M Arrest Triggered by Natural Borneol

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Proteomic analysis of G2/M arrest triggered by natural borneol/ curcumin in HepG2 cells, the importance of ROS-p53 pathway Jianping Chen, Li Lin, Jianyu Su, Bing Li, Xia Zhang, and Tianfeng Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01773 • Publication Date (Web): 06 Jun 2015 Downloaded from http://pubs.acs.org on June 9, 2015

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Journal of Agricultural and Food Chemistry

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Proteomic analysis of G2/M arrest triggered by natural borneol/curcumin in

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HepG2 cells, the importance of ROS-p53 pathway

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Jianping Chen , Lin Li , Jianyu Su

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† College of Light Industry and Food Sciences, South China University of Technology,

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Guangzhou 510640, China

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‡ Department of Chemistry, Jinan University, Guangzhou, 510632, China

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§ College of Food Science and Technology, Guangdong Ocean University, Zhanjiang

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524088, China

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# Guangdong Hua Qing Yuan Biological Technology Co., Ltd., Meizhou, 514600,

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†,§

†

*,†,#

†

†

, Bing Li , Xia Zhang , Tianfeng Chen

*,‡,#

China

11 12 13 14 15 16

Correspondence: Dr. Jianyu Su and Tianfeng Chen. College of Light Industry and

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Food Sciences, South China University of Technology, Wu Shan Road 381,

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Guangzhou 510640, China. *E-mail: [email protected], [email protected], Tel:

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(+86) 20 87113252. Fax: (+86) 20 87113252.

20 21 22 23

ACS Paragon Plus Environment


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ABSTRACT: Curcumin (Cur), an active ingredient from the rhizome of the plant,

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Curcuma longa, has wide anticancer activities. However, due to its poor solubility and

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hence poor absorption, Cur has limited clinical applications. It is therefore important

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to develop an effective method to improve its absorption. Natural borneol (NB), a

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terpene and bicyclic organic compound, has been extensively used as a food additive

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and our previous studies show that it can improve the uptake of Cur in cancer cells.

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However, the anticancer mechanism of NB/Cur remains unclear. In this study, the

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effects of NB/Cur on HepG2 cells were investigated by proteomic analysis. The

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results

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MALDI-TOF-MS were significantly changed after NB/Cur treated HepG2 cells for

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24 h. Moreover, 17 proteins increased and 12 proteins decreased significantly.

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Biological progress categorization demonstrated that the identified proteins were

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mainly associated with cell cycle and apoptosis (28.1%). Subcellular location

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categorization exhibited that the identified proteins were mainly located in nucleus

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(28.1%) and mitochondrion (21.9%). Among of all proteins, we selected three

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differentially proteins (hnRNPC1/C2, NPM and PSMA5), which were associated with

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the p53 pathway. Down-regulation of hnRNPC1/C2 and NPM contributed to the

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enhancement of phosphorylated p53. Activated p53 and down-regulation of PSMA5

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resulted in an increase in p21 protein. Further studies showed that NB/Cur induced

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reactive oxygen species (ROS) generation, indicating that ROS might be upstream of

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the G2/M arrest signaling pathway. In summary, the results exhibited that the whole

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proteomic response of HepG2 cells to NB/Cur, which might lead to a better

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understanding of its underlying anticancer mechanisms.

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KEYWORDS: Curcumin, Natural borneol, G2/M phase, ROS-p53 pathway,

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Proteomics

showed

that

32

differentially

expressed


proteins

identified

by

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INTRODUCTION

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Plant phytochemicals are known to be effective chemopreventive agents against

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various types of cancer

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and have received great attention for various pharmacological activities such as

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antioxidative activities, anticancer activities and other beneficial health effects3. The

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polyphenolic curcumin (Cur), an active ingredient from the rhizome of the plant,

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Curcuma longa, has been identified as a both cancer chemoprevent and

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chemotherapeutic agent. 4, 5 In vitro and in vivo studies reported that Cur could inhibit

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the growth of a variety of cancer cell lines, including human acute myelogenous

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leukemia cells6, osteosarcoma cells7, human hepatoma cells8, human lung

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adenocarcinoma cells9-11, prostate cancer cells12, 13 and so on. However, its clinical

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applications remain limited due to its poor solubility and hence poor absorption. It is

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therefore important to develop an effective method to improve its absorption.

