# iTRAQ-Based Proteomic Analysis of Ginsenoside F2 on Human Gastric Carcinoma Cells SGC7901

Authors: Qian Mao; Pin-Hu Zhang; Jie Yang; Jin-Di Xu; Ming Kong; Hong Shen; He Zhu; Min Bai; Li Zhou; Guang-Fu Li; Qiang Wang; Song-Lin Li
Journal: Evidence-Based Complementary and Alternative Medicine
 (2016)

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## Abstract

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 F2 treatment, 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 F2 treatment downregulated PRR5 and RPS15 and upregulated RPL26, which are implicated in ribosomal protein-p53 signaling pathway. F2 also inhibited CISD2, Bcl-xl, and NLRX1, which are associated with autophagic pathway. Furthermore, it was demonstrated that F2 treatment increased Atg5, Atg7, Atg10, and PUMA, the critical downstream effectors of ribosomal protein-p53 signaling pathway, and Beclin-1, UVRAG, and AMBRA-1, the important molecules 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 be the most significantly regulated biological process by F2 treatment in SGC7901 cells, which provided valuable insights into the deep understanding of the molecular mechanisms of F2 for gastric cancer treatment.


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## Body

## 1. Introduction

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide. Annually it results in approximately 700,000 deaths [1]. Currently, chemotherapy has proved to decrease the rate of recurrence and improve overall survival; however, the drug resistance and serious toxic side effects largely reduce therapeutic efficacy and quality of life in patients [2, 3]. In recent years, compounds of natural products have caught wide attention due to their promising anticancer effects and minimal side effects [4–7]. Therefore, it is very necessary to develop new optimal anticancer agent from natural resource [3].Ginsenosides, the major bioactive constituents in ginseng, have been demonstrated to exert potential anticancer ability [4, 5]. Exploration of ginsenoside as a new anticarcinogenic agent is of much interest [4–7]. Structural-function studies showed that the increased antitumor effect is implicated with the decrease of its sugar number [5]. Sugar moiety at C-6 significantly reduces the anticancer activities of ginsenosides. Ginsenoside F2 (see structure in Figure 1), a protopanaxdiol type ginsenoside with one sugar molecular at C-3 and one sugar molecule at C-20, has been shown to be potent in inhibiting tumorigenesis in several different cancers including gastric tumor and glioblastoma multiforme [6, 7]. Recently, ourin vitro andin vivo studies demonstrated that ginsenoside F2 possesses anticancer effects in human gastric carcinoma cells SGC7901 [6]. However, the involved exact mechanisms of ginsenoside F2 on SGC7901 cancer cells at proteome level have not been systemically investigated.Figure 1
Structure of ginsenoside F2.Advancements in the field of proteomics have made it possible to accurately monitor and quantitatively detect the changes of protein expression in response to drug treatment. The achieved data provide valuable insights into the molecular mechanisms of disease and help to identify therapeutic targets [8]. Isobaric tag for relative and absolute quantification (iTRAQ) is a robust mass spectrometry technique that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation. iTRAQ with multiplexing capability up to eight distinct samples in a single experiment and relatively higher sensitivity has gained significant interest in the field of quantitative proteomics. In the present study, SGC7901 cells treated by lower dose and a shorter duration than that in previous report were analyzed by iTRAQ-based proteomics integrated with bioinformatics using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Cluster of Orthologous Groups (COG) of proteins database. And network analysis was applied to identify critical molecules which are involved in anticancer mechanisms of ginsenoside F2 in gastric SGC7901 cells. General molecular biological techniques such as western blot were utilized for validation.


## 2. Materials and Methods

### 2.1. Reagents and Antibodies

Ginsenoside F2 was isolated previously from leaves ofPanax ginseng by a series of chromatographic procedures [9]. Ginsenoside F2 has a molecular mass of 784 Da and was isolated with 98% purity. Primary antibodies 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 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Atg5, Atg7, and Atg10 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).


### 2.2. Cell Culture and Treatment

SGC7901 cells were purchased from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), 100μg/mL streptomycin, and 100 μg/mL penicillin and grown at 37°C in 5% carbon dioxide.


### 2.3. Protein Preparation

In one of our recent reports [6], we have shown that the IC50 of ginsenoside F2 is in <50 μM in 24 hours. In order to characterize ginsenoside F2-related mechanism it is imperative to use samples that are at the early stages of ginsenoside F2 treatment. So, a lower dose than the IC50 (20 μM) and a shorter duration (12 hours in the study) were chosen in the study. The treated (20 μM) and untreated SGC7901 cells were suspended in the lysis buffer and sonicated in ice. The proteins were reduced with 10 μM DTT (final concentration) at 56°C for 1 h and then alkylated by 55 mM iodoacetamide (IAM) (final concentration) in the darkroom for 1 h. The reduced and alkylated protein mixtures were precipitated by adding 4x volume of chilled acetone at −20°C overnight. After centrifugation at 4°C, 30 000 ×g, the pellet was dissolved in 0.5 M triethylammonium bicarbonate (TEAB) (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging at 30000 ×g at 4°C, the supernatants were collected, and the total protein concentration was determined using a Bradford protein assay kit (BioRad, Hercules, CA, USA). The proteins in the supernatant were kept at −80°C for further analysis.


### 2.4. iTRAQ Labeling and SCX Fractionation

Total protein (100μg) was taken out of each sample solution and then the protein was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein : trypsin = 30 : 1 at 37°C for 16 hours. iTRAQ labeling was performed according to the iTRAQ Reagents-8plex labeling manual (AB SCIEX, Madrid, Spain). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL isopropanol. iTRAQ labels 113 were used to label control sample separately, and 115 and 117 were used to label twice F2-treated samples for duplicated experiment. The peptides were labeled with the isobaric tags, incubated at room temperature for 2 h. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation.The mixed peptides were fractionated by strong cation exchange (SCX) chromatography on a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan). The iTRAQ labeled peptide mixtures were reconstituted with 4 mL buffer A (25 mM NaH2PO4 in 25% acetonitrile, pH 2.7) and loaded onto a 4.6 × 250 mm Ul tremex SCX column containing 5 μm particles (Phenomenex). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, 5–60% buffer B (25 mM NaH2PO4, 1 M KCl in 25% acetonitrile, pH 2.7) for 27 min, and 60–100% buffer B for 1 min. The system was then maintained at 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected at 1-minute intervals. The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex), and vacuum-dried. The cleaned 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

Each fraction was resuspended in buffer A (2% acetonitrile, 0.1% FA) and centrifuged at 20 000 ×g for 10 min. In each fraction, the final concentration of peptide was about 0.5μg/μL. 10 μL supernatant was loaded on a LC-20AD nano-HPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column. Then, the peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μL/min for 4 min; then the 44 min gradient was run at 300 nL/min starting from 2 to 35% B (98% acetonitrile, 0.1% FA), followed by 2-minute linear gradient to 80%, maintenance at 80% B for 4 min. Initial chromatographic conditions were restored in 1 min.Data acquisition was performed with tandem mass spectrometry (MS/MS) in a Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 70 000. Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27.0; ion fragments were detected in the Orbitrap at a resolution of 17500. In the octopole collision cell, the ten most intense peptide ions (charge states≥ 2) were sequentially isolated to a maximum target value of 5 × 105 by pAGC and fragmented HCD. A data-dependent procedure that alternated between one MS scan and 15 MS/MS scans was applied for the 15 most abundant precursor ions above a threshold ion count of 20000 in the MS survey scan with a following Dynamic Exclusion duration of 15 s. The electrospray voltage applied was 1.6 kV. Automatic gain control (AGC) was used to optimize the spectra generated by the Orbitrap. A sweeping collision energy setting of 35±5 eV was applied to all precursor ions for collision-induced dissociation. The AGC target for full MS was 3e6 and 1e5 for MS2. For MS scans, them/z scan range was 350 to 2000 Da. For MS2 scans, them/z scan range was 100–1800 Da. The iTRAQ experiments were performed as three technical replicates to gather reliable quantitative information.


### 2.6. Data Analysis

Raw data files acquired from the Orbitrap were converted into MGF files using Proteome Discoverer 1.2 (PD 1.2, Thermo) [5600 msconverter] and the MGF files were searched. Protein identifications were performed by using Mascot search engine (Matrix Science, London, UK; version 2.3.02) against database containing 143397 sequences.For protein identification and quantification, a peptide mass tolerance of 20 ppm was allowed for intact peptide masses and 0.05 Da for fragmented ions, with allowance for one missed cleavage in the trypsin digests. Carbamidomethylation of cysteine was considered a fixed modification, and the conversion ofN-terminal glutamine to pyroglutamic acid and methionine oxidation were considered variable modifications. All identified peptides had an ion score above the Mascot peptide identity threshold, and a protein was considered identified if at least one such unique peptide match was apparent for the protein. To reduce the probability of false peptide identification, only peptides at the 95% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We set a 1.2-fold change as the threshold and a p value must be below 0.05 to identify significant changes.


### 2.7. Function Method Description

Functional annotations of the proteins were conducted using Blast2 GO program against the nonredundant protein database (NR; NCBI). The KEGG database (http://www.genome.jp/kegg/) and the COG database (http://www.ncbi.nlm.nih.gov/COG/) were used to classify and group these identified proteins.GO is an international standardization of gene function classification system. It provides a set of dynamic updating controlled vocabulary to describe genes and gene products attributes in the organism. GO has 3 ontologies which can describe molecular function, cellular component, and biological process, respectively.COG is the database for protein orthologous classification. Every protein in COG is supposed to derive from a same protein ancestor.KEGG PATHWAY is a collection of manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line).


### 2.8. Western Blot

Western blot analyses were performed to confirm the presence of differentially expressed proteins. After the treatment of the indicated concentration of ginsenoside F2 (10, 20, and 40 μM) for 12 h, cells were harvested, washed with cold PBS (pH 7.4), and lysed with ice-cold lysis buffer (50 μM Tris-HCl, 150 μM NaCl, 1 μM EGTA, 1 μM EDTA, 20 μM NaF, 100 μM Na3VO4, 1%NP40, 1 μM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, pH 7.4) for 30 min and centrifuged at 12 000 ×g for 30 min at 4°C. The protein concentration of the clear supernatant was quantified using Bio-Rad Protein Assay Kit.Approximately 30μg of protein was loaded into a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Thereafter, proteins were electrophoretically transferred to nitrocellulose membrane and nonspecific sites were blocked with 5% skimmed milk in 1% Tween-20 (Sigma-Aldrich) in 20 μM TBS (pH 7.5) and reacted 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 three times (5 min each), the membrane was then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody. The signal was observed and developed with Kodak film by exposure to enhanced chemiluminescence (ECL) plus western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA).


### 2.9. Statistical Analysis

For cell-based assay, experiments were performed in duplicate and three independent experiments were performed. Western blot analyses of differential protein expressions were validated on cell lysates from three biological replicates. Statistical significance was analyzed using Student’st-test or ANOVA test by using GraphPad Prism v4.0 software (GraphPad Software, San Diego, CA, USA). Statistical significance is expressed as p∗∗∗<0.001; p∗∗<0.01; p∗<0.05.


## 2.1. Reagents and Antibodies

Ginsenoside F2 was isolated previously from leaves ofPanax ginseng by a series of chromatographic procedures [9]. Ginsenoside F2 has a molecular mass of 784 Da and was isolated with 98% purity. Primary antibodies 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 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Atg5, Atg7, and Atg10 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).


## 2.2. Cell Culture and Treatment

SGC7901 cells were purchased from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), 100μg/mL streptomycin, and 100 μg/mL penicillin and grown at 37°C in 5% carbon dioxide.


