Proteomic Study Reveals a Co-occurrence of Gallic Acid-Induced

Article pubs.acs.org/JAFC

Proteomic Study Reveals a Co-occurrence of Gallic Acid-Induced Apoptosis and Glycolysis in B16F10 Melanoma Cells Cheng Liu,†,§ Jen-Jie Lin,# Zih-Yan Yang,⊥ Chi-Chu Tsai,○ Jue-Liang Hsu,*,¶,∥ and Yu-Jen Wu*,Δ,∥ †

Hyperbaric Oxygen Therapy Center and Division of Plastic Surgery, Chi Mei Medical Center, Tainan 710, Taiwan Department of Electrical Engineering, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan # Graduate Institute of Veterinary Medicine, ⊥Graduate Institute of Food Science, and ¶Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 91202, Taiwan ○ Kaohsiung District Agricultural Improvement Station, Pingtung 900, Taiwan Δ Department of Beauty Science, Meiho University, Pingtung 91202, Taiwan §

S Supporting Information *

ABSTRACT: Gallic acid (GA) has long been associated with a wide range of biological activities. In this study, its antitumor effect against B16F10 melanoma cells was demonstrated by MTT assay, cell migration assay, wound-healing assay, and flow cytometric analysis. GA with a concentration >200 μM shows apoptotic activity toward B16F10 cells. According to Western blotting data, overexpressions of cleaved forms of caspase-9, caspase-3, and PARP-1 and pro-apoptotic Bax and Bad, accompanied by underexpressed anti-apoptotic Bcl-2 and Bcl-xL indicate that GA induces B16F10 cell apoptosis via mitochondrial pathway. The 2-DE based comparative proteomics was further employed in B16F10 cells with and without GA treatment for a large-scale protein expression profiling. A total of 41 differential protein spots were quantified, and their identities were characterized using LC-MS/MS analysis and database matching. In addition to some regulated proteins that were associated with apoptosis, interestingly, some identified proteins involved in glycolysis such as glucokinase, α-enolase, aldolase, pyruvate kinase, and GAPDH were simultaneously up-regulated, which reveals that the GA-induced cellular apoptosis in B16 melanoma cells is associated with metabolic glycolysis. KEYWORDS: gallic acid, apoptosis, mitochondrial dysfunction, proteomics, 2-DE, glycolysis

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INTRODUCTION Gallic acid (GA), a trihydroxybenzoic acid, is classified as a type of phenolic compound and commonly found in free form or as part of hydrolyzable tannins in gallnuts, grape seeds and skin, tea leaves, and wine.1 It has long been associated with a wide range of biological activities, such as antioxidant,2 antimicrobial,3 antimalarial,4 anti-inflammatory,5 and antidiabetic actions.6 Due to their potent free radical scavenging ability, most phenolic compounds have been regarded as antioxidants.7 However, unlike other radical scavengers such as glutathione peroxidase and superoxide dismutase, which can reduce glutathione and protect cells from oxidative stress and even apoptosis,8 GA has been reported to have both pro-oxidant and antioxidant properties,2 and its pro-oxidant property is supported by the observation of activated oxygen species formation under GA stimulation.9 GA has been shown to have cytotoxic effects against a broad range of tumor cells such as lung cancer, breast cancer, intestinal cancer, and gastric cancer.10−13 Recently, GA has been shown to have a potent chemoprevention effect against melanoma, mainly due to its dual prooxidant and antioxidant properties.14,15 Melanoma, a malignancy of pigment-producing cells (melanocytes), is a tough skin cancer that is notoriously aggressive and also shows high chemotherapy resistance.16 GA shows cytotoxic effect against A375.S2 human melanoma cells through caspase-dependent and -independent apoptosis14 or the octyl, decyl, dodecyl, and © XXXX American Chemical Society

tetradecyl gallates against melanoma cells via glutathione depletion and the downstream inhibition of γ-glutamylcystein synthase activity.15 Beyond the cytotoxic effect, GA also shows other biological functions toward melanoma cells, such as antimelanogenic property17 and cell adhesion inhibition.15 Although the biological functions induced by GA have been extensively studied, the correlations between each function or the comprehensive signaling network for the melanoma cells upon GA induction so far are not well-understood. Comparative proteomics is well-suited to a large-scale protein profiling, and therefore it has been regarded as a useful tool to explore the protein−protein interaction network, which is feasible to correlate the individual biological effects stimulated by natural products.18 According to Kim’s study, GA showed a significant antimelanogenic activity toward B16F10 cells, which have been used as a typical cellular model to screen skin-whitening agents.17 However, the further impact of GA on this cell line has not been well studied. In this study, we used the MTT assay, wound-healing assay, and cell migration assay to evaluate the antitumor effect of GA toward B16F10 cells. The mechanism of its cytotoxic effect was further determined Received: August 22, 2014 Revised: November 13, 2014 Accepted: November 14, 2014

