Proteomic Identification of eEF1A1 as a Molecular Target of 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., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00573 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Journal of Agricultural and Food Chemistry

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, Li Li* College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, P.R. China

*Corresponding authors: Hongyi Qi: College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing 400716, China. E-mail: [email protected]; Tel./Fax: +86-23-68251225 Li Li: College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing 400716, China. E-mail: [email protected]; Tel./Fax: +86-23-68251225

Running title: eEF1A1 identified as anti-metastasis target of curcumol

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ACS Paragon Plus Environment


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ABSTRACT

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Curcumol, a major volatile component in Rhizoma Curcumae, exhibited potent

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anti-metastatic effect on breast cancer cells. However, its molecular mechanism

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remains poorly understood. In this study, we employed a 2-DE based proteomics to

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investigate the cellular targets of curcumol in MDA-MB-231 cells, and identified 10

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differentially expressed proteins. Moreover, Gene Ontology analysis revealed that

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those proteins mainly get involved in 9 types of cellular components, 7 different

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biological processes and 9 kinds of molecular functions, and 35 pathways (p < 0.05)

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were enriched by KEGG pathway analysis. Specially, eEF1A1, a well-characterized

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actin binding protein, draws our attention. Curcumol decreased eEF1A1 expression at

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both mRNA and protein levels. EEF1A1 expression was shown to be correlated with

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the invasiveness of cancer cells. Importantly, overexpression of eEF1A1 significantly

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reversed the inhibition of curcumol on the invasion and adhesion of MDA-MB-231

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cells (p < 0.05). Together, our data suggest that eEF1A1 may be a potential molecular

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target underlying the anti-metastatic effect of curcumol.

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KEYWORDS: curcumol, proteomics, breast cancer, metastasis, eEF1A1

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INTRODUCTION

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Breast cancer is the most common form of malignant tumor among women. In United

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States, it’s estimated that the new diagnoses of breast cancer in women rank the first

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place in all new cancer diagnoses and the breast cancer deaths in women is next only

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to cancer of the lung and bronchus in 2016 1. In China, breast cancer is also the

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leading cause of malignant tumors in women in the statistical years of 2000 to 20112, 3.

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However, the majority of deaths caused by breast cancer in women are not due to the

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primary tumor itself, but are the result of metastasis to other organs, such as lung and

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brain, in the body4. It has been shown that there was a 5-year survival rate of 98% for

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women with localized breast cancer, whereas only 27% for women with distant

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metastasis 5. Thus, targeting metastasis process, such as motility, invasion into the

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second site and adhesion profile to other tissues, may provide an alternative way for

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breast cancer therapy.

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Rhizoma Curcumae should be the dry rhizomes derived from Curcuma wenyujin Y.

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H. Chen et C. Ling, Curcuma phaeocaulis Val., and Curcuma kwangsiensis S. G. Lee

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et C. F. Liang., which belong to the genus Curcuma in the family Zingiberaceae, as

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recorded in Chinese Pharmacopoeia 2015 edition 6. Rhizoma Curcumae is a common

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Chinese herbal medicine with a history of more than one thousand years. Moreover, it

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is also frequently used as health food supplement in many Chinese families 7.

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Curcumol with the structure of a guaiane-type sesquiterpenoid hemiketal is one of the

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main components in the volatile oil of Rhizoma Curcumae, while curcumin, the 3



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principal curcuminoid, exists as a non-volatile component in Rhizoma Curcumae 8. As

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a well-known natural compound, curcumin has been widely studied in the past

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decades, whereas the activity of curcumol is still largely unknown. In recent years, the

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volatile oil of plants in the genus Curcuma attracted extensive attention for the potent

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repellent, larvicidal and insecticidal activities

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essential oils that produced by plants to dissuade insects and other herbivores from

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eating them may have beneficial applications in the prevention and treatment of

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various human tumors

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prescribed for the treatment of gynecological cancer in Chinese clinical practice.