## 2.3. Protein Preparation

In one of our recent reports [6], we have shown that the IC50 of ginsenoside F2 is in <50 μM in 24 hours. In order to characterize ginsenoside F2-related mechanism it is imperative to use samples that are at the early stages of ginsenoside F2 treatment. So, a lower dose than the IC50 (20 μM) and a shorter duration (12 hours in the study) were chosen in the study. The treated (20 μM) and untreated SGC7901 cells were suspended in the lysis buffer and sonicated in ice. The proteins were reduced with 10 μM DTT (final concentration) at 56°C for 1 h and then alkylated by 55 mM iodoacetamide (IAM) (final concentration) in the darkroom for 1 h. The reduced and alkylated protein mixtures were precipitated by adding 4x volume of chilled acetone at −20°C overnight. After centrifugation at 4°C, 30 000 ×g, the pellet was dissolved in 0.5 M triethylammonium bicarbonate (TEAB) (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging at 30000 ×g at 4°C, the supernatants were collected, and the total protein concentration was determined using a Bradford protein assay kit (BioRad, Hercules, CA, USA). The proteins in the supernatant were kept at −80°C for further analysis.


## 2.4. iTRAQ Labeling and SCX Fractionation

Total protein (100μg) was taken out of each sample solution and then the protein was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein : trypsin = 30 : 1 at 37°C for 16 hours. iTRAQ labeling was performed according to the iTRAQ Reagents-8plex labeling manual (AB SCIEX, Madrid, Spain). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL isopropanol. iTRAQ labels 113 were used to label control sample separately, and 115 and 117 were used to label twice F2-treated samples for duplicated experiment. The peptides were labeled with the isobaric tags, incubated at room temperature for 2 h. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation.The mixed peptides were fractionated by strong cation exchange (SCX) chromatography on a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan). The iTRAQ labeled peptide mixtures were reconstituted with 4 mL buffer A (25 mM NaH2PO4 in 25% acetonitrile, pH 2.7) and loaded onto a 4.6 × 250 mm Ul tremex SCX column containing 5 μm particles (Phenomenex). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, 5–60% buffer B (25 mM NaH2PO4, 1 M KCl in 25% acetonitrile, pH 2.7) for 27 min, and 60–100% buffer B for 1 min. The system was then maintained at 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected at 1-minute intervals. The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex), and vacuum-dried. The cleaned 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

Each fraction was resuspended in buffer A (2% acetonitrile, 0.1% FA) and centrifuged at 20 000 ×g for 10 min. In each fraction, the final concentration of peptide was about 0.5μg/μL. 10 μL supernatant was loaded on a LC-20AD nano-HPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column. Then, the peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μL/min for 4 min; then the 44 min gradient was run at 300 nL/min starting from 2 to 35% B (98% acetonitrile, 0.1% FA), followed by 2-minute linear gradient to 80%, maintenance at 80% B for 4 min. Initial chromatographic conditions were restored in 1 min.Data acquisition was performed with tandem mass spectrometry (MS/MS) in a Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 70 000. Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27.0; ion fragments were detected in the Orbitrap at a resolution of 17500. In the octopole collision cell, the ten most intense peptide ions (charge states≥ 2) were sequentially isolated to a maximum target value of 5 × 105 by pAGC and fragmented HCD. A data-dependent procedure that alternated between one MS scan and 15 MS/MS scans was applied for the 15 most abundant precursor ions above a threshold ion count of 20000 in the MS survey scan with a following Dynamic Exclusion duration of 15 s. The electrospray voltage applied was 1.6 kV. Automatic gain control (AGC) was used to optimize the spectra generated by the Orbitrap. A sweeping collision energy setting of 35±5 eV was applied to all precursor ions for collision-induced dissociation. The AGC target for full MS was 3e6 and 1e5 for MS2. For MS scans, them/z scan range was 350 to 2000 Da. For MS2 scans, them/z scan range was 100–1800 Da. The iTRAQ experiments were performed as three technical replicates to gather reliable quantitative information.


## 2.6. Data Analysis

Raw data files acquired from the Orbitrap were converted into MGF files using Proteome Discoverer 1.2 (PD 1.2, Thermo) [5600 msconverter] and the MGF files were searched. Protein identifications were performed by using Mascot search engine (Matrix Science, London, UK; version 2.3.02) against database containing 143397 sequences.For protein identification and quantification, a peptide mass tolerance of 20 ppm was allowed for intact peptide masses and 0.05 Da for fragmented ions, with allowance for one missed cleavage in the trypsin digests. Carbamidomethylation of cysteine was considered a fixed modification, and the conversion ofN-terminal glutamine to pyroglutamic acid and methionine oxidation were considered variable modifications. All identified peptides had an ion score above the Mascot peptide identity threshold, and a protein was considered identified if at least one such unique peptide match was apparent for the protein. To reduce the probability of false peptide identification, only peptides at the 95% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We set a 1.2-fold change as the threshold and a p value must be below 0.05 to identify significant changes.


## 2.7. Function Method Description

Functional annotations of the proteins were conducted using Blast2 GO program against the nonredundant protein database (NR; NCBI). The KEGG database (http://www.genome.jp/kegg/) and the COG database (http://www.ncbi.nlm.nih.gov/COG/) were used to classify and group these identified proteins.GO is an international standardization of gene function classification system. It provides a set of dynamic updating controlled vocabulary to describe genes and gene products attributes in the organism. GO has 3 ontologies which can describe molecular function, cellular component, and biological process, respectively.COG is the database for protein orthologous classification. Every protein in COG is supposed to derive from a same protein ancestor.KEGG PATHWAY is a collection of manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line).


## 2.8. Western Blot

Western blot analyses were performed to confirm the presence of differentially expressed proteins. After the treatment of the indicated concentration of ginsenoside F2 (10, 20, and 40 μM) for 12 h, cells were harvested, washed with cold PBS (pH 7.4), and lysed with ice-cold lysis buffer (50 μM Tris-HCl, 150 μM NaCl, 1 μM EGTA, 1 μM EDTA, 20 μM NaF, 100 μM Na3VO4, 1%NP40, 1 μM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, pH 7.4) for 30 min and centrifuged at 12 000 ×g for 30 min at 4°C. The protein concentration of the clear supernatant was quantified using Bio-Rad Protein Assay Kit.Approximately 30μg of protein was loaded into a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Thereafter, proteins were electrophoretically transferred to nitrocellulose membrane and nonspecific sites were blocked with 5% skimmed milk in 1% Tween-20 (Sigma-Aldrich) in 20 μM TBS (pH 7.5) and reacted 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 three times (5 min each), the membrane was then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody. The signal was observed and developed with Kodak film by exposure to enhanced chemiluminescence (ECL) plus western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA).


