Life Sciences 98 (2014) 12–17
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Proteomics screening of molecular targets of curcumin in mouse brain Zohreh Firouzi a,1, Parisa Lari b,1, Marzieh Rashedinia b,1, Mohammad Ramezani c, Mehrdad Iranshahi d,⁎, Khalil Abnous e,⁎ a
Department of Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
a r t i c l e
i n f o
Article history: Received 12 September 2013 Accepted 23 December 2013 Keywords: Affinity chromatography Curcuma longa L. Curcumin Proteomics Target deconvolution
a b s t r a c t Aims: Curcumin is one of the most important constituent of Curcuma longa L. with antioxidant, anti-inflammatory and anticancer effects. In this study, we investigated potential intracellular targets of curcumin by affinity chromatography based on target deconvolution. Identification of curcumin interacting proteins may help in evaluating biological and side effects of this natural compound. Main methods: Curcumin was immobilized through a linker to sepharose beads as solid matrix. Pull down assay was performed by passing tissue lysate of mouse brain through the column to enrich and purify curcumin interacting proteins. Then proteins were separated using two-dimensional gel electrophoresis and identified using MALDI/TOF/TOF mass spectrometry. Key findings: Our results show that curcumin physically binds to a wide range of cellular proteins including structural proteins, metabolic enzymes and proteins involved in apoptosis pathway. Significance: Finding curcumin interacting proteins may help in understanding a part of curcumin pharmacological effects. © 2014 Elsevier Inc. All rights reserved.
Introduction Curcumin, a polyphenol compound, is an active biological constituent of the perennial herb Curcuma longa L. Turmeric is an Indian spice derived from the rhizomes of the plant from Zingiberaceae family. Turmeric has a long history of use in Ayurvedic medicine as a treatment for inflammatory conditions (Goel et al., 2008). Recently several studies have been performed on curcumin to reveal its cellular targets. For example, targets of curcumin in neuroblastoma cell line have been already identified (D'Agnano et al., 2012). Numerous studies revealed that curcumin has antioxidant and radical scavenger, anti-inflammatory, anti-infectious, cardioprotective and anticancer properties (Calabrese et al., 2008). Moreover, curcumin, as an antioxidant, could potentially inhibit lipid peroxidation in rat liver microsomes, erythrocyte membranes and brain homogenates (Chan et al., 2005; Ak and Gülçin, 2008). Various studies have shown that cardioprotective, neuroprotective, memory enhancing, and antiaging effects of curcumin are related to its high antioxidant capacity (Thiyagarajan and Sharma, 2004; Miriyala et al., 2007).
⁎ Corresponding authors. Tel.: +98 511 882 3268; fax: +98 511 882 3251. E-mail addresses: [email protected] (M. Iranshahi), [email protected] (K. Abnous). 1 Contributed equally to this project. 0024-3205/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.200
In addition curcumin has the ability to inhibit the proliferation of an extremely wide array of cancer cell types. Other study showed that curcumin inhibited tumor necrosis factor (TNF-α) and induced apoptosis in PC-12 cells through modulating mRNA expression of Bcl-2 family proteins (Shishodia et al., 2007). Previous studies have shown that curcumin has a neuroprotective effect in some central nervous system such as cerebral ischemia, traumatic brain injury and memory impairment (Miriyala et al., 2007; Shishodia et al., 2007). Curcumin may show its neuroprotective effects through some mechanisms including anti-oxidation, anti-apoptosis, anti-inflammation, anti-amyloidogenic, metal chelating properties, preventing BBB damage, and reducing edema. Some clinical trials, such as application of curcumin Alzheimer's disease, are currently in progress (Sun et al., 2011). Mechanistic study of curcumin may provide a deeper understanding of the therapeutic potential of curcumin and other curcuminoids in neurological disorders (Baum et al., 2008). In this study affinity chromatography based target deconvolution was chosen to find potential targets of curcumin in the brain. Target deconvolution is a high-throughput technique to discover drug targets and predict potential therapeutic and side effects as well as toxicity of chemicals in early study of a new drug development (Terstappen et al., 2007; Raida, 2011). Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. In our study,
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curcumin was covalently coupled to epoxy activated sepharose as chromatographic matrix through phenolic group (Lee and Bogyo, 2013). Following the affinity chromatography-based purification of curcumin targets, proteins were separated using two-dimensional gel electrophoresis and were identified using, matrix-assisted laser desorption/ionization/time of flight (MALDI/TOF/TOF) and MASCOT database.
fluoride using a Polytron Homogenizer (Kinematica, Switzerland). After sonication (UP100H, Hielscher) for 40 s, homogenates were centrifuged (Hettich Universal 320R, Germany) at 25,000 g for 10 min at 4 °C and total protein contents in supernatants were determined using a Bradford protein assay kit (BioRad, #500-0002). The same amount of proteins was applied for each test (Osawa et al., 1995).
Material and methods
Tissue extracts were incubated with control beads for 30 min at 4 °C. During this contiguous the proteins that capable to link to the epoxy activated sepharose were out, while proteins that can be band to curcumin were free in supernatant. After centrifugation at 1000 g for 1 min, supernatants were collected and transferred to curcumin coupled beads and incubated for 30 min at 4 °C. Beads were washed four times, each time with 2 mL of binding buffer, and supernatants were discarded. Then curcumin interacting proteins were eluted with 2 mL of 2 M NaCl. The later step was repeated 3 times and fractions were pooled and dialyzed for against ddwater for 3 days at 4 °C using a membrane with a 2000 Da cutoff (Spectra, USA). Desalted samples were freeze dried and stored at − 80 °C until use (Kunnumakkara et al., 2008).
