Proteomic Identification of eEF1A1 as a Molecular Target of Curcumol

Mar 27, 2017 - were enriched by KEGG pathway analysis. Specially, eEF1A1, a well-characterized actin binding protein, draws our attention. Curcumol ...
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Proteomic Identification of eEF1A1 as a Molecular Target of Curcumol for Suppressing Metastasis of MDA-MB-231 Cells Hongyi Qi,* Ling Ning, Zanyang Yu, Guojun Dou, and Li Li*

J. Agric. Food Chem. 2017.65:3074-3082. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/30/18. For personal use only.

College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, P.R. China ABSTRACT: Curcumol, a major volatile component in Rhizoma Curcumae, exhibits a potent antimetastatic effect on breast cancer cells. However, its molecular mechanism remains poorly understood. In this study, we employed two-dimensional gel electrophoresis-based proteomics to investigate the cellular targets of curcumol in MDA-MB-231 cells and identified 10 differentially expressed proteins. Moreover, Gene Ontology analysis revealed that these proteins are mainly involved in nine types of cellular components, seven different biological processes, and nine kinds of molecular functions, and 35 pathways (p < 0.05) were enriched by KEGG pathway analysis. Specially, eEF1A1, a well-characterized actin binding protein, draws our attention. Curcumol decreased eEF1A1 expression at both mRNA and protein levels. EEF1A1 expression was shown to be correlated with the invasiveness of cancer cells. Importantly, overexpression of eEF1A1 significantly reversed the inhibition of curcumol regarding the invasion and adhesion of MDA-MB-231 cells (p < 0.05). Together, our data suggest that eEF1A1 may be a potential molecular target underlying the antimetastatic effect of curcumol. KEYWORDS: curcumol, proteomics, breast cancer, metastasis, eEF1A1

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INTRODUCTION Breast cancer is the most common form of malignant tumor among women. In the United States, it is estimated that new diagnoses of breast cancer in women ranks first among all new cancer diagnoses, and deaths caused by breast cancer in women is next only to cancers of the lung and bronchus in 2016.1 In China, breast cancer is also the leading cause of malignant tumors in women in the years 2000−2011.2,3 However, the majority of deaths caused by breast cancer in women are not due to the primary tumor itself but rather are the result of metastasis to other organs, such as lung and brain, in the body.4 It has been shown that there was a 5-year survival rate of 98% for women with localized breast cancer, whereas only 27% for women with distant metastasis.5 Thus, targeting the metastasis process, such as motility, invasion into the second site, and adhesion profile to other tissues, may provide an alternative method for breast cancer therapy. Rhizoma Curcumae are the dry rhizomes derived from Curcuma wenyujin Y. H. Chen et C. Ling, Curcuma phaeocaulis Val., and Curcuma kwangsiensis S. G. Lee et C. F. Liang, which belong to the genus Curcuma in the family Zingiberaceae, as recorded in Chinese Pharmacopoeia 2015 edition.6 Rhizoma Curcumae is a common Chinese herbal medicine with a history of more than 1000 years. Moreover, it is also frequently used as health food supplement in many Chinese families.7 Curcumol with the structure of a guaiane-type sesquiterpenoid hemiketal is one of the main components in the volatile oil of Rhizoma Curcumae, whereas curcumin, the principal curcuminoid, exists as a nonvolatile component in Rhizoma Curcumae.8 As a wellknown natural compound, curcumin has been widely studied over the past few decades, whereas the activity of curcumol is still largely unknown. In recent years, the volatile oil of plants in the genus Curcuma has attracted extensive attention for the potent repellent, larvicidal, and insecticidal activities.9−13 Emerging evidence suggests that essential oils that produced © 2017 American Chemical Society

by plants to dissuade insects and other herbivores from eating them may have beneficial applications in the prevention and treatment of various human tumors.14−16 Interestingly, Rhizoma Curcumae has been traditionally prescribed for the treatment of gynecological cancer in Chinese clinical practice. Moreover, a growing body of scientific reports also support that the volatile oil of Rhizoma Curcumae processes anticancer potential as evidenced by data obtained from cancer cell lines and xenografted mice.8 It is noteworthy that curcumol was shown to inhibit gynecological tumor cells, such as MCF-7, MDA-MB-231, HeLa, and OV-UL-2.8,17 Our recent investigation also revealed that curcumol effectively inhibited the migration, invasion, and adhesion of breast cancer cells.2 However, the molecular targets regulated by curcumol for these activities are still largely unknown and require further in-depth investigation. To comprehensively identify the potential molecular targets responsible for the antimetastatic effect of curcumol, we applied a proteomic and bioinformatic approach in the current study. First, we compared the proteomic profiles of highly invasive MDA-MB-231 cells treated with curcumol or vehicle, and then differentially expressed proteins were identified by MALDITOF mass spectrometry. All of the differentially expressed proteins were analyzed using bioinformatic technology, including Gene Ontology (GO) and KEGG pathway enrichment analyses. Finally, the key molecular targets were verified by confirmation of their expression level and biological role. Received: Revised: Accepted: Published: 3074