## 2.9. Statistical Analysis

For cell-based assay, experiments were performed in duplicate and three independent experiments were performed. Western blot analyses of differential protein expressions were validated on cell lysates from three biological replicates. Statistical significance was analyzed using Student’st-test or ANOVA test by using GraphPad Prism 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 F2 at a dose of 20 μM for 12 hours. The harvested proteins are used to perform iTRAQ for quantifying the difference of total 31853 peptides and 5411 proteins in SGC7901 cells with or without treatment. Finally, 205 proteins were screened out in terms of the change in their expression level which meet our predefined criteria of p<0.05 with relative expression levels at least >1.2-fold (Table 1) or <0.83-fold (Table 2) (both 113/115 and 113/117) in ginsenoside F2-treated group compared with the control group. The protein properties, including pI, molecular weight (MW), and number of residues were calculated by Mascot. The results are highly reproducible in two individual experiments.Table 1
Differentially upregulated (>1.20-fold) proteins identified by iTRAQ in F2 treated SGC7901 cells.
Rank # Accession Gene symbol (GN) Definition (description) Score Mass Cov% Ration COG function-description Up 1 sp|P07305-2 H1F0 Isoform 2 of histone H1.0 51 35582 13 2.11 — Up 2 sp|P20962 PTMS Parathymosin 503 15782 23.5 1.32 — Up 3 tr|B8ZWD1 DBI Diazepam binding inhibitor, splice form 1A(2) 121 15706 28.9 1.31 Acyl-CoA-binding protein Up 4 sp|Q16576 RBBP7 Histone-binding protein RBBP7 877 55737 24.5 1.25 FOG: WD40 repeat Up 5 sp|P46779-2 RPL28 Isoform 2 of 60S ribosomal protein L28 524 22107 27.6 1.35 — Up 6 tr|B2R514 — cDNA, FLJ92300, Homo sapiens COP9 subunit 6 (MOV34 homolog, 34 kD) (COPS6), mRNA 74 39068 20.2 1.22 Predicted metal-dependent protease of the PAD1/JAB1 superfamily Up 7 tr|B3KY12 — cDNA FLJ46581 fis, clone THYMU3043200, highly similar to splicing factor 3A subunit 3 527 71859 22 1.24 Splicing factor 3a, subunit 3 Up 8 sp|Q71DI3 HIST2H3A Histone H3.2 617 19694 26.5 1.40 Histones H3 and H4 Up 9 tr|Q9P0H9 RER1 RER1 protein 118 28927 22 1.26 Golgi protein involved in Golgi-to-ER retrieval Up 10 tr|A8K3Q9 — cDNA FLJ76611, highly similar to Homo sapiens ribosomal protein L14 (RPL14), mRNA 781 35114 25.9 2.24 Ribosomal protein L14E/L6E/L27E Up 11 sp|Q9Y3A2 UTP11L Probable U3 small nucleolar RNA-associated protein 11 94 44174 21.7 1.30 Uncharacterized conserved protein Up 12 tr|F2Z388 RPL35 60S ribosomal protein L35 99 15372 32.3 1.35 Ribosomal protein L29 Up 13 sp|Q9NZZ3 CHMP5 Charged multivesicular body protein 5 268 32218 21 1.42 — Up 14 tr|B2R4D8 — 60S ribosomal protein L27 398 23061 36 1.28 Ribosomal protein L14E/L6E/L27E Up 15 tr|M0QXF7 C19orf10 UPF0556 protein C19orf10 (fragment) 265 11851 25 1.24 — Up 16 tr|D3DV26 S100A10 S100 calcium binding protein A10 (annexin II ligand, calpactin I, light polypeptide (P11)), isoform CRA_b (fragment) 134 27935 8.3 1.21 — Up 17 tr|H7C2N1 PTMA Thymosin alpha-1 (fragment) 117 18283 8.8 1.30 — Up 18 tr|G2XKQ0 — Sumo13 60 14938 11.9 1.22 Ubiquitin-like protein (sentrin) Up 19 tr|I3L1Y9 FLYWCH2 FLYWCH family member 2 99 19302 47.2 1.45 — Up 20 tr|M0R210 RPS16 40S ribosomal protein S16 1105 19391 57.4 1.27 Ribosomal protein S9 Up 21 sp|O43715 TRIAP1 TP53-regulated inhibitor of apoptosis 1 82 12050 18.4 1.36 — Up 22 sp|P49207 RPL34 60S ribosomal protein L34 187 18684 20.5 1.66 Ribosomal protein L34E Up 23 sp|Q92522 H1FX Histone H1x 342 35250 25.4 1.33 — Up 24 tr|J3KRX5 RPL17 60S ribosomal protein L17 (fragment) 795 27382 38.5 1.26 Ribosomal protein L22 Up 25 sp|P02795 MT2A Metallothionein-2 104 9915 52.5 1.42 — Up 26 tr|Q6FIE5 PHP14 PHP14 protein 72 17301 8.8 1.27 — Up 27 tr|A0PJ62 RPL14 RPL14 protein (fragment) 536 21409 43.5 2.85 Ribosomal protein L14E/L6E/L27E Up 28 tr|G3XAA2 MAP4K4 Mitogen-activated protein kinase kinase kinase kinase 4 142 156989 2.7 1.24 Serine/threonine protein kinase Up 29 tr|C9JNW5 RPL24 60S ribosomal protein L24 666 24642 32 1.67 Ribosomal protein L24E Up 30 sp|Q13951 CBFB Core-binding factor subunit beta 197 24461 18.1 1.20 — Up 31 tr|D3DUE6 N-PAC Cytokine-like nuclear factor n-pac, isoform CRA_c 219 76728 14.5 1.24 3-Hydroxyisobutyrate dehydrogenase and related beta-hydroxy acid dehydrogenases Up 32 tr|K7EKW4 ISOC2 Isochorismatase domain-containing protein 2, mitochondrial (fragment) 130 21202 17.4 1.34 Amidases related to nicotinamidase Up 33 sp|Q9NQ55-2 PPAN Isoform 2 of Suppressor of SWI4 1 homolog 73 63713 10.7 1.37 — Up 34 tr|B3KMF8 — cDNA FLJ10869 fis, clone NT2RP4001677 127 12398 27.7 1.28 — Up 35 sp|P62424 RPL7A 60S ribosomal protein L7a 613 42316 27.1 1.78 Ribosomal protein HS6-type (S12/L30/L7a) Up 36 tr|B4E0X1 — Beta-2-microglobulin 185 17093 13.1 1.25 — Up 37 tr|H0Y7A7 CALM2 Calmodulin (fragment) 735 24209 30.5 1.26 Ca2+-binding protein (EF-Hand superfamily) Up 38 tr|J3KTJ8 RPL26 60S ribosomal protein L26 (fragment) 363 15545 34 1.24 Ribosomal protein L24 Up 39 tr|B4DJM5 — cDNA FLJ61294, highly similar to keratin, type I cytoskeletal 17 326 21291 24.9 1.46 — Up 40 sp|Q9Y3C1 NOP16 Nucleolar protein 16 79 27925 20.8 1.24 — Up 41 sp|Q16543 CDC37 Hsp90 cochaperone Cdc37 384 57730 29.6 1.22 — Up 42 sp|P16401 HIST1H1B Histone H1.5 801 42644 17.3 2.38 — Up 43 sp|Q07866-3 KLC1 Isoform G of kinesin light chain 1 642 81828 23.9 1.24 FOG: TPR repeat Up 44 tr|B4DKJ4 — cDNA FLJ57738, highly similar to translationally controlled tumor protein 344 19250 32.4 1.28 —Table 2
Differentially downregulated (<0.83-fold) proteins identified by iTRAQ in F2 treated SGC7901 cells.
Rank # Accession Gene symbol (GN) Definition (description) Score Mass Cov% Ration COG function-description Down 1 tr|F5H740 VDAC3 Voltage-dependent anion-selective channel protein 3 1114 39598 41.5 0.81 — Down 2 sp|Q9H845 ACAD9 Acyl-CoA dehydrogenase family member 9, mitochondrial 311 81512 21.9 0.69 Acyl-CoA dehydrogenases Down 3 sp|Q969S9-2 GFM2 Isoform 2 of ribosome-releasing factor 2, mitochondrial 153 94059 5.1 0.80 Translation elongation factors (GTPases) Down 4 sp|P35908 KRT2 Keratin, type II cytoskeletal 2 epidermal 338 76630 18.2 0.67 Myosin heavy chain Down 5 tr|B7Z8A2 — cDNA FLJ51671, highly similar to prenylcysteine oxidase (EC 1.8.3.5) 492 63740 23.8 0.83 — Down 6 sp|Q9Y512 SAMM50 Sorting and assembly machinery component 50 homolog 170 59339 18.6 0.76 Outer membrane protein/protective antigen OMA87 Down 7 sp|Q6ZNW5 GDPGP1 GDP-D-glucose phosphorylase 1 118 45302 8.6 0.78 — Down 8 sp|P51970 NDUFA8 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 72 25720 15.1 0.68 — Down 9 tr|B4DRW0 — cDNA FLJ58125, highly similar to copper-transporting ATPase 1 (EC 3.6.3.4) 102 61873 6.1 0.78 Cation transport ATPase Down 10 tr|Q8NBW7 KDELR1 ER lumen protein retaining receptor 51 20327 12.7 0.73 ER lumen protein retaining receptor Down 11 tr|B2R6F5 — cDNA, FLJ92928, highly similar to Homo sapiens retinitis pigmentosa 2 (X-linked recessive) (RP2), mRNA 59 47451 2.3 0.82 — Down 12 tr|Q2VIN3 — RBM1 (fragment) 1232 45756 26.8 0.81 RNA-binding proteins (RRM domain) Down 13 sp|P14174 — Macrophage migration inhibitory factor 608 13856 17.4 0.71 — Down 14 tr|B2R6S4 — cDNA, FLJ93089, highly similar to Homo sapiens NCK adaptor protein 1 (NCK1), mRNA 137 53755 18.3 0.83 — Down 15 sp|Q16822 PCK2 Phosphoenolpyruvate carboxykinase [GTP], mitochondrial 1795 78784 41.6 0.74 Phosphoenolpyruvate carboxykinase (GTP) Down 16 tr|E9PM12 TCIRG1 V-type proton ATPase 116 kDa subunit a isoform 3 (fragment) 63 25815 13.3 0.74 Archaeal/vacuolar-type H+-ATPase subunit I Down 17 sp|Q2T9J0-2 TYSND1 Isoform 2 of peroxisomal leader peptide-processing