A

dx.doi.org/10.1021/jf504035s | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

using a DNA laddering assay, flow cytometric analysis, and Western blotting. To investigate the global influences within melanoma cells caused by GA induction, two-dimensional gel electrophoresis (2-DE) was used to monitor comprehensive protein expression comparatively from B16F10 melanoma cells with and without GA treatment. Using this method, quantitative analysis of protein expression profiling on a large scale can be achieved by intensity comparison of protein spots stained by silver or Coomassie blue on the gels of control and experimental samples. Differentially displayed proteins can be further hydrolyzed using in-gel digestion, and the protein identities can be identified using tandem mass spectrometry (MS/MS) and database matching. In this study, the regulated proteins from B16F10 melanoma cells under GA stimulation were uncovered, and their relative expressions were validated by Western blotting. The functions of the differentially displayed proteins were further correlated for exploring a more comprehensive mechanism underlying GA-mediated biological action.

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of morphological changes of B16F10 cells with and without GA stimulation. Cell Migration Assay and Wound-Healing Assay. To validate GA’s cytotoxic effect, B16F10 melanoma cells were seeded onto polycarbonate (PC) membranes (8.0 μm, BD Biosciences, San Jose, CA, USA) in the culture inserts. Then these inserts were transferred into wells with DMEM containing 10% FBS. B16F10 cells with or without GA treatment were incubated for 24 h. After removal of cells on the upper site, migrated cells on the lower site were fixed by 100% methanol and stained using Giemsa (Merck, Darmstadt, Germany). The migrated cells were photographed and counted at 100× magnification under microscope. GA’s cytotoxic effect was also examined using the wound-healing assay. B16F10 cells were seeded in 6-well plates to allow the cells to grow to conflux. A pipet tip was used to create an artificial scratch/wound in each well. Detached B16F10 cells were removed by washing with PBS. Images of control and treated samples at different concentrations (0, 100, 200, and 400 μM of GA) were acquired at 0, 6, 12, and 24 h after the treatments. Analysis of DNA Fragmentation by Agarose Gel Electrophoresis. B16F10 cells (2 × 105 cells/well) were incubated in a medium containing GA with various concentrations (0, 100, 200, and 400 μM) for 24 h. The cells were then washed using cold PBS buffer and kept in a lysis buffer containing 10 mM EDTA, 50 mM Tris-HCl (pH at pH 8.0), 0.5 g/L proteinase K, and 0.25% NP-40 at 50 °C for 2 h. The precipitated DNA was collected using ethanol at 25 °C. The resulting DNA pellets were washed with 70% ice-cold ethanol and then dissolved in TE buffer (0.6 g/mL RNase A and 1 mM EDTA in 10 mM Tris-HCl at pH 8.0), and the DNA mixture was further incubated at 37 °C for 1 h. The DNA separation was performed using horizontal electrophoresis at 200 V on a 1.5% agarose gel using tris/ acetic acid/EDTA (TAE) as the running buffer. Finally, the DNA gel was stained with ethidium bromide (EtBr) and visualized under a 2 UV transilluminator (MacroVue UV-20 Hoefer). Analysis of Cell Cycle Distribution and Apoptosis. B16F10 cells were treated with DMSO (0.5%) or with various concentrations (0, 100, 200, and 400 μM) of GA for 24 h. After treatment, the cells were collected and the reagents were removed by washing with PBS twice. Then the cells were fixed in 70% ethanol overnight. The phase distribution of cell DNA content was determined using PI (Sigma) staining. The cells were precipitated using centrifugation at 700 rpm for 5 min at 4 °C, and the resulting cell pellet was stained with 10 μg/ mL of PI (in PBS buffer containing 10 μg/mL of RNase A) for 15 min at room temperature in the dark. The apoptotic cells induced by GA were observed using annexin V staining. B16F10 cells at a density of 1 × 106 cells per 100 mm Petri dish were treated with DMSO (0.5%) or GA at various concentrations (0, 100, 200, and 400 μM) for 24 h and subsequently labeled with 10 μg/mL of annexin V−FITC individually. The analysis of cell cycle distribution and apoptosis of GA-treated B16F10 cells were performed using FACScan flow cytometer (BectonDickinson, Mansfield, MA, USA) coupled with Cell-Quest software (Becton-Dickinson). Protein Extraction and Quantification. B16F10 cells were treated with solvent vehicle (0.5% DMSO) or indicated concentrations of GA for 24 h, and then the cells were lysed using cell extraction buffer (BioSource International), and protease inhibitor cocktail (Sigma) was added to avoid protein degradation. The protein precipitation from supernatant was carried out using 10% TCA/ acetone solution containing 20 mM DTT overnight (−20 °C). The protein pellet was collected, and the supernatant was discarded after centrifugation at 8000 rpm for 30 min at 4 °C. The resulting pellet was dissolved in a rehydration buffer that contained 6 M urea, 2 M thiourea, 0.5% CHAPS, 0.002% bromophenol blue, 20 mM DTT, and 0.5% IPG buffer. The quantification of protein content was performed using a 2-D Quant Kit (GE Healthcare). Here, the calibration curve was established using bovine serum albumin (BSA). Two-Dimensional Gel Electrophoresis. Two-dimensional gel electrophoresis procedure was carried out according to a previous study.19 The isoelectric focusing (IEF) was performed on GE Healthcare Ettan IPGphor 3 at 20 °C with a current limit of 30 A. Protein mixture was dissolved in a rehydration buffer and applied on

