research article itraq-based proteomic analysis of

Research ArticleiTRAQ-Based Proteomic Analysis of Ginsenoside F2 onHuman Gastric Carcinoma Cells SGC7901

Qian Mao,1 Pin-Hu Zhang,2 Jie Yang,3 Jin-Di Xu,1 Ming Kong,1 Hong Shen,1

He Zhu,1 Min Bai,1 Li Zhou,1 Guang-Fu Li,4 Qiang Wang,3 and Song-Lin Li1

1Department of Pharmaceutical Analysis & Metabolomics, Affiliated Hospital of Integrated Traditional Chinese andWestern Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, China2Jiangsu Center for New Drug Screening & National New Drug Screening Laboratory, China Pharmaceutical University,Nanjing 210009, China3Department of Chinese Medicines Analysis, China Pharmaceutical University, Nanjing 210009, China4Department of Surgery, The Medical University of South Carolina, Charleston, SC 29466, USA

Correspondence should be addressed to Qiang Wang; [email protected] and Song-Lin Li; [email protected]

Received 12 May 2016; Revised 4 August 2016; Accepted 25 August 2016

Academic Editor: Isabel Andujar

Copyright © 2016 Qian Mao et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ginsenoside F2(F2), a protopanaxdiol type of saponin, was reported to inhibit human gastric cancer cells SGC7901. To better

understand the molecular mechanisms of F2, an iTRAQ-based proteomics approach was applied to define protein expression

profiles in SGC7901 cells in response to lower dose (20 𝜇M) and shorter duration (12 hour) of F2treatment, compared with

previous study. 205 proteins were screened in terms of the change in their expression level which met our predefined criteria.Further bioinformatics and experiments demonstrated that F

2treatment downregulated PRR5 and RPS15 and upregulated RPL26,

which are implicated in ribosomal protein-p53 signaling pathway. F2also inhibited CISD2, Bcl-xl, andNLRX1, which are associated

with autophagic pathway. Furthermore, it was demonstrated that F2treatment increased Atg5, Atg7, Atg10, and PUMA, the

critical downstream effectors of ribosomal protein-p53 signaling pathway, and Beclin-1, UVRAG, and AMBRA-1, the importantmolecules in Bcl-xl/Beclin-1 pathway. The 6 differentially abundant proteins, PRR5, CISD2, Bcl-xl, NLRX1, RPS15, and RPL26,were confirmed by western blot. Taken together, ribosomal protein-p53 signaling pathway and Bcl-xl/Beclin-1 pathway might bethe most significantly regulated biological process by F

2treatment in SGC7901 cells, which provided valuable insights into the deep

understanding of the molecular mechanisms of F2for gastric cancer treatment.

1. Introduction

Gastric cancer is the fifth most common cancer and the thirdleading cause of cancer-related death worldwide. Annuallyit results in approximately 700,000 deaths [1]. Currently,chemotherapy has proved to decrease the rate of recurrenceand improve overall survival; however, the drug resistanceand serious toxic side effects largely reduce therapeuticefficacy and quality of life in patients [2, 3]. In recent years,compounds of natural products have caught wide attentiondue to their promising anticancer effects and minimal sideeffects [4–7]. Therefore, it is very necessary to develop newoptimal anticancer agent from natural resource [3].

Ginsenosides, the major bioactive constituents in gin-seng, have been demonstrated to exert potential anticancerability [4, 5]. Exploration of ginsenoside as a new anti-carcinogenic agent is of much interest [4–7]. Structural-function studies showed that the increased antitumor effectis implicated with the decrease of its sugar number [5]. Sugarmoiety at C-6 significantly reduces the anticancer activitiesof ginsenosides. Ginsenoside F

2(see structure in Figure 1), a

protopanaxdiol type ginsenoside with one sugar molecular atC-3 and one sugar molecule at C-20, has been shown to bepotent in inhibiting tumorigenesis in several different cancersincluding gastric tumor and glioblastoma multiforme [6, 7].Recently, our in vitro and in vivo studies demonstrated that

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016, Article ID 2635483, 21 pageshttp://dx.doi.org/10.1155/2016/2635483

2 Evidence-Based Complementary and Alternative Medicine

O

H

HO

H

O

O

OHOH

OH

HO

HO

HO

HO

HO

HO

Figure 1: Structure of ginsenoside F2.

ginsenoside F2possesses anticancer effects in human gastric

carcinoma cells SGC7901 [6]. However, the involved exactmechanisms of ginsenoside F

2on SGC7901 cancer cells at

proteome level have not been systemically investigated.Advancements in the field of proteomics have made it

possible to accurately monitor and quantitatively detect thechanges of protein expression in response to drug treatment.The achieved data provide valuable insights into the molec-ular mechanisms of disease and help to identify therapeutictargets [8]. Isobaric tag for relative and absolute quantifica-tion (iTRAQ) is a robust mass spectrometry technique thatallows quantitative comparison of protein abundance bymea-suring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation. iTRAQ with multiplexingcapability up to eight distinct samples in a single experimentand relatively higher sensitivity has gained significant interestin the field of quantitative proteomics. In the present study,SGC7901 cells treated by lower dose and a shorter durationthan that in previous report were analyzed by iTRAQ-based proteomics integrated with bioinformatics using GeneOntology (GO), Kyoto Encyclopedia of Genes and Genomes(KEGG), and Cluster of Orthologous Groups (COG) ofproteins database. And network analysis was applied toidentify critical molecules which are involved in anticancermechanisms of ginsenoside F

2in gastric SGC7901 cells.

General molecular biological techniques such as western blotwere utilized for validation.

2. Materials and Methods

2.1. Reagents and Antibodies. Ginsenoside F2was isolated

previously from leaves of Panax ginseng by a series of chro-matographic procedures [9]. Ginsenoside F

2has a molecular

mass of 784Da and was isolated with 98% purity. Primaryantibodies of PRR5, CISD2, Bcl-2L, NLRX1, RPS15, RPL26,p53, PUMA, Beclin-1, UVRAG, AMBRA-1, mTOR, LC3-II,LC3-I, and 𝛽-actin together with all secondary antibodieswere purchased from Cell Signaling Technology (Danvers,MA, USA). The Atg5, Atg7, and Atg10 antibodies wereobtained from Santa Cruz Biotechnology (Santa Cruz, CA,USA).

2.2. Cell Culture and Treatment. SGC7901 cells were pur-chased from American Type Culture Collection and main-tained in Dulbecco’s modified Eagle’s medium (Hyclone)

supplemented with 10% fetal bovine serum (FBS), 100 𝜇g/mLstreptomycin, and 100 𝜇g/mL penicillin and grown at 37∘C in5% carbon dioxide.

2.3. Protein Preparation. In one of our recent reports [6],we have shown that the IC

50of ginsenoside F

2is in <50 𝜇M

in 24 hours. In order to characterize ginsenoside F2-related

mechanism it is imperative to use samples that are at theearly stages of ginsenoside F

2treatment. So, a lower dose

than the IC50

(20𝜇M) and a shorter duration (12 hours inthe study) were chosen in the study. The treated (20𝜇M) anduntreated SGC7901 cells were suspended in the lysis bufferand sonicated in ice. The proteins were reduced with 10 𝜇MDTT (final concentration) at 56∘C for 1 h and then alkylatedby 55mM iodoacetamide (IAM) (final concentration) in thedarkroom for 1 h.The reduced and alkylated proteinmixtureswere precipitated by adding 4x volume of chilled acetone at−20∘C overnight. After centrifugation at 4∘C, 30 000×g, thepellet was dissolved in 0.5M triethylammonium bicarbonate(TEAB) (Applied Biosystems, Milan, Italy) and sonicated inice. After centrifuging at 30000×g at 4∘C, the supernatantswere collected, and the total protein concentration was deter-mined using a Bradford protein assay kit (BioRad, Hercules,CA,USA).Theproteins in the supernatantwere kept at−80∘Cfor further analysis.

2.4. iTRAQ Labeling and SCX Fractionation. Total protein(100 𝜇g) was taken out of each sample solution and then theprotein was digested with Trypsin Gold (Promega, Madison,WI, USA) with the ratio of protein : trypsin = 30 : 1 at 37∘Cfor 16 hours. iTRAQ labeling was performed according to theiTRAQReagents-8plex labeling manual (AB SCIEX,Madrid,Spain). Briefly, one unit of iTRAQ reagent was thawed andreconstituted in 24𝜇L isopropanol. iTRAQ labels 113 wereused to label control sample separately, and 115 and 117were used to label twice F

2-treated samples for duplicated

experiment. The peptides were labeled with the isobarictags, incubated at room temperature for 2 h. The labeledpeptide mixtures were then pooled and dried by vacuumcentrifugation.

Themixed peptideswere fractionated by strong cation ex-change (SCX) chromatography on a LC-20AB HPLC Pumpsystem (Shimadzu, Kyoto, Japan).The iTRAQ labeled peptidemixtures were reconstituted with 4mL buffer A (25mMNaH2PO4in 25% acetonitrile, pH 2.7) and loaded onto a 4.6×

Evidence-Based Complementary and Alternative Medicine 3

250mm Ul tremex SCX column containing 5𝜇m particles(Phenomenex). The peptides were eluted at a flow rate of1mL/minwith a gradient of buffer A for 10min, 5–60% bufferB (25mMNaH

2PO4, 1MKCl in 25% acetonitrile, pH 2.7) for

27min, and 60–100% buffer B for 1min.The system was thenmaintained at 100% buffer B for 1min before equilibratingwith buffer A for 10min prior to the next injection. Elutionwas monitored by measuring the absorbance at 214 nm, andfractions were collected at 1-minute intervals. The elutedpeptides were pooled into 20 fractions, desalted with aStrata X C18 column (Phenomenex), and vacuum-dried. Thecleaned fractions were then lyophilized again and stored at−20∘C until analyzed by mass spectrometry.

2.5. LC-ESI-MS/MS Analysis Based on Q EXACTIVE. Eachfraction was resuspended in buffer A (2% acetonitrile, 0.1%FA) and centrifuged at 20 000×g for 10min. In each fraction,the final concentration of peptide was about 0.5 𝜇g/𝜇L.10 𝜇L supernatant was loaded on a LC-20AD nano-HPLC(Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cmC18trap column. Then, the peptides were eluted onto a 10 cmanalytical C18 column (inner diameter 75 𝜇m) packed in-house. The samples were loaded at 8 𝜇L/min for 4min; thenthe 44min gradient was run at 300 nL/min starting from 2to 35% B (98% acetonitrile, 0.1% FA), followed by 2-minutelinear gradient to 80%, maintenance at 80% B for 4min.Initial chromatographic conditions were restored in 1min.