protease 96 43618 9.8 0.67 — Down 18 tr|J3KPX7 PHB2 Prohibitin-2 1543 39466 51.8 0.82 Membrane protease subunits, stomatin/prohibitin homologs Down 19 tr|Q8NCF7 — cDNA FLJ90278 fis, clone NT2RP1000325, highly similar to phosphate carrier protein, mitochondrial precursor 517 48576 26.9 0.81 — Down 20 tr|B4E0R0 — cDNA FLJ54220, highly similar to Long-chain-fatty-acid-CoA ligase 1 (EC 6.2.1.3) 100 88560 6.2 0.74 Long-chain acyl-CoA synthetases (AMP-forming) Down 21 tr|B3KRY3 — cDNA FLJ35079 fis, clone PLACE6005283, highly similar to lysosome-associated membrane glycoprotein 1 319 48851 11.1 0.79 — Down 22 tr|B3KU09 — cDNA FLJ39034 fis, clone NT2RP7008085, highly similar to Homo sapiens ring finger protein 123 (RNF123), mRNA 110 166029 2.4 0.78 — Down 23 sp|Q9BVV7 TIMM21 Mitochondrial import inner membrane translocase subunit Tim21 86 35219 13.7 0.82 — Down 24 sp|Q9UMY1 NOL7 Nucleolar protein 7 148 39504 12.5 0.78 — Down 25 sp|Q9UNN8 PROCR Endothelial protein C receptor 103 27909 15.1 0.80 — Down 26 sp|Q86SF2 GALNT7 N-Acetylgalactosaminyltransferase 7 95 89410 9.9 0.81 — Down 27 tr|I3L0U2 PRSS21 Testisin (fragment) 115 27083 14.7 0.82 Secreted trypsin-like serine protease Down 28 tr|B7ZLP5 SAFB SAFB protein 557 121835 13 0.83 — Down 29 tr|F2Z3N7 TMEM106B Transmembrane protein 106B 135 12975 12.5 0.82 — Down 30 tr|B7Z361 — Reticulon 166 27838 12.2 0.76 — Down 31 tr|H0Y6F2 PRR5 Proline-rich protein 5 (fragment) 57 39929 2.3 0.78 — Down 32 sp|Q7Z7E8 UBE2Q1 Ubiquitin-conjugating enzyme E2 Q1 92 54711 1.9 0.76 — Down 33 tr|A8K4K9 — cDNA FLJ76169 146 42007 8.8 0.83 — Down 34 sp|P13645 KRT10 Keratin, type I cytoskeletal 10 382 66321 21.6 0.55 — Down 35 sp|Q8N5K1 CISD2 CDGSH iron-sulfur domain-containing protein 2 167 20364 26.7 0.81 — Down 36 sp|Q8NI27 THOC2 THO complex subunit 2 282 241732 8.7 0.83 — Down 37 tr|B4DEP8 — cDNA FLJ56960, highly similar to Homo sapiens phosphatidylinositol 4-kinase type II (PI4KII), mRNA 127 61711 9.8 0.76 — Down 38 sp|Q5BKZ1 ZNF326 DBIRD complex subunit ZNF326 145 78123 7.9 0.78 — Down 39 tr|Q8IW24 EXOC5 Exocyst complex component 5 108 99962 9.3 0.82 — Down 40 tr|B3KMG6 — cDNA FLJ10939 fis, clone OVARC1001065, highly similar to Homo sapiens MTERF domain containing 1 (MTERFD1), mRNA 117 43225 9.8 0.76 — Down 41 sp|Q8NBM4-2 UBAC2 Isoform 2 of ubiquitin-associated domain-containing protein 2 150 37306 18.1 0.83 — Down 42 sp|Q8NGA1 OR1M1 Olfactory receptor 1M1 76 39512 2.2 0.69 — Down 43 tr|E9PN17 ATP5L ATP synthase subunit g, mitochondrial 366 11489 63.2 0.82 — Down 44 tr|B2R686 TGOLN2 Trans-golgi network protein 2, isoform CRA_a 166 61093 13 0.79 — Down 45 tr|B4DIR5 — cDNA FLJ56026 51 143728 1.7 0.74 — Down 46 tr|J3KS15 ICT1 Peptidyl-tRNA hydrolase ICT1, mitochondrial (fragment) 169 26740 26 0.82 Protein chain release factor B Down 47 tr|F5H0F9 ANAPC5 Anaphase-promoting complex subunit 5 72 98300 7.5 0.82 — Down 48 tr|C8C504 HBB Beta-globin 1233 20056 29.9 0.21 — Down 49 tr|B2R921 — cDNA, FLJ94171, highly similar to Homo sapiens solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 15 (SLC25A15), nuclear gene encoding mitochondrial protein, mRNA 53 39308 9 0.77 — Down 50 sp|Q9Y613 FHOD1 FH1/FH2 domain-containing protein 1 255 141625 8.8 0.81 — Down 51 sp|Q92643 PIGK GPI-anchor transamidase 110 51592 10.9 0.77 Glycosylphosphatidylinositol transamidase (GPIT), subunit GPI8 Down 52 tr|A4FTY4 TXNRD2 TXNRD2 protein 331 41672 24.6 0.79 Pyruvate/2-oxoglutarate dehydrogenase complex, dihydrolipoamide dehydrogenase (E3) component, and related enzymes Down 53 tr|D3DP46 SPCS3 Signal peptidase complex subunit 3 homolog (S. cerevisiae), isoform CRA_a 147 24007 18.9 0.82 — Down 54 sp|Q9Y5Q9 GTF3C3 General transcription factor 3C polypeptide 3 154 117216 7.8 0.79 — Down 55 sp|P60468 SEC61B Protein transport protein Sec61 subunit beta 192 11546 37.5 0.72 — Down 56 sp|Q5RI15-2 — Isoform 2 of cytochrome c oxidase protein 20 homolog 106 17682 20 0.83 — Down 57 sp|Q9P206-2 — Isoform 2 of uncharacterized protein KIAA1522 146 128602 6.5 0.73 — Down 58 sp|Q86YN1 DOLPP1 Dolichyldiphosphatase 1 64 28953 5.5 0.69 Membrane-associated phospholipid phosphatase Down 59 sp|O00165-2 — Isoform 2 of HCLS1-associated protein X-1 111 34281 16 0.81 — Down 60 tr|B4E303 — cDNA FLJ57449, highly similar to Notchless homolog 1 127 54134 16.5 0.82 FOG: WD40 repeat Down 61 sp|O00194 RAB27B Ras-related protein Rab-27B 56 29688 14.2 0.77 GTPase SAR1 and related small G proteins Down 62 tr|B4DI41 MBD1 Methyl-CpG-binding domain protein 1 72 87409 1.8 0.80 — Down 63 tr|B0UXB6 ABHD16A Abhydrolase domain-containing protein 16A 129 73275 10.3 0.83 Hydrolases of the alpha/beta superfamily Down 64 sp|Q5T8D3-2 — Isoform 2 of Acyl-CoA-binding domain-containing protein 5 148 64353 11.6 0.72 Acyl-CoA-binding protein Down 65 tr|B4DNZ6 GTF2H3 General transcription factor IIH subunit 3 48 37020 4.5 0.79 RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB4 Down 66 sp|Q96FQ6 S100A16 Protein S100-A16 346 15197 22.3 0.83 — Down 67 tr|B4DSE1 — cDNA FLJ55364, highly similar to CRSP complex subunit 6 55 84524 3.7 0.73 — Down 68 tr|J3KNX9 MYO18A Unconventional myosin-XVIIIa 157 282257 3.5 0.72 Myosin heavy chain Down 69 tr|B4DMK6 — cDNA FLJ60055, highly similar toRattus norvegicus Ssu72 RNA polymerase II CTD phosphatase homolog, mRNA 51 23745 13.5 0.82 RNA polymerase II-interacting protein involved in transcription start site selection Down 70 tr|G3V1A0 TRAPPC4 HCG38438, isoform CRA_b 51 14838 20.5 0.81 — Down 71 tr|B1AHA8 HMOX1 Heme oxygenase 1 (fragment) 53 25525 15.5 0.83 Heme oxygenase Down 72 sp|Q9Y3B3-2 TMED7 Isoform 2 of transmembrane emp24 domain-containing protein 7 193 24908 28.2 0.82 — Down 73 tr|G3V1U5 GOLT1B Golgi transport 1 homolog B (S. cerevisiae), isoform CRA_c 167 9121 20.3 0.77 Membrane protein involved in Golgi transport Down 74 tr|B1PBA3 — SKNY protein 148 109440 8.4 0.81 — Down 75 sp|Q15061 WDR43 WD repeat-containing protein 43 138 91327 5.6 0.83 FOG: WD40 repeat Down 76 tr|D3DUJ0 AFG3L2 AFG3 ATPase family gene 3-like 2 (yeast), isoform CRA_a (fragment) 695 103842 21.2 0.83 ATP-dependent Zn proteases Down 77 tr|B2RBL9 — cDNA, FLJ95582, highly similar to Homo sapiens breast cancer antiestrogen resistance 1 (BCAR1), mRNA 204 104223 6 0.79 — Down 78 sp|Q3SXM5-2 — Isoform 2 of inactive hydroxysteroid dehydrogenase-like protein 1 170 35499 13.5 0.83 Short-chain dehydrogenases of various substrate specificities Down 79 sp|O43920 NDUFS5 NADH dehydrogenase [ubiquinone] iron-sulfur protein 5 106 16388 11.3 0.74 — Down 80 tr|H0YG20 MAN1B1 Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (fragment) 155 90816 8.2 0.80 — Down 81 tr|Q0KKI6 — Immunoglobulin light chain (fragment) 66 28559 8.2 0.80 — Down 82 sp|P62244 RPS15A 40S ribosomal protein S15a 1521 18594 66.2 0.82 Ribosomal protein S8 Down 83 tr|B4DL07 — cDNA FLJ53353, highly similar to ATP-binding cassette subfamily D member 3 398 92669 16.7 0.81 ABC-type uncharacterized transport system, permease, and ATPase components Down 84 tr|B4DR67 ALG5 Dolichyl-phosphate beta-glucosyltransferase 66 32213 10.9 0.81 Glycosyltransferases involved in cell wall biogenesis Down 85 tr|Q9BTT5 — Similar to NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 (39 kD) (fragment) 189 45471 21 0.75 Predicted nucleoside-diphosphate-sugar epimerases Down 86 tr|Q5U0H8 — Myelin protein zero-like 1 55 34725 4.8 0.74 — Down 87 sp|Q5SY16 NOL9 Polynucleotide 5-hydroxyl-kinase NOL9 109 91782 7.4 0.79 Predicted GTPase or GTP-binding protein Down 88 sp|O15173-2 PGRMC2 Isoform 2 of membrane-associated progesterone receptor component 2 620 30166 26.3 0.75 — Down 89 sp|Q5VT52-3 RPRD2 Isoform 3 of regulation of nuclear pre-mRNA domain-containing