MATERIALS AND METHODS

Materials. Protease inhibitor cocktail, Dulbecco’s modified Eagle medium (DMEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Gibco (Carlsbad, CA, USA). Cell extraction buffer was obtained from BioSource International (Camarillo, CA, USA). The immobilized pH gradient (IPG) strips and sampling buffer were from GE Healthcare (Buckinghamshire, UK). Gallic acid (GA), iodoacetamide (IAA), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), trichloroacetic acid (TCA), propidium iodide (PI), RNase A, Triton X-100, cyclosporine A (CyA), and trifluoperazine (TFZ) (mitochondrial permeability transition inhibitors) were obtained from Sigma. Formic acid (FA), acetone, acetonitrile (ACN), ammonium bicarbonate (ABC), and 1,4-dithiothreitol (DTT) were obtained from J. T. Baker (Phillipsburg, NJ, USA). Trypsin used in in-gel digestion was purchased from Promega (Madison, WI, USA). Rabbit anti-mouse TCP1, PRDX-1, Rho-GDI, G-3-P DHase, SOD, aldolase, HSP 60, HSC 71, VDAC-1, ATPase, enolase, and glucokinase antibodies were purchased from ProteinTech Group (Chicago, IL, USA). Antibodies for apoptosis-related proteins such as cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, PARP-1, Bad, AIF, Bax, cytochrome c, Mcl-1, Bcl-xL, and Bcl-2 were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-mouse β-actin antibodies (used to recognize β-actin as the internal control) were obtained from Sigma. Horseradish peroxidase conjugated IgG (goat anti-rabbit) was purchased from Millipore (Bellerica, MA, USA). Chemiluminescent HRP substrate and polyvinylidene difluoride (PVDF) membranes were obtained from Pierce (Rockford, IL, USA). The water used in this study was produced using a Milli-Q (Millipore) water purification system. Cell Culture. The mouse melanoma cell line (B16F10) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in 75 cm2 flasks at approximately 1 × 106 cells/mL cell density with 90% MEM, 10% FBS, penicillin/streptomycin (100 μg/mL streptomycin and 100 U/ mL penicillin), and 2 mM L-glutamine, and the cells were incubated at 37 °C in 100% humidity, 5% CO2−95% air. Viability Assay. B16F10 cells (2 × 105 cells/well) were placed onto 12-well plates and incubated at 37 °C for 24 h prior to be treated with GA at different concentrations (0, 50, 100, 200, and 400 μM) for various time periods (0, 6, 12, and 24 h). Cell viabilities under different conditions were determined using the MTT assay. A control was carried out using DMSO (0.5%); 0.5% of DMSO did not show any significant impact on cell viability. After incubation, cells were photographed using contrast phase microscope for the observation B



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Figure 1. Evaluation of cytotoxicity and antimigratory effects of GA at different concentrations on B16F10 cells: (A) MTT assay for B16F10 cells treated with GA at 50−400 μM for 24 h (data are presented as the mean ± SEM from three independent experiments); (B) population and morphological changes of B16F10 cells treated with GA at different concentrations for 24 h; (C) wound-healing assay at different concentrations and time periods; (D) DNA laddering assay of B16F10 cells treated with GA at different concentrations (M, markers). an IPG strip. An 11 cm IPG strip (pI 3−10) was rehydrated at 30 V for 12 h, and then proteins with different pI values were separated and focused using different stages of applied voltages according to the program 200 V (0−2 h), 500 V (2−4 h), 1000 V (4−6 h), 4000 V (6− 7 h), 8000 V (7−11 h), until the total voltage (V) × hour (h) reached 39400. The equilibrated strip was placed on the top of a SDS-PAGE gel (12.5%), sealed with 0.5% agarose, and then the gel electrophoresis was performed under SE 600 Ruby (Hoefer, Holliston, MA, USA) at 150 V for 6.5 h. After that, the 2-DE gel was stained by silver, and the resulting silver-stained gel was scanned and analyzed using PDQuest image analysis software (Bio-Rad). 2-DE images were obtained from triplicates for each sample and normalized prior to statistical analysis. Protein spots (the same pI and Mw) in experimental and control samples showing more than 1.5-fold intensity difference were regarded as statistically significant. In-Gel Digestion. A protein spot of interest was excised, cut into pieces, and immersed in 100 μL of 25 mM ammonium bicarbonate (ABC) (pH 8.5) containing 50 mM DTT at 37 °C for 1 h. After removal of supernatant, the gel pieces were resuspended in 100 μL of 100 mM iodoacetamide (IAA) in 25 mM ABC (pH 8.5) and then shaken for 30 min at room temperature in the dark. After removal of supernatant, the gel pieces were soaked in 100 μL of 50% acetonitrile in 25 mM ABC (pH 8.5) for 30 min, and then the buffer was completely removed by vacuum. The gel pieces were rehydrated and soaked in 100 μL of 25 mM ABC (pH 8.5) containing 0.1 μg of trypsin. The trypsin digestion was performed at 37 °C for 16 h. The reaction was quenched using 5 μL of 5% formic acid, and the resulting peptide mixture was obtained after removal of buffer. LC-MS/MS Analysis and Database Search. The peptide mixture obtained from in-gel digestion was separated by a reversed phase C18 column (PepMap100 C18, 150 mm length, 2.1 mm i.d., 2.6 μm particle size) on a nano LC (Agilent 1200) system with a gradient from 5 to 70% acetonitrile containing 0.1% formic acid over 90 min at 300 nL/min. The separated peptides were online analyzed using an AB