Materials and reagents Reagents were obtained from Sigma (USA). Deionized water was used for all experiments and all other chemical substances were purchased from Merck (Germany). Animals Animal study was approved by the Mashhad University of Medical Sciences Ethics Committee (#87639). Male BALB/c mice weighing 20–25 g were obtained from the animal house of Mashhad University of Medical Sciences. Mice were kept in a room at 25 ± 2 °C on a 12-h light/dark period and let to eat and drink ad libitum. 6 mice were sacrificed by decapitation and brain tissues were rinsed using 0.9% normal saline solution. Then tissues were quickly frozen in liquid nitrogen and stored at − 80 °C until further processing. Curcumin preparation Curcumin 65–70% was purchased from Sigma (Cat. # C 1386). The curcumin 65–70% further purification was performed according to the previous work using silica column chromatography and dichloromethane as solvent (Péret-Almeida et al., 2005). The isolated curcumin was also crystallized with methanol–water. TLC experiments showed that the curcumin was completely free of demethoxycurcumin and bisdemethoxycurcumin (Moghaddam et al., 2009). Preparation of curcumin-activated sepharose 6-B complex Curcumin was immobilized on epoxy activated sepharose 6-B as chromatographic matrix, according to previously described method (Conboy et al., 2009). 0.5 g of epoxy activated sepharose 6B (sigma, USA) was suspended in 50 mL deionized water to swell and washed five times with 50 mL coupling buffer containing 50% dimethylformamide, 0.1 M Na2CO3, 10 mM NaOH at 4 °C. Swallowed beads were immediately coupled to curcumin. Briefly, curcumin solution (20 mM solution in 50% dimethylformamide, 0.1 M Na 2CO3 , 10 mM NaOH) was added to beads with the ratio of 2:1 (v: v) and incubated overnight at 30 °C. Unreacted epoxy groups were blocked by incubation and shaking beads with 1 M ethanolamine at 30 °C overnight. Control beads were prepared by incubation of the swallowed epoxy activated sepharose 6-B with 1 M ethanolamine (pH 11). The control and curcumin coupled beads were finally washed in three cycles of alternating low pH (0.1 M acetate buffer, pH = 4) and high pH (0.1 M Tris–HCl buffer, pH 8, containing 0.1 M NaCl) buffers and stored at 4 °C until use (Conboy et al., 2009). Separation of target proteins 200 mg of brain tissues was homogenized in 1 mL lysis buffer containing 50 mM Tris pH 7.4, 2 mM ethylene glycol tetraacetic acid, 2 mM ethylenediaminetetraacetic acid, 2 mM Na 3 VO 4, 1% Triton X-100 and 10 mM 2-mercaptoethanol, 2 μL complete protease inhibitor cocktail (Sigma P8340, USA), 0.2 (w/v) 1 mM phenylmethylsulfonyl
Affinity chromatography
2D gel electrophoresis Freeze-dried samples were dissolved in rehydration buffer containing 6 M urea, 2 M thiourea, 2% 3-[(3 cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 20% Bio-Lyte (BioRad) and concentration was adjusted to 1 μg protein/1 μL. For isoelectric focusing (IEF) experiment, 125 μL of lysate was loaded to 7 cm non linear immobilized pH gradient (IPG) strips (pH range: 3–10, BioRad). To separate curcumin target proteins based on their isoelectric point (pI), IPG strips were actively rehydrated for 12 h at 50 V and subsequently isoelectric focusing was carried out using PROTEAN IEF CELL (BioRad) at 4000 V for 11 h. The IPG strips were equilibrated in buffer containing 50 mM Tris–HCl, 6 M urea, 30% glycerol and 2.5% sodium dodecyl sulfate (SDS), 1.5% DTT for 20 min. For the second dimension, strips were placed on 12% SDS-PAGE and electrophoresis was carried out to separate proteins according to molecular weight. Gels were stained by MS-compatible silver staining method and protein spots were excised and transferred to Center of Genomic Sciences at University of Hong Kong for in gel digestion and curcumin targets identification (Rashedinia et al., 2013). In gel digestion Gel slices were destained and dehydrated in acetonitrile for 30 min and dried in vacufuge. Protein reduction was performed by incubation of gel slices in 10 mM DTT and alkylated by incubation in 55 mM iodoacetamide in dark at room temperature. Gel pieces were washed and diluted with 10 mM ammonium bicarbonate before digestion. Gel pieces were covered with 12.5 ng/μL of trypsin and incubated 30 min at 4 °C. Gel pieces were incubated by 20 μL of 10 mM ammonium bicarbonate overnight at 37 °C. 100 μL stop solution containing 50% acetonitrile and 5% formic acid was added. Peptide extraction was performed in 3 steps. 100 mL of 100 mM ammonium bicarbonate in step 1, 100 mL extraction solutions (50% acetonitrile and 5% formic acid) in step 2 and 150 mL extraction solution in step 3 were added. To extract peptides, all fractions were pooled and dried down in vacufuge and then resuspended in 0.1% formic acid. Samples were desalted using ZipTip® μC-18 (Millipore). Eluted samples were stored at − 20 °C until use. Samples were prepared in MALDI matrix containing 10 mg/mL α-Cyano-4-hydroxycinnamic acid in 50% water/acetonitrile and 0.1% formic acid and dried before applying matrix.