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Moreover, a growing body of scientific reports also support that the volatile oil of

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Rhizoma Curcumae processes anti-cancer potential as evidenced by data obtained

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from cancer cell lines and xenografted mice 8. It is noteworthy that curcumol was

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shown to inhibit gynecological tumor cells, such as MCF-7, MDA-MB-231, HeLa,

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and OV-UL-28, 17. Our recent investigation also revealed that curcumol effectively

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inhibited the migration, invasion and adhesion of breast cancer cells2. However, the

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molecular targets regulated by curcumol for these activities are still largely unknown

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and need to further in-depth investigation.

9-13

. Emerging evidence suggests that

14-16

. Interestingly, Rhizoma Curcumae has been traditionally

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To comprehensively identify the potential molecular targets responsible for the

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anti-metastatic effect of curcumol, a proteomic and bioinformatic approach was

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applied in the current study. First, we compared the proteomic profiles of highly

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invasive MDA-MB-231 cells respectively treated with curcumol and vehicle, and then

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differentially expressed proteins were identified by MALDI-TOF mass spectrometry. 4


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All the differentially expressed proteins were analyzed using bioinformatic technology,

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including Gene Ontology (GO) analysis and KEGG pathway enrichment analysis.

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Finally, the key molecular targets were verified by confirmation of their expression

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level and biological role.

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MATERIALS AND METHODS

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Chemicals and Antibodies. Curcumol with a purity more than 98 % was obtained

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from Must Bio-Technology Co., Ltd (Chengdu, China) and stored at -20 °C before

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use. Anti-eEF1A1 and Anti-β-actin antibodies were from Santa Cruz Biotechnology

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(Santa Cruz, California, USA).

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Cell culture. Human breast cancer cells MDA-MB-231 were obtained from ATCC

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(Manassas, VA, USA). Cells were cultured in DMEM supplemented with 10 % FBS,

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2mM L-glutamine, 40 U/ml penicillin and 40 µg/ml streptomycin at 37 °C in a 5 %

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CO2 and 95 % humidified atmosphere.

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Two-dimensional gel electrophoresis (2-DE) and gel analysis. MDA-MB-231

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cells were treated with curcumol (20 µg/ml) or vehicle alone for 24 h. The proteins

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were isolated with FOCUSTM Mammalian protein extraction kit (Sangon, Shanghai,

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China) and resolved by 2-DE according to the instruction provided by manufacturer

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(GE healthcare, USA). The isoelectric focusing (IEF) was performed with a precast

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immobilized pH gradient (IPG) strip (24 cm, pH 3.0-10.0, linear gradient). 1200 µg of

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cellular proteins were loaded onto an IPG strip, allowed to swell for 12 h and then

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rehydrated for 24 h. The isoelectric focusing was conducted at 300 V for 0.5 h, at 700 5



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V for 0.5 h, followed by 1.5 h at 1,500 V, a gradient to 9,000 V for 3 h, and then at

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9,000 V for 4 h. All of the IEF steps were conducted at 20 °C. After the

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first-dimensional IEF, the IPG gel strips were equilibrated in 2D Equilibration Buffer

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(Sangon, Shanghai, China) for 15 min. The free thiol groups were alkylated with an

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equilibration solution containing 2.5% iodoacetamide for another 20 min. After the

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IPG strips were placed on the top of polyacrylamide gel slab, the proteins were

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separated in the second dimension in 12.5% SDS gels conduced at a current setting of

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15 mA/gel for the initial 0.5 h and at 30 mA/gel thereafter; the temperature was

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maintained at 20 °C. The experiments were carried out in triplicate.

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The proteins resolved in the gel were visualized by the modified CBB R-250

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staining method. The stained gels were scanned using ImageScanner III LabScan 6.0

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(GE Healthcare) and analyzed using ImageMaster 2D Platinum Software (Version 5.0,

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GE Healthcare) following the user’s manual.

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In-gel digestion and mass spectrometry. The protein spots of interest were

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excised from the 2D gels. After destaining, the spots were digested with trypsin

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(sequencing grade, GE Healthcare), and the peptides were isolated following a

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previous report

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solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1%

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trifluoroacetic acid and spotted onto the MALDI sample plates. The dried spots were

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analyzed using a Ultraflex-III (Bruker) MALDI TOF/TOF mass spectrometer and

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subjected to peptide mass fingerprinting. The MS spectra were searched against NCBI

18

. Then, the samples were mixed with an equal volume of matrix

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database using Mascot Daemon (Matrix Science, London, UK) as a client attached to

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the Mascot search protocol. The database searches had peptide mass tolerance set at

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approximately±0.1 Da and one missed cleavage site.