SCIEX QTRAP 5500 Q mass spectrometer (Applied Biosystems, Foster City CA, USA). The LC-MS/MS was performed under a positive survey scan in the range of m/z 100−1000. The raw data were processed into WIFF format with Analyst 1.5.1. The database-assisted protein identification was carried out using Mascot search engine v2.3 (Matrix Science, UK) with the following search parameters: (1) protein database, Swiss-Prot; (2) taxonomy, Mus musculus (house mouse); (3) allowed trypsin missed cleavage, 1; (4) precursor and product ion mass tolerance, 2 Da/1 Da; (5) fixed modification, carbamidomethyl (C); (6) variable modifications, oxidation (M); (7) significance threshold, p < 0.05. Proteins with scores higher than the threshold were regarded as significant hits. Western Blotting Analysis. GA-treated samples and controls were separated on SDS-PAGE gels, and the separated proteins on gels were transferred to PVDF membranes (Millipore) for 1.5 h at a fixed current of 400 mA under Transphor TE 62 (Hoefer). The membranes were then incubated with mouse antibodies of caspase-3/cleaved caspase-3, caspase-9/cleaved caspase-9, cleaved poly-ADP ribose polymerase (PARP), cytochrome c, AIF, Bcl-2, Bcl-xL, Mcl-1, Bax, p-Bad, Bad, TCP1, PRDX-1, Rho-GDI, G-3-P DHase, SOD, aldolase, HSP 60, HSC 71, VDAC-1, ATPase, α-enolase, glucokinase, and βactin at 4 °C for 2 h or overnight. The membranes were washed three times using PBST (10 mM NaH2PO4, 130 mM NaCl, 0.05% Tween 20), and then the primary antibodies were recognized and probed with the secondary antibodies (goat anti-rabbit) conjugated with horseradish peroxidase in blocking solution (1:5000) for 1 h. After removal of the excess secondary antibodies and blocking solution, the membranes were washed with PBST three times. The chemiluminescence on the blot was visualized through the redox reaction between horseradish peroxidase and substrate (ECL Western blotting reagent, Pierce). Statistical Analysis. Experiments were done in triplicate or quaduplicate, and data of MTT assay, cell migration assay, and flow cytometric analysis were expressed as the mean ± SE. For the C



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Figure 2. Cell cycle distribution and apoptosis: (A) flow cytometric measurement of cell cycle distribution of B16F10 cells treated with GA at different concentrations; (B) quantitative data obtained according to the DNA content measurement in panel A; (C) annexin V-FITC/PI analysis of B16F10 cells treated with GA at different concentrations. determination of significant differences (p ≤ 0.05) between experimental groups, one-way ANOVA followed by the Tukey− Kramer test, which was carried out on GraphPad InStat 3 (San Diego, CA, USA), was used in this study.

apoptosis-mediated cell death. Some phenomena such as mitochondrial dysfunction, intracellular Ca2+ level increase, and DNA fragmentation were associated with this GA-induced apoptosis.20,21 In fact, the effect of GA and its alkyl esters varies toward different tumor cell lines and results in cell-specific behavior. For example, the apoptotic DNA ladder fragmentation can be observed when mouse B cell lymphoma WEHI 231 cell line is stimulated by GA; however, in the case of GA-treated fibroblast L929 cells, some morphological changes such as cell shrinkage, chromatin condensation, and apoptotic body formation can be simultaneously observed, whereas the DNA fragmentation characteristic does not occur.20 In this study, GA induced the breakdown of chromatin, resulting in apparent DNA fragmentation in B16F10 cells. GA-Induced Apoptosis of B16 Cells. To investigate the effect of GA on cell cycle, B16F10 cells pretreated with GA at various concentrations (0, 100, 200, and 400 μM) were analyzed using annexin V−FITC and PI staining on flow cytometry (Becton-Dickinson). Apparently, B16F10 cells at sub-G1 phase were increased dose-dependently, which indicated that the accumulation of small DNA fragments was caused by apoptosis. The percentage of 400 μM GA-treated B16F10 cells at sub-G1 was enhanced to 41% while the control was only 6% (Figure 2A,B). The GA-induced apoptosis of B16F10 cells was further determined using the flow cytometer based-annexin V staining. Under fluorescent microscopy, the formation of apoptotic bodies indicated that GA concentrations of 200 and 400 μM dose-dependently increased the percentage of apoptotic cells (Figure 2C). These results all indicated that the cytotoxic effect of GA against B16F10 cells was due to its apparent apoptosis induction. According to the hypothesis