Data acquisition was performed with tandem mass spec-trometry (MS/MS) in a Q EXACTIVE (Thermo FisherScientific, San Jose, CA) coupled online to the HPLC.Intact peptides were detected in the Orbitrap at a resolutionof 70 000. Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with anormalized collision energy setting of 27.0; ion fragmentswere detected in the Orbitrap at a resolution of 17500. Inthe octopole collision cell, the ten most intense peptide ions(charge states ≥ 2) were sequentially isolated to a maximumtarget value of 5× 105 by pAGCand fragmentedHCD.Adata-dependent procedure that alternated between one MS scanand 15MS/MS scans was applied for the 15 most abundantprecursor ions above a threshold ion count of 20000 inthe MS survey scan with a following Dynamic Exclusionduration of 15 s. The electrospray voltage applied was 1.6 kV.Automatic gain control (AGC) was used to optimize thespectra generated by the Orbitrap. A sweeping collisionenergy setting of 35 ± 5 eV was applied to all precursor ionsfor collision-induced dissociation. The AGC target for fullMS was 3e6 and 1e5 for MS2. For MS scans, the m/z scanrange was 350 to 2000Da. For MS2 scans, them/z scan rangewas 100–1800Da. The iTRAQ experiments were performedas three technical replicates to gather reliable quantitativeinformation.

2.6. Data Analysis. Raw data files acquired from theOrbitrapwere converted intoMGF files using ProteomeDiscoverer 1.2(PD 1.2,Thermo) [5600msconverter] and theMGF files weresearched. Protein identifications were performed by usingMascot search engine (Matrix Science, London, UK; version2.3.02) against database containing 143397 sequences.

For protein identification and quantification, a peptidemass tolerance of 20 ppm was allowed for intact peptidemasses and 0.05Da for fragmented ions, with allowance foronemissed cleavage in the trypsin digests. Carbamidomethy-lation of cysteine was considered a fixed modification, andthe conversion of N-terminal glutamine to pyroglutamicacid and methionine oxidation were considered variablemodifications. All identified peptides had an ion score abovethe Mascot peptide identity threshold, and a protein wasconsidered identified if at least one such unique peptidematch was apparent for the protein. To reduce the probabilityof false peptide identification, only peptides at the 95%confidence interval by a Mascot probability analysis greaterthan “identity” were counted as identified. The quantitativeprotein ratios were weighted and normalized by the medianratio in Mascot. We set a 1.2-fold change as the threshold anda 𝑝 value must be below 0.05 to identify significant changes.

2.7. Function Method Description. Functional annotations ofthe proteins were conducted using Blast2 GO programagainst the nonredundant protein database (NR; NCBI). TheKEGGdatabase (http://www.genome.jp/kegg/) and the COGdatabase (http://www.ncbi.nlm.nih.gov/COG/) were used toclassify and group these identified proteins.

GO is an international standardization of gene functionclassification system. It provides a set of dynamic updatingcontrolled vocabulary to describe genes and gene productsattributes in the organism. GO has 3 ontologies whichcan describe molecular function, cellular component, andbiological process, respectively.

COG is the database for protein orthologous classifica-tion. Every protein in COG is supposed to derive from a sameprotein ancestor.

KEGG PATHWAY is a collection of manually drawnpathway maps representing our knowledge on the molecularinteraction and reaction networks.Molecules are representedas nodes, and the biological relationship between two nodesis represented as an edge (line).

2.8. Western Blot. Western blot analyses were performedto confirm the presence of differentially expressed proteins.After the treatment of the indicated concentration of gin-senoside F

2(10, 20, and 40 𝜇M) for 12 h, cells were harvested,

washed with cold PBS (pH 7.4), and lysed with ice-cold lysisbuffer (50 𝜇M Tris-HCl, 150 𝜇M NaCl, 1 𝜇M EGTA, 1 𝜇MEDTA, 20 𝜇M NaF, 100 𝜇M Na

3VO4, 1%NP40, 1 𝜇M PMSF,

10 𝜇g/mL aprotinin, and 10 𝜇g/mL leupeptin, pH 7.4) for30min and centrifuged at 12 000×g for 30min at 4∘C. Theprotein concentration of the clear supernatant was quantifiedusing Bio-Rad Protein Assay Kit.

Approximately 30 𝜇g of protein was loaded into a 10–15% sodiumdodecyl sulfate polyacrylamide gel electrophore-sis (SDS–PAGE). Thereafter, proteins were electrophoreti-cally transferred to nitrocellulose membrane and nonspe-cific sites were blocked with 5% skimmed milk in 1%Tween-20 (Sigma-Aldrich) in 20 𝜇M TBS (pH 7.5) andreacted with a primary polyclonal antibody, PRR5, CISD2,Bcl-2L, NLRX1, RPS15, RPL26, p53, Atg5, Atg7, Atg10, LC3-II, LC3-I PUMA, Beclin-1, UVRAG, and mTOR and 𝛽-actin


for 4 h at room temperature. After washing with TBS threetimes (5min each), the membrane was then incubated withalkaline phosphatase-conjugated goat anti-rabbit secondaryantibody. The signal was observed and developed withKodak film by exposure to enhanced chemiluminescence(ECL) plus western Blotting Detection Reagents (AmershamBiosciences, Piscataway, NJ, USA).

2.9. Statistical Analysis. For cell-based assay, experimentswere performed in duplicate and three independent experi-ments were performed. Western blot analyses of differentialprotein expressions were validated on cell lysates from threebiological replicates. Statistical significance was analyzedusing Student’s t-test or ANOVA test by using GraphPadPrism v4.0 software (GraphPad Software, San Diego, CA,USA). Statistical significance is expressed as ∗∗∗𝑝 < 0.001;∗∗𝑝 < 0.01; ∗𝑝 < 0.05.

3. Results

3.1. Proteome Analysis. Human gastric carcinoma cells(SGC7901) are treated with ginsenoside F

2at a dose of

20𝜇M for 12 hours. The harvested proteins are used toperform iTRAQ for quantifying the difference of total 31853peptides and 5411 proteins in SGC7901 cells with or withouttreatment. Finally, 205 proteins were screened out in termsof the change in their expression level which meet ourpredefined criteria of 𝑝 < 0.05 with relative expressionlevels at least >1.2-fold (Table 1) or <0.83-fold (Table 2) (both113/115 and 113/117) in ginsenoside F

2-treated group compared

with the control group. The protein properties, includingpI, molecular weight (MW), and number of residues werecalculated by Mascot. The results are highly reproducible intwo individual experiments.

3.2. Classification of Differentially Expressed Proteins. Firstly,screened proteins were functionally catalogued with GOand WEGO to three different groups (Figures 2 and 3(a)):biological process (BP), cellular component (CC), andmolec-ular function (MF). As shown in Figure 2, the proteins areinvolved in BP including cellular process (13.44%), metabolicprocess (11.16%), single-organism process (10.36%), biolog-ical regulation (8.06%), and regulation of biological pro-cess (7.59%). The identified proteins separated accordingto CC include cell (19.40%), cell part (19.40%), organelle(16.68%), organelle part (12.46%), membrane (7.97%), andmacromolecular complex (7.94%). MF of the proteins wasclassified and large groupswere found to be binding (50.59%),catalytic activity (27.97%), enzyme regulator activity (3.94%),transporter activity (3.84%), and structuralmolecular activity(3.43%).

Further COG function classification revealed that post-translational modification, protein turnover, and ribosomalstructure biogenesis were major function of the screened205 proteins (Figure 3(b)). In each category of BP, CC, andMF, top twenty proteins which generated bigger difference inresponse to ginsenoside F

2treatment are listed in Figure 4.

KEGG is a publicly available pathway database and couldprovide biologists excellent resources to attain a deeper

understanding of biological mechanisms in response to dif-ferent treatments. Protein analysis through KEGG indicatedthat 205 differentially expressed proteins were involved in128 different pathways (data not shown). The connectiondegree between proteins is calculated by protein-proteininteraction network analysis and the results are shown inFigure 5. Among these proteins, PRR5, RPS15, and RPL26were found in ribosomal protein signaling pathway; CISD2,Bcl-xl, and NLRX1 were found in Beclin-1/Bcl-xL pathway.Therefore, PRR5, RPS15, RPL26, CISD2, Bcl-xl, and NLRX1were selected for further validation and study in order to pro-vide a comprehensive perspective for elucidating underlyingmolecular mechanisms of ginsenoside F

2.

3.3. Western Blot Analysis

3.3.1. For Verification. To validate the information obtainedfrom the iTRAQ-based quantitative proteomics study andbioinformatics analysis, the screened proteins with strongresponse to ginsenoside F

2treatment were further confirmed

by western blot. As shown in Figure 6, ginsenoside F2

significantly reduced protein expressions of PRR5, CISD2,Bcl-xl, NLRX1, and RPS15 (𝑝 < 0.01) and enhanced theexpression of the RPL26 (𝑝 < 0.01) in SGC7901 cells incomparison with the treatment with vehicle control.