protein 2 295 177879 4.5 0.82 — Down 90 sp|Q8TC12 RDH11 Retinol dehydrogenase 11 494 41238 14.5 0.76 Dehydrogenases with different specificities (related to short-chain alcohol dehydrogenases) Down 91 tr|B4DZ55 — cDNA FLJ52097, weakly similar to Homo sapiens transmembrane and tetratricopeptide repeat containing 1 (TMTC1), mRNA 164 126875 10.1 0.79 FOG: TPR repeat Down 92 tr|J3KQA9 MTUS2 Microtubule-associated tumor suppressor candidate 2 150 181383 0.6 0.77 — Down 93 sp|Q96MG7 NDNL2 Melanoma-associated antigen G1 58 41645 7.6 0.72 — Down 94 tr|H3BQH3 KLHDC4 Kelch domain-containing protein 4 (fragment) 107 47359 10.7 0.83 — Down 95 tr|J3KN00 NDUFA13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 258 28599 23.3 0.81 — Down 96 sp|Q8NF37 LPCAT1 Lysophosphatidylcholine acyltransferase 1 708 67346 15.7 0.82 1-Acyl-sn-glycerol-3-phosphate acyltransferase Down 97 sp|Q9Y5P4-2 COL4A3BP Isoform 2 of collagen type IV alpha-3-binding protein 82 81121 6.7 0.80 — Down 98 tr|Q5T8U5 SURF4 Surfeit 4 418 22863 39.8 0.81 Predicted membrane protein Down 99 sp|P26599-2 PTBP1 Isoform 2 of polypyrimidine tract-binding protein 1 570 69515 16.2 0.82 — Down 100 sp|Q8NC56 LEMD2 LEM domain-containing protein 2 137 63423 7.4 0.76 — Down 101 tr|Q2Q9H2 G6PD Glucose-6-phosphate 1-dehydrogenase (fragment) 2165 64315 58.3 0.80 Glucose-6-phosphate 1-dehydrogenase Down 102 sp|P21796 VDAC1 Voltage-dependent anion-selective channel protein 1 2340 38777 62.9 0.80 — Down 103 tr|J3KNH7 SENP3 Sentrin-specific protease 3 88 73986 7.7 0.78 Protease, Ulp1 family Down 104 sp|A6NHL2-2 TUBAL3 Isoform 2 of tubulin alpha chain-like 3 768 51287 11.8 0.79 Tubulin Down 105 tr|B4DR71 — cDNA FLJ57078, highly similar to Homo sapiens opioid receptor, sigma 1 (OPRS1), transcript variant 1, mRNA 63 18151 8.4 0.83 — Down 106 sp|Q5JRA6-2 MIA3 Isoform 2 of melanoma inhibitory activity protein 3 415 249369 7.8 0.80 — Down 107 tr|J9ZVQ3 APOE Apolipoprotein E (fragment) 171 30543 12.2 0.79 — Down 108 tr|G5E9V5 MRPS22 28S ribosomal protein S22, mitochondrial 224 49264 17.3 0.77 — Down 109 tr|B7Z7X8 ATL2 Atlastin-2 112 76668 10.8 0.82 — Down 110 sp|P54709 ATP1B3 Sodium/potassium-transporting ATPase subunit beta-3 243 39135 17.9 0.83 — Down 111 tr|Q6IBK3 SCAMP2 SCAMP2 protein 258 39155 9.7 0.81 — Down 112 tr|A4LAA3 ATRX Alpha thalassemia/mental retardation syndrome X-linked 129 374604 2.5 0.81 Superfamily II DNA/RNA helicases, SNF2 family Down 113 sp|Q9UK59 DBR1 Lariat debranching enzyme 203 72182 14.5 0.80 — Down 114 tr|B4DI61 — cDNA FLJ58182, highly similar to protein CYR61 68 50414 6.4 0.70 — Down 115 tr|H3BNF1 CLN6 Ceroid-lipofuscinosis neuronal protein 6 300 12918 20 0.80 — Down 116 tr|E7ERK9 EIF2B4 Translation initiation factor eIF-2B subunit delta 170 71199 8.8 0.79 Translation initiation factor 2B subunit, eIF-2B alpha/beta/delta family Down 117 tr|H0Y8C3 MTCH1 Mitochondrial carrier homolog 1 (fragment) 97 50964 12.9 0.81 — Down 118 tr|B2RMV2 CYTSA CYTSA protein 52 149539 2.5 0.79 Ca2+-binding actin-bundling protein fimbrin/plastin (EF-hand superfamily) Down 119 tr|I3L1P8 SLC25A11 Mitochondrial 2-oxoglutarate/malate carrier protein (fragment) 470 37200 35.5 0.83 — Down 120 sp|Q8NBU5-2 ATAD1 Isoform 2 of ATPase family AAA domain-containing protein 1 124 40468 11.1 0.72 ATPases of the AAA+ class Down 121 sp|Q9Y3E7 CHMP3 Charged multivesicular body protein 3 102 32415 14.4 0.83 Conserved protein implicated in secretion Down 122 sp|P02763 ORM1 Alpha-1-acid glycoprotein 1 262 28288 20.4 0.80 — Down 123 tr|Q53F51 — FGF intracellular binding protein isoform b variant (fragment) 165 48798 12 0.83 — Down 124 sp|Q3ZAQ7 VMA21 Vacuolar ATPase assembly integral membrane protein VMA21 241 12868 24.8 0.81 — Down 125 tr|B2R6X8 — cDNA, FLJ93169, highly similar to Homo sapiens GPAA1P anchor attachment protein 1 homolog (yeast) (GPAA1), mRNA 106 72151 7.6 0.80 — Down 126 sp|Q9P0S9 TMEM14C Transmembrane protein 14C 45 12774 8.9 0.70 — Down 127 sp|P08779 KRT16 Keratin, type I cytoskeletal 16 630 57054 23.9 0.62 — Down 128 sp|Q86UT6-2 NLRX1 Isoform 2 of NLR family member X1 75 110309 4.1 0.71 — Down 129 tr|Q59E99 — Thrombospondin 1 variant (fragment) 153 155789 3.4 0.68 — Down 130 sp|Q8WXH0-2 SYNE2 Isoform 2 of nesprin-2 149 986758 1.1 0.82 Ca2+-binding actin-bundling protein fimbrin/plastin (EF-hand superfamily) Down 131 sp|P78310-2 CXADR Isoform 2 of coxsackievirus and adenovirus receptor 47 47491 3.8 0.74 — Down 132 tr|B2R995 — Malic enzyme 98 77738 5.8 0.83 Malic enzyme Down 133 tr|Q5QP56 BCL2L1 Bcl-2-like protein 1 (fragment) 98 21810 23.2 0.82 — Down 134 tr|H0YK72 SEC11A SEC11-like 1 (S. cerevisiae), isoform CRA_a 247 22018 16.5 0.81 Signal peptidase I Down 135 tr|B4DDH8 — cDNA FLJ55184, highly similar to Homo sapiens leukocyte receptor cluster (LRC) member 4 (LENG4), mRNA 137 54865 8.8 0.79 Predicted membrane protein Down 136 sp|Q9UJS0-2 SLC25A13 Isoform 2 of calcium-binding mitochondrial carrier protein Aralar2 719 86824 17.5 0.82 — Down 137 tr|A8KAK5 — cDNA FLJ77399, highly similar to Homo sapiens cofactor required for Sp1 transcriptional activation, subunit 2, 150 kDa (CRSP2), mRNA 85 182987 3.4 0.82 — Down 138 tr|H0YEF3 RNASEH2C Ribonuclease H2 subunit C (fragment) 76 18856 25.3 0.77 — Down 139 tr|Q5QNZ2 ATP5F1 ATP synthase F(0) complex subunit B1, mitochondrial 406 27794 47.7 0.82 — Down 140 sp|Q6UW68 TMEM205 Transmembrane protein 205 165 23294 15.9 0.82 — Down 141 tr|B3KPJ4 PHC2 Polyhomeotic-like protein 2 193 59764 9.3 0.79 — Down 142 tr|H0Y4D4 ACAA1 3-Ketoacyl-CoA thiolase, peroxisomal (fragment) 131 30218 12.7 0.78 Acetyl-CoA acetyltransferase Down 143 tr|Q4G0F4 POLRMT DNA-directed RNA polymerase 167 159664 4.6 0.81 Mitochondrial DNA-directed RNA polymerase Down 144 tr|Q6FGZ3 EPHX1 EPHX1 protein (fragment) 519 62281 14.9 0.77 Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) Down 145 tr|B4DVN1 — cDNA FLJ52214, highly similar to DnaJ homolog subfamily B member 6 90 37740 8.6 0.70 DnaJ-class molecular chaperone with C-terminal Zn finger domain Down 146 sp|Q92667-2 AKAP1 A-kinase anchor protein 1, mitochondrial 66 111940 4.9 Down 147 sp|O00483 NDUFA4 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4 165 11855 46.9 0.83 — Down 148 sp|Q9NTJ5 SACM1L Phosphatidylinositide phosphatase SAC1 179 77476 18.2 0.83 Phosphoinositide polyphosphatase (Sac family) Down 149 tr|B3KVC5 — cDNA FLJ16380 fis, clone TLIVE2002882, weakly similar to imidazolonepropionase (EC 3.5.2.7) 41 53582 3.3 0.83 Imidazolonepropionase and related amidohydrolases Down 150 tr|B7ZLI5 FAM98C Family with sequence similarity 98, member C 72 41696 9.5 0.68 — Down 151 tr|B7Z6F5 YIPF1 Protein YIPF1 64 40866 2.7 0.61 — Down 152 sp|Q6NVY1-2 HIBCH Isoform 2 of 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial 101 46543 19.2 0.82 Enoyl-CoA hydratase/carnitine racemase Down 153 tr|U3KQJ1 POLDIP2 Polymerase delta-interacting protein 2 282 46395 26.4 0.76 Uncharacterized protein affecting Mg2+/Co2+ transport Down 154 tr|D6RGZ2 THOC3 THO complex subunit 3 172 12690 36.2 0.75 — Down 155 tr|A0S0T0 ATP6 ATP synthase subunit a 128 26896 4.4 0.78 F0F1-type ATP synthase, subunit a Down 156 tr|G3V2U7 ACYP1 Acylphosphatase 85 17520 14.7 0.80 acylphosphatases Down 157 sp|Q9ULG6-2 CCPG1 Isoform 2 of cell cycle progression protein 1 79 93313 4.1 0.81 — Down 158 tr|H7BXZ6 RHOT1 Mitochondrial Rho GTPase 142 81600 5.9 0.77 GTPase SAR1 and related small G proteins Down 159 sp|Q14151 SAFB2 Scaffold attachment factor B2 461 129824 13 0.83 — Down 160 sp|Q96LD4 TRIM47 Tripartite motif-containing protein 47 138 75838 7.8 0.81 — Down 161 tr|A8K2K2 — cDNA FLJ76494, highly similar to Homo sapiens GTPBP2 GTP-binding like protein 2 137 64767 11.7 0.83 GTPase