1, 2

. Polyphenolics occur widely in the edible plant kingdom

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Borneol, a terpene and bicyclic organic compound, has been widely applied in

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food, pharmaceutical, and perfume industries, especially in Tradition Chinese

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medicine14. Generally, there are two different types of borneol: synthetic borneol (SB)

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and natural borneol (NB). SB is racemate, including (+)-borneol and (−)-isoborneol,

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while NB only contains (+)-borneol15. Studies have reported that isoborneol exhibited

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more mucosa stimulus and hepatotoxicity than (+)-borneol

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with SB, NB, a safer form of borneol, has been widely used in candies, beverages,

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baked goods, chewing gum and other foods

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NB is a good penetration enhancer, as evidenced by improving the brain 18, eye 19, and

16

. Therefore, compared

17

. Meanwhile, studies have shown that



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nasal16 bioavailabilities of many poorly permeable drugs. Therefore, NB would be

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possibly used as an effective penetration enhancer to improve cellular uptake of Cur.

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Our previous studies have demonstrated that NB could improve the cellular uptake of

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Cur in cancer cells20. However, the underlying anticancer mechanisms of NB and Cur

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in combination have not been explored.

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Proteomics has been used in cancer research and helps us to analyze the changes

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in proteins related to intracellular regulatory pathways5, 21-23. Moreover, it also been

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considered as a powerful tool in comprehensive characterization of proteins in

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response to drug treatment 24, 25. Therefore, the proteomic platform may be one of the

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most effective methods to investigate the molecular mechanisms of anticancer drug26.

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In the present study, two-dimensional polyacrylamide gel electrophoresis (2-DE) is

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used to investigate differentially expressed proteins in HepG2 cells under different

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treatment conditions and 2-DE integrated with MALDI-TOF-MS has been used to

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further identify anticancer mechanism of NB and Cur in combination by comparative

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proteomics approach.

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MATERIALS AND METHODS

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Materials. Curcumin (Cur), 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium

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-bromide

(MTT), propidium iodide (PI) and bicinchoninic acid (BCA) kit were bought

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from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Natural borneol (NB) was

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obtained from the Natural Institute for the Control of Pharmaceuticals and Biological

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Products, Beijing, China. All reagents used in two-dimensional electrophoresis (2-DE)

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were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Antibodies against


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p53, p21, cdc 2, and cyclin B1 were purchased from Cell Signaling Technology

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(Beverly, MA, USA).

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Cell Culture. The human hepatocellular carcinoma cell line HepG2 was purchased

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from American Type Culture Collection (ATCC, Manassas, VA, USA). The human

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fetal liver cell line LO2 was obtained from Nanjing KeyGEN Biotech. CO., LTD.

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(Nanjing, China). Both the cell lines were cultured in DMEM medium with 10% fetal

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bovine serum, 100 units/ml penicillin and 50 units/ml streptomycin at 37°C in a

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humidified (5% CO2, 95% air) atmosphere.

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Cell Viability Assay. The HepG2 cells with a density of 2×104 cells/well and LO2

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cells with a density of 5×104 cells/well were seeded in 96-well culture plates for 24 h.

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The cells were pre-treated with or without NB at different concentrations for 12 h and

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co-incubated with or without Cur at different concentrations for different periods of

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time. Cell viability was determined by MTT assay as previously described 27.

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Flow Cytometric Analysis. The HepG2 cells were harvested after treatment of NB

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(20 µg/ml)/Cur (20 µM) for 24 h, washed with PBS and fixed with 70% cold ethanol

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at -20 °C overnight. The fixed cells were then stained with propidium iodide (PI) for

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30 min in darkness. The cell cycle distribution was monitored by flow cytometric

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analysis as previously described by Chen et al 28.

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2-D SDS-PAGE Analysis. The HepG2 cells were treated with NB (20 µg/ml), Cur

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(20 µM) and NB (20 µg/ml)/Cur (20 µM) for 24 h, respectively. Then, the cells were

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washed with phosphate buffered saline (PBS) and were lysed with 1000 µl Alklysis

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buffer

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3-[(3-cholamidopropyl)]dimethylammonio-1-propanesulfonate (CHAPs), and 1 mM

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phenylmethanesulfonyl fluoride (PMSF). The cell lysates were treated with

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ultrasound to break nucleic acid and centrifuged at 12000 r/min for 20 min at 4 °C,

including

20

mM

Tris,

7

M

Urea,


2

M

Thiourea,

2%


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and the supernatant was transferred into new EP tubes. Proteins from the supernatant

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were precipitated by acetone and then kept at -20 °C overnight. The protein

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concentration was measured using the Bradford assay with bovine serum albumin

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(BSA) as the standard sample for normalization. The protein samples were diluted to

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450 µl rehydration buffer solution containing 7 M Urea, 2 M Thiourea, 4% CHAPs,

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1% dithiothreitol (DTT), 1% immobilized pH gradient (IPG) buffer and a trace of 1×

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bromophenol blue. Then, the samples were applied to immobilized dry IPG strips

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(linear pH gradient from pH 4-7, 24 cm) and rehydrated for 12 h. The samples were

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then focused for 30 min at 300 V, 30 min at 700 V, 90 min at 1500 V, 3 h gradient at

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9000 V and 4 h at 9000 V, for a total of 52 KVh, using the Ettan IPGphor 3 isoelectric

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focusing (IEF) system (GE Health care, Little Chalfont, Buckinghamshire, UK).