3.3.2. For Determining the Expression of Apoptosis andAutophagic Proteins. As shown in Figure 6, ginsenoside F

2

suppressed the expression of mTOR and upregulated theexpression of p53 in a dose-dependent manner. Atg5, Atg7,Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1 are knownto be modulated by p53 or Bcl-xl signaling, which maytrigger apoptosis or autophagy. Therefore, we proceeded tocheck the expressions of Atg5, Atg7, Atg10, PUMA, Beclin-1,UVRAG, and AMBRA-1. As shown in Figure 7, ginsenosideF2upregulated the expressions of these proteins in a dose-

dependent manner. LC3 is now widely used to monitorautophagy. During autophagy, the cytoplasmic form LC3-Iis processed and recruited to phagophores, where LC3-II isgenerated by site-specific proteolysis and lipidation at the C-terminus. Thus, the amount of LC3-II positively correlateswith the number of autophagosomes [10]. We examined theeffect of F

2on LC3 conversion in SGC7901 cells. Western blot

analysis showed that F2treatment resulted in dose-dependent

accumulation of LC3-II and reduction of LC3-I (Figure 7).The conversion of LC3-I to LC3-II suggested F

2treatment

induces autophagy.In the present study, combination of iTRAQ-based pro-

teomics method with bioinformatics was used to identifycritical molecules in SGC7901 cancer cells in response to gin-senoside F

2treatment. Ginsenoside F

2generated significant

change of protein profile in SGC7901 cells. Some of themhave been demonstrated to participate in either apoptosis orautophagy responses, suggesting that the antitumor mecha-nisms of ginsenoside F

2in SGC7901 cells are involved in both

apoptosis and autophagy.The current findings demonstrate that ginsenoside F

2

impacts distinct signaling pathways and induces broadchange in the protein profile of SGC7901 cells. Overall, 205


Table1:Differentia

llyup

regu

lated(>1.2

0-fold)p

roteinsidentified

byiTRA

Qin

F 2tre

ated

SGC7

901cells.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Up1

sp|P07305-2

H1F0

Isoform

2of

histo

neH1.0

5135582

132.11

—Up2

sp|P20962

PTMS

Parathym

osin

503

15782

23.5

1.32

—Up3

tr|B8Z

WD1

DBI

Diazepam

bind

inginhibitor,spliceform

1A(2)

121

15706

28.9

1.31

Acyl-C

oA-binding

protein

Up4

sp|Q

16576

RBBP

7Histon

e-bind

ingproteinRB

BP7

877

55737

24.5

1.25

FOG:W

D40

repeat

Up5

sp|P46

779-2

RPL2

8Isoform

2of

60Srib

osom

alproteinL2

8524

22107

27.6

1.35

—

Up6

tr|B2R

514

—cD

NA,FLJ92300,Hom

osapiensC

OP9

subu

nit6

(MOV34

homolog

,34k

D)(CO

PS6),m

RNA

743906

820.2

1.22

Predictedmetal-dependent

protease

ofthe

PAD1/JAB1

superfa

mily

Up7

tr|B3K

Y12

—cD

NAFL

J46581

fis,clone

THYM

U3043200,high

lysim

ilartosplicingfactor

3Asubu

nit3

527

71859

221.2

4Splicingfactor

3a,sub

unit3

Up8

sp|Q

71DI3

HIST2

H3A

Histon

eH3.2

617

19694

26.5

1.40

Histon

esH3andH4

Up9

tr|Q

9P0H

9RE

R1RE

R1protein

11828927

221.2

6Golgiproteininvolved

inGolgi-to

-ER

retrieval

Up10

tr|A8K

3Q9

—cD

NAFL

J76611,highlysim

ilartoHom

osapiens

ribosom

alproteinL14(RPL

14),mRN

A781

35114

25.9

2.24

Ribo

somalproteinL14E

/L6E

/L27E

Up11

sp|Q

9Y3A

2UTP

11L

Prob

ableU3sm

alln

ucleolar

RNA-

associated

protein11

9444

174

21.7

1.30

Uncharacterized

conservedprotein

Up12

tr|F2Z

388

RPL35

60Srib

osom

alproteinL35

9915372

32.3

1.35

Ribo

somalproteinL2

9Up13

sp|Q

9NZZ

3CH

MP5

Chargedmultiv

esicular

body

protein5

268

32218

211.4

2—

Up14

tr|B2R

4D8

—60Srib

osom

alproteinL2

7398

23061

361.2

8Ribo

somalproteinL14E

/L6E

/L27E

Up15

tr|M

0QXF

7C19orf10

UPF

0556

proteinC19orf10

(fragment)

265

11851

251.2

4—

Up16

tr|D3D

V26

S100A10

S100

calcium

bind

ingproteinA10

(ann

exin

IIligand,

calpactin

I,light

polypeptide(

P11)),iso

form

CRA

b(fr

agment)

134

27935

8.3

1.21

—

Up17

tr|H

7C2N

1PT

MA

Thym

osin

alph

a-1(fragment)

11718283

8.8

1.30

—Up18

tr|G2X

KQ0

—Sumo13

6014938

11.9

1.22

Ubiqu

itin-lik

eprotein

(sentrin)

Up19

tr|I3

L1Y9

FLYW

CH2

FLYW

CHfamily

mem

ber2

9919302

47.2

1.45

—Up20

tr|M

0R210

RPS16

40Srib

osom

alproteinS16

1105

19391

57.4

1.27

Ribo

somalproteinS9

Up21

sp|O

43715

TRIAP1

TP53-regulated

inhibitoro

fapo

ptosis1

8212050

18.4

1.36

—Up22

sp|P49207

RPL3

460Srib

osom

alproteinL3

4187

18684

20.5

1.66

Ribo

somalproteinL3

4EUp23

sp|Q

92522

H1FX

Histon

eH1x

342

35250

25.4

1.33

—Up24

tr|J3

KRX5

RPL17

60Srib

osom

alproteinL17(fr

agment)

795

27382

38.5

1.26

Ribo

somalproteinL2

2Up25

sp|P02795

MT2

AMetallothionein-2

104

9915

52.5

1.42

—Up26

tr|Q

6FIE5

PHP14

PHP14protein

7217301

8.8

1.27

—Up27

tr|A0P

J62

RPL14

RPL14protein(fr

agment)

536

2140

943.5

2.85

Ribo

somalproteinL14E

/L6E

/L27E

Up28

tr|G3X

AA2

MAP4

K4Mito

gen-activ

ated

proteinkinase

kinase

kinase

kinase

4142

156989

2.7

1.24

Serin

e/threon

inep

rotein

kinase


Table1:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Up29

tr|C9JNW5

RPL24

60Srib

osom

alproteinL24

666

2464

232

1.67

Ribo

somalproteinL24E

Up30

sp|Q

13951

CBFB

Core-bind

ingfactor

subu

nitb

eta

197

24461

18.1

1.20

—

Up31

tr|D3D

UE6

N-PAC

Cytokine-like

nucle

arfactor

n-pac,iso

form

CRA

c219

76728

14.5

1.24

3-Hydroxyiso

butyratedehydrogenasea

ndrelated

beta-hydroxy

acid

dehydrogenases

Up32

tr|K7E

KW4

ISOC2

Isocho

rismatased

omain-containing

protein2,

mito

chon

drial(fragment)

130

21202

17.4

1.34

Amidases

related

tonicotin

amidase

Up33

sp|Q

9NQ55-2

PPAN

Isoform

2of

Supp

ressor

ofSW

I41h

omolog

7363713

10.7

1.37

—Up34

tr|B3K

MF8

—cD

NAFL

J10869fis,clone

NT2

RP40

01677

127

12398

27.7

1.28

—Up35

sp|P62424

RPL7

A60Srib

osom

alproteinL7

a613

42316

27.1

1.78

Ribo

somalproteinHS6-ty

pe(S12/L30/L7a)

Up36

tr|B4E

0X1

—Be

ta-2-m

icroglob

ulin

185

17093

13.1

1.25

—Up37

tr|H

0Y7A

7CA

LM2

Calm

odulin

(fragment)

735

24209

30.5

1.26

Ca2+-binding

protein(EF-Handsuperfa

mily)

Up38

tr|J3

KTJ8

RPL2

660Srib

osom

alproteinL2

6(fr

agment)

363

15545

341.2

4Ribo

somalproteinL24

Up39

tr|B4D

JM5

—cD

NAFL

J61294,highlysim

ilartokeratin

,typeI

cytoskele

tal17

326

21291

24.9

1.46

—

Up40

sp|Q

9Y3C

1NOP16

Nucleolar

protein16

7927925

20.8

1.24

—Up41

sp|Q

16543

CDC3

7Hsp90

cochaperon

eCdc37

384

57730

29.6

1.22

—Up42

sp|P1640

1HIST1H1B

Histon

eH1.5

801

4264

417.3

2.38

—Up43

sp|Q

07866-3

KLC1

Isoform

Gof

kinesin

light

chain1

642

81828

23.9

1.24

FOG:T

PRrepeat

Up44

tr|B4D

KJ4

—cD

NAFL

J57738,highlysim

ilartotransla

tionally

controlledtumor

protein

344

19250

32.4

1.28

—


Table2:Differentia

llydo

wnregulated

(<0.83-fo

ld)p

roteinsidentified

byiTRA

Qin

F 2tre

ated

SGC7

901cells.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n1

tr|F5H

740

VDAC

3Vo

ltage-dependent

anion-selectivec

hann

elprotein3

1114

39598

41.5

0.81

—Dow

n2

sp|Q

9H845

ACAD9

Acyl-C

oAdehydrogenasefam

ilymem

ber9

,mito

chon

drial

311

81512

21.9

0.69

Acyl-C

oAdehydrogenases

Dow

n3

sp|Q

969S9-2

GFM

2Isoform

2of

ribosom

e-releasingfactor

2,mito

chon

drial

153

94059

5.1

0.80

Transla

tionelo

ngationfactors

(GTP

ases)

Dow

n4

sp|P35908

KRT2

Keratin

,typeIIc

ytoskeletal2

epidermal

338

76630

18.2

0.67

Myosin

heavychain

Dow

n5

tr|B7Z

8A2

—cD

NAFL

J51671,highlysim

ilartoprenylcyste

ineo

xidase

(EC1.8

.3.5)

492

63740

23.8

0.83

—

Dow

n6

sp|Q

9Y512

SAMM50

Sortingandassemblymachinery

compo

nent

50ho

molog

170

59339

18.6

0.76

Outer

mem

branep

rotein/protective

antig

enOMA87

Dow

n7

sp|Q

6ZNW5

GDPG

P1GDP-D-glucose

phosph

orylase1

118

45302

8.6

0.78

—

Dow

n8

sp|P51970

NDUFA

8NADHdehydrogenase[ub

iquino

ne]1

alph

asub

complex

subu

nit8

7225720

15.1

0.68

—

Dow

n9

tr|B4D

RW0

—cD

NAFL

J58125,highlysim

ilartocopp

er-tr

ansportin

gAT

Pase

1(EC

3.6.3.4)

102

61873

6.1

0.78

Catio

ntransportA

TPase

Dow

n10

tr|Q

8NBW

7KD

ELR1

ERlumen

proteinretainingreceptor

5120327

12.7

0.73

ERlumen

proteinretainingreceptor

Dow

n11

tr|B2R

6F5

—cD

NA,FLJ92928,high

lysim

ilartoHom

osapiensretinitis

pigm

entosa

2(X

-link

edrecessive)(RP2

),mRN

A59

47451

2.3

0.82

—

Dow

n12

tr|Q

2VIN

3—

RBM1(fragment)