### 3.2. Classification of Differentially Expressed Proteins

Firstly, screened proteins were functionally catalogued with GO and WEGO to three different groups (Figures2 and 3(a)): biological process (BP), cellular component (CC), and molecular function (MF). As shown in Figure 2, the proteins are involved in BP including cellular process (13.44%), metabolic process (11.16%), single-organism process (10.36%), biological regulation (8.06%), and regulation of biological process (7.59%). The identified proteins separated according to CC include cell (19.40%), cell part (19.40%), organelle (16.68%), organelle part (12.46%), membrane (7.97%), and macromolecular complex (7.94%). MF of the proteins was classified and large groups were found to be binding (50.59%), catalytic activity (27.97%), enzyme regulator activity (3.94%), transporter activity (3.84%), and structural molecular activity (3.43%).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.
(a) (b) (c)Figure 3
WEGO (a) and COG (b) assay of the 205 differentially expressed proteins.
(a) (b)Further COG function classification revealed that posttranslational modification, protein turnover, and ribosomal structure biogenesis were major function of the screened 205 proteins (Figure3(b)). In each category of BP, CC, and MF, top twenty proteins which generated bigger difference in response to ginsenoside F2 treatment are listed in Figure 4.Figure 4
GO annotation of the final selected differentially expressed proteins. The top 20 components for BP (a), CC (b), and MF (c) of the selected differentially expressed proteins are shown along with their enrichment score, represented as ap value.
(a) (b) (c)KEGG is a publicly available pathway database and could provide biologists excellent resources to attain a deeper understanding of biological mechanisms in response to different treatments. Protein analysis through KEGG indicated that 205 differentially expressed proteins were involved in 128 different pathways (data not shown). The connection degree between proteins is calculated by protein-protein interaction network analysis and the results are shown in Figure5. Among these proteins, PRR5, RPS15, and RPL26 were 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 NLRX1 were selected for further validation and study in order to provide a comprehensive perspective for elucidating underlying molecular mechanisms of ginsenoside F2.Figure 5
The protein-protein interaction network of the differentially expressed proteins identified. Red triangle denotes upregulated proteins; green triangle denotes downregulated protein.


### 3.3. Western Blot Analysis

#### 3.3.1. For Verification

To validate the information obtained from the iTRAQ-based quantitative proteomics study and bioinformatics analysis, the screened proteins with strong response to ginsenoside F2 treatment 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 the expression of the RPL26 (p<0.01) in SGC7901 cells in comparison with the treatment with vehicle control.Figure 6
Western blot validations of RPS15, RPL26, PRR5, CISD2, NLRX1, p53, PUMA, mTOR, and Bcl-xl in SGC7901 cells with different concentrations of ginsenoside F2. 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: accumulated results show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as p∗∗<0.01, p∗<0.05.
(a) (b)


#### 3.3.2. For Determining the Expression of Apoptosis and Autophagic Proteins

As shown in Figure6, ginsenoside F2 suppressed the expression of mTOR and upregulated the expression of p53 in a dose-dependent manner. Atg5, Atg7, Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1 are known to be modulated by p53 or Bcl-xl signaling, which may trigger apoptosis or autophagy. Therefore, we proceeded to check the expressions of Atg5, Atg7, Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1. As shown in Figure 7, ginsenoside F2 upregulated the expressions of these proteins in a dose-dependent manner. LC3 is now widely used to monitor autophagy. During autophagy, the cytoplasmic form LC3-I is processed and recruited to phagophores, where LC3-II is generated by site-specific proteolysis and lipidation at the C-terminus. Thus, the amount of LC3-II positively correlates with the number of autophagosomes [10]. We examined the effect of F2 on LC3 conversion in SGC7901 cells. Western blot analysis showed that F2 treatment resulted in dose-dependent accumulation of LC3-II and reduction of LC3-I (Figure 7). The conversion of LC3-I to LC3-II suggested F2 treatment induces autophagy.Figure 7
Effect of ginsenoside F2 on 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 representative western blot analysis. β-actin was used as the loading control. Right panel: accumulated results show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as p∗∗<0.01, p∗<0.05.
(a) (b)In the present study, combination of iTRAQ-based proteomics method with bioinformatics was used to identify critical molecules in SGC7901 cancer cells in response to ginsenoside F2 treatment. Ginsenoside F2 generated significant change of protein profile in SGC7901 cells. Some of them have been demonstrated to participate in either apoptosis or autophagy responses, suggesting that the antitumor mechanisms of ginsenoside F2 in SGC7901 cells are involved in both apoptosis and autophagy.The current findings demonstrate that ginsenoside F2 impacts distinct signaling pathways and induces broad change in the protein profile of SGC7901 cells. Overall, 205 differentially expressed proteins were identified with ≥95% confidence in ginsenoside F2 treated group. Application of a ratio of 1.2-fold change as criteria resulted in 44 and 161 differentially abundant proteins in SGC7901 cells.In our study, some proteins that were significantly altered by ginsenoside F2 show close relationship of protein-protein interaction (Figure 5). Ribosomal proteins, such as RPS15 and 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 of autophagy or apoptosis. The changes of these six potential proteins were verified by western blot analysis.Ribosomal proteins (RPs) are considered to have diverse extra ribosomal functions, ranging from cell cycle progression to cell death and to malignant transformation and cellular metabolism [11]. Relevantly, a number of RPs have been shown to bind to MDM2, the inhibitor of p53 (murine double minute 2, and also HDM2 for its human ortholog), and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and activation, then triggering apoptosis or autophagy [11]. Following the treatment of ginsenoside F2 in SGC7901 cells, the levels of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14, RPL24, RPL7A, and RPL26 were increased, whereas that of RPS15 reduced. Although the functions of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14, RPL24, and RPL7A have not been well studied, RPL26, a positive regulator of p53, was found to increase the translational rate of p53 mRNA by binding to its 50 untranslated region [12] and, in this case, MDM2 acts as an ubiquitin E3 ligase for ubiquitylation and degradation of RPL26 [18]. Thus, under the treatment of ginsenoside F2, the increased level of RPL26 indicated that RPL26 may inhibit MDM2 and subsequently activate p53. RPS15, identified as a direct p53 transcriptional target, was thought to activate p53 by repressing MDM2 activity [19]. Interestingly, in our study, the level of RPS15 reduced in SGC7901 followed by ginsenoside F2 treatment, suggesting that the roles of RPS15 and RPL26 involved in the anticancer mechanism of ginsenoside F2 are different, which warrant further investigation.mTOR, existing in two multiprotein complexes, mTORC1 and mTORC2, regulates cell growth in response to a variety of cellular signals derived from growth factors and environmental stress [20]. mTORC2 is a kinase complex comprised of mTOR, PRR5, Rictor, mSin1, and mLST8/GbL. The expression level of PRR5 is correlated with that of mTORC2. Recent study showed that mTORC2 is implicated in actin cytoskeleton regulation, as well as phosphorylation of Akt [13]. Although TOR kinase has been largely attributed as a negative regulator of autophagy through TORC1, resent study indicated that mTORC2 was an independent positive regulator of autophagy during amino acid starvation [21]. In the present study, ginsenoside F2 decreased level of PPR5, indicated that ginsenoside F2 may 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 may activate the AMPK pathway and inhibit the mTOR pathway, subsequently triggering autophagy. p53 may also transactivate multiple genes with proautophagic roles, including proapoptotic Bcl-2 proteins (Bax, PUMA) [22, 23]. In this network, PUMA induces the noncanonical autophagy pathway regulated via Atg5, Atg7, and Atg10. PUMA’s initiation of autophagy promotes cytochrome c release, which then leads to apoptosis [22]. Interestingly, in our previous work, increasing level of cytochrome c and decreased mitochondrial transmembrane potential (MTP) were observed [6]. In present study, decreased expressions of PRR5 and RPL26 were found, which implied that ginsenoside F2 might trigger p53 signal pathway. It was reported that western blot analyses tended to show greater differential abundance compared with iTRAQ analyses [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 and reduced level of P53 and mTORC2 suggested that ginsenoside F2 may 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]. Our results showed that CISD2 was significantly decreased in ginsenoside F2 treated group, confirmed by western blot analysis. Recent work identified CISD2 as a Bcl-xl binding partner at a branch point between autophagy and apoptosis, life and death, under nutrient-deprived and oxidative stress conditionsin vivo cells [25, 26]. Bcl-xl, also called Bcl-2L, is known to function through inhibition of the autophagy effector and tumor suppressor Beclin-1 [15]. CISD2 is required in this pathway for Bcl-xl to functionally antagonize Beclin-1-dependent autophagy. In our study, the expression of Bcl-xl decreased, confirmed by western blot analysis. Thus, CISD2 may be a Bcl-xl-associated cofactor that targets Bcl-2 for the autophagy pathway.During initiation of autophagosome formation, after release from Bcl-xl, Beclin-1 functions as a platform by binding to class III PI3K/vacuolar protein sorting-34 (Vps34), UV-resistance-associated gene (UVRAG), activating molecule in Beclin-1-regulated autophagy (AMBRA-1) [15, 26, 27]. Previous studies have shown that binding of Beclin-1 to Bcl-2/Bcl-xl inhibits the autophagic function of Beclin-1, suggesting that Beclin-1 might have a role in the convergence between autophagy and apoptotic cell death [22]. For confirming the Beclin-1/Bcl-xl pathway, western blot was employed. The expressions of Beclin-1, UVRAG, and AMBRA-1 were increased, while Bcl-xl was decreased, which suggested that ginsenoside F2 may induce autophagy via Bcl-xl/Beclin-1 pathway.NLRX1, a mitochondrial NOD-like receptor that amplifies apoptosis by inducing reactive oxygen species production, is an important component of TLR mediated inflammatory pathways [13, 16]. Recent evidence suggested that upregulated expression of NLRX1 may synergistically regulate metabolism and autophagy for highly invasive growth of 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. In our iTRAQ results, expression of NLRX1 was significantly decreased in SGC7901 cells treated with ginsenoside F2. The phenomenon suggested different role of NLRX1 involved in the ginsenoside F2 treatment that may be different from that of published reports [16, 17], though the mechanism needs further research.Mai et al. reported that F2 induces apoptotic cell death accompanied by protective autophagy in breast cancer stem cells [28]. In one of our previous studies, we found that F2 induces apoptosis by causing an accumulation of ROS and activating the apoptosis signaling pathway [6]. However, there was no report systemically comparing differently regulated proteins and building a network of F2-treated cancer cells at proteome level. In the current study, by the close look at cellular mechanisms at proteome level, we clearly identified the distinct pattern of cellular responses for the F2-treated cells, and 6 differentially regulated proteins were identified, which provide useful information on elucidating the anticancer mechanism of F2 to SGC7901 cells. Moreover, the integration of networks and pathway with the proteomic data enhanced our understanding of the functional relationship of proteome changes caused by the compound.