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Protein data bioinformatic analysis. The successfully identified proteins were

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subjected to Gene Ontology (GO) analysis and pathway enrichment analysis. The GO

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analysis was performed against the DAVID database (http:// david.abcc.ncifcrf.gov/).

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The pathway enrichment used the KEGG database (http://www.kegg.jp/) and

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analyzed the significance of pathway.

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RT-PCR analysis. Total RNA was isolated from MDA-MB-231 cells with a

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TriZol RNA extraction reagent (Invitrogen, CA, USA) and reverse transcribed by

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using SuperScript® III reverse transcription reagent (Invitrogen, CA, USA) as

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described by manufacturer. Forward and reverse primers for EEF1A119, NRAS20 and

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ACAT221 were listed in Table 1. PCR amplification was set as followings: after an

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initial denaturation at 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and

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72 °C for 60 s. The reaction ended with a final extension step at 72 °C for 5 min. The

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PCR products were then resolved by 1.2% agarose gels, stained with ethidium bromide,

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and visualized by UV.

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Plasmids and Transient Transfection. The pcMV3-eEF1A1 and pcMV3-vector

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were obtained from Sino Biological Inc. (Beijing, China). Cells (50 % confluence)

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were transfected with 2.5 µg DNA using transfection reagent Lipofectamine 2000

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(Invitrogen, USA) following the protocol provided by manufacturer. Transfected cells 7



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were first cultured in antibiotic-free medium for 6 h and then in fresh medium for 24 h,

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followed by further drug treatments.

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Western blotting analysis. Cells were lysed on ice with 1RIPA buffer (Cell

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signaling, MA, USA) containing complete proteinase inhibitors. Thirty microgram of

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protein samples were applied for SDS-polyacrylamide gel electrophoresis and the

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blotting was subsequently performed with a polyvinylidene fluoride (PVDF)

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membrane. The blots were then incubated with 5% BSA in TRIS phosphate-buffered

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saline solution containing Tween-20 (0.1%) (TBST) for 1 h at room temperature. The

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primary antibodies diluted in blocking buffer were incubated with corresponding blots

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overnight at 4 °C. The horseradish peroxidase-conjugated secondary antibodies were

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incubated with corresponding blots for 1 h at room temperature. Chemiluminescent

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visualization was performed by the gel imaging system (Tanon, China) with a

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commercial kit (GE Healthcare, Sweden). Band intensity was quantified and analyzed

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by the software of this gel imaging system. The intensity of each protein was first

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normalized to that of the corresponding β-actin and then each was further normalized

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to control.

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Boyden chamber invasion assay. The invasion ability of MDA-MB-231 cells was

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evaluated by a Boyden chamber with Matrigel-coated polycarbonate filters with 8 µm

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pore size in the Transwell (Corning, NY, USA). After pre-treatment with curcumol

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(20 µg/ml) or vehicle for 24 h, the cells with a density of 1×105 cells/well were seeded

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in the upper chamber of the Transwell and cultured with serum-free medium. The 8


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lower chamber was added with the medium supplemented with 10% FBS as a

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chemoattractive agent. Then, the cells in the upper surface of the filter membrane

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were carefully cleaned with a cotton swab after 24 h incubation. The cells that had

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crossed the Matrigel to the lower surface of the membrane were fixed with 100 %

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methanol and stained with 0.05% crystal violet. Different fields were randomly

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chosen and captured under fluorescence microscopy (OLYMPUS IX71) at 100×

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magnification, and the cells were scored by manual counting. Inhibition rate was

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quantified with cells treated vehicle representing 100 %.

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Adhesion assay. MDA-MB-231 cells after pre-treatment were seeded onto a

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96-well plate coated with Matrigel. Following 4 h incubation, attached cells were

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fixed in 100 % methanol and stained with 0.05 % crystal violet solution. Different

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fields were randomly chosen and captured using fluorescence microscopy

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(OLYMPUS IX71) at 100× magnification. Then, attached cells were scored by

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manual counting. Inhibition rate was quantified with cells treated vehicle representing

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100%.