1232

45756

26.8

0.81

RNA-

bind

ingproteins

(RRM

domain)

Dow

n13

sp|P14174

—Macroph

agem

igratio

ninhibitory

factor

608

13856

17.4

0.71

—

Dow

n14

tr|B2R

6S4

—cD

NA,FLJ93089,high

lysim

ilartoHom

osapiensN

CKadaptorp

rotein

1(NCK

1),m

RNA

137

53755

18.3

0.83

—

Dow

n15

sp|Q

16822

PCK2

Phosph

oeno

lpyruvatec

arbo

xykinase

[GTP

],mito

chon

drial

1795

78784

41.6

0.74

Phosph

oeno

lpyruvatec

arbo

xykinase

(GTP

)

Dow

n16

tr|E9P

M12

TCIRG1

V-type

proton

ATPase

116kD

asub

unitaisoform

3(fr

agment)

6325815

13.3

0.74

Archaeal/v

acuo

lar-type

H+-ATP

ase

subu

nitI

Dow

n17

sp|Q

2T9J0-2

TYSN

D1

Isoform

2of

peroxisomalleader

peptide-processin

gprotease

9643618

9.80.67

—

Dow

n18

tr|J3

KPX7

PHB2

Proh

ibitin-2

1543

3946

651.8

0.82

Mem

branep

roteases

ubun

its,

stomatin/prohibitin

homologs

Dow

n19

tr|Q

8NCF

7—

cDNAFL

J90278

fis,clone

NT2

RP1000325,high

lysim

ilarto

phosph

atec

arrie

rprotein,m

itochon

drialprecursor

517

48576

26.9

0.81

—

Dow

n20

tr|B4E

0R0

—cD

NAFL

J54220,highlysim

ilarto

Long

-chain-fa

tty-acid-CoA

ligase1

(EC6.2.1.3

)100

88560

6.2

0.74

Long

-chain

acyl-C

oAsynthetases

(AMP-form

ing)

Dow

n21

tr|B3K

RY3

—cD

NAFL

J35079

fis,clone

PLAC

E6005283,highlysim

ilarto

lysosome-associated

mem

braneg

lycoprotein1

319

48851

11.1

0.79

—

Dow

n22

tr|B3K

U09

—cD

NAFL

J39034

fis,clone

NT2

RP7008085,high

lysim

ilarto

Hom

osapiensringfin

gerp

rotein

123(RNF123),mRN

A110

166029

2.4

0.78

—

Dow

n23

sp|Q

9BVV7

TIMM21

Mito

chon

drialimpo

rtinnerm

embranetranslocase

subu

nit

Tim21

8635219

13.7

0.82

—


Table2:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n24

sp|Q

9UMY1

NOL7

Nucleolar

protein7

148

39504

12.5

0.78

—Dow

n25

sp|Q

9UNN8

PROCR

Endo

thelialprotein

Creceptor

103

27909

15.1

0.80

—Dow

n26

sp|Q

86SF2

GALN

T7N-Acetylgalactosaminyltransfe

rase

795

89410

9.90.81

—Dow

n27

tr|I3

L0U2

PRSS21

Testisin

(fragment)

115

27083

14.7

0.82

Secreted

trypsin

-like

serin

eprotease

Dow

n28

tr|B7Z

LP5

SAFB

SAFB

protein

557

121835

130.83

—Dow

n29

tr|F2Z

3N7

TMEM

106B

Transm

embranep

rotein

106B

135

12975

12.5

0.82

—Dow

n30

tr|B7Z

361

—Re

ticulon

166

27838

12.2

0.76

—Dow

n31

tr|H

0Y6F

2PR

R5Proline-ric

hprotein5(fr

agment)

5739929

2.3

0.78

—Dow

n32

sp|Q

7Z7E

8UBE

2Q1

Ubiqu

itin-conjugatingenzymeE

2Q1

9254711

1.90.76

—Dow

n33

tr|A8K

4K9

—cD

NAFL

J76169

146

42007

8.8

0.83

—Dow

n34

sp|P1364

5KR

T10

Keratin

,typeI

cytoskele

tal10

382

66321

21.6

0.55

—Dow

n35

sp|Q

8N5K

1CI

SD2

CDGSH

iron-sulfu

rdom

ain-containing

protein2

167

20364

26.7

0.81

—Dow

n36

sp|Q

8NI27

THOC2

THOcomplex

subu

nit2

282

241732

8.7

0.83

—

Dow

n37

tr|B4D

EP8

—cD

NAFL

J56960,highlysim

ilartoHom

osapiens

phosph

atidylinosito

l4-kinasetypeII(PI4K

II),mRN

A127

61711

9.80.76

—

Dow

n38

sp|Q

5BKZ

1ZN

F326

DBIRD

complex

subu

nitZ

NF326

145

78123

7.90.78

—Dow

n39

tr|Q

8IW24

EXOC5

Exocystcom

plex

compo

nent

5108

99962

9.30.82

—

Dow

n40

tr|B3K

MG6

—cD

NAFL

J10939fis,clone

OVA

RC1001065,high

lysim

ilarto

Hom

osapiensM

TERF

domaincontaining

1(MTE

RFD1),

mRN

A117

43225

9.80.76

—

Dow

n41

sp|Q

8NBM

4-2

UBA

C2Isoform

2of

ubiquitin

-associateddo

main-containing

protein2

150

37306

18.1

0.83

—

Dow

n42

sp|Q

8NGA1

OR1M1

Olfactoryreceptor

1M1

7639512

2.2

0.69

—Dow

n43

tr|E9P

N17

ATP5

LAT

Psynthase

subu

nitg

,mito

chon

drial

366

11489

63.2

0.82

—Dow

n44

tr|B2R

686

TGOLN

2Trans-golgin

etworkprotein2,iso

form

CRA

a166

61093

130.79

—Dow

n45

tr|B4D

IR5

—cD

NAFL

J56026

51143728

1.70.74

—Dow

n46

tr|J3

KS15

ICT1

Peptidyl-tR

NAhydrolaseICT

1,mito

chon

drial(fragment)

169

26740

260.82

ProteinchainreleasefactorB

Dow

n47

tr|F5H

0F9

ANAPC

5Anaph

ase-prom

otingcomplex

subu

nit5

7298300

7.50.82

—Dow

n48

tr|C8C

504

HBB

Beta-globin

1233

20056

29.9

0.21

—

Dow

n49

tr|B2R

921

—

cDNA,FLJ94171,high

lysim

ilartoHom

osapienssolute

carrierfam

ily25

(mito

chon

drialcarrie

r;ornithine

transporter)mem

ber15(SLC

25A15),nu

clear

gene

encoding

mito

chon

drialprotein,m

RNA

5339308

90.77

—

Dow

n50

sp|Q

9Y613

FHOD1

FH1/F

H2do

main-containing

protein1

255

141625

8.8

0.81

—

Dow

n51

sp|Q

92643

PIGK

GPI-ancho

rtransam

idase

11051592

10.9

0.77

Glycosylpho

sphatid

ylinosito

ltransamidase(GPIT),sub

unitGPI8

Dow

n52

tr|A4F

TY4

TXNRD

2TX

NRD

2protein

331

41672

24.6

0.79

Pyruvate/2-oxoglutarate

dehydrogenasec

omplex,

dihydrolipoamided

ehydrogenase

(E3)

compo

nent,and

related

enzymes


Table2:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n53

tr|D3D

P46

SPCS

3Sign

alpeptidasec

omplex

subu

nit3

homolog

(S.cerevisiae),

isoform

CRA

a147

24007

18.9

0.82

—

Dow

n54

sp|Q

9Y5Q

9GTF

3C3

Generaltranscrip

tionfactor

3Cpo

lypeptide3

154

117216

7.80.79

—Dow

n55

sp|P60

468

SEC6

1BProteintransportp

rotein

Sec61sub

unitbeta

192

11546

37.5

0.72

—Dow

n56

sp|Q

5RI15-2

—Isoform

2of

cytochromec

oxidasep

rotein

20ho

molog

106

17682

200.83

—Dow

n57

sp|Q

9P206-2

—Isoform

2of

uncharacteriz

edproteinKIAA1522

146

128602

6.5

0.73

—

Dow

n58

sp|Q

86YN

1DOLP

P1Dolichyldipho

sphatase

164

28953

5.5

0.69

Mem

brane-associated

phosph

olipid

phosph

atase

Dow

n59

sp|O

00165-2

—Isoform

2of

HCL

S1-associatedproteinX-

1111

34281

160.81

—Dow

n60

tr|B4E

303

—cD

NAFL

J57449,highlysim

ilartoNotchlessho

molog

1127

54134

16.5

0.82

FOG:W

D40

repeat

Dow

n61

sp|O

00194

RAB2

7BRa

s-relatedproteinRa

b-27B

5629688

14.2

0.77

GTP

aseS

AR1

andrelatedsm

allG

proteins

Dow

n62

tr|B4D

I41

MBD

1Methyl-C

pG-binding

domainprotein1

728740

91.8

0.80

—

Dow

n63

tr|B0U

XB6

ABH

D16A

Abhydrolased

omain-containing

protein16A

129

73275

10.3

0.83

Hydrolaseso

fthe

alph

a/beta

superfa

mily

Dow

n64

sp|Q

5T8D

3-2

—Isoform

2of

Acyl-C

oA-binding

domain-containing

protein

5148

64353

11.6

0.72

Acyl-C

oA-binding

protein

Dow

n65

tr|B4D

NZ6

GTF

2H3

Generaltranscrip

tionfactor

IIHsubu

nit3

4837020

4.5

0.79

RNApo

lymeraseIItranscriptio

ninitiation/nu

cleotidee

xcision

repair

factor

TFIIH,sub

unitTF

B4Dow

n66

sp|Q

96FQ

6S100A16

ProteinS100-A16

346

15197

22.3

0.83

—Dow

n67

tr|B4D

SE1

—cD

NAFL

J55364

,highlysim

ilartoCR

SPcomplex

subu

nit6

5584524

3.7

0.73

—Dow

n68

tr|J3

KNX9

MYO

18A

Uncon

ventionalm

yosin

-XVIIIa

157

282257

3.5

0.72

Myosin

heavychain

Dow

n69

tr| B4D

MK6

—cD

NAFL

J60055,highlysim

ilartoRa

ttusn

orvegicusS

su72

RNApo

lymeraseIIC

TDph

osph

ataseh

omolog

,mRN

A51

23745

13.5

0.82

RNApo

lymeraseII-interactingprotein

involved

intranscrip

tionsta

rtsite

selection

Dow

n70

tr|G3V

1A0

TRAPP

C4HCG

38438,iso

form

CRA

b51

14838

20.5

0.81

—Dow

n71

tr|B1A

HA8

HMOX1

Hem

eoxygenase

1(fragment)