## 3.1. Proteome Analysis

Human gastric carcinoma cells (SGC7901) are treated with ginsenoside F2 at a dose of 20 μM for 12 hours. The harvested proteins are used to perform iTRAQ for quantifying the difference of total 31853 peptides and 5411 proteins in SGC7901 cells with or without treatment. Finally, 205 proteins were screened out in terms of the change in their expression level which meet our predefined criteria of p<0.05 with relative expression levels at least >1.2-fold (Table 1) or <0.83-fold (Table 2) (both 113/115 and 113/117) in ginsenoside F2-treated group compared with the control group. The protein properties, including pI, molecular weight (MW), and number of residues were calculated by Mascot. The results are highly reproducible in two individual experiments.Table 1
Differentially upregulated (>1.20-fold) proteins identified by iTRAQ in F2 treated SGC7901 cells.
Rank # Accession Gene symbol (GN) Definition (description) Score Mass Cov% Ration COG function-description Up 1 sp|P07305-2 H1F0 Isoform 2 of histone H1.0 51 35582 13 2.11 — Up 2 sp|P20962 PTMS Parathymosin 503 15782 23.5 1.32 — Up 3 tr|B8ZWD1 DBI Diazepam binding inhibitor, splice form 1A(2) 121 15706 28.9 1.31 Acyl-CoA-binding protein Up 4 sp|Q16576 RBBP7 Histone-binding protein RBBP7 877 55737 24.5 1.25 FOG: WD40 repeat Up 5 sp|P46779-2 RPL28 Isoform 2 of 60S ribosomal protein L28 524 22107 27.6 1.35 — Up 6 tr|B2R514 — cDNA, FLJ92300, Homo sapiens COP9 subunit 6 (MOV34 homolog, 34 kD) (COPS6), mRNA 74 39068 20.2 1.22 Predicted metal-dependent protease of the PAD1/JAB1 superfamily Up 7 tr|B3KY12 — cDNA FLJ46581 fis, clone THYMU3043200, highly similar to splicing factor 3A subunit 3 527 71859 22 1.24 Splicing factor 3a, subunit 3 Up 8 sp|Q71DI3 HIST2H3A Histone H3.2 617 19694 26.5 1.40 Histones H3 and H4 Up 9 tr|Q9P0H9 RER1 RER1 protein 118 28927 22 1.26 Golgi protein involved in Golgi-to-ER retrieval Up 10 tr|A8K3Q9 — cDNA FLJ76611, highly similar to Homo sapiens ribosomal protein L14 (RPL14), mRNA 781 35114 25.9 2.24 Ribosomal protein L14E/L6E/L27E Up 11 sp|Q9Y3A2 UTP11L Probable U3 small nucleolar RNA-associated protein 11 94 44174 21.7 1.30 Uncharacterized conserved protein Up 12 tr|F2Z388 RPL35 60S ribosomal protein L35 99 15372 32.3 1.35 Ribosomal protein L29 Up 13 sp|Q9NZZ3 CHMP5 Charged multivesicular body protein 5 268 32218 21 1.42 — Up 14 tr|B2R4D8 — 60S ribosomal protein L27 398 23061 36 1.28 Ribosomal protein L14E/L6E/L27E Up 15 tr|M0QXF7 C19orf10 UPF0556 protein C19orf10 (fragment) 265 11851 25 1.24 — Up 16 tr|D3DV26 S100A10 S100 calcium binding protein A10 (annexin II ligand, calpactin I, light polypeptide (P11)), isoform CRA_b (fragment) 134 27935 8.3 1.21 — Up 17 tr|H7C2N1 PTMA Thymosin alpha-1 (fragment) 117 18283 8.8 1.30 — Up 18 tr|G2XKQ0 — Sumo13 60 14938 11.9 1.22 Ubiquitin-like protein (sentrin) Up 19 tr|I3L1Y9 FLYWCH2 FLYWCH family member 2 99 19302 47.2 1.45 — Up 20 tr|M0R210 RPS16 40S ribosomal protein S16 1105 19391 57.4 1.27 Ribosomal protein S9 Up 21 sp|O43715 TRIAP1 TP53-regulated inhibitor of apoptosis 1 82 12050 18.4 1.36 — Up 22 sp|P49207 RPL34 60S ribosomal protein L34 187 18684 20.5 1.66 Ribosomal protein L34E Up 23 sp|Q92522 H1FX Histone H1x 342 35250 25.4 1.33 — Up 24 tr|J3KRX5 RPL17 60S ribosomal protein L17 (fragment) 795 27382 38.5 1.26 Ribosomal protein L22 Up 25 sp|P02795 MT2A Metallothionein-2 104 9915 52.5 1.42 — Up 26 tr|Q6FIE5 PHP14 PHP14 protein 72 17301 8.8 1.27 — Up 27 tr|A0PJ62 RPL14 RPL14 protein (fragment) 536 21409 43.5 2.85 Ribosomal protein L14E/L6E/L27E Up 28 tr|G3XAA2 MAP4K4 Mitogen-activated protein kinase kinase kinase kinase 4 142 156989 2.7 1.24 Serine/threonine protein kinase Up 29 tr|C9JNW5 RPL24 60S ribosomal protein L24 666 24642 32 1.67 Ribosomal protein L24E Up 30 sp|Q13951 CBFB Core-binding factor subunit beta 197 24461 18.1 1.20 — Up 31 tr|D3DUE6 N-PAC Cytokine-like nuclear factor n-pac, isoform CRA_c 219 76728 14.5 1.24 3-Hydroxyisobutyrate dehydrogenase and related beta-hydroxy acid dehydrogenases Up 32 tr|K7EKW4 ISOC2 Isochorismatase domain-containing protein 2, mitochondrial (fragment) 130 21202 17.4 1.34 Amidases related to nicotinamidase Up 33 sp|Q9NQ55-2 PPAN Isoform 2 of Suppressor of SWI4 1 homolog 73 63713 10.7 1.37 — Up 34 tr|B3KMF8 — cDNA FLJ10869 fis, clone NT2RP4001677 127 12398 27.7 1.28 — Up 35 sp|P62424 RPL7A 60S ribosomal protein L7a 613 42316 27.1 1.78 Ribosomal protein HS6-type (S12/L30/L7a) Up 36 tr|B4E0X1 — Beta-2-microglobulin 185 17093 13.1 1.25 — Up 37 tr|H0Y7A7 CALM2 Calmodulin (fragment) 735 24209 30.5 1.26 Ca2+-binding protein (EF-Hand superfamily) Up 38 tr|J3KTJ8 RPL26 60S ribosomal protein L26 (fragment) 363 15545 34 1.24 Ribosomal protein L24 Up 39 tr|B4DJM5 — cDNA FLJ61294, highly similar to keratin, type I cytoskeletal 17 326 21291 24.9 1.46 — Up 40 sp|Q9Y3C1 NOP16 Nucleolar protein 16 79 27925 20.8 1.24 — Up 41 sp|Q16543 CDC37 Hsp90 cochaperone Cdc37 384 57730 29.6 1.22 — Up 42 sp|P16401 HIST1H1B Histone H1.5 801 42644 17.3 2.38 — Up 43 sp|Q07866-3 KLC1 Isoform G of kinesin light chain 1 642 81828 23.9 1.24 FOG: TPR repeat Up 44 tr|B4DKJ4 — cDNA FLJ57738, highly similar to translationally controlled tumor protein 344 19250 32.4 1.28 —Table 2
Differentially downregulated (<0.83-fold) proteins identified by iTRAQ in F2 treated SGC7901 cells.
Rank # Accession Gene symbol (GN) Definition (description) Score Mass Cov% Ration COG function-description Down 1 tr|F5H740 VDAC3 Voltage-dependent anion-selective channel protein 3 1114 39598 41.5 0.81 — Down 2 sp|Q9H845 ACAD9 Acyl-CoA dehydrogenase family member 9, mitochondrial 311 81512 21.9 0.69 Acyl-CoA dehydrogenases Down 3 sp|Q969S9-2 GFM2 Isoform 2 of ribosome-releasing factor 2, mitochondrial 153 94059 5.1 0.80 Translation elongation factors (GTPases) Down 4 sp|P35908 KRT2 Keratin, type II cytoskeletal 2 epidermal 338 76630 18.2 0.67 Myosin heavy chain Down 5 tr|B7Z8A2 — cDNA FLJ51671, highly similar to prenylcysteine oxidase (EC 1.8.3.5) 492 63740 23.8 0.83 — Down 6 sp|Q9Y512 SAMM50 Sorting and assembly machinery component 50 homolog 170 59339 18.6 0.76 Outer membrane protein/protective antigen OMA87 Down 7 sp|Q6ZNW5 GDPGP1 GDP-D-glucose phosphorylase 1 118 45302 8.6 0.78 — Down 8 sp|P51970 NDUFA8 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 72 25720 15.1 0.68 — Down 9 tr|B4DRW0 — cDNA FLJ58125, highly similar to copper-transporting ATPase 1 (EC 3.6.3.4) 102 61873 6.1 0.78 Cation transport ATPase Down 10 tr|Q8NBW7 KDELR1 ER lumen protein retaining receptor 51 20327 12.7 0.73 ER lumen protein retaining receptor Down 11 tr|B2R6F5 — cDNA, FLJ92928, highly similar to Homo sapiens retinitis pigmentosa 2 (X-linked recessive) (RP2), mRNA 59 47451 2.3 0.82 — Down 12 tr|Q2VIN3 — RBM1 (fragment) 1232 45756 26.8 0.81 RNA-binding proteins (RRM domain) Down 13 sp|P14174 — Macrophage migration inhibitory factor 608 13856 17.4 0.71 — Down 14 tr|B2R6S4 — cDNA, FLJ93089, highly similar to Homo sapiens NCK adaptor protein 1 (NCK1), mRNA 137 53755 18.3 0.83 — Down 15 sp|Q16822 PCK2 Phosphoenolpyruvate carboxykinase [GTP], mitochondrial 1795 78784 41.6 0.74 Phosphoenolpyruvate carboxykinase (GTP) Down 16 tr|E9PM12 TCIRG1 V-type proton ATPase 116 kDa subunit a isoform 3 (fragment) 63 25815 13.3 0.74 Archaeal/vacuolar-type H+-ATPase subunit I Down 17 sp|Q2T9J0-2 TYSND1 Isoform 2 of peroxisomal leader peptide-processing protease 96 43618 9.8 0.67 — Down 18 tr|J3KPX7 PHB2 Prohibitin-2 1543 39466 51.8 0.82 Membrane protease subunits, stomatin/prohibitin homologs Down 19 tr|Q8NCF7 — cDNA FLJ90278 fis, clone NT2RP1000325, highly similar to phosphate carrier protein, mitochondrial precursor 517 48576 26.9 0.81 — Down 20 tr|B4E0R0 — cDNA FLJ54220, highly similar to Long-chain-fatty-acid-CoA ligase 1 (EC 6.2.1.3) 100 88560 6.2 0.74 Long-chain acyl-CoA synthetases (AMP-forming) Down 21 tr|B3KRY3 — cDNA FLJ35079 fis, clone PLACE6005283, highly similar to lysosome-associated membrane glycoprotein 1 319 48851 11.1 0.79 — Down 22 tr|B3KU09 — cDNA FLJ39034 fis, clone NT2RP7008085, highly similar to Homo sapiens ring finger protein 123 (RNF123), mRNA 110 166029 2.4 0.78 — Down 23 sp|Q9BVV7 TIMM21 Mitochondrial import inner membrane translocase subunit Tim21 86 35219 13.7 0.82 — Down 24 sp|Q9UMY1 NOL7 Nucleolar protein 7 148 39504 12.5 0.78 — Down 25 sp|Q9UNN8 PROCR Endothelial protein C receptor 103 27909 15.1 0.80 — Down 26 sp|Q86SF2 GALNT7 N-Acetylgalactosaminyltransferase 7 95 89410 9.9 0.81 — Down 27 tr|I3L0U2 PRSS21 Testisin (fragment) 115 27083 14.7 0.82 Secreted trypsin-like serine protease Down 28 tr|B7ZLP5 SAFB SAFB protein 557 121835 13 0.83 — Down 29 tr|F2Z3N7 TMEM106B Transmembrane protein 106B 135 12975 12.5 0.82 — Down 30 tr|B7Z361 — Reticulon 166 27838 12.2 0.76 — Down 31 tr|H0Y6F2 PRR5 Proline-rich protein 5 (fragment) 57 39929 2.3 0.78 — Down 32 sp|Q7Z7E8 UBE2Q1 Ubiquitin-conjugating enzyme E2 Q1 92 54711 1.9 0.76 — Down 33 tr|A8K4K9 — cDNA FLJ76169 146 42007 8.8 0.83 — Down 34 sp|P13645 KRT10 Keratin, type I cytoskeletal 10 382 66321 21.6 0.55 — Down 35 sp|Q8N5K1 CISD2 CDGSH iron-sulfur domain-containing protein 2 167 20364 26.7 0.81 — Down 36 sp|Q8NI27 THOC2 THO complex subunit 2 282 241732 8.7 0.83 — Down 37 tr|B4DEP8 — cDNA FLJ56960, highly similar to Homo sapiens phosphatidylinositol 4-kinase type II (PI4KII), mRNA 127 61711 9.8 0.76 — Down 38 sp|Q5BKZ1 ZNF326 DBIRD complex subunit ZNF326 145 78123 7.9 0.78 — Down 39 tr|Q8IW24 EXOC5 Exocyst complex component 5 108 99962 9.3 0.82 — Down 40 tr|B3KMG6 — cDNA FLJ10939 fis, clone OVARC1001065, highly similar to Homo sapiens MTERF domain containing 1 (MTERFD1), mRNA 117 43225 9.8 0.76 — Down 41 sp|Q8NBM4-2 UBAC2 Isoform 2 of ubiquitin-associated domain-containing protein 2 150 37306 18.1 0.83 — Down 42 sp|Q8NGA1 OR1M1 Olfactory receptor 1M1 76 39512 2.2 0.69 — Down 43 tr|E9PN17 ATP5L ATP synthase subunit g, mitochondrial 366 11489 63.2 0.82 — Down 44 tr|B2R686 TGOLN2 Trans-golgi network protein 2, isoform CRA_a 166 61093 13 0.79 — Down 45 tr|B4DIR5 — cDNA FLJ56026 51 143728 1.7 0.74 — Down 46 tr|J3KS15 ICT1 Peptidyl-tRNA hydrolase ICT1, mitochondrial (fragment) 169 26740 26 0.82 Protein chain release factor B Down 47 tr|F5H0F9 ANAPC5 Anaphase-promoting complex subunit 5 72 98300 7.5 0.82 — Down 48 tr|C8C504 HBB Beta-globin 1233 20056 29.9 0.21 — Down 49 tr|B2R921 — cDNA, FLJ94171, highly similar to Homo sapiens solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 15 (SLC25A15), nuclear gene encoding mitochondrial protein, mRNA 53 39308 9 0.77 — Down 50 sp|Q9Y613 FHOD1 FH1/FH2 domain-containing protein 1 255 141625 8.8 0.81 — Down 51 sp|Q92643 PIGK GPI-anchor transamidase 110 51592 10.9 0.77 Glycosylphosphatidylinositol transamidase (GPIT), subunit GPI8 Down 52 tr|A4FTY4 TXNRD2 TXNRD2 protein 331 41672 24.6 0.79 Pyruvate/2-oxoglutarate dehydrogenase complex, dihydrolipoamide dehydrogenase (E3) component, and related enzymes Down 53 tr|D3DP46 SPCS3 Signal peptidase complex subunit 3 homolog (S. cerevisiae), isoform CRA_a 147 24007 18.9 0.82 — Down 54 sp|Q9Y5Q9 GTF3C3 General transcription factor 3C polypeptide 3 154 117216 7.8 0.79 — Down 55 sp|P60468 SEC61B Protein transport protein Sec61 