February 6, 2017 March 24, 2017 March 27, 2017 March 27, 2017 DOI: 10.1021/acs.jafc.7b00573 J. Agric. Food Chem. 2017, 65, 3074−3082

Article

Journal of Agricultural and Food Chemistry

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MATERIALS AND METHODS

Table 1. Primers Used for Semi-Quantitative RT-PCR

Chemicals and Antibodies. Curcumol with a purity of more than 98% was obtained from Must Bio-Technology Co., Ltd. (Chengdu, China) and stored at −20 °C before use. Anti-eEF1A1 and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell Culture. Human breast cancer cells MDA-MB-231 were obtained from ATCC (Manassas, VA, USA). Cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 40 U/ml penicillin, and 40 μg/mL of streptomycin at 37 °C in a 5% CO2 and 95% humidified atmosphere. Two-Dimensional Gel Electrophoresis (2-DE) and Gel Analysis. MDA-MB-231 cells were treated with curcumol (20 μg/ mL) or vehicle alone for 24 h. The proteins were isolated with FOCUSTM Mammalian protein extraction kit (Sangon, Shanghai, China) and resolved by 2-DE according to the instructions provided by manufacturer (GE Healthcare, USA). The isoelectric focusing (IEF) was performed with a precast immobilized pH gradient (IPG) strip (24 cm, pH 3.0−10.0, linear gradient), and 1200 μg of cellular proteins were loaded onto an IPG strip, allowed to swell for 12 h, and then rehydrated for 24 h. The isoelectric focusing was conducted at 300 V for 0.5 h, at 700 V for 0.5 h, at 1,500 V for 1.5 h, over a gradient to 9,000 V for 3 h, and then at 9,000 V for 4 h. All of the IEF steps were conducted at 20 °C. After the first-dimensional IEF, the IPG gel strips were equilibrated in 2D equilibration buffer (Sangon, Shanghai, China) for 15 min. The free thiol groups were alkylated with an equilibration solution containing 2.5% iodoacetamide for another 20 min. After the IPG strips were placed on the top of the polyacrylamide gel slab, the proteins were separated in the second dimension in 12.5% SDS gels conducted at a current setting of 15 mA/gel for the initial 0.5 h and at 30 mA/gel thereafter; the temperature was maintained at 20 °C. The experiments were carried out in triplicate. The proteins resolved in the gel were visualized by the modified CBB R-250 staining method. The stained gels were scanned using ImageScanner III LabScan 6.0 (GE Healthcare) and analyzed using ImageMaster 2D Platinum Software (Version 5.0, GE Healthcare) following the user’s manual. In-Gel Digestion and Mass Spectrometry. The protein spots of interest were excised from the 2D gels. After destaining, the spots were digested with trypsin (sequencing grade, GE Healthcare), and the peptides were isolated following a previous report.18 Then, the samples were mixed with an equal volume of matrix solution of αcyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid and spotted onto the MALDI sample plates. The dried spots were analyzed using an Ultraflex-III (Bruker) MALDI TOF/ TOF mass spectrometer and subjected to peptide mass fingerprinting. The MS spectra were searched against the NCBI database using Mascot Daemon (Matrix Science, London, UK) as a client attached to the Mascot search protocol. The database searches had peptide mass tolerance set at approximately ±0.1 Da and one missed cleavage site. Protein Data Bioinformatic Analysis. The successfully identified proteins were subjected to Gene Ontology (GO) and pathway enrichment analyses. The GO analysis was performed against the DAVID database (http://david.abcc.ncifcrf.gov/). The pathway enrichment used the KEGG database (http://www.kegg.jp/) and analyzed the significance of the pathway. RT-PCR Analysis. Total RNA was isolated from MDA-MB-231 cells with a TriZol RNA extraction reagent (Invitrogen, CA, USA) and reverse transcribed using SuperScript III reverse transcription reagent (Invitrogen, CA, USA) as described by the manufacturer. Forward and reverse primers for EEF1A1,19 NRAS20 and ACAT221 are listed in Table 1. PCR amplification was set as follows: after an initial denaturation at 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s. The reaction ended with a final extension step at 72 °C for 5 min. The PCR products were then resolved by 1.2% agarose gels, stained with ethidium bromide, and visualized by UV. Plasmids and Transient Transfection. The pcMV3-eEF1A1 and pcMV3 vectors were obtained from Sino Biological Inc. (Beijing, China). Cells (50% confluence) were transfected with 2.5 μg of DNA