5325525

15.5

0.83

Hem

eoxygenase

Dow

n72

sp|Q

9Y3B

3-2

TMED

7Isoform

2of

transm

embranee

mp24do

main-containing

protein7

193

24908

28.2

0.82

—

Dow

n73

tr|G3V

1U5

GOLT

1BGolgitransport1

homolog

B(S.cerevisiae),iso

form

CRA

c167

9121

20.3

0.77

Mem

branep

rotein

involved

inGolgi

transport

Dow

n74

tr|B1PBA

3—

SKNYprotein

148

109440

8.4

0.81

—Dow

n75

sp|Q

15061

WDR4

3WDrepeat-con

tainingprotein43

138

91327

5.6

0.83

FOG:W

D40

repeat

Dow

n76

tr|D3D

UJ0

AFG

3L2

AFG

3AT

Pase

family

gene

3-lik

e2(yeast)

,isoform

CRA

a(fr

agment)

695

103842

21.2

0.83

ATP-depend

entZ

nproteases

Dow

n77

tr|B2R

BL9

—cD

NA,FLJ95582,high

lysim

ilartoHom

osapiensb

reast

cancer

antiestr

ogen

resis

tance1

(BCA

R1),mRN

A204

104223

60.79

—

Dow

n78

sp|Q

3SXM

5-2

—Isoform

2of

inactiv

ehydroxyste

roid

dehydrogenase-lik

eprotein1

170

35499

13.5

0.83

Short-c

hain

dehydrogenases

ofvario

ussubstrates

pecificities


Table2:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n79

sp|O

43920

NDUFS


iquino

ne]iron-sulfu

rprotein

5106

16388

11.3

0.74

—

Dow

n80

tr|H

0YG20

MAN1B1

Endo

plasmicretic

ulum

manno

syl-o

ligosaccharide

1,2-alpha-m

anno

sidase(fragment)

155

90816

8.2

0.80

—

Dow

n81

tr|Q

0KKI

6—

Immun

oglobu

linlight

chain(fr

agment)

6628559

8.2

0.80

—Dow

n82

sp|P62244

RPS15A

40Srib

osom

alproteinS15a

1521

18594

66.2

0.82

Ribo

somalproteinS8

Dow

n83

tr|B4D

L07

—cD

NAFL

J53353,highlysim

ilartoAT

P-bind

ingcassette

subfam

ilyDmem

ber3

398

92669

16.7

0.81

ABC

-type

uncharacteriz

edtransport

syste

m,permease,and

ATPase

compo

nents

Dow

n84

tr|B4D

R67

ALG

5Dolichyl-p

hosphatebeta-glucosyltransfe

rase

6632213

10.9

0.81

Glycosyltransfe

rasesinvolvedin

cell

wallbiogenesis

Dow

n85

tr|Q

9BTT

5—

SimilartoNADHdehydrogenase(ub

iquino

ne)1

alph

asubcom

plex,9

(39k

D)(fragment)

189

45471

210.75

Predicted

nucle

oside-diph

osph

ate-sugar

epim

erases

Dow

n86

tr|Q

5U0H

8—

Myelin

proteinzero-like

155

34725

4.8

0.74

—

Dow

n87

sp|Q

5SY16

NOL9

Polynu

cleotide5

-hydroxyl-k

inaseN

OL9

109

91782

7.40.79

PredictedGTP

aseo

rGTP

-binding

protein

Dow

n88

sp|O

15173-2

PGRM

C2Isoform

2of

mem

brane-associated

progesterone

receptor

compo

nent

2620

30166

26.3

0.75

—

Dow

n89

sp|Q

5VT5

2-3

RPRD

2Isoform

3of

regu

latio

nof

nucle

arpre-mRN

Ado

main-containing

protein2

295

177879

4.5

0.82

—

Dow

n90

sp|Q

8TC1

2RD

H11

Retin

oldehydrogenase11

494

41238

14.5

0.76

Dehydrogenasesw

ithdifferent

specificitie

s(related

toshort-c

hain

alcoho

ldehydrogenases)

Dow

n91

tr|B4D

Z55

—cD

NAFL

J52097,w

eaklysim

ilartoHom

osapiens

transm

embranea

ndtetratric

opeptid

erepeatcon

taining1

(TMTC

1),m

RNA

164

126875

10.1

0.79

FOG:T

PRrepeat

Dow

n92

tr|J3

KQA9

MTU

S2Microtubu

le-associatedtumor

supp

ressor

cand

idate2

150

181383

0.6

0.77

—Dow

n93

sp|Q

96MG7

NDNL2

Mela

noma-associated

antig

enG1

584164

57.6

0.72

—Dow

n94

tr|H

3BQH3

KLHDC4

Kelch

domain-containing

protein4(fr

agment)

107

47359

10.7

0.83

—Dow

n95

tr|J3

KN00

NDUFA

13NADHdehydrogenase(ub

iquino

ne)1

alph

asub

complex,13

258

28599

23.3

0.81

—

Dow

n96

sp|Q

8NF37

LPCA

T1Lysoph

osph

atidylcholinea

cyltransfe

rase

1708

67346

15.7

0.82

1-Acyl-sn-glycerol-3-pho

sphate

acyltransfe

rase

Dow

n97

sp|Q

9Y5P

4-2

COL4

A3B

PIsoform

2of

collagentype

IValph

a-3-bind

ingprotein

8281121

6.7

0.80

—Dow

n98

tr|Q

5T8U

5SU

RF4

Surfe

it4

418

22863

39.8

0.81

Predictedmem

branep

rotein

Dow

n99

sp|P26599-2

PTBP

1Isoform

2of

polypyrim

idinetract-binding

protein1

570

69515

16.2

0.82

—Dow

n100

sp|Q

8NC5

6LE

MD2

LEM

domain-containing

protein2

137

63423

7.40.76

—Dow

n101

tr|Q

2Q9H

2G6P

DGlucose-6-pho

sphate1-d

ehydrogenase

(fragment)

2165

64315

58.3

0.80

Glucose-6-pho

sphate1-d

ehydrogenase

Dow

n102

sp|P21796

VDAC

1Vo

ltage-dependent

anion-selectivec

hann

elprotein1

2340

38777

62.9

0.80

—Dow

n103

tr|J3

KNH7

SENP3

Sentrin

-specific

protease

388

73986

7.70.78

Protease,U

lp1fam

ily


Table2:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n104

sp|A6N

HL2

-2TU

BAL3

Isoform

2of

tubu

linalph

achain-like

3768

51287

11.8

0.79

Tubu

lin

Dow

n105

tr|B4D

R71

—cD

NAFL

J57078,highlysim

ilartoHom

osapienso

pioid

receptor,sigma1

(OPR

S1),transcrip

tvariant

1,mRN

A63

18151

8.4

0.83

—

Dow

n106

sp|Q

5JRA

6-2

MIA3

Isoform

2of

melanom

ainh

ibito

ryactiv

ityprotein3

415

249369

7.80.80

—Dow

n107

tr|J9

ZVQ3

APO

EAp

olipop

rotein

E(fr

agment)

171

30543

12.2

0.79

—Dow

n108

tr|G5E

9V5

MRP

S22

28Srib

osom

alproteinS22,mito

chon

drial

224

49264

17.3

0.77

—Dow

n109

tr|B7Z

7X8

ATL2

Atlastin-2

112

7666

810.8

0.82

—Dow

n110

sp|P54709

ATP1B3

Sodium

/potassiu

m-tr

ansportin

gAT

Pase

subu

nitb

eta-3

243

39135

17.9

0.83

—Dow

n111

tr|Q

6IBK

3SC

AMP2

SCAMP2

protein

258

39155

9.70.81

—

Dow

n112

tr|A4L

AA3

ATRX

Alpha

thalassemia/m

entalretardatio

nsynd

romeX

-link

ed129

3746

042.5

0.81

Superfa

mily

IIDNA/RNAhelicases,

SNF2

family

Dow

n113

sp|Q

9UK5

9DBR

1Laria

tdebranching

enzyme

203

72182

14.5

0.80

—Dow

n114

tr|B4D

I61

—cD

NAFL

J58182,highlysim

ilartoproteinCY

R61

6850414

6.4

0.70

—Dow

n115

tr|H

3BNF1

CLN6

Ceroid-lip

ofuscino

sisneuron

alprotein6

300

12918

200.80

—

Dow

n116

tr|E7E

RK9

EIF2B4

Transla

tioninitiationfactor

eIF-2B

subu

nitd

elta

170

71199

8.8

0.79

Transla

tioninitiationfactor

2Bsubu

nit,eIF-2B

alph

a/beta/deltafam

ilyDow

n117

tr|H

0Y8C

3MTC

H1

Mito

chon

drialcarrie

rhom

olog

1(fragment)

975096

412.9

0.81

—

Dow

n118

tr|B2R

MV2

CYTS

ACY

TSAprotein

52149539

2.5

0.79

Ca2+-binding

actin

-bun

dlingprotein

fimbrin/plastin

(EF-hand

superfa

mily)

Dow

n119

tr|I3

L1P8

SLC2

5A11

Mito

chon

drial2-oxoglutarate/malatec

arrie

rprotein

(fragment)

470

37200

35.5

0.83

—

Dow

n120

sp|Q

8NBU

5-2

ATAD1

Isoform

2of

ATPase

family

AAAdo

main-containing

protein1

124

4046

811.1

0.72

ATPaseso

fthe

AAA+cla

ss

Dow

n121

sp|Q

9Y3E

7CH

MP3

Chargedmultiv

esicular

body

protein3

102

32415

14.4

0.83

Con

served

proteinim

plicated

insecretion

Dow

n122

sp|P02763

ORM

1Alpha-1-

acid

glycop

rotein

1262

28288

20.4

0.80

—

Dow

n123

tr|Q

53F51

—FG

Fintracellularb

inding

proteiniso

form

bvaria

nt(fr

agment)