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RESULTS AND DISCUSSION Antitumor Effects of GA on B16F10 Cells. B16F10 cells were treated with GA of various concentrations (0, 50, 100, 200, 400 μM) for 24 h. The MTT assay showed that significant cell death occurred (around 40%) when 400 μM GA was added (Figure 1A). The morphological change and reduced population of GA-treated cells observed by inverted light microscopy also displayed a similar trend (Figure 1B), which indicated that GA at a concentration of 400 μM can inhibit the proliferation of B16F10 cells. This result was similar to that reported by Locatelli et al.15 The migratory capability of cells was measured by cell migration and wound-healing assay. After treatment with GA of various concentrations (50, 200, and 400 μM) in different time periods (6, 12, and 24 h), suppression of B16F10 cell migration was observed, and the migration suppression was also dose-dependent (Figure 1C and Supporting Information Figure S1). To study whether cell death was caused by apoptosis or not, the cells were treated with 0, 100, 200, and 400 μM GA for 24 h. After incubation, the cellular DNA was extracted and analyzed using agarose gel electrophoresis. As shown in Figure 1D, 400 μM GA induced the chromatin breakdown and the degraded DNA fragments resulting in ladder-like gel patterns, which indicated that cell death occurred via apoptosis instead of necrosis. According to a previous study, the cytotoxicities of GA and its derivatives toward other cell lines were mainly proposed to be involved in D



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Figure 3. Expressions of apoptosis-related proteins in GA-treated B16F10 cells: (A) Western blotting analysis of caspase-3/cleaved caspase-3, caspase-9/cleaved caspase-9, cleaved-poly-ADP ribose polymerase (PARP), cytochrome c (cytosolic), and apoptosis-inducing factor (AIF) in B16F10 cells treated with GA at different concentrations; (B) Western blotting analysis of Bcl-2, Bcl-xL, induced myeloid leukemia cell differentiation protein (MCl-1), Bax, p-Bad, and Bad in B16F10 cells treated with GA at different concentrations; (C) cell viabilities (%, compared with the control) of GA (400 μM)-induced cell apoptosis with and without the mitochondrial permeability transition inhibitors cyclosporine A (CyA) or trifluoperazine (TFZ); (D) expression levels of Bcl2, Bcl-xl, Bax, and Bad proteins in the GA (400 μM)-induced B16F10 cells with and without mitochondrial permeability transition inhibitors. β-Actin was used as the internal control.

Similarly, the increase of cleaved form of caspase-9 was also found at 37 kDa after GA treatment; the enhancement of cytosolic cytochrome c released from mitochondria, which activated caspase-dependent cell death, was simultaneously observed. Interestingly, the decrease of PARP-1 (116 kDa) was accompanied by dose-dependent increases of cleaved PARP-1 (89 kDa) and AIF after GA treatment (Figure 3A). This implied that the PARP-1 activation propagated to mitochondria, triggered the AIF translocation from mitochondria to the nucleus, and induced chromatin condensation and DNA fragmentation. The overexpression of AIF in the nucleus suggested that GA-induced apoptosis was also through a caspase-independent apoptosis. Unlike other cancer cell lines in which the GA-induced apoptosis was reported to be occurred solely via a caspase-dependent pathway, GA-induced apoptosis involved in B16F10 cells was simultaneously through caspasedependent and -independent pathways, which is consistent with the phenomenon occurring in the other melanoma cell, A375.S2.14 To further verify this conclusion, the expression levels of some mitochondrial cell death effectors such as proapoptotic B-cell lymphoma-2 (Bcl-2) family members Bax, Bak, and Bad were simultaneously evaluated with the increase of GA concentration. Meanwhile, the expression of anti-apoptotic Bcl2, Bcl-xL, Mcl-1, and p-Bad was simultaneously suppressed (Figure 3B). The combined effects promoted the mitochondrial outer membrane permeablization and the release of cytochrome c from mitochondria into the cytosol and led to the