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Statistical analysis. The experimental data were expressed as means± SD for three

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independent experiments. A ANOVA test was used to calculate the significant

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difference in the study. A p-value of less than 0.05 was considered to be statistically

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significant.

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RESULTS

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Proteomic identification of the proteins mostly affected by curcumol in 9



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MDA-MB-231 cells. In most recent study, we demonstrated that curcumol suppressed

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breast cancer cell metastasis 2. To find the potential molecular targets underlying

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the anti-metastatic effect of curcumol, a 2D gel electrophoresis technique was used in

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this study to identify the differential proteins in MDA-MB-231 cells treated with

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vehicle or 20 µg/ml of curcumol, which was found to efficiently reduce the metastasis

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of MDA-MB-231 cells with no cytotoxicity 2. Figure 1 showed the representative gels

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with marked protein spots. After detection, editing and matching of the spots, the

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intensity of protein spots in the gel with curcumol-treated cells was quantified and

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compared with that in the gel with vehicle-treated cells using ImageMaster 2D platinum

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5.0 software (GE Healthcare). We have identified 19 spots as differentially expressed

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after curcumol treatment by applying a threshold of 1.2-fold variation. There are 16

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down-regulated proteins and 3 up-regulated proteins. The spots are marked in Figure

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1 with circle, arrows and numbers. Then, these spots were isolated and digested with

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trypsin. The proteins were identified by MALDI-TOF-MS. Eventually, 10 proteins

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were successfully identified. The related information for each protein was

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summarized in Table 2.

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Gene Ontology (GO) analysis. The differentially expressed proteins were

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subjected to GO analysis to classify the cellular components, biological processes and

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molecular functions. As shown in Figure 2A, the identified proteins are mainly located

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in various cellular organelles (76 %), including nucleus (32 %), mitochondrion (8 %),

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endoplasmic reticulum (4 %), Golgi apparatus (8 %), lysosome (4 %), ribosome (4 %)

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and cytoskeleton (16 %). The rest were from plasma membrane (12 %) and cytosol 10


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(12 %). Moreover, the identified proteins were shown to participate in a variety of

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biological processes, especially tumor biology (Figure 2B), such as cellular process

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(proliferation, differentiation, cycle, death, autophagy and adhesion, etc.), metabolic

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process (metabolism of amino acid, lipid, DNA, etc.), response to stress and signal

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transduction. Figure 2C showed that these proteins are correlated with various

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molecular functions, mainly including catalytic activity (18 %), RNA binding (14 %),

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enzyme binding (14 %), ion binding (14 %), and transcription regulator activity (14 %).

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Pathway enrichment of the identified proteins. We then further analyzed the

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biological pathways involving in these differentially expressed proteins. Totally, 78

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KEGG pathways were enriched for these identified proteins (data not shown). The top

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35 pathways (P < 0.05) were shown in Table 3. It can be seen that a number of

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metabolic pathways were modulated in MDA-MB-231 treated with curcumol, such as

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butanoate metabolism, glyoxylate and dicarboxylate metabolism, propanoate

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metabolism, tryptophan metabolism, pyruvate metabolism, fatty acid metabolism and

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central carbon metabolism in cancer. Notably, several cancer-related pathways are

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also interfered by curcumol, such as thyroid cancer, bladder cancer, acute myeloid

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leukemia, endometrial cancer, non-small cell lung cancer, glioma, melanoma, renal

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cell carcinoma, chronic myeloid leukemia.

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Verification of Differentially Expressed Proteins by RT-PCR. Among the 10

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identified proteins, we found that three of them may be correlated to anti-metastatic

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effect of curcumol on breast cancer cells. These target proteins include: elongation 11



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factor 1-alpha 1 (eEF1A1/EF-1α), GTPase NRas (NRAS) and Acetyl-CoA

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acetyltransferase, cytosolic (ACAT2). First, we confirmed the effect of curcumol on

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these three differentially expressed proteins by enlarging the corresponding protein

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spots image (Figure 3A). Then, we further examined their expression at

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transcriptional level by RT-PCR after MDA-MB-231 cells were treated with vehicle

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or curcumol (10, 20 and 40 µg/ml) for 24 h. As a result, , curcumol decreased the

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mRNA level of eEF1A1 and NRAS and increased the expression of ACAT2

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compared with those treated with vehicle (Figure 3B), which showed a consistent

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trend with that of the corresponding proteins.