165

48798

120.83

—

Dow

n124

sp|Q

3ZAQ

7VMA21

Vacuolar

ATPase

assemblyintegralmem

branep

rotein

VMA21

241

12868

24.8

0.81

—

Dow

n125

tr|B2R

6X8

—cD

NA,FLJ93169,high

lysim

ilartoHom

osapiensG

PAA1P

anchor

attachmentp

rotein

1hom

olog

(yeast)

(GPA

A1),

mRN

A106

72151

7.60.80

—

Dow

n126

sp|Q

9P0S9

TMEM

14C

Transm

embranep

rotein

14C

4512774

8.9

0.70

—Dow

n127

sp|P08779

KRT16

Keratin

,typeI

cytoskele

tal16

630

57054

23.9

0.62

—Dow

n128

sp|Q

86UT6

-2NLR

X1Isoform

2of

NLR

family

mem

berX

175

110309

4.1

0.71

—Dow

n129

tr|Q

59E9

9—

Thrombo

spon

din1v

ariant

(fragment)

153

155789

3.4

0.68

—

Dow

n130sp|Q

8WXH

0-2

SYNE2

Isoform

2of

nesprin

-2149

986758

1.10.82

Ca2+-binding

actin

-bun

dlingprotein

fimbrin/plastin

(EF-hand

superfa

mily)


Table2:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n131

sp|P78310-2

CXADR

Isoform

2of

coxsackieviru

sand

adenoviru

sreceptor

4747491

3.8

0.74

—Dow

n132

tr|B2R

995

—Malicenzyme

9877738

5.8

0.83

Malicenzyme

Dow

n133

tr|Q

5QP5

6BC

L2L1

Bcl-2

-like

protein1(fragment)

9821810

23.2

0.82

—Dow

n134

tr|H

0YK7

2SE

C11A

SEC1

1-like1

(S.cerevisiae),iso

form

CRA

a247

22018

16.5

0.81

Sign

alpeptidaseI

Dow

n135

tr|B4D

DH8

—cD

NAFL

J55184,highlysim

ilartoHom

osapiensleuko

cyte

receptor

cluste

r(LR

C)mem

ber4

(LEN

G4),m

RNA

137

54865

8.8

0.79

Predictedmem

branep

rotein

Dow

n136

sp|Q

9UJS0-2

SLC2

5A13

Isoform

2of

calcium-binding

mito

chon

drialcarrie

rprotein

Aralar2

719

86824

17.5

0.82

—

Dow

n137

tr|A8K

AK5

—cD

NAFL

J77399,highlysim

ilartoHom

osapiensc

ofactor

requ

iredforS

p1transcrip

tionalactivation,

subu

nit2,

150k

Da(

CRSP

2),m

RNA

85182987

3.4

0.82

—

Dow

n138

tr|H

0YEF

3RN

ASE

H2C

Ribo

nucle

aseH

2subu

nitC

(fragment)

7618856

25.3

0.77

—Dow

n139

tr|Q

5QNZ2

ATP5

F1AT

Psynthase

F(0)

complex

subu

nitB

1,mito

chon

drial

406

27794

47.7

0.82

—Dow

n140

sp|Q

6UW68

TMEM

205

Transm

embranep

rotein

205

165

23294

15.9

0.82

—Dow

n141

tr|B3K

PJ4

PHC2

Polyho

meotic

-like

protein2

193

59764

9.30.79

—Dow

n142

tr|H

0Y4D

4AC

AA1

3-Ke

toacyl-C

oAthiolase,peroxiso

mal(fr

agment)

131

30218

12.7

0.78

Acetyl-C

oAacetyltransfe

rase

Dow

n143

tr|Q

4G0F

4PO

LRMT

DNA-

directed

RNApo

lymerase

167

159664

4.6

0.81

Mito

chon

drialD

NA-

directed

RNA

polymerase

Dow

n144

tr|Q

6FGZ3

EPHX1

EPHX1

protein(fr

agment)

519

62281

14.9

0.77

Predictedhydrolases

oracyltransfe

rases(alph

a/betahydrolase

superfa

mily)

Dow

n145

tr|B4D

VN1

—cD

NAFL

J52214,highlysim

ilartoDnaJh

omolog

subfam

ilyBmem

ber6

9037740

8.6

0.70

DnaJ-cla

ssmolecular

chaperon

ewith

C-term

inalZn

fingerd

omain

Dow

n146

sp|Q

92667-2

AKA

P1A-

kinase

anchor

protein1,mito

chon

drial

66111

940

4.9

Dow

n147

sp|O

00483

NDUFA


iquino

ne]1

alph

asub

complex

subu

nit4

165

11855

46.9

0.83

—

Dow

n148

sp|Q

9NTJ5

SACM

1LPh

osph

atidylinositide

phosph

ataseS

AC1

179

77476

18.2

0.83

Phosph

oino

sitidep

olypho

sphatase

(Sac

family)

Dow

n149

tr|B3K

VC5

—cD

NAFL

J16380fis,clone

TLIV

E2002882,w

eaklysim

ilarto

imidazolon

epropion

ase(EC

3.5.2.7)

4153582

3.3

0.83

Imidazolon

epropion

asea

ndrelated

amidoh

ydrolases

Dow

n150

tr|B7Z

LI5

FAM98C

Family

with

sequ

ence

similarity98,m

emberC

7241696

9.50.68

—Dow

n151

tr|B7Z

6F5

YIPF

1ProteinYIPF

164

40866

2.7

0.61

—

Dow

n152

sp|Q

6NVY1-2

HIBCH

Isoform

2of

3-hydroxyisobu

tyryl-C

oAhydrolase,

mito

chon

drial

101

46543

19.2

0.82

Enoyl-C

oAhydratase/carnitine

racemase

Dow

n153

tr|U3K

QJ1

POLD

IP2

Polymerased

elta-interactingprotein2

282

46395

26.4

0.76

Uncharacterized

proteinaffectin

gMg2+/C

o2+transport

Dow

n154

tr|D6R

GZ2

THOC3

THOcomplex

subu

nit3

172

12690

36.2

0.75

—Dow

n155

tr|A0S0T

0AT

P6AT

Psynthase

subu

nita

128

26896

4.4

0.78

F0F1-ty

peAT

Psynthase,sub

unita

Dow

n156

tr|G3V

2U7

ACYP

1Ac

ylph

osph

atase

8517520

14.7

0.80

acylph

osph

atases

Dow

n157

sp|Q

9ULG

6-2

CCPG

1Isoform

2of

cellcycle

progressionprotein1

7993313

4.1

0.81

—


Table2:Con

tinued.

Rank

#Ac

cession

Genes

ymbo

l(GN)

Definitio

n(descriptio

n)Score

Mass

Cov%

Ratio

nCO

Gfunctio

n-descrip

tion

Dow

n158

tr|H

7BXZ

6RH

OT1

Mito

chon

drialR

hoGTP

ase

142

8160

05.9

0.77

GTP

aseS

AR1

andrelatedsm

allG

proteins

Dow

n159

sp|Q

14151

SAFB

2ScaffoldattachmentfactorB

2461

129824

130.83

—Dow

n160

sp|Q

96LD

4TR

IM47

Tripartitem

otif-containing

protein47

138

75838

7.80.81

—

Dow

n161

tr|A8K

2K2

—cD

NAFL

J76494,highlysim

ilartoHom

osapiensG

TPBP

2GTP

-binding

likep

rotein

2137

64767

11.7

0.83

GTP

ase


Biological adhesion (0.66%)Biological regulation (8.06%)Cell killing (0.06%)Cellular component organization or

Cellular process (13.44%)Developmental process (3.85%)Establishment of localization (4.07%)Growth (0.70%)Immune system process (1.76%)Localization (4.76%)Locomotion (1.03%)Metabolic process (11.16%)Multiorganism process (1.82%)Multicellular organismal process (4.51%)Negative regulation of biological process (3.07%)Positive regulation of biological process (3.54%)

Regulation of biological process (7.59%)Reproduction (1.84%)Reproductive process (1.73%)Response to stimulus (6.23%)Rhythmic process (0.15%)Signaling (4.15%)Single-organism process (10.36%)

biogenesis (5.46%)

(a)

Cell (19.40%)Cell junction (0.83%)Cell part (19.40%)Extracellular matrix (0.22%)Extracellular matrix part (0.12%)Extracellular region (1.10%)Extracellular region part (0.65%)Macromolecular complex (7.94%)Membrane (7.97%)Membrane part (5.24%)Membrane-enclosed lumen (7.09%)Nucleoid (0.16%)Organelle (16.68%)Organelle part (12.46%)Synapse (0.44%)Synapse part (0.29%)Virion (0.00%)Virion part (0.00%)

(b)

Antioxidant activity (0.46%)Binding (50.59%)Catalytic activity (27.97%)Channel regulator activity (0.25%)Chemoattractant activity (0.06%)Electron carrier activity (0.85%)Enzyme regulator activity (3.94%)Metallochaperone activity (0.04%)Molecular transducer activity (2.27%)Nucleic acid binding transcription factor activity (2.02%)Nutrient reservoir activity (0.01%)Protein binding transcription factor activity (2.45%)Protein tag (0.01%)Receptor activity (1.51%)Receptor regulator activity (0.01%)Structural molecule activity (3.43%)Translation regulator activity (0.19%)Transporter activity (3.84%)

(c)

Figure 2: Classification of identified proteins. (a)The biological processes (BPs), (b) cellular components (CCs), and (c) molecular functions(MFs) of the total identified proteins classified by GO database.