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Mass analysis and data base searching for protein identification Mass analysis was performed using 4800 MALDI-TOF/TOF analyzer (ABI) in positive ion reflector mode. MASCOT (version 2.1.0, Matrix Science, London, UK) was used to analyze Mass Data. Data were BLASTed against both NCBInr and Swiss-Prot database. MASCOT parameters were set as follow: Fixed modification: Carbamidomethyl (Cysteine), Variable modification: Oxidation (Methionine), Taxonomy: mouse, MS/MS fragment tolerance: ±0.2 Da, Precursor mass tolerance: 75 ppm, Peptide charge: +1, monoisotopic. MASCOT cutoff scores N30 and confidence interval (C.I.) N 95% were accepted. Classifications and network interaction analysis for curcumin target proteins PANTHER (Protein analysis through evolutionary relationships) classification system (http://www.pantherdb.org) was used for the classification of proteins according to biological function and process. Moreover, STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins) as a biological database and web resource (http:// www.string.embl.de) was used to demonstrate protein–protein interaction of identified curcumin target proteins. Results In order to determine novel targets of curcumin in the brain tissue, curcumin was immobilized on beads and pull down assay was performed, then proteins were separated and identified by 2D gel electrophoresis and MS analysis respectively as described in Material and methods section. Several spots on silver-stained 2D gels were detected as targets of curcumin but most of them were too low in abundance to let identification by mass spectrometry (Fig. 1). 11 proteins were identified among the detected spots by MALDI-TOF-TOF. MASCOT search engine was used for data analysis. All confidence intervals of identified proteins were 100%. The information of identified proteins including Protein name/gene names, Sample name, Swiss-Prot accession number, Confidence interval(C.I.%), Protein score, Sequence coverage (%), theoretical MW (Da)/pI, Unique peptides detected, sequence of identified peptide with the highest ion score were listed in Table 1. Swiss-Prot accession numbers of curcumin target proteins
were submitted to PANTHER database and categorized based on biological process (Fig. 2). The identified proteins were: Creatine kinase, gamma enolase, annexin 5, phosphoglycerate mutase 1, 14-3-3 delta alpha (14-3-3 alpha and delta are the phosphorylated forms of Raf-activating 14-3-3 beta and zeta), tubulin beta, beta actin like protein, 14-3-3 tata and gamma, peroxiredoxin 2, alpha enolase, fructose bisphosphate aldolase A. The proteins were divided in 6 classes including generation of precursor metabolites and energy, response to stimuli, cellular immune system process, metabolic process, cellular component organization, and cell process. Most of the identified proteins were classified in the metabolic process. Connectivity of identified proteins was demonstrated using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) (Fig. 3A). Moreover categories with more than 20 assigned proteins were shown (Fig. 3B). Discussion Utilizing a proteomics approach and affinity chromatography, we have identified 11 proteins which can be considered as potential curcumin target proteins in the brain tissue. While earlier studies have already identified some of these proteins as curcumin target, results of this study revealed some of novel curcumin target proteins for the first time in brain tissue. According to previous studies, curcumin can directly bind transcription factors, growth factors, kinases, inflammatory cytokines, adhesion molecules, apoptosis-related proteins and others (Goel et al., 2008). The novelty of this study, compared to earlier studies, was that we identified the targets of curcumin in the whole brain tissue. Moreover, some of the identified targets, such as annexin 5, creatine kinase, and phospho glycerate mutase 1, have been reported for the first time. Because of phenolic hydroxyl group in its chemical structure, curcumin has a powerful antioxidant activity (Calabrese et al., 2008; Osawa et al., 1995). Here we attached curcumin to epoxy group on stationary phase through phenolic hydroxyl group on curcumin. Because of excess molar amount of curcumin, it was predicted that one curcumin molecule can couple to each bead. Our data also showed that curcumin bound to a variety of cellular proteins such as metabolic enzymes, structural proteins, antioxidant and proteins that are involved in the apoptosis pathway. We found that shock cognate 71 kDa (Hsc70) was one of the target proteins of curcumin. Hsp70 is classified as a
Fig. 1. SDS-PAGE and two-dimensional gel electrophoresis of curcumin targets in mouse brain.
6.8e−12 K.SLSQNYGVLKNDEGIAYR.G 2(2) 21936.1/5.2 14 281 Q61171 B14
100
1.3e−07 1.3e−05 1.3e−09 R.FSGWYDADLSPAGHEEAKR.G K.ADDGRPFPQVIK.S R.DNAGAATEEFIKR.A 3(3) 2(2) 6(6) 28927.9/6.67 39787.4/8.31 39769.4/6.67 16 7 10 374 209 326 Q9DBJ1 P05064 P05063 B11 B12 B13
100 100 100
6.4e−19 K.GNDISSGTVLSDYVGSGPPSGTGLHR.Y 2(2) 20988.4/5.19 17 287 P70296 B10
100
0.006 4.8e−08 4.1e−12 1.9e−13 2.6e−08 1.1e−10 R.YLTVAAVFR.G R.FPGQLNADLR.K R.YITGDQLGALYQDFVR.N K.SYELPDGQVITIGNER.F K.LKDDEVAQLR.K K.SVTEQGAELSNEER.N 2(1) 4(3) 5(4) 4(4) 2(2) 1(1) 50010.1/4.78 50255.2/4.79 47609.1/4.99 42051.9/5.28 36834.1/5.7 27924.8/4.7 4 8 17 12 5 5 119 276 551 501 243 162 Q9D6F9 P68372 P17183 P60710 P16125 P63101 B3 B4 B5 B7 B8 B9
100 100 100 100 100 100
5.1e−11 K.STAGDTHLGGEDFDNR.M 4(4) 71082.3/5.37 8 583 100 P63017 B1
Heat shock cognate 71 kDa protein/(Hsc70) Tubulin beta-4 chain/TBB4 Tubulin beta-2C chain/TBB2C Gamma-enolase/ENOG Actin, cytoplasmic 1/ACTB L-Lactate dehydrogenase B chain/LDHB Protein kinase c inhibitor protein 1/1433Z Phosphatidylethanolamine-binding protein 1/PEBP1 Phosphoglycerate mutase 1/PGAM Fructose-bisphosphate aldolase/ALDOA Fructose-bisphosphate aldolase C/ALDOC Peroxiredoxin-2/Prdx2
Swiss-Prot accession number Sample name (on Fig. 1) Protein name/gene names
Table 1 Identified curcumin target proteins in mice brain by MALDI/TOF/TOF.