proposed by Serrano et al., GA can induce apoptosis not only because it can promote the generation of reactive oxygen species (ROS) but also as a consequence of its inhibitory activity toward protein tyrosine kinases (PTKs).21 In the study of anticancer effects of GA on A549 human lung adenocarcinoma cells, Maurya et al. presented that GA treatment simultaneously decreased mitochondrial membrane potential and increased intracellular ROS, and the caspase activity measurement further showed the activation of caspase-3 but not caspase-8, indicating the involvement of intrinsic cell apoptosis.10 Ou et al. presented that GA can induce G2/M phase cell cycle arrest in human bladder transitional carcinoma cells through regulating 14-3-3β release from activation of Cdc25C and Chk2.22 In normal human lymphocytes, however, GA shows anti-apoptotic ability via Bcl-2 independent mechanism.23 Therefore, the detailed mechanism of apoptosis induction by GA in different tumor cells so far is not welldefined. Western Blot of Apoptosis Related Proteins. To determine the mechanism for GA-induced apoptosis, we further analyzed the expression levels of several apoptotic markers including cytosolic cytochrome c, caspase-3, caspase-9, cleaved PARP, and apoptosis-inducing factor (AIF) from GAtreated B16F10 cells. Western blot showed dose-dependent down-regulations of pro-caspase-9 and pro-caspase-3. Expression of caspase-3 (17 kDa proteolytic fragments) was elevated when the concentration of GA increased to 200 μM or higher. E



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Figure 4. 2-DE maps of B16F10 cells treated with 400 μM GA and the control sample processed in different pI ranges: (A) control and (B) treated with 400 μM GA for 24 h in a range of pI 4−7; (C) control and (D) treated samples in pI 3−10. Protein spots marked on the maps were considered differentially expressed and identified by LC-MS/MS. The results are representative of three independent runs. Solid arrows denote up-regulated proteins, and dashed ones denote down-regulated proteins.

10. The quantitative analysis of protein spots in 2-DE maps between the GA-treated and the control samples was achieved using PDQuest image analysis software (Bio-Rad). Proteins with >1.5-fold spot intensity difference between the experimental and the control samples were defined as differentially expressed proteins. The differentially expressed proteins were hydrolyzed using in-gel trypsin digestion followed by LC-MS/ MS analysis. The database-assisted protein identification was achieved using the MASCOT protein identification search engine. A total of 41 differentially expressed proteins were quantified and identified. Using a 1.5-fold intensity difference compared to control as threshold, 25 spots were overexpressed and 16 spots were underexpressed. The identified proteins with their MASCOT scores, identified peptide sequences, calculated Mw, pI, coverages, and folds of change in expression level (upregulation or down-regulation) are shown in Table 1. Some upregulated proteins such as 60 kDa heat shock protein (HSP60), heat shock cognate 71 kDa protein (HSC 71), nucleophosmin (NPM), peroxiredoxin-1 (PRDX-1), succinyl-CoA ligase (SCSβG), poly(rC)-binding protein 1 (PCBP1), superoxide dismutase (SOD), voltage-dependent anion-selective channel protein 1 (VDAC-1), and voltage-dependent anion-selective channel protein 2 (VDAC-2) have been reported to be associated with apoptosis.24−27 Among these up-regulated proteins, HSP60 acts as a chaperone protein to maintain proper folding and assembling of polypeptide chains in

subsequent activation of caspase-3 and the executioner caspases. The data all indicated that GA induced B16F10 cell apoptosis via a mitochondrial intrinsic pathway. To further confirm that GA-induced cell apoptosis is through a mitochondria-related apoptotic pathway, B16F10 cells were pretreated with either cyclosporine A (CyA) or trifluoperazine (TFZ) (both are mitochondrial permeability transition inhibitors) prior to the GA treatment. The results showed that GA-induced cell apoptosis was significantly decreased when CyA or TFZ was added (Figure 3C). In addition, several mitochondrial-related apoptotic proteins, including Bcl2, Bcl-xl, Bax, and Bad proteins, were estimated by Western blotting, respectively. The expressions of Bcl2 and Bcl-xl were promoted when the cells were pretreated with CyA and TFZ. By contrast, the expression levels of Bax and Bad were decreased in the GAtreated cells when the cells were pretreated with CyA and TFZ (Figure 3D). Taken together, these results suggested that mitochondria dysfunction-mediated apoptotic pathway was involved in GA-induced cell apoptosis. To further uncover the pathway networks associated with this apoptosis, a comprehensive proteome study was used to globally analyze proteins regulated in GA-induced B16F10 cells. Proteomic Analysis of B16F10 Cells with GA Stimulation. Protein spots from samples with and without 400 μM GA treatment were visualized by silver staining on 2-DE gels, as shown in Figure 4A,B with pI 4−7 and Figure 4C,D with pI 3− F



