ-lactone unity of GTN. Recently, GTN has been reported to strongly inhibit the exportin (CRM1), the export receptor, which involves in the transports of many nuclear export proteins (Wach et al., 2010). From the X-ray crystallography study, α, β-unsaturated ester of the styryl lactone GTN covalently binds to hydrophobic binding grove of exportin and blocks the exportin-dependent nuclear export that may account for the GTN-mediated cytotoxicity (Gademann, 2011). GSK-3β is considered as a multifunctional serine/threonine kinase in the cytoplasm, which is a negative regulator of β-catenin degradation in Wnt pathway (Caspi et al., 2008). However, it was also reported to play an important role in the nucleus by forming nuclear complexes of GSK-3β with β-catenin and results in the reduction of TCF-mediated transcription (Caspi et al., 2008). In the present study, there is a possibility that 5GTN, which contains
similar α, β-unsaturated δ-lactone to GTN, might inhibit the exportin-mediated GSK-3β export from the nucleus. However, 5GTN did not alter total GSK-3β, but only phosphorylated GSK-3β was decreased. It is likely that 5GTN targeted on the destruction of βcatenin protein by increasing the functional activity of GSK-3β, may not relate to its expression. In addition, this effect of 5GTN on GSK-3β activity was also confirmed by the lacking of the effect in S33Y, which is a mutated β-catenin insensitive to GSK-3β-mediated phosphorylation. Hence, the activation of GSK-3β might lead to the decrease of β-catenin, which subsequently down regulated the β-catenin/TCF transcriptional activity and the expression of target genes involving with cell proliferation, cell cycle, and apoptosis. However, further experiments are required to clarify the nuclear action of 5GTN in Wnt/β-catenin signaling pathway. Although 5GTN essentially displayed Wnt inhibitory activity in the present study, the styryl lactone structure and Michael acceptor capacity of goniothalamin have been reported to have a wide range of biological activities including anti-inflammation and genotoxicity (Seyed et al., 2014). Those alternative mechanism of actions of styryl lactone should not be excluded from its anticancer activity. In summary, Wnt/β-catenin signaling pathway is the molecular target for anticancer activity of 5GTN in breast cancer cells. 5GTN inhibited the β-catenin-mediated the expression of cancer cell proliferation related target genes and therefore induced apoptosis. This pathway may serve as a promising chemotherapeutic intervention pathway for this anticancer compound.
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Fig. 7. Effect of 5GTN on the expression of GSK-3β protein. (A) MCF-7 and MDA-MB-231 cells were treated with 5GTN for 24 h and then Western blotting was performed for phosphorylated GSK-3β and total GSK-3β proteins. Expression of β-actin was used as protein loading control. (B) Bar graphs represent the mean normalized densitometry values of phosphorylated GSK-3β and total GSK-3β ratio. (C) GSK-3β-dependent inhibitory effect of 5GTN on Wnt/β-catenin signaling. HEK293 cells were transiently transfected with either TOPflash, β-catenin-FLAG or constitutively active β-catenin mutant S33Y, which is insensitive to GSK-3β-mediated phosphorylation, and Renilla luciferase vectors. Cells were then treated with 5GTN for 24 h. Luciferase assay was determined and results are expressed as relative luciferase units compared with the cell transfected with pcDNA3.1-transfected cells. Values are means 7 S.E.M of three independent experiments. *Po 0.05 and **Po 0.01; significantly different from the vehicle control.
Acknowledgment This research project is supported by the Thailand Research Fund (TRF)-Mahidol University through the Royal Golden Jubilee Ph.D. Program (PHD/0170/2552 to PP and NB), the research grants from Mahidol University, and the TRF through the International Research Network (IRN-58W0004), and the plant compounds from the Center of Excellence for Innovation in Chemistry (PERCHCIC). We are grateful to Prof. Chumpol Pholpramool for his critical
reading of the manuscript.
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