Protein score C.I. %
Protein score
Sequence coverage (%)
Theoretical MW (Da)/pI
Unique peptides detected
Peptide sequence
Probability/expectation value
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chaperone family member with anti apoptotic effects. HSPs help the folding of misfolded proteins by preventing their aggregation. Previous studies demonstrated that Hsp70 was up-regulated in human neuroblastoma and schwannoma cell lines (Angelo et al., 2011; D'Agnano et al., 2012). Peroxiredoxin-2 (PRDX2) was another identified target of curcumin. Peroxiredoxin-2 reduces the level of H2O2 and other reactive oxygen species in cells. However curcumin has been recognized to be an antioxidant agent by reducing reactive oxygen species (ROS) in normal cells, numerous studies demonstrated that curcumin-induced apoptosis was due to oxidative stress and ROS production in transformed cells. In cancer cells curcumin increases ROS by irreversibly inhibiting of TrxR activity and increasing NADPH oxidase activity. Increase in ROS induces cell death in cancerous cell. This effect may explain, anti cancer activity of curcumin (D'Agnano et al., 2012; Jung et al., 2005). Whereas, curcumin improves antioxidant defense in normal cells through increase in glutathione (GSH) content by increasing catalase activity (Jung et al., 2005; Calabrese et al., 2008). Moreover, previous studies have shown overexpression of peroxiredoxin enzymes in transformed cells. Curcumin has anticancer activity because of activation of NF-E2-related factor 2 (Nrf2) followed by down-regulation of PRDX enzymes and ROSmediated DR5 (death receptor 5) upregulation. Apoptosis induced by curcumin is inhibited by the ectopic expression of peroxiredoxin II (Jung et al., 2005; D'Agnano et al., 2012). Besides, protein kinase C inhibitor protein was identified as another target of curcumin. It has been shown that a variety of antioxidant agents inhibit cellular protein kinase C. Polyphenolic compounds such as curcumin selectively react with the catalytic domain of PKC, inhibit cellular PKC and interfere with the action of tumor promoters. On the contrary, oxidants stimulate PKC through interaction with the regulatory domain and produce signal for cell growth and tumor promotion (Lin et al., 1997; Gopalakrishna and Jaken, 2000). Furthermore, earlier studies, as we found, have shown that lactate dehydrogenase (LDH) was a target of curcumin. LDH is a metabolic enzyme that involved in the conversion of pyruvate to lactate. LDH is extremely expressed by tumor cells. Cai and colleagues have also revealed that curcumin might induce decrease of LDH. This metabolic dysfunction could induce apoptosis in cancer cells (Kounelakis et al., 2010; Cai et al., 2013). This study indicated that phosphatidylethanolamine-binding protein (PEBP) was another target of curcumin. Downregulation of PEBP, also called Raf-1 kinase inhibitor protein, protects cancer cells against death in the metastatic cells. Binding of antioxidants, such as curcumin, can restore PEBP expression and suppress cancer metastasis (Keller et al., 2004; Fu et al., 2006). Microtubules and actin filaments are two major cytoskeletal components in all eukaryotic cells which are vital for the maintenance of cell morphology, intracellular transport, cell division and cell motility (Zhou and Giannakakou, 2005). According to our results curcumin binds to tubulin beta-4 chain (TBB4), tubulin beta-2c chain (TBB2C) and cytoplasmic actin (ACTB). It has been reported that curcumin binds to tubulin and inhibits its polymerization in vitro (Banerjee et al., 2010). Moreover, curcumin has shown some anti-proliferative effect in HeLa and MCF-7 cells through perturbation of microtubule structure and dynamics (Gupta et al., 2006). Our results showed that four proteins of curcumin targets are involved in glycolysis process, including gamma-enolase (ENOG), fructose-bisphosphate aldolase (ALDOA), fructose-bisphosphate aldolase C (ALDOC) and phosphoglycerate mutase 1 (PGAM). In agreement with our results, it has been reported that glycolysis inhibited and several enzymes associated in glycolysis/gluconeogenesis were down-regulated after neuroblastoma cell lines treated with curcumin (D'Agnano et al., 2012). In fact increasing glucose uptake and high level of aerobic glycolysis are characteristics of a wide spectrum of human cancers (Warburg, 1956). Based on these considerations, aerobic glycolysis targets are the other mechanism of antitumor effect of curcumin, and also it can be a promising agent for anticancer treatment. In vitro model of blood–brain barrier showed that curcumin can pass the blood–brain barrier (Perry et al., 2010).
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enolase, fructose bisphosphate aldolase A, annexin 5, creatine kinase, 14-3-3 delta alpha (14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta, 14-3-3 tata and gamma), peroxiredoxin 2. Conflict of interest The authors have declared no conflict of interest.
Acknowledgments and funding The authors are thankful to the vice chancellor of research at the Mashhad University of Medical Sciences for the financial support of this project. This study was part of the Pharm.D thesis of MMA. References
Fig. 2. Biological process classification curcumin target proteins in mice brain by PANTHER classification system.