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Analysis of eEF1A1 Protein Expression. As accumulating evidence demonstrated

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that eEF1A1 played a pivotal role in increasing metastatic propensity of tumor cells

220

22-24

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protein level of eEF1A1 using a specific antibody in a group of human cell lines. As

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shown in Figure 4A, a relatively high expression of eEF1A1 protein was detected in

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HL-60 and MDA-MB-231 cells and a moderate level was shown in HEK 293T and

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U87 cells, whereas a very weak level was shown in Kasumi-1, MCF-7 and U251 cells.

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Then, we examined the protein expression of eEF1A1 after 24 h treatment of

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MDA-MB-231 cells by vehicle or curcumol (10, 20 and 40 µg/ml). Figure 4B showed

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that a high basal protein level of eEF1A1 was observed in MDA-MB-231 cells treated

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by vehicle, whereas the protein level of eEF1A1 significantly decreased after

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curcumol treatment.

, we thereafter payed our special attention to eEF1A1. First, we determined the

12


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Overexpression of eEF1A1 reversed the inhibitory effect on invasion and

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adhesion by curcumol. Our recent investigation demonstrated that curcumol

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effectively suppressed the migration, invasion and adhesion of breast cancer cells at

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non-cytotoxic concentrations 2. To verify the role of eEF1A1 in the anti-metastatic

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effect of curcumol, we first transfected MDA-MB-231 cells with pcMV-vector or

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pcMV-eEF1A1. As shown in Figure 5A, a remarkable overexpression of eEF1A1

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protein was observed in MDA-MB-231 cells with pcMV-eEF1A1 transfection

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compared to that with pcMV-vector transfection. Then, the invasion assay was

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performed with Matrigel-coated 24-well Boyden chambers after MDA-MB-231 cells

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with pcMV-vector or pcMV-eEF1A1 transfection were pre-treated by curcumol for 24

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h. As shown in Figure 5B, curcumol (20 µg/mL) significantly blocked the invasion of

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MDA-MB-231 cells transfected with only pcMV-vector compared to that of the same

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cells treated with vehicle (P < 0.05). Notably, overexpression of eEF1A1 by

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transfecting pcMV-eEF1A1 greatly promoted the invasion of MDA-MB-231 cells

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compared with that of cells transfected with only pcMV-vector (P < 0.001).

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Importantly, overexpression of eEF1A1 remarkably attenuated the inhibitory effect of

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curcumol on the invasion of MDA-MB-231 cells (P < 0.05). Furthermore, our results

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also showed that overexpression of eEF1A1 facilitated the adhesion of MDA-MB-231

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cells onto the Matrigel (P < 0.01), whereas the inhibitory effect of curcumol on the

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adhesion was also significantly reversed by overexpression of eEF1A1 (P < 0.05)

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(Figure 5C).

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DISCUSSION 13



252

Curcumol is one of volatile components in Rhizoma Curcumae, which is well-known

253

as herbal medicine and health food supplement in China. Recent investigations

254

showed that curcumol exhibited inhibitory effect on various gynecological tumor

255

cells8,

256

remarkably suppressed the metastatic process of breast cancer cells.2 In this study, a

257

proteomic and bioinformatic strategy was applied to comprehensively elucidate the

258

molecular targets responsible for the anti-metastatic effect of curcumol. First, a 2D gel

259

electrophoresis was used to separate the proteins extracted from the highly invasive

260

MDA-MB-231 cells treated by curcumol and vehicle, respectively. Nineteen proteins

261

were identified as differentially expressed by comparing the protein profiles. Of these

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19 proteins, 10 proteins influenced by curcumol were finally identified by

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MALDI-TOF-MS.