a

gene

product length

EEF1A1

229 bp

NRAS

428 bp

ACAT2

318 bp

GAPDH

202 bp

primer sequencesa F 5′-AACATTGTCGTCATTGGACA-3′ R 5′-ACTTGCTGGTCTCAAAT TTC-3′ F5′-GGAGCTTGAGGTTCTTGCTGGTGTG3′ R 5′-GCCAGTTCGTGGGCTTGTTTT-3′ F5′-CCAGAACAGGACAGAGAATGC-3′ R 5′-GAAGGCTCCACACCCACTT-3′ F5′-TGTTGCCATCAATGACCCCTT-3′ R 5′-CTCCACGACGTACTCAGCG-3′

F: forward primer; R: reverse primer.

using the transfection reagent Lipofectamine 2000 (Invitrogen, USA) following the protocol provided by the manufacturer. Transfected cells were first cultured in antibiotic-free medium for 6 h and then in fresh medium for 24 h, followed by further drug treatments. Western Blotting Analysis. Cells were lysed on ice with 1× RIPA buffer (Cell Signaling, MA, USA) containing complete proteinase inhibitors. Thirty micrograms of protein samples were applied for SDS-polyacrylamide gel electrophoresis, and the blotting was subsequently performed with a polyvinylidene fluoride (PVDF) membrane. The blots were then incubated with 5% BSA in TRIS phosphate-buffered saline solution containing Tween-20 (0.1%) (TBST) for 1 h at room temperature. The primary antibodies diluted in blocking buffer were incubated with corresponding blots overnight at 4 °C. The horseradish peroxidase-conjugated secondary antibodies were incubated with corresponding blots for 1 h at room temperature. Chemiluminescent visualization was performed by the gel imaging system (Tanon, China) with a commercial kit (GE Healthcare, Sweden). Band intensity was quantified and analyzed by the software of this gel imaging system. The intensity of each protein was first normalized to that of the corresponding β-actin and then each was further normalized to control. Boyden Chamber Invasion Assay. The invasion ability of MDAMB-231 cells was evaluated by a Boyden chamber with Matrigelcoated polycarbonate filters with 8 μm pore size in the Transwell (Corning, NY, USA). After pretreatment with curcumol (20 μg/mL) or vehicle for 24 h, the cells with a density of 1 × 105 cells/well were seeded in the upper chamber of the Transwell and cultured with serum-free medium. To the lower chamber was added medium supplemented with 10% FBS as a chemoattractive agent. Then, the cells in the upper surface of the filter membrane were carefully cleaned with a cotton swab after 24 h incubation. The cells that had crossed the Matrigel to the lower surface of the membrane were fixed with 100% methanol and stained with 0.05% crystal violet. Different fields were randomly chosen and captured under fluorescence microscopy (OLYMPUS IX71) at 100× magnification, and the cells were scored by manual counting. Inhibition rate was quantified with cells treating the vehicle as representing 100%. Adhesion Assay. MDA-MB-231 cells after pretreatment were seeded onto a 96-well plate coated with Matrigel. Following a 4 h incubation, attached cells were fixed in 100% methanol and stained with 0.05% crystal violet solution. Different fields were randomly chosen and captured using fluorescence microscopy (OLYMPUS IX71) at 100× magnification. Then, attached cells were scored by manual counting. Inhibition rate was quantified with cells treating the vehicle as representing 100%. Statistical Analysis. The experimental data were expressed as means ± SD for three independent experiments. An ANOVA test was used to calculate the significant difference in the study. A p-value of less than 0.05 was considered to be statistically significant.