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Focused IPG strips were equilibrated in 8 mL sodium dodecyl sulfonate (SDS)

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equilibration buffer I (6 M Urea, 30% (v/v) Glycerol, 2% (w/v) SDS, 50 mM Tris-HCl,

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pH 8.8, 0.002% bromophenol blue, and 100 mM DTT) for 15 min and then

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equilibrated for an additional 15 min in SDS equilibration buffer II (6 M Urea,

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30%(v/v) Glycerol, 2% (w/v) SDS, 50 mM Tris-HCl, pH 8.8, 0.002% bromophenol

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blue, and 250 mM iodoacetamide). After equilibration, 12.5% SDS-PAGE was

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performed

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Chalfont, Buckinghamshire, UK). The 2-DE gel was then visualized using sliver

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staining.

on

an

Ettan

DALTsix

system

(GE

Health

care,

Little

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Protein Identification by MALDI-TOF-MS Analysis. The stained 2-DE gels

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were scanned with UMAX Powerlook1100 scanner (UMAX) and analyzed using

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ImageMaster 2D platinum 5.0 (GE Healthcare, Little Chalfont, Buckinghamshire, UK)

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according to the manufacturer’s protocol. The spots of interest were manually excised

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from the silver-stained gel and subjected to in-gel trypsin digestion. After digestion,


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the peptides in gel pieces were then extracted using extraction buffer containing 2.5%

144

trifluoroacetic acid (TFA) and 90% acetonitrile (ACN). The extracts were dried

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completely using a DZF-6020 vacuum drying system (Yiheng instrument Co., LTD.,

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Shanghai, China). The peptides were redissolved using 0.1% TFA in 30% ACN. Then,

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0.5 µl of matrix (5mg/ml α-cyano-4-hydroxycinnamic acid [CHCA] in 50% ACN and

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0.1% TFA) was added into 0.8 µl of peptide solution before spotting on the target

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plate. Protein identification was carried out on an Ultraflex III MALDI-TOF mass

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spectrometric analysis (Bruker Dalton, Billerica, MA, USA). The peptides were

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detected in 355 nm UV wavelength and were accelerated with a 20000 V voltage. The

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peptides were scanned in the mass range of 700-3200 Da. Peptide masses were

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matched with the theoretical peptides of all proteins in the NCBI database using the

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BioTools (Bruker Dalton, Billerica, MA, USA) software. The following parameters

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were used for the database searching: peptides mass range of 800-4000 Da, first grade

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mass tolerance of 50 ppm, second grade mass tolerance of 0.5 Da, a maximum of one

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missed cleavage, monoisotopic masses, methionine oxsidation as a variable

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modification, cysteine iodoacetamide as a global modification.

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Western Blot Analysis. Total cellular proteins were harvested by incubating the

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cells in the lysis buffer obtained from Cell Signaling Technology. The protein

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concentrations were determined by BCA kit (Sigma-Aldrich, St. Louis, MO, USA)

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according to the manufacturer’s protocols. Equal amounts of protein (30 µg) loaded

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per lane were run on 10% SDS-PAGE, and transferred to nitrocellulose membrane at

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110 V for 1 hour. Following blocked with 5% nonfat milk in TBST buffer (20 mM

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Tris-HCl [pH 7.4], 137 mM NaCl, and 0.1% Tween-20) for 2 hours, the membranes

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were then incubated with primary antibodies at 1:1000 dilution in 5% nonfat milk

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over night at 4 °C, and then incubated with secondary antibodies conjugated with



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horseradish peroxidase at 1:2000 dilution for 1 hour at room temperature. Protein

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bands were visualized on X-ray ﬁlm using an enhanced chemiluminescence system

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(Kodak, Rochester, NY, USA).

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Measurement of ROS Generation. The intracellular production of reactive

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oxygen species (ROS) was measured by dihydroethidium (DHE) fluorescence assay

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according to the previously description20.

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Statistical Analysis. Experiments were carried out at least in triplicate and repeated

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three times. All data were expressed as mean ±S.D. Statistical analysis was performed

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using SPSS statistical package (SPSS 13.0 for Windows; SPSS, Inc. Chicago, IL,

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USA). The difference between two groups was analyzed by two-tailed Student’s t-test.

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The difference between three or more groups was analyzed by one-way analysis of

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variance multiple comparisons. Differences with P

M Arrest Triggered by Natural Borneol

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