Consequently curcumin can be a potential candidate for chemoprevention and therapeutic indications in brain cancer because of passing blood–brain barrier which affects on aforementioned findings in this study. Conclusion Over the last 60 years, more than 3000 studies have demonstrated that curcumin has antioxidant, antibacterial, antifungal, antiviral, antiinflammatory, antiproliferative, pro-apoptotic and anti-atherosclerotic effects. In this paper affinity-based target purification showed that curcumin can physically interact with proteins involved in antioxidant, glycolytic and metabolic activities may explain some of its anticarcinogenic and pharmacological effects (Zhou et al., 2011). Some of our identified targets were reported for the first time; annexin 5, creatine kinase, phosphoglycerate mutase 1. Other targets that previously were identified are tubulin beta, beta actin like protein, alpha enolase, gamma
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Proteomics screening of molecular targets of curcumin in mouse brain Zohreh Firouzi a,1, Parisa Lari b,1, Marzieh Rashedinia b,1, Mohammad Ramezani c, Mehrdad Iranshahi d,⁎, Khalil Abnous e,⁎ a
Department of Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
a r t i c l e
i n f o
Article history: Received 12 September 2013 Accepted 23 December 2013 Keywords: Affinity chromatography Curcuma longa L. Curcumin Proteomics Target deconvolution
a b s t r a c t Aims: Curcumin is one of the most important constituent of Curcuma longa L. with antioxidant, anti-inflammatory and anticancer effects. In this study, we investigated potential intracellular targets of curcumin by affinity chromatography based on target deconvolution. Identification of curcumin interacting proteins may help in evaluating biological and side effects of this natural compound. Main methods: Curcumin was immobilized through a linker to sepharose beads as solid matrix. Pull down assay was performed by passing tissue lysate of mouse brain through the column to enrich and purify curcumin interacting proteins. Then proteins were separated using two-dimensional gel electrophoresis and identified using MALDI/TOF/TOF mass spectrometry. Key findings: Our results show that curcumin physically binds to a wide range of cellular proteins including structural proteins, metabolic enzymes and proteins involved in apoptosis pathway. Significance: Finding curcumin interacting proteins may help in understanding a part of curcumin pharmacological effects. © 2014 Elsevier Inc. All rights reserved.
Introduction Curcumin, a polyphenol compound, is an active biological constituent of the perennial herb Curcuma longa L. Turmeric is an Indian spice derived from the rhizomes of the plant from Zingiberaceae family. Turmeric has a long history of use in Ayurvedic medicine as a treatment for inflammatory conditions (Goel et al., 2008). Recently several studies have been performed on curcumin to reveal its cellular targets. For example, targets of curcumin in neuroblastoma cell line have been already identified (D'Agnano et al., 2012). Numerous studies revealed that curcumin has antioxidant and radical scavenger, anti-inflammatory, anti-infectious, cardioprotective and anticancer properties (Calabrese et al., 2008). Moreover, curcumin, as an antioxidant, could potentially inhibit lipid peroxidation in rat liver microsomes, erythrocyte membranes and brain homogenates (Chan et al., 2005; Ak and Gülçin, 2008). Various studies have shown that cardioprotective, neuroprotective, memory enhancing, and antiaging effects of curcumin are related to its high antioxidant capacity (Thiyagarajan and Sharma, 2004; Miriyala et al., 2007).
⁎ Corresponding authors. Tel.: +98 511 882 3268; fax: +98 511 882 3251. E-mail addresses: [email protected] (M. Iranshahi), [email protected] (K. Abnous). 1 Contributed equally to this project. 0024-3205/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.200
In addition curcumin has the ability to inhibit the proliferation of an extremely wide array of cancer cell types. Other study showed that curcumin inhibited tumor necrosis factor (TNF-α) and induced apoptosis in PC-12 cells through modulating mRNA expression of Bcl-2 family proteins (Shishodia et al., 2007). Previous studies have shown that curcumin has a neuroprotective effect in some central nervous system such as cerebral ischemia, traumatic brain injury and memory impairment (Miriyala et al., 2007; Shishodia et al., 2007). Curcumin may show its neuroprotective effects through some mechanisms including anti-oxidation, anti-apoptosis, anti-inflammation, anti-amyloidogenic, metal chelating properties, preventing BBB damage, and reducing edema. Some clinical trials, such as application of curcumin Alzheimer's disease, are currently in progress (Sun et al., 2011). Mechanistic study of curcumin may provide a deeper understanding of the therapeutic potential of curcumin and other curcuminoids in neurological disorders (Baum et al., 2008). In this study affinity chromatography based target deconvolution was chosen to find potential targets of curcumin in the brain. Target deconvolution is a high-throughput technique to discover drug targets and predict potential therapeutic and side effects as well as toxicity of chemicals in early study of a new drug development (Terstappen et al., 2007; Raida, 2011). Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. In our study,
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curcumin was covalently coupled to epoxy activated sepharose as chromatographic matrix through phenolic group (Lee and Bogyo, 2013). Following the affinity chromatography-based purification of curcumin targets, proteins were separated using two-dimensional gel electrophoresis and were identified using, matrix-assisted laser desorption/ionization/time of flight (MALDI/TOF/TOF) and MASCOT database.
fluoride using a Polytron Homogenizer (Kinematica, Switzerland). After sonication (UP100H, Hielscher) for 40 s, homogenates were centrifuged (Hettich Universal 320R, Germany) at 25,000 g for 10 min at 4 °C and total protein contents in supernatants were determined using a Bradford protein assay kit (BioRad, #500-0002). The same amount of proteins was applied for each test (Osawa et al., 1995).
Material and methods
Tissue extracts were incubated with control beads for 30 min at 4 °C. During this contiguous the proteins that capable to link to the epoxy activated sepharose were out, while proteins that can be band to curcumin were free in supernatant. After centrifugation at 1000 g for 1 min, supernatants were collected and transferred to curcumin coupled beads and incubated for 30 min at 4 °C. Beads were washed four times, each time with 2 mL of binding buffer, and supernatants were discarded. Then curcumin interacting proteins were eluted with 2 mL of 2 M NaCl. The later step was repeated 3 times and fractions were pooled and dialyzed for against ddwater for 3 days at 4 °C using a membrane with a 2000 Da cutoff (Spectra, USA). Desalted samples were freeze dried and stored at − 80 °C until use (Kunnumakkara et al., 2008).