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RESULTS Proteomic Identification of the Proteins Most Affected by Curcumol in MDA-MB-231 Cells. In the 3075

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Figure 1. Representative 2-DE maps of curcumol or vehicle-treated MDA-MB-231 cells. Cells were treated with 20 μg/mL of curcumol or vehicle for 24 h. The differentially expressed protein spots are marked on the maps, which are representative of three independent runs.

Table 2. MALDI/MS Identification of the Protein Spots in Response to Curcumol

a

spot no.

fold change

ther. Mr /pIa

protein description

accession no.

pep. no.b

score

A03 A06 A11 A12 A13 A14 A15 A16 B01 B03

−1.44 −1.25 −1.44 −1.29 −1.26 −1.46 −1.62 −10000 1.57 1.24

57.96/5.24 50.14/9.10 31.71/6.55 20.68/5.23 21.28/4.76 20.90/5.01 14.26/9.60 62.06/5.14 51.25/5.39 41.35/6.46

60 kDa heat shock protein elongation factor 1-alpha 1 39S ribosomal protein L46 chromobox protein homologue 3 proteasome subunit beta type-9 GTPase NRas activated RNA polymerase II transcriptional coactivator p15 keratin, type I cytoskeletal 9 keratin, type II cytoskeletal 7 acetyl-CoA acetyltransferase, cytosolic

P10809 P68104 Q9h2W6 Q13185 P28065 P01111 P53999 P35527 P08729 Q9BWD1

14 4 8 5 13 8 11 13 13 8

324 58 132 94 297 181 163 102 243 250

Theoretical molecular weight (kDa) and pI from the ExPASy database. bThe number of unique peptides identified by MS/MS sequencing.

most recent study, we demonstrated that curcumol suppressed breast cancer cell metastasis.2 For the potential molecular targets underlying the antimetastatic effect of curcumol to be elucidated, a 2D gel electrophoresis technique was used in this study to identify the differential proteins in MDA-MB-231 cells treated with vehicle or 20 μg/mL of curcumol, which was found to efficiently reduce the metastasis of MDA-MB-231 cells with no cytotoxicity.2 Figure 1 shows representative gels with marked protein spots. After detection, editing, and matching of the spots, the intensity of protein spots in the gel with curcumol-treated cells was quantified and compared with that in the gel with vehicle-treated cells using ImageMaster 2D platinum 5.0 software (GE Healthcare). We identified 19 spots as differentially expressed after curcumol treatment by applying a threshold of 1.2-fold variation. There are 16 down-regulated proteins and 3 up-regulated proteins. The spots are marked in Figure 1 with circles, arrows, and numbers. Then, these spots were isolated and digested with trypsin. The proteins were identified by MALDI-TOF-MS. Eventually, 10 proteins were successfully identified. The related information for each protein is summarized in Table 2. Gene Ontology (GO) Analysis. The differentially expressed proteins were subjected to GO analysis to classify the cellular components, biological processes, and molecular functions. As shown in Figure 2A, the identified proteins are mainly located in various cellular organelles (76%), including

nucleus (32%), mitochondrion (8%), endoplasmic reticulum (4%), Golgi apparatus (8%), lysosome (4%), ribosome (4%), and cytoskeleton (16%). The rest were from the plasma membrane (12%) and cytosol (12%). Moreover, the identified proteins were shown to participate in a variety of biological processes, especially tumor biology (Figure 2B), such as cellular process (proliferation, differentiation, cycle, death, autophagy, adhesion, etc.), metabolic process (metabolism of amino acid, lipid, DNA, etc.), response to stress, and signal transduction. Figure 2C shows that these proteins are correlated with various molecular functions, mainly including catalytic activity (18%), RNA binding (14%), enzyme binding (14%), ion binding (14%), and transcription regulator activity (14%). Pathway Enrichment of the Identified Proteins. We then further analyzed the biological pathways involving in these differentially expressed proteins. In total, 78 KEGG pathways were enriched for these identified proteins (data not shown). The top 35 pathways (p < 0.05) are shown in Table 3. It can be seen that a number of metabolic pathways were modulated in MDA-MB-231 cells treated with curcumol, including the metabolism of butanoate, glyoxylate, dicarboxylate, propanoate, tryptophan, pyruvate, fatty acids, and central in cancer. Notably, several cancer-related pathways are also interfered with by curcumol, such as thyroid cancer, bladder cancer, acute myeloid leukemia, endometrial cancer, nonsmall cell lung cancer, 3076