100

10

1

0.1

Perc

ent o

f pro

tein

s

Num

ber o

f pro

tein

s

Homo01

5115

511

51

0

Mol

ecul

ar tr

ansd

ucer

activ

ityM

etal

loch

aper

one a

ctiv

ityEn

zym

e reg

ulat

or ac

tivity

Elec

tron

carr

ier a

ctiv

ityCh

emoa

ttrac

tant

activ

ityCh

anne

l reg

ulat

or ac

tivity

Cata

lytic

activ

ityBi

ndin

gA

ntio

xida

nt ac

tivity

Biol

ogic

al ad

hesio

nBi

olog

ical

regu

latio

nC

ell k

illin

g

Cel

lula

r pro

cess

Dev

elopm

enta

l pro

cess

Cell

ular

com

pone

nt o

rgan

izat

ion

or b

ioge

nesis

Loca

lizat

ion

Imm

une s

yste

m p

roce

ssG

row

thEs

tabl

ishm

ent o

f loc

aliz

atio

n

Sing

le-o

rgan

ism p

roce

ssSi

gnal

ing

Rhyt

hmic

pro

cess

Resp

onse

to st

imul

usRe

prod

uctiv

e pro

cess

Repr

oduc

tion

Regu

latio

n of

bio

logi

cal p

roce

ssPo

sitiv

e reg

ulat

ion

of b

iolo

gica

l pro

cess

Neg

ativ

e reg

ulat

ion

of b

iolo

gica

l pro

cess

Mul

ticel

lula

r org

anism

al p

roce

ssM

ultio

rgan

ism p

roce

ssM

etab

olic

pro

cess

Loco

mot

ion

Virio

n pa

rtVi

rion

Syna

pse p

art

Syna

pse

Org

anel

le p

art

Org

anel

leN

ucle

oid

Mem

bran

e-en

close

d lu

men

Mem

bran

e par

tM

embr

ane

Mac

rom

olec

ular

com

plex

Extr

acel

lula

r reg

ion

part

Extr

acel

lula

r reg

ion

Extr

acel

lula

r mat

rix p

art

Extr

acel

lula

r mat

rixC

ell p

art

Cel

l jun

ctio

nC

ell

Nuc

leic

acid

bin

ding

tran

scrip

tion

fact

or ac

tivity

Nut

rient

rese

rvoi

r act

ivity

Prot

ein

bind

ing

tran

scrip

tion

fact

or ac

tivity

Prot

ein

tag

Rece

ptor

activ

ityRe

cept

or re

gulat

or ac

tivity

Stru

ctur

al m

olec

ule a

ctiv

ityTr

ansla

tion

regu

lator

activ

ityTr

ansp

orte

r act

ivity

Biological process Cellular component Molecular function

(a)

0

200

400

600

800

Function class

Num

ber o

f pro

tein

s

A B C D E F GH I J K L MNO P Q R S T U V Y Z

A: RNA processing and modificationB: chromatin structure and dynamicsC: energy production and conversion

E: amino acid transport and metabolismF: nucleotide transport and metabolismG: carbohydrate transport and metabolismH: coenzyme transport and metabolismI: lipid transport and metabolismJ: translation, ribosomal structure, and biogenesisK: transcriptionL: replication, recombination, and repair

M: cell wall/membrane/envelope biogenesisN: cell motilityO: posttranslational modification, and protein turnover, chaperonesP: inorganic ion transport and metabolismQ: secondary metabolites biosynthesis, transport and catabolismR: general function prediction onlyS: function unknownT: signal transduction mechanismsU: intracellular trafficking, secretion, and vesicular transportV: defense mechanismsY: nuclear structureZ: cytoskeleton

COG function classification of homo01 sequence

D: cell cycle control, cell division, andchromosome partitioning

(b)

Figure 3: WEGO (a) and COG (b) assay of the 205 differentially expressed proteins.


Protein localization to endoplasmic reticulumSRP-dependent cotranslational protein targeting to membrane

Macromolecular complex disassemblyProtein targeting to ER

Establishment of protein localization to endoplasmic reticulumCotranslational protein targeting to membrane

Translational terminationProtein complex disassembly

Cellular protein complex disassemblyViral genome expression

Viral transcriptionProtein targeting to membrane

TransportEstablishment of localization

Cellular component disassemblySingle-organism transport

Translational elongationLocalization

Protein localization to organelleRibosomal large subunit biogenesis

Biological process

20 40 60 800Number of proteins

≤ 0.001p value

(a)

Intrinsic to membraneIntegral to membrane

Membrane partOrganelle membrane

Endoplasmic reticulum membraneCytosolic large ribosomal subunit

Nuclear outer membrane-endoplasmic reticulum membrane networkLarge ribosomal subunit

Endoplasmic reticulum partEndoplasmic reticulum

Cytosolic ribosomeOrganelle part

Intracellular organelle partMitochondrial membrane

Ribosomal subunitmembrane

Mitochondrial partMitochondrial envelope

Organelle outer membraneOuter membrane

Cellular component

50 100 1500Number of proteins

≤ 0.001p value

(b)

Structural constituent of ribosomeGrowth factor binding

Structural molecule activityGPI anchor binding

NADH dehydrogenase activityNADH dehydrogenase (ubiquinone) activity

NADH dehydrogenase (quinone) activityAnion transmembrane transporter activity

Ion transmembrane transporter activityTransmembrane transporter activity

Substrate-specific transmembrane transporter activityGPI-anchor transamidase activity

Porin activityWide pore channel activity

Primary active transmembrane transporter activityP-P-bond-hydrolysis-driven transmembrane transporter activity

G-protein coupled receptor activityOxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor

Substrate-specific transporter activityOxidoreductase activity, acting on NAD(P)H

Molecular function

10 20 30 400Number of proteins

0.01 <

0.001 < ≤ 0.01p value≤ 0.05p value

≤ 0.001p value

(c)

Figure 4: GO annotation of the final selected differentially expressed proteins. The top 20 components for BP (a), CC (b), and MF (c) of theselected differentially expressed proteins are shown along with their enrichment score, represented as a 𝑝 value.

differentially expressed proteins were identified with ≥95%confidence in ginsenoside F

2treated group. Application of

a ratio of 1.2-fold change as criteria resulted in 44 and 161differentially abundant proteins in SGC7901 cells.

In our study, some proteins that were significantly alteredby ginsenoside F

2show close relationship of protein-protein

interaction (Figure 5). Ribosomal proteins, such as RPS15and RPL26, exert critical roles in MDM2-p53 signal pathway[11, 12]. PRR5 [13], CISD2 [14], Bcl-xl [15], and NLRX1 [16, 17]have been reported to play a key role in the regulation ofautophagy or apoptosis. The changes of these six potentialproteins were verified by western blot analysis.

Ribosomal proteins (RPs) are considered to have diverseextra ribosomal functions, ranging from cell cycle progres-sion to cell death and to malignant transformation and cel-lular metabolism [11]. Relevantly, a number of RPs have been

shown to bind toMDM2, the inhibitor of p53 (murine doubleminute 2, and also HDM2 for its human ortholog), andinhibit MDM2 E3 ligase activity, leading to p53 stabilizationand activation, then triggering apoptosis or autophagy [11].Following the treatment of ginsenoside F

2in SGC7901 cells,

the levels of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14,RPL24, RPL7A, and RPL26 were increased, whereas that ofRPS15 reduced. Although the functions of RPL28, RPL34,RPL35, RPS16, RPL17, RPL14, RPL24, and RPL7A have notbeen well studied, RPL26, a positive regulator of p53, wasfound to increase the translational rate of p53 mRNA bybinding to its 50 untranslated region [12] and, in this case,MDM2 acts as an ubiquitin E3 ligase for ubiquitylation anddegradation of RPL26 [18]. Thus, under the treatment ofginsenoside F

2, the increased level of RPL26 indicated that

RPL26 may inhibit MDM2 and subsequently activate p53.


UpregulatedDownregulated

ATP5L

ATP5F1

MT-ATP6

TMEM14C

CISD2EXOC5

GLYR1

SPECC1L

RER1

AFG3L2ISOC2

ICT1

YIPF1

TPT1

GFM2

PRS15 EMSG00000215472

RPS11RPS16

RPS20 RPL35RPS14

RPL7A

SEC11A

RPL26

RPL24RPL14RPL27A

RPL28SPCS3

SEC61BRPL34

GALNT7

FLYWCH2 LEMD2

RPRD2

TRIAP1

SLC25A3

TRAPPC4

ZNF326

AKAP1

DBR1MTUS2

THOC2HAX1

C12orf23

ACYP1

NOL9 THOC3

EIF2B4ORM1

HSDL1RTN3COL4A3BP

PGRMC2PCK2RP2

RNF23ME3

G6PD NLRX1MRPS22

TMEM205FAM98C

LPCAT1SCAMP2 S100A10

TCIRG1

KRT16

NDNL2

VDAC3

MBOAT7

COPS6

PCYOX1

C19orf10LAMP1

TGOLN2

MED17

APOE

THBS1

CYR61NCK1

MAP4K4CHMP3

MED14

KLC1

MIF

HMOX1TMTC3

BCL2L1DBI

FHOD1

SYNE2

CALM1CDC37

UBE2IHSP9

DNAJB6

CHMP5

SENP3

MYO18A

SUM01

TUBAL3

MBD1SAFB2PPAN

BCAR1NOL7

SLC25A13

NOP16

UTP11L

KIAA1522ABHD16A

WDR43

SAFB

RBMXL1

SF3A1SA3A2

SA3A3PTBP1

NLE1

ATRX

PRR5

HB3

EPHX1

DOLPP1

ATAD1

TIRAP3

ENSG00000228325CCPG1 COX20

MIA3MQZL1

S100A16

CXADR ATP1B3 UBE2Q1 CBFB

SLC25A15KRT2ALG5

SLC25A11

FIBP

KRT10 TMEM106B

NDUFA9SMPD4NDFA8

NDUFS5PRS21

NDUFA13NDUFA4

SIGMAR1 TXNRD2

H1F0

H1FX

HIST1H1B

ATL2 GDPGP1 RDH11

GTPBP2 RNASEH2C KRT17

MTCH1 AMDHD1 KLHDC4

VNA21 ATP71 GOLT1B

FBXO3

MT2A

B2MNPL2

OR1M1HIBCH

TYSND1 ACSL1 ACAD9

ABCD3 ACAA1

SAMM50

CLN6

PROCR

VDAC1

PHB2 MAN1B1SURF4

TIMM21

GPAA1PTMARHOT1

TRIM47

EMC2 PIGTPIGK

ACBD5

POLRMTPTMS

RAB27B

MTERFD1

PHPT1 GTF3C3

SSU72RBBP7

HIST2H3D

GTF2H3

ANAPC5

PHC2 SACM1LUBAC2

HEATR7A

Figure 5: The protein-protein interaction network of the differentially expressed proteins identified. Red triangle denotes upregulatedproteins; green triangle denotes downregulated protein.