Materials and reagents Reagents were obtained from Sigma (USA). Deionized water was used for all experiments and all other chemical substances were purchased from Merck (Germany). Animals Animal study was approved by the Mashhad University of Medical Sciences Ethics Committee (#87639). Male BALB/c mice weighing 20–25 g were obtained from the animal house of Mashhad University of Medical Sciences. Mice were kept in a room at 25 ± 2 °C on a 12-h light/dark period and let to eat and drink ad libitum. 6 mice were sacrificed by decapitation and brain tissues were rinsed using 0.9% normal saline solution. Then tissues were quickly frozen in liquid nitrogen and stored at − 80 °C until further processing. Curcumin preparation Curcumin 65–70% was purchased from Sigma (Cat. # C 1386). The curcumin 65–70% further purification was performed according to the previous work using silica column chromatography and dichloromethane as solvent (Péret-Almeida et al., 2005). The isolated curcumin was also crystallized with methanol–water. TLC experiments showed that the curcumin was completely free of demethoxycurcumin and bisdemethoxycurcumin (Moghaddam et al., 2009). Preparation of curcumin-activated sepharose 6-B complex Curcumin was immobilized on epoxy activated sepharose 6-B as chromatographic matrix, according to previously described method (Conboy et al., 2009). 0.5 g of epoxy activated sepharose 6B (sigma, USA) was suspended in 50 mL deionized water to swell and washed five times with 50 mL coupling buffer containing 50% dimethylformamide, 0.1 M Na2CO3, 10 mM NaOH at 4 °C. Swallowed beads were immediately coupled to curcumin. Briefly, curcumin solution (20 mM solution in 50% dimethylformamide, 0.1 M Na 2CO3 , 10 mM NaOH) was added to beads with the ratio of 2:1 (v: v) and incubated overnight at 30 °C. Unreacted epoxy groups were blocked by incubation and shaking beads with 1 M ethanolamine at 30 °C overnight. Control beads were prepared by incubation of the swallowed epoxy activated sepharose 6-B with 1 M ethanolamine (pH 11). The control and curcumin coupled beads were finally washed in three cycles of alternating low pH (0.1 M acetate buffer, pH = 4) and high pH (0.1 M Tris–HCl buffer, pH 8, containing 0.1 M NaCl) buffers and stored at 4 °C until use (Conboy et al., 2009). Separation of target proteins 200 mg of brain tissues was homogenized in 1 mL lysis buffer containing 50 mM Tris pH 7.4, 2 mM ethylene glycol tetraacetic acid, 2 mM ethylenediaminetetraacetic acid, 2 mM Na 3 VO 4, 1% Triton X-100 and 10 mM 2-mercaptoethanol, 2 μL complete protease inhibitor cocktail (Sigma P8340, USA), 0.2 (w/v) 1 mM phenylmethylsulfonyl
Affinity chromatography
2D gel electrophoresis Freeze-dried samples were dissolved in rehydration buffer containing 6 M urea, 2 M thiourea, 2% 3-[(3 cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 20% Bio-Lyte (BioRad) and concentration was adjusted to 1 μg protein/1 μL. For isoelectric focusing (IEF) experiment, 125 μL of lysate was loaded to 7 cm non linear immobilized pH gradient (IPG) strips (pH range: 3–10, BioRad). To separate curcumin target proteins based on their isoelectric point (pI), IPG strips were actively rehydrated for 12 h at 50 V and subsequently isoelectric focusing was carried out using PROTEAN IEF CELL (BioRad) at 4000 V for 11 h. The IPG strips were equilibrated in buffer containing 50 mM Tris–HCl, 6 M urea, 30% glycerol and 2.5% sodium dodecyl sulfate (SDS), 1.5% DTT for 20 min. For the second dimension, strips were placed on 12% SDS-PAGE and electrophoresis was carried out to separate proteins according to molecular weight. Gels were stained by MS-compatible silver staining method and protein spots were excised and transferred to Center of Genomic Sciences at University of Hong Kong for in gel digestion and curcumin targets identification (Rashedinia et al., 2013). In gel digestion Gel slices were destained and dehydrated in acetonitrile for 30 min and dried in vacufuge. Protein reduction was performed by incubation of gel slices in 10 mM DTT and alkylated by incubation in 55 mM iodoacetamide in dark at room temperature. Gel pieces were washed and diluted with 10 mM ammonium bicarbonate before digestion. Gel pieces were covered with 12.5 ng/μL of trypsin and incubated 30 min at 4 °C. Gel pieces were incubated by 20 μL of 10 mM ammonium bicarbonate overnight at 37 °C. 100 μL stop solution containing 50% acetonitrile and 5% formic acid was added. Peptide extraction was performed in 3 steps. 100 mL of 100 mM ammonium bicarbonate in step 1, 100 mL extraction solutions (50% acetonitrile and 5% formic acid) in step 2 and 150 mL extraction solution in step 3 were added. To extract peptides, all fractions were pooled and dried down in vacufuge and then resuspended in 0.1% formic acid. Samples were desalted using ZipTip® μC-18 (Millipore). Eluted samples were stored at − 20 °C until use. Samples were prepared in MALDI matrix containing 10 mg/mL α-Cyano-4-hydroxycinnamic acid in 50% water/acetonitrile and 0.1% formic acid and dried before applying matrix.