DOI: 10.1021/acs.jafc.7b00573 J. Agric. Food Chem. 2017, 65, 3074−3082

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Journal of Agricultural and Food Chemistry

Figure 2. Gene Ontology (GO) classification of the differentially expressed proteins caused by curcumol. The proteins are grouped into three GO terms: (A) cellular component, (B) biological process, and (C) molecular function.

expression of ACAT2 compared with those treated with vehicle (Figure 3B), which showed a consistent trend with that of the corresponding proteins. Analysis of eEF1A1 Protein Expression. As accumulating evidence demonstrated that eEF1A1 played a pivotal role in increasing the metastatic propensity of tumor cells,22−24 we thereafter payed special attention to eEF1A1. First, we determined the protein level of eEF1A1 using a specific antibody in a group of human cell lines. As shown in Figure 4A, a relatively high expression of eEF1A1 protein was detected in HL-60 and MDA-MB-231 cells and a moderate level was shown in HEK 293T and U87 cells, whereas a very weak level was shown in Kasumi-1, MCF-7, and U251 cells. Then, we examined the protein expression of eEF1A1 after 24 h treatment of MDA-MB-231 cells by vehicle or curcumol (10, 20, and 40 μg/mL). Figure 4B showed that a high basal protein level of eEF1A1 was observed in MDA-MB-231 cells treated with vehicle, whereas the protein level of eEF1A1 was significantly decreased after curcumol treatment. Overexpression of eEF1A1 Reversed the Inhibitory Effect of Invasion and Adhesion by Curcumol. Our recent investigation demonstrated that curcumol effectively suppressed the migration, invasion, and adhesion of breast cancer cells at noncytotoxic concentrations.2 To verify the role of eEF1A1 in the antimetastatic effect of curcumol, we first transfected MDA-MB-231 cells with pcMV or pcMV-eEF1A1. As shown in Figure 5A, remarkable overexpression of eEF1A1 protein was observed in MDA-MB-231 cells with pcMVeEF1A1 transfection compared to that with pcMV transfection. Then, the invasion assay was performed with Matrigel-coated 24-well Boyden chambers after MDA-MB-231 cells with pcMV or pcMV-eEF1A1 transfection were pretreated with curcumol for 24 h. As shown in Figure 5B, curcumol (20 μg/mL) significantly blocked the invasion of MDA-MB-231 cells transfected with only pcMV compared to that of the same cells treated with vehicle (p < 0.05). Notably, overexpression of eEF1A1 by transfecting pcMV-eEF1A1 greatly promoted the invasion of MDA-MB-231 cells compared with that of cells transfected with only pcMV (p < 0.001). Importantly, overexpression of eEF1A1 remarkably attenuated the inhibitory effect of curcumol on the invasion of MDA-MB-231 cells (p < 0.05). Furthermore, our results also showed that overexpression of eEF1A1 facilitated the adhesion of MDA-MB-231 cells onto the Matrigel (p < 0.01), whereas the inhibitory effect of curcumol on the adhesion was also significantly reversed by overexpression of eEF1A1 (p < 0.05) (Figure 5C).

glioma, melanoma, renal cell carcinoma, and chronic myeloid leukemia. Verification of Differentially Expressed Proteins by RT-PCR. Among the 10 identified proteins, we found that three of them may be correlated to the antimetastatic effect of curcumol on breast cancer cells. These target proteins include elongation factor 1-alpha 1 (eEF1A1/EF-1α), GTPase NRas (NRAS), and acetyl-CoA acetyltransferase, cytosolic (ACAT2). First, we confirmed the effect of curcumol on these three differentially expressed proteins by enlarging the corresponding protein spot images (Figure 3A). Then, we further examined their expression at the transcriptional level by RT-PCR after MDA-MB-231 cells were treated with vehicle or curcumol (10, 20, and 40 μg/mL) for 24 h. As a result, curcumol decreased the mRNA level of eEF1A1 and NRAS and increased the

DISCUSSION Curcumol is one of the volatile components in Rhizoma Curcumae, which is well-known as a herbal medicine and health food supplement in China. Recent investigations showed that curcumol exhibited an inhibitory effect on various gynecological tumor cells.8,17 The most recent study from our group demonstrated that curcumol remarkably suppressed the metastatic process of breast cancer cells.2 In this study, a proteomic and bioinformatic strategy was applied to comprehensively elucidate the molecular targets responsible for the antimetastatic effect of curcumol. First, 2D gel electrophoresis was used to separate the proteins extracted from the highly invasive MDA-MB-231 cells treated by curcumol and vehicle. Nineteen proteins were identified as differentially expressed by comparing the protein profiles. Of