17

. The most recent study from our group demonstrated that curcumol

264

To obtain a comprehensive insight into the identified proteins, bioinformation of

265

identified 10 proteins including GO analysis and KEGG pathway analysis was

266

performed. The GO analysis indicated that the identified proteins mainly distributed

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in 9 types of cellular components, participated in 7 different biological processes and

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exhibited 9 kinds of molecular functions. The KEGG pathway enrichment analysis

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showed that the identified proteins are involved in numerous signaling pathways. Of

270

note, NRAS (P01111) was found to be included in various cancer-related pathways,

271

such as thyroid cancer, bladder cancer, acute myeloid leukemia, endometrial cancer,

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non-small cell lung cancer, glioma, melanoma, renal cell carcinoma, chronic myeloid

273

leukemia. In addition, ACAT2 (Q9BWD1) was involved in diverse metabolism 14


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pathways, such as: butanoate metabolism, glyoxylate and dicarboxylate metabolism,

275

propanoate metabolism, tryptophan metabolism, pyruvate metabolism, fatty acid

276

metabolism. It has been reported that down-regulation of NRAS was closely

277

correlated with the anti-metastatic effect of isothiouronium salts in melanoma cells 25,

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miR-515-5p in breast cancer cells

279

Additionally, a strong correlation was found among metastasis, ACAT2 and

280

hydroxyl-coenzyme A dehydrogenase, alpha subunit (HADHA) down-expression and

281

poorer prognosis on survival of clear cell renal cell carcinoma (ccRCC) 28.

26

and introduction of let-7i to mutant p53 cells 27.

282

Moreover, another protein, eEF1A1 (P68104), drew our special attention. EEF1A1

283

is a member of the abundant evolutionarily conserved elongation factor proteins that

284

normally responsible for binding aminoacyl-tRNA to the ribosome during protein

285

synthesis 29. Thus, eEF1A1 was found to be involved in the pathway of RNA transport

286

(hsa03013) according to the KEGG pathway enrichment analysis. However,

287

accumulating evidence indicated that eEF1A1 played a pivotal role in the increase of

288

metastatic propensity of tumor cells. Generally, the metastatic process is regarded as

289

similar to the chemotaxis of motile cells along gradients of cytokines

290

cellular motile machinery consisted of the actin cytoskeleton play a crucial role in the

291

chemotactic response and thereby it may be also very important for metastasis

292

More specifically, it’s believed that the stabilization of polarized surface projections,

293

which contain newly polymerized actin, by actin binding proteins will prompt the cell

294

to move in the specific direction of the cytokine gradient

295

also been reported as a well-characterized actin binding protein in addition to

30, 31

. The

23

.

23, 32

15


. Notably, eEF1A1 has


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functioning as a polypeptide elongation factor

297

co-localizes with filamentous actin (F-actin) in cells and its distribution is associated

298

with changes in the organization of the actin cytoskeleton during chemotaxis

299

Importantly, the overexpression of eEF1A1 mRNA has been correlated with increased

300

metastatic propensity

301

RNA interference (RNAi) inhibited the proliferation, invasion, and migration of

302

prostate cancer cells

303

biomarker related to the metastatic progression of human prostate cancer

304

eEF1A1 appears to be a promising target for suppressing the metastasis of aggressive

305

tumor cells.

33

. It has been shown that eEF1A1

34

.

22

. Recent study revealed that downregulation of eEF1A1 by

23

. Moreover, eEF1A1 was identified as a candidate serum 24

. Thus,

306

To evaluate whether protein changes discovered by proteomics are correlated with

307

the alterations of those mRNAs at the transcription level, eEF1A1, NRAS and

308

ACAT2 were determined by RT-PCR. Our results demonstrated that mRNA level of

309

eEF1A1 and NRAS was reduced by curcumol, while ACAT2 level was enhanced by

310

curcumol, which is accordant with that of the corresponding protein counterparts.