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Table 1. Regulated Proteins Identified Using 2-DE-Based Comparative Proteomics spot no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 a

protein name (short name) phosphatidylethanolamine-binding protein 1 (PEBP1) T-complex protein 1 subunit epsilon (TCP-1) 60 kDa heat shock protein (HSP60) succinyl-CoA ligase (SCS-βG) vimentin 60 kDa heat shock protein (HSP60) Rho GDP-dissociation inhibitor 1 (Rho-GDI) heat shock cognate 71 kDa protein (HSC 71) inorganic pyrophosphatase 2 (PPase 2) mannose-6-phosphate (perilipin-3) peroxiredoxin-6 (PRDX-6) glucose-6-phosphate 1-dehydrogenase (G6PD) proteasome subunit α type 2 isovaleryl-CoA dehydrogenase (IVD) vacuolar ATP synthase catalytic subunit A 60S acidic ribosmal protein P0 protein disulfide-isomerase precursor (PDI) nucleophosmin (NPM) import inner membrane translocase subunit (Timm50) peroxiredoxin-1 (PRDX-1) phosphoenolpyruvate carboxykinase (PEPCK-M) aspartate aminotransferase (transaminase A) voltage-dependent anion-selective channel protein 2 (VDAC-2) poly(rC)-binding protein 1 (PCBP1) G-3-P dehydrogenase (GAPDH) α-enolase (enolase) superoxide dismutase (SOD) fructose-bisphosphate aldolase (aldolase 1) fumarate hydratase (Fh1) ATP synthase subunit α (ATPase) pyruvate kinase isozyme M2 (PKM) ChChd3Domain-containing protein (ChChd3) voltage-dependent anion-selective channel protein 1 (VDAC-1) peptidyl-prolyl cis−trans isomerase B (cyclophilin B, CypB, PPlase B) elongation factor 2 (EF-2) phosphoglycerate mutase 1 (PGAM-1) ATP synthase subunit α 26S protease regulatory subunit 8 (mSUG1) serine hydroxymethyltransferase (SHMT) inosine-5′-monophosphate dehydrogenase 2 (IMPD 2) calcyclin-binding protein (CacyBP)

accession no.

calcd Mw/pI

peptides matched

sequence covered (%)

P70296

20.81/5.19

9

41

98

−1.7

P80316 P63038 Q9Z2I8 P20152 P63038 Q99PT1 P63017 Q91VM9 Q9DBG5 O08709 Q00612 P49722 Q9JHI5 P50516 P14869 P09103 Q61937 Q9D880

59.58/5.72 60.9/5.91 46.81/6.58 53.65/5.06 60.9/5.91 23.39/5.12 70.82/5.37 38.09/6.51 47.2/5.45 24.8/5.71 59.0/6.07 25.9/8.39 46.2/8.53 68.2/5.62 34.1/5.91 57.1/4.79 32.54/4.62 39.75/8.26

10 74 12 13 4 16 41 4 36 19 33 4 17 84 42 3 5 13

14 60 27 55 5 45 37 15 21 58 38 16 30 50 42 4 12 19

142 1057 219 1888 77 288 445 51 358 281 502 81 250 1041 483 36 55 228

−2.1 +2.4 +2.1 −1.9 +2.5 −2.2 +3.1 +2.4 +2.3 −2.1 +2.2 +2.1 +2.6 +2.9 −2.8 −1.8 +2.1 −2.2

P35700 Q8BH04 P05201 Q60930

22.16/8.26 70.48/6.92 45.2/6.68 31.71/7.44

13 19 34 13

40 25 43 26

119 265 518 271

+2.5 −2.7 +2.4 +2.1

P60335 P16858 P17182 P09671 P05064 P97807 Q03265 P52480 Q9CRB9 Q60932

37.47/6.66 35.78/8.44 47.1/6.37 24.5/8.8 39.3/8.31 54.33/9.12 59.71/9.22 57.85/7.18 26.31/8.56 32.33/8.55

27 6 10 4 17 40 48 11 6 41

31 21 21 22 31 31 45 17 21 59

624 70 101 66 264 360 737 99 117 470

+2.2 +2.4 +2.9 +3.2 +2.6 +3.4 −2.8 +2.8 +1.7 +2.4

22.69/9.48

33

49

415

+3.4

P58252 Q9DBJ1 Q03265 P62196 P50431 P24547

92.25/6.41 28.81/6.67 59.71/9.22 45.59/7.11 52.55/6.47 55.78/6.84

30 14 3 14 5 32

17 35 5 32 4 35

564 164 52 207 106 720

−1.8 −2.4 −3.4 −2.1 −2.4 +1.7

Q9CXW3

26.49/7.63

14

45

200

−2.1

MASCOT score

regulation (-fold change)a

Regulations (-fold changes) of differentially expression proteins are expressed at 24 h of treatment of gallic acid.

mitochondria, and overexpression of HSP60 is regarded as sustaining cellular survival under toxic or stressful conditions;24 HSC 71 also acts as a chaperone involved in cell proliferation, differentiation, and tumorigenesis. Its increasing level in B16F10 cells after treatment with 400 μM GA suggested the association with the responses of either apoptosis or cell survival.25 Similarly, the expressions of some down-regulated proteins such as T-complex protein 1 subunit ε (TCP-1), vimentin, Rho GDP-dissociation inhibitor 1 (Rho-GDI), peroxiredoxin-6 (PRDX-6), elongation factor 2 (EF-2), and calcyclin-binding protein (CacyBP) are also consistent with those involved in apoptosis reported in the literature.18,28−31