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Journal of Agricultural and Food Chemistry Table 3. Significantly Enriched KEGG Pathways of Differentially Expressed Proteins no.

pathway name

ID

genes

count

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

legionellosis synthesis and degradation of ketone bodies terpenoid backbone biosynthesis butanoate metabolism glyoxylate and dicarboxylate metabolism propanoate metabolism thyroid cancer tryptophan metabolism fat digestion and absorption pyruvate metabolism lysine degradation proteasome bladder cancer fatty acid degradation valine, leucine and isoleucine degradation fatty acid metabolism type I diabetes mellitus acute myeloid leukemia endometrial cancer nonsmall cell lung cancer VEGF signaling pathway RNA degradation long-term depression Fc epsilon RI signaling pathway glioma melanoma central carbon metabolism in cancer B cell receptor signaling pathway longevity regulating pathway−multiple species long-term potentiation renal cell carcinoma prolactin signaling pathway chronic myeloid leukemia EGFR tyrosine kinase inhibitor resistance gap junction

hsa05134 hsa00072 hsa00900 hsa00650 hsa00630 hsa00640 hsa05216 hsa00380 hsa04975 hsa00620 hsa00310 hsa03050 hsa05219 hsa00071 hsa00280 hsa01212 hsa04940 hsa05221 hsa05213 hsa05223 hsa04370 hsa03018 hsa04730 hsa04664 hsa05214 hsa05218 hsa05230 hsa04662 hsa04213 hsa04720 hsa05211 hsa04917 hsa05220 hsa01521 hsa04540

P68104, P10809 Q9BWD1 Q9BWD1 Q9BWD1 Q9BWD1 Q9BWD1 P01111 Q9BWD1 Q9BWD1 Q9BWD1 Q9BWD1 P28065 P01111 Q9BWD1 Q9BWD1 Q9BWD1 P10809 P01111 P01111 P01111 P01111 P10809 P01111 P01111 P01111 P01111 P01111 P01111 P01111 P01111 P01111 P01111 P01111 P01111 P01111

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

these 19 proteins, 10 were finally identified by MALDI-TOFMS. For comprehensive insight into the identified proteins to be obtained, bioinformation on the 10 identified proteins including GO and KEGG pathway analyses was obtained. GO analysis indicated that the identified proteins were mainly distributed into nine types of cellular components, participating in seven different biological processes and exhibiting nine kinds of molecular functions. KEGG pathway enrichment analysis showed that the identified proteins are involved in numerous signaling pathways. Of note, NRAS (P01111) was found to be included in various cancer-related pathways, such as thyroid cancer, bladder cancer, acute myeloid leukemia, endometrial cancer, nonsmall cell lung cancer, glioma, melanoma, renal cell carcinoma, and chronic myeloid leukemia. In addition, ACAT2 (Q9BWD1) was involved in diverse metabolic pathways, such as those of butanoate, glyoxylate, dicarboxylate, propanoate, tryptophan, pyruvate, and fatty acids. It has been reported that downregulation of NRAS was closely correlated with the antimetastatic effect of isothiouronium salts in melanoma cells,25 miR-515-5p in breast cancer cells26 and introduction of let-7i to mutant p53 cells.27 Additionally, a strong correlation was found among metastasis, ACAT2, and hydroxyl-coenzyme A dehydrogenase, alpha subunit (HADHA) downexpression

p-value 5.24 6.52 1.22 1.68 1.72 2.02 2.13 2.17 2.21 2.32 2.51 2.70 2.74 2.77 2.89 3.00 3.11 3.37 3.37 3.52 3.74 3.89 3.97 4.00 4.08 4.08 4.23 4.26 4.30 4.34 4.41 4.48 4.56 4.59 4.96

× × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × ×

10−04 10−03 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02 10−02

and poorer prognosis on survival of clear cell renal cell carcinoma (ccRCC).28 Moreover, another protein, eEF1A1 (P68104), drew our special attention. EEF1A1 is a member of the abundant evolutionarily conserved elongation factor proteins that are normally responsible for binding aminoacyl-tRNA to the ribosome during protein synthesis.29 Thus, eEF1A1 was found to be involved in the pathway of RNA transport (hsa03013) according to KEGG pathway enrichment analysis. However, accumulating evidence indicates that eEF1A1 plays a pivotal role in increasing the metastatic propensity of tumor cells. Generally, the metastatic process is regarded as similar to the chemotaxis of motile cells along gradients of cytokines.30,31 The cellular motile machinery consisted of the actin cytoskeleton playing a crucial role in the chemotactic response, and thus, it may also be very important for metastasis.23 More specifically, it is believed that the stabilization of polarized surface projections, which contain newly polymerized actin, by actin binding proteins will prompt the cell to move in the specific direction of the cytokine gradient.23,32 Notably, eEF1A1 has also been reported as a well-characterized actinbinding protein in addition to functioning as a polypeptide elongation factor.33 It has been shown that eEF1A1 colocalizes with filamentous actin (F-actin) in cells, and its distribution is 3078

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promising target for suppressing the metastasis of aggressive tumor cells. To evaluate whether the protein changes discovered by proteomics are correlated with the alterations of the mRNA levels at the transcription level, we evaluated eEF1A1, NRAS, and ACAT2 by RT-PCR. Our results demonstrated that mRNA levels of eEF1A1 and NRAS were reduced by curcumol, whereas the ACAT2 level was enhanced, which is in accordance with that of the corresponding protein counterparts. Furthermore, to explore the correlation of the eEF1A1 level with invasiveness, we examined eEF1A1 protein expression in a group of human cell lines. As a result, eEF1A1 was detected at various levels, whereas HL-60, MDA-MB-231, and U87 cells showed a higher level of eEF1A1 than their counterparts Kasumi-1, MCF-7, and U251 cells. A previous study demonstrated that estrogen receptor α-negative breast cancer cell line MDA-MB-231 is more invasive than estrogen receptor α-positive MCF-7.35 Similarly, U87 exhibited higher metastatic potential to lungs or other organs than U251 when grown orthotopically.36 Conceivably, eEF1A1 expression is positively correlated with tumor invasiveness. It is worth noting that a moderate level of eEF1A1 was also observed in the normal cell line HEK 293T, which may be interpreted that eEF1A1 can normally function as a housekeeping gene product facilitating the maintenance of cell growth and/or survival.37 To understand the pharmacological regulation of eEF1A1 expression, we determined the influence of curcumol on eEF1A1 protein level. Expectedly, curcumol obviously inhibited the expression of eEF1A1. Our previous study demonstrates that curcumol blocked breast cancer cell metastasis by suppressing MMP-9, which was related to inhibition of the NF-κB signaling pathway by curcumol. 2 eEF1A1 has been shown to phosphorylate STAT3 at serine 727, which is necessary for NF-κB/STAT3-stimulated IL-6 expression, indicating eEF1A1 is positively related to the NF-κB signaling pathway. Thus, deserving of further exploration was whether curcumolmediated inhibition of eEF1A1 was correlated with the suppression of metastasis of breast cancer cells.38 To determine the role of eEF1A1 in the antimetastatic effect of curcumol, we overexpressed eEF1A1 in MDA-MB-231 cells with pcMVeEF1A1 transfection. Then, we evaluated the effect of eEF1A1 overexpression and/or curcumol intervention on the invasion and adhesion ability of MDA-MB-231 cells. Moreover, considering the similarity of the metastatic process to the chemotaxis and potential role of eEF1A1 in the organization of the actin cytoskeleton during chemotaxis, a Boyden chamber invasion assay was performed with complete medium with 10% FBS as chemoattractive agent. Consequently, overexpression of eEF1A1 promoted both invasion and adhesion ability of MDAMB-231 cells, suggesting the critical role of eEF1A1 in the increased metastatic propensity, which is consistent with previous reports.22,23 Importantly, overexpression of eEF1A1 reversed the inhibitory effect of curcumol on the invasion and adhesion of MDA-MB-231 cells, indicating that eEF1A1 may be a key molecular target responsible for the antimetastatic effect of curcumol. Similarly, a previous study demonstrated that narciclasine, an Amaryllidaceae isocarbostyril, at nontoxic doses enhanced the survival of mice bearing metastatic apoptosis-resistant melanoma xenografts in their brain by targeting eEF1A1.39 This study together with our previous study2 demonstrate that curcumol ranging from 10 to 40 μg/ mL remarkably suppressed metastasis and inhibited eEF1A1 expression in breast cancer cells without causing obvious

Figure 3. Confirmation of eEF1A1, NRAS, and ACAT2 expression by RT-PCR. (A) Enlarged spots of the three proteins. (B) mRNA level of the three genes. MDA-MB-231 cells were treated with vehicle or curcumol at the indicated concentrations for 24 h.