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Mass analysis and data base searching for protein identification Mass analysis was performed using 4800 MALDI-TOF/TOF analyzer (ABI) in positive ion reflector mode. MASCOT (version 2.1.0, Matrix Science, London, UK) was used to analyze Mass Data. Data were BLASTed against both NCBInr and Swiss-Prot database. MASCOT parameters were set as follow: Fixed modification: Carbamidomethyl (Cysteine), Variable modification: Oxidation (Methionine), Taxonomy: mouse, MS/MS fragment tolerance: ±0.2 Da, Precursor mass tolerance: 75 ppm, Peptide charge: +1, monoisotopic. MASCOT cutoff scores N30 and confidence interval (C.I.) N 95% were accepted. Classifications and network interaction analysis for curcumin target proteins PANTHER (Protein analysis through evolutionary relationships) classification system (http://www.pantherdb.org) was used for the classification of proteins according to biological function and process. Moreover, STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins) as a biological database and web resource (http:// www.string.embl.de) was used to demonstrate protein–protein interaction of identified curcumin target proteins. Results In order to determine novel targets of curcumin in the brain tissue, curcumin was immobilized on beads and pull down assay was performed, then proteins were separated and identified by 2D gel electrophoresis and MS analysis respectively as described in Material and methods section. Several spots on silver-stained 2D gels were detected as targets of curcumin but most of them were too low in abundance to let identification by mass spectrometry (Fig. 1). 11 proteins were identified among the detected spots by MALDI-TOF-TOF. MASCOT search engine was used for data analysis. All confidence intervals of identified proteins were 100%. The information of identified proteins including Protein name/gene names, Sample name, Swiss-Prot accession number, Confidence interval(C.I.%), Protein score, Sequence coverage (%), theoretical MW (Da)/pI, Unique peptides detected, sequence of identified peptide with the highest ion score were listed in Table 1. Swiss-Prot accession numbers of curcumin target proteins
were submitted to PANTHER database and categorized based on biological process (Fig. 2). The identified proteins were: Creatine kinase, gamma enolase, annexin 5, phosphoglycerate mutase 1, 14-3-3 delta alpha (14-3-3 alpha and delta are the phosphorylated forms of Raf-activating 14-3-3 beta and zeta), tubulin beta, beta actin like protein, 14-3-3 tata and gamma, peroxiredoxin 2, alpha enolase, fructose bisphosphate aldolase A. The proteins were divided in 6 classes including generation of precursor metabolites and energy, response to stimuli, cellular immune system process, metabolic process, cellular component organization, and cell process. Most of the identified proteins were classified in the metabolic process. Connectivity of identified proteins was demonstrated using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) (Fig. 3A). Moreover categories with more than 20 assigned proteins were shown (Fig. 3B). Discussion Utilizing a proteomics approach and affinity chromatography, we have identified 11 proteins which can be considered as potential curcumin target proteins in the brain tissue. While earlier studies have already identified some of these proteins as curcumin target, results of this study revealed some of novel curcumin target proteins for the first time in brain tissue. According to previous studies, curcumin can directly bind transcription factors, growth factors, kinases, inflammatory cytokines, adhesion molecules, apoptosis-related proteins and others (Goel et al., 2008). The novelty of this study, compared to earlier studies, was that we identified the targets of curcumin in the whole brain tissue. Moreover, some of the identified targets, such as annexin 5, creatine kinase, and phospho glycerate mutase 1, have been reported for the first time. Because of phenolic hydroxyl group in its chemical structure, curcumin has a powerful antioxidant activity (Calabrese et al., 2008; Osawa et al., 1995). Here we attached curcumin to epoxy group on stationary phase through phenolic hydroxyl group on curcumin. Because of excess molar amount of curcumin, it was predicted that one curcumin molecule can couple to each bead. Our data also showed that curcumin bound to a variety of cellular proteins such as metabolic enzymes, structural proteins, antioxidant and proteins that are involved in the apoptosis pathway. We found that shock cognate 71 kDa (Hsc70) was one of the target proteins of curcumin. Hsp70 is classified as a
Fig. 1. SDS-PAGE and two-dimensional gel electrophoresis of curcumin targets in mouse brain.
6.8e−12 K.SLSQNYGVLKNDEGIAYR.G 2(2) 21936.1/5.2 14 281 Q61171 B14
100
1.3e−07 1.3e−05 1.3e−09 R.FSGWYDADLSPAGHEEAKR.G K.ADDGRPFPQVIK.S R.DNAGAATEEFIKR.A 3(3) 2(2) 6(6) 28927.9/6.67 39787.4/8.31 39769.4/6.67 16 7 10 374 209 326 Q9DBJ1 P05064 P05063 B11 B12 B13
100 100 100
6.4e−19 K.GNDISSGTVLSDYVGSGPPSGTGLHR.Y 2(2) 20988.4/5.19 17 287 P70296 B10
100
0.006 4.8e−08 4.1e−12 1.9e−13 2.6e−08 1.1e−10 R.YLTVAAVFR.G R.FPGQLNADLR.K R.YITGDQLGALYQDFVR.N K.SYELPDGQVITIGNER.F K.LKDDEVAQLR.K K.SVTEQGAELSNEER.N 2(1) 4(3) 5(4) 4(4) 2(2) 1(1) 50010.1/4.78 50255.2/4.79 47609.1/4.99 42051.9/5.28 36834.1/5.7 27924.8/4.7 4 8 17 12 5 5 119 276 551 501 243 162 Q9D6F9 P68372 P17183 P60710 P16125 P63101 B3 B4 B5 B7 B8 B9
100 100 100 100 100 100
5.1e−11 K.STAGDTHLGGEDFDNR.M 4(4) 71082.3/5.37 8 583 100 P63017 B1
Heat shock cognate 71 kDa protein/(Hsc70) Tubulin beta-4 chain/TBB4 Tubulin beta-2C chain/TBB2C Gamma-enolase/ENOG Actin, cytoplasmic 1/ACTB L-Lactate dehydrogenase B chain/LDHB Protein kinase c inhibitor protein 1/1433Z Phosphatidylethanolamine-binding protein 1/PEBP1 Phosphoglycerate mutase 1/PGAM Fructose-bisphosphate aldolase/ALDOA Fructose-bisphosphate aldolase C/ALDOC Peroxiredoxin-2/Prdx2
Swiss-Prot accession number Sample name (on Fig. 1) Protein name/gene names
Table 1 Identified curcumin target proteins in mice brain by MALDI/TOF/TOF.