RPS15, identified as a direct p53 transcriptional target, wasthought to activate p53 by repressing MDM2 activity [19].Interestingly, in our study, the level of RPS15 reduced inSGC7901 followed by ginsenoside F

2treatment, suggesting

that the roles of RPS15 and RPL26 involved in the anticancermechanism of ginsenoside F

2are different, which warrant

further investigation.

mTOR, existing in twomultiprotein complexes, mTORC1and mTORC2, regulates cell growth in response to a vari-ety of cellular signals derived from growth factors andenvironmental stress [20]. mTORC2 is a kinase complexcomprised of mTOR, PRR5, Rictor, mSin1, and mLST8/GbL.The expression level of PRR5 is correlated with that ofmTORC2. Recent study showed that mTORC2 is implicated


PRR5

CISD2

NLRX-1

0 10 20 40

RPL26

RPS15

40 20 10 0

p53

PUMA

4020100

𝛽-actin

𝛽-actin

mTOR

40 20 10 0

Bcl-xl

F2 concentration (𝜇M)




5

2

1

40 20 10 0

3

A

4020100

n

n

R

40 2010 0

l




𝛽-actin

𝛽-actin

(a)

Control10𝜇M

20𝜇M40𝜇M

Control10𝜇M

20𝜇M40𝜇M

PRR5 CISD2 NLRX-1

∗

∗

∗∗

∗∗

∗∗ ∗∗

RPL26 RPS150.0

0.5

1.0

1.5

2.0

Fold

chan

ge (r

elat

ive i

nten

sity)

0.0

0.5

1.0

1.5

Fold

chan

ge (r

elat

ive i

nten

sity)

Control

0

2

4

6

8

Fold

chan

ge (r

elat

ive i

nten

sity)

PUMAP53

10𝜇M20𝜇M40𝜇M

mTOR Bcl-xl0.0

0.5

1.0

1.5

Fold

chan

ge (r

elat

ive i

nten

sity)

Control10𝜇M

20𝜇M40𝜇M

∗

∗

∗∗

∗∗

∗∗

∗∗

(b)

Figure 6: Western blot validations of RPS15, RPL26, PRR5, CISD2, NLRX1, p53, PUMA, mTOR, and Bcl-xl in SGC7901 cells with differentconcentrations of ginsenoside F

2. 1 × 106 SGC7901 cells are seeded in 6-well plate for overnight. On day 2, the cultured cells are treated with

different concentration ginsenoside F2. 12 hours after treatment, the protein is prepared by lysating cells with RIPA buffer for performing

western blot analysis. Left panel: the representative western blot analysis. 𝛽-actin was used as the loading control. Right panel: accumulatedresults show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as ∗∗𝑝 < 0.01, ∗𝑝 < 0.05.


UVRAG

40 20 10 0

40 20 10 0

Beclin-1

AMBRA-1


Atg10

Atg7

Atg5

LC3-IILC3-I


402010 0


𝛽-actin

𝛽-actin

(a)

2

4

6

8

UVRAGBeclin-1 AMBRA-10

10

Fold

chan

ge (r

elat

ive i

nten

sity)

Control10𝜇M

20𝜇M40𝜇M

Control10𝜇M

20𝜇M40𝜇M

∗

∗

∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗∗∗

0

1

2

3

4

5

Fold

chan

ge (r

elat

ive i

nten

sity)

Atg10Atg7Atg5LC3-IILC3-I

(b)

Figure 7: Effect of ginsenoside F2on the expression of Beclin-1, UVRAG, AMBRA-1, Atg5, Atg7, Atg10, LC3 I, and LC3-II. 1 × 106 SGC7901

cells are seeded in 6-well plate for overnight. On day 2, the cultured cells are treated with different concentration ginsenoside F2. 12 hours

after treatment, the protein is prepared by lysating cells with RIPA buffer for performing western blot analysis. Left panel: the representativewestern blot analysis. 𝛽-actin was used as the loading control. Right panel: accumulated results show the relative protein density. Error barsrepresent means ± SEMs. Significant difference is expressed as ∗∗𝑝 < 0.01, ∗𝑝 < 0.05.

in actin cytoskeleton regulation, as well as phosphorylationof Akt [13]. Although TOR kinase has been largely attributedas a negative regulator of autophagy through TORC1, resentstudy indicated that mTORC2 was an independent positiveregulator of autophagy during amino acid starvation [21]. Inthe present study, ginsenoside F

2decreased level of PPR5,

indicated that ginsenoside F2may inhibit the expression of

PRR5, and consequently inhibited mTORC2.Recent study indicated that p53 can be a positive or

negative regulator of autophagy. In the nucleus, p53 mayactivate the AMPK pathway and inhibit the mTOR pathway,subsequently triggering autophagy. p53 may also transac-tivate multiple genes with proautophagic roles, includingproapoptotic Bcl-2 proteins (Bax, PUMA) [22, 23]. In thisnetwork, PUMA induces the noncanonical autophagy path-way regulated via Atg5, Atg7, and Atg10. PUMA’s initiationof autophagy promotes cytochrome c release, which thenleads to apoptosis [22]. Interestingly, in our previous work,increasing level of cytochrome c and decreased mitochon-drial transmembrane potential (MTP) were observed [6].In present study, decreased expressions of PRR5 and RPL26were found, which implied that ginsenoside F

2might trigger

p53 signal pathway. It was reported that western blot analyses

tended to show greater differential abundance comparedwithiTRAQanalyses [24].Thus, the expressions of p53, Atg5, Atg7,Atg10, and PUMA were validated by western blot analyses.The increased level of Atg5 Atg7, Atg10, and PUMA andreduced level of P53 andmTORC2 suggested that ginsenosideF2may initiate autophagy by ribosomal protein-p53 signaling

pathway.CISD2, also known as NAF-1, Miner1, Eris, and Noxp70,

is a member of the 2Fe-2S cluster NEET family [25]. Ourresults showed that CISD2 was significantly decreased inginsenoside F

2treated group, confirmed by western blot

analysis. Recent work identified CISD2 as a Bcl-xl bindingpartner at a branch point between autophagy and apoptosis,life and death, under nutrient-deprived and oxidative stressconditions in vivo cells [25, 26]. Bcl-xl, also called Bcl-2L, isknown to function through inhibition of the autophagy effec-tor and tumor suppressor Beclin-1 [15]. CISD2 is required inthis pathway for Bcl-xl to functionally antagonize Beclin-1-dependent autophagy. In our study, the expression of Bcl-xldecreased, confirmed by western blot analysis. Thus, CISD2may be a Bcl-xl-associated cofactor that targets Bcl-2 for theautophagy pathway.


During initiation of autophagosome formation, afterrelease from Bcl-xl, Beclin-1 functions as a platform bybinding to class III PI3K/vacuolar protein sorting-34(Vps34), UV-resistance-associated gene (UVRAG), activat-ing molecule in Beclin-1-regulated autophagy (AMBRA-1)[15, 26, 27]. Previous studies have shown that binding ofBeclin-1 to Bcl-2/Bcl-xl inhibits the autophagic function ofBeclin-1, suggesting that Beclin-1 might have a role in theconvergence between autophagy and apoptotic cell death[22]. For confirming the Beclin-1/Bcl-xl pathway, westernblot was employed. The expressions of Beclin-1, UVRAG,and AMBRA-1 were increased, while Bcl-xl was decreased,which suggested that ginsenoside F

2may induce autophagy

via Bcl-xl/Beclin-1 pathway.NLRX1, a mitochondrial NOD-like receptor that ampli-

fies apoptosis by inducing reactive oxygen species produc-tion, is an important component of TLR mediated inflam-matory pathways [13, 16]. Recent evidence suggested thatupregulated expression of NLRX1 may synergistically regu-late metabolism and autophagy for highly invasive growthof the autophagy addicted MDA-MB-231 breast cancer cells[16]. And it acted as tumor suppressor by regulating TNF-𝛼 induced apoptosis and metabolism in cancer cells. Inour iTRAQ results, expression of NLRX1 was significantlydecreased in SGC7901 cells treated with ginsenoside F

2. The

phenomenon suggested different role of NLRX1 involved inthe ginsenoside F

2treatment that may be different from that

of published reports [16, 17], though the mechanism needsfurther research.

Mai et al. reported that F2induces apoptotic cell death

accompanied by protective autophagy in breast cancer stemcells [28]. In one of our previous studies, we found thatF2induces apoptosis by causing an accumulation of ROS

and activating the apoptosis signaling pathway [6]. However,there was no report systemically comparing differently reg-ulated proteins and building a network of F

2-treated cancer

cells at proteome level. In the current study, by the close lookat cellularmechanisms at proteome level, we clearly identifiedthe distinct pattern of cellular responses for the F

2-treated

cells, and 6 differentially regulated proteins were identified,which provide useful information on elucidating the anti-cancer mechanism of F

2to SGC7901 cells. Moreover, the

integration of networks and pathway with the proteomic dataenhanced our understanding of the functional relationship ofproteome changes caused by the compound.

4. Conclusions

In conclusion, 44 upregulated proteins and 161 downregu-lated proteinswere discovered by iTRAQanalysis in SGC7901cells treated with lower dose and shorter duration of ginseno-side F

2, compared with our previous study. 6 differentially

abundant common proteins, PRR5, CISD2, Bcl-xl, NLRX1,RPS15, and RPL26, were confirmed by western blot analysis.Ribosomal protein-p53 signaling pathway and Bcl-xl/Beclin-1 pathway might be significantly regulated biological processby ginsenoside F

2treatment in SGC7901 cells. Althoughmore

work is required to find out the precise role of targetedproteins, our data lead to a better understanding of the

molecular mechanisms of ginsenoside F2for gastric cancer

treatment.

Abbreviations

iTRAQ: Isobaric tag for relative and absolutequantification

KEGG: Kyoto Encyclopedia of Genes andGenomes

COG: Cluster of orthologous groups of proteinsGo: Gene OntologyFBS: Fetal bovine serumSCX: Strong cation exchangeHCD: High-energy collision dissociationAGC: Automatic gain controlNR: Nonredundant protein databaseSDS-PAGE: Sodium dodecyl sulfate polyacrylamide

gel electrophoresisECL: Enhanced chemiluminescenceBP: Biological processCC: Cellular componentMF: Molecular functionRPs: Ribosomal proteinsMTP: Mitochondrial transmembrane potentialVps34: Vacuolar protein sorting-34UVRAG: UV-resistance-associated geneAMBRA-1: Activating molecule in Beclin-1-regulated

autophagy.

Competing Interests

The authors declare that there is no conflict of interests.

Acknowledgments

This work was supported by the Natural Science Founda-tion of China (nos. 81573596, 81503191, 81274018, 81373946,and 81303221) and National High Technology Research andDevelopment Plan of China (863 Plan) (2014AA022204).

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