Figure 4. Analysis of eEF1A1 protein expression. (A) Detection of eEF1A1 protein expression in various cell lines. The cell lines as indicated were seeded and cultured for 24 h. (B) Detection of eEF1A1 protein level in MDA-MB-231 cells with the treatment of curcumol as indicated for 24 h. The protein was extracted and analyzed using Western blotting. The Western blots were representative of three independent experiments.

associated with changes in the organization of the actin cytoskeleton during chemotaxis.34 Importantly, the overexpression of eEF1A1 mRNA has been correlated with an increased metastatic propensity.22 A recent study revealed that downregulation of eEF1A1 by RNA interference (RNAi) inhibited the proliferation, invasion, and migration of prostate cancer cells.23 Moreover, eEF1A1 was identified as a candidate serum biomarker related to the metastatic progression of human prostate cancer.24 Thus, eEF1A1 appears to be a 3079

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Journal of Agricultural and Food Chemistry

Figure 5. Overexpression of eEF1A1 reversed the inhibitory effect on invasion and adhesion by curcumol. (A) The level of eEF1A1 was measured by Western blotting after MDA-MB-231 cells were transfected with pcMV or pcMV-eEF1A1 for 24 h. (B) Boyden chamber invasion assay. After being transfected with pcMV or pcMV-eEF1A1 for 24 h, MDA-MB-231 cells were pretreated with curcumol (20 μg/mL) or vehicle for 24 h and then seeded in the chamber with coated Matrigel of a 24-transwell plate for another 24 h. The invasion assay was then conducted according to the procedure in the Materials and Methods. (C) Adhesion assay. After being transfected with pcMV or pcMV-eEF1A1 for 24 h, MDA-MB-231 cells were preincubated with curcumol (20 μg/mL) or vehicle for 24 h and seeded onto a 96-well plate coated with Matrigel for 4 h. Then, attached cells were stained and counted according to the procedure in the Materials and Methods. Randomly chosen fields were obtained using an optical microscope (100× magnification). Values represent mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control.

cytotoxicity. It has been shown that the plasma concentration of curcumol reached almost 50 μg/mL after rats were intravenously administered a single dose of 30 mg/kg of curcumol.40 However, requiring further investigation is whether the effective plasma concentration range of curcumol can be achieved after oral administration of Rhizoma Curcumae or curcumol and whether a lower concentration of curcumol is still effective for metastasis inhibition, especially for in vivo studies. In conclusion, our proteomic analysis demonstrated that curcumol altered the expression of several biologically important proteins and diverse signaling pathways in MDAMB-231 cells. In particular, eEF1A1, a well-characterized actin binding protein, was discovered to be reduced by curcumol at

both mRNA and protein levels. Moreover, the eEF1A1 level was shown to be correlated to the invasiveness of cancer cells. Finally, we found that eEF1A1 played a critical role in the inhibition of curcumol on the invasion and adhesion of MDAMB-231 cells. These results suggest that eEF1A1 may be a potential molecular target of curcumol, which will provide a better understanding of the antimetastatic effect of curcumol.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; Tel./Fax: +86-23-68251225. *E-mail: [email protected]; Tel./Fax: +86-23-68251225. 3080

DOI: 10.1021/acs.jafc.7b00573 J. Agric. Food Chem. 2017, 65, 3074−3082

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Journal of Agricultural and Food Chemistry ORCID

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Hongyi Qi: 0000-0003-0564-3892 Funding

This work was supported by National Science Foundation of China projects (Nos. 81373903 and 81202946), Chongqing Project of Science and Technology Talent Cultivation (cstc2013kjrc-qnrc1002), the Key Project of Fundamental Research Fund for the Central Universities (XDJK2016B040 and XDJK2016B034) and National Training Program of Innovation and Entrepreneurship for Undergraduates (201510635019). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 2-DE, two-dimensional gel electrophoresis; GO, Gene Ontology; IPG, immobilized pH gradient; IEF, isoelectric focusing; RT-PCR, reverse transcriptase-polymerase chain reaction; eEF1A1, elongation factor 1-alpha 1; NRAS, GTPase NRas; ACAT2, acetyl-CoA acetyltransferase, cytosolic

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