311

Furthermore, to explore the correlation of eEF1A1 level with invasiveness, we

312

examined the eEF1A1 protein expression in a group of human cell lines. As a result,

313

eEF1A1 was detected at various level, whereas HL-60, MDA-MB-231 and U87 cells

314

showed higher level of eEF1A1 than their counterparts Kasumi-1, MCF-7 and U251

315

cells. Previous study demonstrated that estrogen receptor α-negative breast cancer cell

316

line MDA-MB-231 is more invasive than estrogen receptor α-positive MCF-7

317

Similarly, U87 exhibited higher metastatic potential to lungs or other organs than 16


35

.

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318

U251 when orthotopically grown

319

correlated with the tumor invasiveness. It is worth noting that a moderate level of

320

eEF1A1 was also observed in normal cell line HEK 293T, which may be interpreted

321

as that eEF1A1 can normally function as a housekeeping gene product facilitating the

322

maintenance of cell growth and/or survival

323

regulation of eEF1A1 expression, we determined the influence of curcumol on

324

eEF1A1 protein level. Expectedly, curcumol obviously inhibited the expression of

325

eEF1A1. Our previous study demonstrates that curcumol blocked breast cancer cell

326

metastasis through suppressing MMP-9, which was related to inhibition of NF-κB

327

signaling pathway by curcumol2. eEF1A1 has been shown to phosphorylate STAT3 at

328

serine 727, which is necessary for NF-κB/STAT3-stimulated IL-6 expression,

329

indicating eEF1A1 is positively related to NF-κB signaling pathway. Thus, it is

330

deserved to be further explored that whether curcumol-mediated inhibition of eEF1A1

331

is correlated with the suppression of metastasis of breast cancer cells38. To determine

332

the role of eEF1A1 in the anti-metastatic effect of curcumol, we overexpressed

333

eEF1A1 level in MDA-MB-231 cells with pcMV-eEF1A1 transfection. Then, we

334

evaluated the effect of eEF1A1 overexpression and/or curcumol intervention on the

335

invasion and adhesion ability of MDA-MB-231 cells. Moreover, considering the

336

similarity of the metastatic process to the chemotaxis and the potential role of eEF1A1

337

in the organization of the actin cytoskeleton during chemotaxis, the Boyden chamber

338

invasion assay was performed with complete medium with 10% FBS as

339

chemoattractive agent. Consequently, overexpression of eEF1A1 promoted both

36

. Conceivably, eEF1A1 expression is positively

37

. To understand the pharmacological

17



Page 18 of 38

340

invasion and adhesion ability of MDA-MB-231 cells, suggesting the critical role of

341

eEF1A1 in the increase of metastatic propensity, which is consistent with previous

342

reports

343

curcumol on the invasion and adhesion of MDA-MB-231 cells, indicating eEF1A1

344

may be a key molecular target responsible for the anti-metastatic effect of curcumol.

345

Similarly, previous study demonstrated that narciclasine, an Amaryllidaceae

346

isocarbostyril, at nontoxic doses enhanced the survival of mice bearing metastatic

347

apoptosis-resistant melanoma xenografts in their brain by targeting eEF1A1

348

study together with our previous study2 demonstrate that curcumol ranging from 10 to

349

40 µg/ml remarkably suppressed metastasis and inhibited eEF1A1 expression in

350

breast cancer cells without causing obvious cytotoxicity. It has been shown that the

351

plasma concentration of curcumol reached almost 50 µg/ml after rats were

352

intravenously administered a single dose of 30 mg/kg curcumol40. However, it is still

353

needed for further investigation that whether the effective plasma concentration range

354

of curcumol can be achieved after oral administration of Rhizoma Curcumae or

355

curcumol and whether lower concentration of curcumol is still effective on metastasis

356

inhibition, especially in in vivo study.

22, 23

. Importantly, overexpression of eEF1A1 reversed the inhibitory effect of

39

. This

357

In conclusion, our proteomic analysis demonstrated that curcumol altered the

358

expression of several biologically important proteins and diverse signaling pathways

359

in MDA-MB-231 cells. Especially, eEF1A1, a well-characterized actin binding

360

protein, was discovered to be reduced by curcumol at both mRNA and protein levels.

361

Moreover, eEF1A1 level was shown to be correlated to the invasiveness of cancer 18


Page 19 of 38


362

cells. Finally, we found that eEF1A1 played a critical role in the inhibition of

363

curcumol on the invasion and adhesion of MDA-MB-231 cells. These results suggest

364

that eEF1A1 may be a potential molecular target of curcumol, which will provide a

365

better understanding of the anti-metastatic effect of curcumol.