The identities and relative expressions of some regulated proteins under GA treatment with various concentrations were further validated by Western blotting (Figure 5). HSP60, HSC 71, VDAC-1, SOD, and PRDX-1 were dose-dependently upregulated, and their expression levels upon 400 μM GA treatment were consistent with those measured by 2-DE experiment. Meanwhile, TCP1, Rho-GDI, and ATPase were simultaneously down-regulated, which showed trends similar to those using a proteomics approach. In addition to these regulated proteins, which were regarded as being associated with apoptosis, interestingly, some proteins involved in glycolysis such as α-enolase, aldolase, pyruvate kinase, and G



Article

pathway. 2-DE-based comparative proteomics showed that most regulated proteins were associated with apoptosis, consistent with the Western blot data. Notably, the proteomics data also revealed that most proteins involved in glycolysis were simultaneously up-regulated in this GA-induced cell apoptosis. The correlation between glycolysis and apoptosis was further confirmed by Western blot of glucokinase, α-enolase, aldolase, and GAPDH.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 5. Validations of some regulated proteins identified from 2-DE experiment. Western blotting analysis of TCP1, PRDX-1, Rho-GDI, G-3-P DHase, SOD, aldolase, HSP 60, HSC 71, VDAC-1, ATPase, enolase, and glucokinase in B16F10 cells treated with increasing concentrations of GA. β-Actin was used as the internal control.

Figure S1: Cell migration assay of GA. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(J.-L.H.) E-mail: [email protected]. *(Y.-J.W.) Mail: 23 Pingkung Road, Neipu Hsiang, Pingtung 91202, Taiwan. Phone: +886-8-7799821, ext 8613. Fax: +8868-7797821. E-mail: [email protected] or x00002180@ meiho.edu.tw.

GAPDH were simultaneously up-regulated during the GAinduced apoptosis according to 2-DE experiment. The dosedependent effects of GA on these glycolytic proteins were further confirmed by Western blotting, as shown in Figure 5. The result indicated that enolase, aldolase, pyruvate kinase, and GAPDH were up-regulated dose-dependently in GA-induced B16F10 cells, which implied that GA plays a dual role in both glycolysis and apoptosis. To further validate the activation of glycolysis, the expression of glucokinase, the key glycolysis enzyme, was also examined. The expression of glucokinase was up-regulated accordingly (Figure 5), which further provided evidence for this correlation. As we know, the oxidative phosphorylation of cancer cells is usually suppressed when they are under stressful condition, such as under GA stimulation. The energy supply of cancer cells shifts from oxidative phosphorylation to glycolysis, aiming to produce sufficient ATP, which is known as the Warburg effect.32 The overexpression of glycolytic proteins in GA-treated B16F10 cells may be due to this effect. Typically, glycolysis and apoptosis are considered as major but independent pathways that are critical for cell survival. Through comparative proteomics, an association between GA-induced apoptosis and metabolic glycolysis in B16F10 melanoma cells was identified and correlated in this study. The interaction between GA-induced apoptosis and metabolic glycolysis may be also explained by the following two reasons: (1) Danial et al. combined the study of proteomics, genetics, and physiology to conclude that BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis,33 which suggests that GA triggered the intrinsic apoptosis accompanied by glycolysis; (2) GA induces intensive glycolysis and produces a highly reactive metabolite methylglyoxal (MG), which leads to B16F10 cell apoptosis via a mitochondrial intrinsic pathway.34 However, these two reasons can not fully explain the co-occurrence of GA-induced apoptosis and metabolic glycolysis in B16F10 melanoma cells, so further exploration and validation experiments are required. In this study, we comprehensively investigated the cytotoxicity on B16F10 melanoma cells caused by gallic acid with a concentration >200 μM using the MTT assay, woundhealing assay, and cell migration assay. The result of DNA laddering assay and flow cytometry assay further indicated that cell death occurred via apoptosis instead of necrosis. The overexpressions of cleaved forms of caspase-9, caspase-3, and PARP-1 and pro-apoptic Bax and Bad, accompanied by underexpressed anti-apoptotic Bcl-2 and Bcl-XL, indicated that GA induced B16F10 cell apoptosis via a mitochondrial

Author Contributions ∥

J.-L.H. and Y.-J.W. contributed equally to this work.

Funding

This work was supported by Taiwan NSC grants (NSC 1022313-B-276-001-MY3 for Dr. Yu-Jen Wu; NSC 102-2113-M020-001-MY2 for Dr. Jue-Liang Hsu). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the MS technical assistance of TzuChun Lin in the Food Science and Nutrition Department in Meiho University of Science and Technology and Dr. Ann Na Gong for English editing.

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REFERENCES

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Proteomic Study Reveals a Co-occurrence of Gallic Acid-Induced

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