Protein score C.I. %
Protein score
Sequence coverage (%)
Theoretical MW (Da)/pI
Unique peptides detected
Peptide sequence
Probability/expectation value
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chaperone family member with anti apoptotic effects. HSPs help the folding of misfolded proteins by preventing their aggregation. Previous studies demonstrated that Hsp70 was up-regulated in human neuroblastoma and schwannoma cell lines (Angelo et al., 2011; D'Agnano et al., 2012). Peroxiredoxin-2 (PRDX2) was another identified target of curcumin. Peroxiredoxin-2 reduces the level of H2O2 and other reactive oxygen species in cells. However curcumin has been recognized to be an antioxidant agent by reducing reactive oxygen species (ROS) in normal cells, numerous studies demonstrated that curcumin-induced apoptosis was due to oxidative stress and ROS production in transformed cells. In cancer cells curcumin increases ROS by irreversibly inhibiting of TrxR activity and increasing NADPH oxidase activity. Increase in ROS induces cell death in cancerous cell. This effect may explain, anti cancer activity of curcumin (D'Agnano et al., 2012; Jung et al., 2005). Whereas, curcumin improves antioxidant defense in normal cells through increase in glutathione (GSH) content by increasing catalase activity (Jung et al., 2005; Calabrese et al., 2008). Moreover, previous studies have shown overexpression of peroxiredoxin enzymes in transformed cells. Curcumin has anticancer activity because of activation of NF-E2-related factor 2 (Nrf2) followed by down-regulation of PRDX enzymes and ROSmediated DR5 (death receptor 5) upregulation. Apoptosis induced by curcumin is inhibited by the ectopic expression of peroxiredoxin II (Jung et al., 2005; D'Agnano et al., 2012). Besides, protein kinase C inhibitor protein was identified as another target of curcumin. It has been shown that a variety of antioxidant agents inhibit cellular protein kinase C. Polyphenolic compounds such as curcumin selectively react with the catalytic domain of PKC, inhibit cellular PKC and interfere with the action of tumor promoters. On the contrary, oxidants stimulate PKC through interaction with the regulatory domain and produce signal for cell growth and tumor promotion (Lin et al., 1997; Gopalakrishna and Jaken, 2000). Furthermore, earlier studies, as we found, have shown that lactate dehydrogenase (LDH) was a target of curcumin. LDH is a metabolic enzyme that involved in the conversion of pyruvate to lactate. LDH is extremely expressed by tumor cells. Cai and colleagues have also revealed that curcumin might induce decrease of LDH. This metabolic dysfunction could induce apoptosis in cancer cells (Kounelakis et al., 2010; Cai et al., 2013). This study indicated that phosphatidylethanolamine-binding protein (PEBP) was another target of curcumin. Downregulation of PEBP, also called Raf-1 kinase inhibitor protein, protects cancer cells against death in the metastatic cells. Binding of antioxidants, such as curcumin, can restore PEBP expression and suppress cancer metastasis (Keller et al., 2004; Fu et al., 2006). Microtubules and actin filaments are two major cytoskeletal components in all eukaryotic cells which are vital for the maintenance of cell morphology, intracellular transport, cell division and cell motility (Zhou and Giannakakou, 2005). According to our results curcumin binds to tubulin beta-4 chain (TBB4), tubulin beta-2c chain (TBB2C) and cytoplasmic actin (ACTB). It has been reported that curcumin binds to tubulin and inhibits its polymerization in vitro (Banerjee et al., 2010). Moreover, curcumin has shown some anti-proliferative effect in HeLa and MCF-7 cells through perturbation of microtubule structure and dynamics (Gupta et al., 2006). Our results showed that four proteins of curcumin targets are involved in glycolysis process, including gamma-enolase (ENOG), fructose-bisphosphate aldolase (ALDOA), fructose-bisphosphate aldolase C (ALDOC) and phosphoglycerate mutase 1 (PGAM). In agreement with our results, it has been reported that glycolysis inhibited and several enzymes associated in glycolysis/gluconeogenesis were down-regulated after neuroblastoma cell lines treated with curcumin (D'Agnano et al., 2012). In fact increasing glucose uptake and high level of aerobic glycolysis are characteristics of a wide spectrum of human cancers (Warburg, 1956). Based on these considerations, aerobic glycolysis targets are the other mechanism of antitumor effect of curcumin, and also it can be a promising agent for anticancer treatment. In vitro model of blood–brain barrier showed that curcumin can pass the blood–brain barrier (Perry et al., 2010).
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enolase, fructose bisphosphate aldolase A, annexin 5, creatine kinase, 14-3-3 delta alpha (14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta, 14-3-3 tata and gamma), peroxiredoxin 2. Conflict of interest The authors have declared no conflict of interest.
Acknowledgments and funding The authors are thankful to the vice chancellor of research at the Mashhad University of Medical Sciences for the financial support of this project. This study was part of the Pharm.D thesis of MMA. References
Fig. 2. Biological process classification curcumin target proteins in mice brain by PANTHER classification system.
Consequently curcumin can be a potential candidate for chemoprevention and therapeutic indications in brain cancer because of passing blood–brain barrier which affects on aforementioned findings in this study. Conclusion Over the last 60 years, more than 3000 studies have demonstrated that curcumin has antioxidant, antibacterial, antifungal, antiviral, antiinflammatory, antiproliferative, pro-apoptotic and anti-atherosclerotic effects. In this paper affinity-based target purification showed that curcumin can physically interact with proteins involved in antioxidant, glycolytic and metabolic activities may explain some of its anticarcinogenic and pharmacological effects (Zhou et al., 2011). Some of our identified targets were reported for the first time; annexin 5, creatine kinase, phosphoglycerate mutase 1. Other targets that previously were identified are tubulin beta, beta actin like protein, alpha enolase, gamma
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