366

AUTHOR INFORMATION

367

Corresponding Author

368

H.Y.Q.*: College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng

369

Road, Beibei District, Chongqing 400716, China. Tel./Fax: +86 23 68251225; E-mail:

370

[email protected]

371

L. L.*: College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road,

372

Beibei District, Chongqing 400716, China; E-mail: [email protected]

373

Funding

374

This work was supported by the NSFC Projects (No. 81373903; No. 81202946),

375

Chongqing

376

(cstc2013kjrc-qnrc1002), the Key Project of Fundamental Research Fund for the

377

Central Universities (XDJK2016B040, XDJK2016B034) and National Training

378

Program of Innovation and Entrepreneurship for Undergraduates (201510635019).

379

Notes

380

The authors declare no competing financial interest.

381

ABBREVIATIONS USED

Project

of

Science

and

technology

19


talent

cultivation


382

2-DE, Two-dimensional gel electrophoresis; GO, Gene Ontology; IPG, immobilized

383

pH gradient; IEF, isoelectric focusing; RT-PCR, Reverse transcriptase-polymerase

384

chain reaction; eEF1A1, elongation factor 1-alpha 1; NRAS, GTPase NRas; ACAT2,

385

Acetyl-CoA acetyltransferase, cytosolic

386

20


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Page 21 of 38


387

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Legends

514

Figure 1. Representative 2-DE maps of curcumol or vehicle-treated MDA-MB-231

515

cells. Cells were treated with 20 µg/ml of curcumol and vehicle for 24 h. The

516

differentially expressed protein spots were marked on the maps. The map are

517

representative of three independent runs.

518 519

Figure 2. Gene Ontology (GO) classification of the differentially expressed proteins

520

caused by curcumol. The proteins were grouped into three GO terms: (A) Cellular

521

component; (B) Biological process; (C) Molecular function.

522 523

Figure 3. Confirmation of eEF1A1, NRAS and ACAT2 expression by RT-PCR. (A)

524

Enlarged spots of the three proteins. (B) mRNA level of the three genes.

525

MDA-MB-231 cells were treated with vehicle or curcumol at indicated concentrations

526

for 24 h, eEF1A1, NRAS and ACAT2 expression were analyzed by RT-PCR.

527 528

Figure 4. Analysis of eEF1A1 protein expression. (A) Detection of eEF1A1 protein

529

expression in various cell lines. The cell lines as indicated were seeded and cultured

530

for 24 h. (B) Detection of eEF1A1 protein level in MDA-MB-231 cells with the

531

treatment of curcumol as indicated for 24 h. The protein was extracted and analyzed

532

using Western blotting. The western blots were a representative of three independent 27



533

experiments.

534 535

Figure 5. Overexpression of eEF1A1 reversed the inhibitory effect on invasion and

536

adhesion by curcumol. (A) Level of eEF1A1 was measured by Western blotting after

537

MDA-MB-231 cells were transfected with pcMV-vector or pcMV-eEF1A1 for 24 h.

538

(B) Boyden chamber invasion assay. After transfected with pcMV-vector or

539

pcMV-eEF1A1 for 24 h, MDA-MB-231 cells were pretreated with curcumol (20

540

µg/ml) or vehicle for 24 h and then seeded in the chamber with coated Matrigel of

541

24-transwell plate for another 24 h. The invasion assay was then conducted according

542

to the procedure in the Materials and Methods. (C) Adhesion assay. After transfected

543

with pcMV-vector or pcMV-eEF1A1 for 24 h, MDA-MB-231 cells were

544

pre-incubated with curcumol (20 µg/ml) or vehicle for 24 h and seeded onto a 96-well

545

plate coated with Matrigel for 4 h. Then, attached cells were stained and counted

546

according to the procedure in the Materials and Methods. Randomly chosen fields

547

were obtained using an optical microscope (100× magnification). Values represent

548

mean ± SD. *p

Proteomic Identification of eEF1A1 as a Molecular Target of Curcumol

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