subunit beta 192 11546 37.5 0.72 — Down 56 sp|Q5RI15-2 — Isoform 2 of cytochrome c oxidase protein 20 homolog 106 17682 20 0.83 — Down 57 sp|Q9P206-2 — Isoform 2 of uncharacterized protein KIAA1522 146 128602 6.5 0.73 — Down 58 sp|Q86YN1 DOLPP1 Dolichyldiphosphatase 1 64 28953 5.5 0.69 Membrane-associated phospholipid phosphatase Down 59 sp|O00165-2 — Isoform 2 of HCLS1-associated protein X-1 111 34281 16 0.81 — Down 60 tr|B4E303 — cDNA FLJ57449, highly similar to Notchless homolog 1 127 54134 16.5 0.82 FOG: WD40 repeat Down 61 sp|O00194 RAB27B Ras-related protein Rab-27B 56 29688 14.2 0.77 GTPase SAR1 and related small G proteins Down 62 tr|B4DI41 MBD1 Methyl-CpG-binding domain protein 1 72 87409 1.8 0.80 — Down 63 tr|B0UXB6 ABHD16A Abhydrolase domain-containing protein 16A 129 73275 10.3 0.83 Hydrolases of the alpha/beta superfamily Down 64 sp|Q5T8D3-2 — Isoform 2 of Acyl-CoA-binding domain-containing protein 5 148 64353 11.6 0.72 Acyl-CoA-binding protein Down 65 tr|B4DNZ6 GTF2H3 General transcription factor IIH subunit 3 48 37020 4.5 0.79 RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB4 Down 66 sp|Q96FQ6 S100A16 Protein S100-A16 346 15197 22.3 0.83 — Down 67 tr|B4DSE1 — cDNA FLJ55364, highly similar to CRSP complex subunit 6 55 84524 3.7 0.73 — Down 68 tr|J3KNX9 MYO18A Unconventional myosin-XVIIIa 157 282257 3.5 0.72 Myosin heavy chain Down 69 tr|B4DMK6 — cDNA FLJ60055, highly similar toRattus norvegicus Ssu72 RNA polymerase II CTD phosphatase homolog, mRNA 51 23745 13.5 0.82 RNA polymerase II-interacting protein involved in transcription start site selection Down 70 tr|G3V1A0 TRAPPC4 HCG38438, isoform CRA_b 51 14838 20.5 0.81 — Down 71 tr|B1AHA8 HMOX1 Heme oxygenase 1 (fragment) 53 25525 15.5 0.83 Heme oxygenase Down 72 sp|Q9Y3B3-2 TMED7 Isoform 2 of transmembrane emp24 domain-containing protein 7 193 24908 28.2 0.82 — Down 73 tr|G3V1U5 GOLT1B Golgi transport 1 homolog B (S. cerevisiae), isoform CRA_c 167 9121 20.3 0.77 Membrane protein involved in Golgi transport Down 74 tr|B1PBA3 — SKNY protein 148 109440 8.4 0.81 — Down 75 sp|Q15061 WDR43 WD repeat-containing protein 43 138 91327 5.6 0.83 FOG: WD40 repeat Down 76 tr|D3DUJ0 AFG3L2 AFG3 ATPase family gene 3-like 2 (yeast), isoform CRA_a (fragment) 695 103842 21.2 0.83 ATP-dependent Zn proteases Down 77 tr|B2RBL9 — cDNA, FLJ95582, highly similar to Homo sapiens breast cancer antiestrogen resistance 1 (BCAR1), mRNA 204 104223 6 0.79 — Down 78 sp|Q3SXM5-2 — Isoform 2 of inactive hydroxysteroid dehydrogenase-like protein 1 170 35499 13.5 0.83 Short-chain dehydrogenases of various substrate specificities Down 79 sp|O43920 NDUFS5 NADH dehydrogenase [ubiquinone] iron-sulfur protein 5 106 16388 11.3 0.74 — Down 80 tr|H0YG20 MAN1B1 Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (fragment) 155 90816 8.2 0.80 — Down 81 tr|Q0KKI6 — Immunoglobulin light chain (fragment) 66 28559 8.2 0.80 — Down 82 sp|P62244 RPS15A 40S ribosomal protein S15a 1521 18594 66.2 0.82 Ribosomal protein S8 Down 83 tr|B4DL07 — cDNA FLJ53353, highly similar to ATP-binding cassette subfamily D member 3 398 92669 16.7 0.81 ABC-type uncharacterized transport system, permease, and ATPase components Down 84 tr|B4DR67 ALG5 Dolichyl-phosphate beta-glucosyltransferase 66 32213 10.9 0.81 Glycosyltransferases involved in cell wall biogenesis Down 85 tr|Q9BTT5 — Similar to NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 (39 kD) (fragment) 189 45471 21 0.75 Predicted nucleoside-diphosphate-sugar epimerases Down 86 tr|Q5U0H8 — Myelin protein zero-like 1 55 34725 4.8 0.74 — Down 87 sp|Q5SY16 NOL9 Polynucleotide 5-hydroxyl-kinase NOL9 109 91782 7.4 0.79 Predicted GTPase or GTP-binding protein Down 88 sp|O15173-2 PGRMC2 Isoform 2 of membrane-associated progesterone receptor component 2 620 30166 26.3 0.75 — Down 89 sp|Q5VT52-3 RPRD2 Isoform 3 of regulation of nuclear pre-mRNA domain-containing protein 2 295 177879 4.5 0.82 — Down 90 sp|Q8TC12 RDH11 Retinol dehydrogenase 11 494 41238 14.5 0.76 Dehydrogenases with different specificities (related to short-chain alcohol dehydrogenases) Down 91 tr|B4DZ55 — cDNA FLJ52097, weakly similar to Homo sapiens transmembrane and tetratricopeptide repeat containing 1 (TMTC1), mRNA 164 126875 10.1 0.79 FOG: TPR repeat Down 92 tr|J3KQA9 MTUS2 Microtubule-associated tumor suppressor candidate 2 150 181383 0.6 0.77 — Down 93 sp|Q96MG7 NDNL2 Melanoma-associated antigen G1 58 41645 7.6 0.72 — Down 94 tr|H3BQH3 KLHDC4 Kelch domain-containing protein 4 (fragment) 107 47359 10.7 0.83 — Down 95 tr|J3KN00 NDUFA13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 258 28599 23.3 0.81 — Down 96 sp|Q8NF37 LPCAT1 Lysophosphatidylcholine acyltransferase 1 708 67346 15.7 0.82 1-Acyl-sn-glycerol-3-phosphate acyltransferase Down 97 sp|Q9Y5P4-2 COL4A3BP Isoform 2 of collagen type IV alpha-3-binding protein 82 81121 6.7 0.80 — Down 98 tr|Q5T8U5 SURF4 Surfeit 4 418 22863 39.8 0.81 Predicted membrane protein Down 99 sp|P26599-2 PTBP1 Isoform 2 of polypyrimidine tract-binding protein 1 570 69515 16.2 0.82 — Down 100 sp|Q8NC56 LEMD2 LEM domain-containing protein 2 137 63423 7.4 0.76 — Down 101 tr|Q2Q9H2 G6PD Glucose-6-phosphate 1-dehydrogenase (fragment) 2165 64315 58.3 0.80 Glucose-6-phosphate 1-dehydrogenase Down 102 sp|P21796 VDAC1 Voltage-dependent anion-selective channel protein 1 2340 38777 62.9 0.80 — Down 103 tr|J3KNH7 SENP3 Sentrin-specific protease 3 88 73986 7.7 0.78 Protease, Ulp1 family Down 104 sp|A6NHL2-2 TUBAL3 Isoform 2 of tubulin alpha chain-like 3 768 51287 11.8 0.79 Tubulin Down 105 tr|B4DR71 — cDNA FLJ57078, highly similar to Homo sapiens opioid receptor, sigma 1 (OPRS1), transcript variant 1, mRNA 63 18151 8.4 0.83 — Down 106 sp|Q5JRA6-2 MIA3 Isoform 2 of melanoma inhibitory activity protein 3 415 249369 7.8 0.80 — Down 107 tr|J9ZVQ3 APOE Apolipoprotein E (fragment) 171 30543 12.2 0.79 — Down 108 tr|G5E9V5 MRPS22 28S ribosomal protein S22, mitochondrial 224 49264 17.3 0.77 — Down 109 tr|B7Z7X8 ATL2 Atlastin-2 112 76668 10.8 0.82 — Down 110 sp|P54709 ATP1B3 Sodium/potassium-transporting ATPase subunit beta-3 243 39135 17.9 0.83 — Down 111 tr|Q6IBK3 SCAMP2 SCAMP2 protein 258 39155 9.7 0.81 — Down 112 tr|A4LAA3 ATRX Alpha thalassemia/mental retardation syndrome X-linked 129 374604 2.5 0.81 Superfamily II DNA/RNA helicases, SNF2 family Down 113 sp|Q9UK59 DBR1 Lariat debranching enzyme 203 72182 14.5 0.80 — Down 114 tr|B4DI61 — cDNA FLJ58182, highly similar to protein CYR61 68 50414 6.4 0.70 — Down 115 tr|H3BNF1 CLN6 Ceroid-lipofuscinosis neuronal protein 6 300 12918 20 0.80 — Down 116 tr|E7ERK9 EIF2B4 Translation initiation factor eIF-2B subunit delta 170 71199 8.8 0.79 Translation initiation factor 2B subunit, eIF-2B alpha/beta/delta family Down 117 tr|H0Y8C3 MTCH1 Mitochondrial carrier homolog 1 (fragment) 97 50964 12.9 0.81 — Down 118 tr|B2RMV2 CYTSA CYTSA protein 52 149539 2.5 0.79 Ca2+-binding actin-bundling protein fimbrin/plastin (EF-hand superfamily) Down 119 tr|I3L1P8 SLC25A11 Mitochondrial 2-oxoglutarate/malate carrier protein (fragment) 470 37200 35.5 0.83 — Down 120 sp|Q8NBU5-2 ATAD1 Isoform 2 of ATPase family AAA domain-containing protein 1 124 40468 11.1 0.72 ATPases of the AAA+ class Down 121 sp|Q9Y3E7 CHMP3 Charged multivesicular body protein 3 102 32415 14.4 0.83 Conserved protein implicated in secretion Down 122 sp|P02763 ORM1 Alpha-1-acid glycoprotein 1 262 28288 20.4 0.80 — Down 123 tr|Q53F51 — FGF intracellular binding protein isoform b variant (fragment) 165 48798 12 0.83 — Down 124 sp|Q3ZAQ7 VMA21 Vacuolar ATPase assembly integral membrane protein VMA21 241 12868 24.8 0.81 — Down 125 tr|B2R6X8 — cDNA, FLJ93169, highly similar to Homo sapiens GPAA1P anchor attachment protein 1 homolog (yeast) (GPAA1), mRNA 106 72151 7.6 0.80 — Down 126 sp|Q9P0S9 TMEM14C Transmembrane protein 14C 45 12774 8.9 0.70 — Down 127 sp|P08779 KRT16 Keratin, type I cytoskeletal 16 630 57054 23.9 0.62 — Down 128 sp|Q86UT6-2 NLRX1 Isoform 2 of NLR family member X1 75 110309 4.1 0.71 — Down 129 tr|Q59E99 — Thrombospondin 1 variant (fragment) 153 155789 3.4 0.68 — Down 130 sp|Q8WXH0-2 SYNE2 Isoform 2 of nesprin-2 149 986758 1.1 0.82 Ca2+-binding actin-bundling protein fimbrin/plastin (EF-hand superfamily) Down 131 sp|P78310-2 CXADR Isoform 2 of coxsackievirus and adenovirus receptor 47 47491 3.8 0.74 — Down 132 tr|B2R995 — Malic enzyme 98 77738 5.8 0.83 Malic enzyme Down 133 tr|Q5QP56 BCL2L1 Bcl-2-like protein 1 (fragment) 98 21810 23.2 0.82 — Down 134 tr|H0YK72 SEC11A SEC11-like 1 (S. cerevisiae), isoform CRA_a 247 22018 16.5 0.81 Signal peptidase I Down 135 tr|B4DDH8 — cDNA FLJ55184, highly similar to Homo sapiens leukocyte receptor cluster (LRC) member 4 (LENG4), mRNA 137 54865 8.8 0.79 Predicted membrane protein Down 136 sp|Q9UJS0-2 SLC25A13 Isoform 2 of calcium-binding mitochondrial carrier protein Aralar2 719 86824 17.5 0.82 — Down 137 tr|A8KAK5 — cDNA FLJ77399, highly similar to Homo sapiens cofactor required for Sp1 transcriptional activation, subunit 2, 150 kDa (CRSP2), mRNA 85 182987 3.4 0.82 — Down 138 tr|H0YEF3 RNASEH2C Ribonuclease H2 subunit C (fragment) 76 18856 25.3 0.77 — Down 139 tr|Q5QNZ2 ATP5F1 ATP synthase F(0) complex subunit B1, mitochondrial 406 27794 47.7 0.82 — Down 140 sp|Q6UW68 TMEM205 Transmembrane protein 205 165 23294 15.9 0.82 — Down 141 tr|B3KPJ4 PHC2 Polyhomeotic-like protein 2 193 59764 9.3 0.79 — Down 142 tr|H0Y4D4 ACAA1 3-Ketoacyl-CoA thiolase, peroxisomal (fragment) 131 30218 12.7 0.78 Acetyl-CoA acetyltransferase Down 143 tr|Q4G0F4 POLRMT DNA-directed RNA polymerase 167 159664 4.6 0.81 Mitochondrial DNA-directed RNA polymerase Down 144 tr|Q6FGZ3 EPHX1 EPHX1 protein (fragment) 519 62281 14.9 0.77 Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) Down 145 tr|B4DVN1 — cDNA FLJ52214, highly similar to DnaJ homolog subfamily B member 6 90 37740 8.6 0.70 DnaJ-class molecular chaperone with C-terminal Zn finger domain Down 146 sp|Q92667-2 AKAP1 A-kinase anchor protein 1, mitochondrial 66 111940 4.9 Down 147 sp|O00483 NDUFA4 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4 165 11855 46.9 0.83 — Down 148 sp|Q9NTJ5 SACM1L Phosphatidylinositide phosphatase SAC1 179 77476 18.2 0.83 Phosphoinositide polyphosphatase (Sac family) Down 149 tr|B3KVC5 — cDNA FLJ16380 fis, clone TLIVE2002882, weakly similar to imidazolonepropionase (EC 3.5.2.7) 41 53582 3.3 0.83 Imidazolonepropionase and related amidohydrolases Down 150 tr|B7ZLI5 FAM98C Family with sequence similarity 98, member C 72 41696 9.5 0.68 — Down 151 tr|B7Z6F5 YIPF1 Protein YIPF1 64 40866 2.7 0.61 — Down 152 sp|Q6NVY1-2 HIBCH Isoform 2 of 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial 101 46543 19.2 0.82 Enoyl-CoA hydratase/carnitine racemase Down 153 tr|U3KQJ1 POLDIP2 Polymerase delta-interacting protein 2 282 46395 26.4 0.76 Uncharacterized protein affecting Mg2+/Co2+ transport Down 154 tr|D6RGZ2 THOC3 THO complex subunit 3 172 12690 36.2 0.75 — Down 155 tr|A0S0T0 ATP6 ATP synthase subunit a 128 26896 4.4 0.78 F0F1-type ATP synthase, subunit a Down 156 tr|G3V2U7 ACYP1 Acylphosphatase 85 17520 14.7 0.80 acylphosphatases Down 157 sp|Q9ULG6-2 CCPG1 Isoform 2 of cell cycle progression protein 1 79 93313 4.1 0.81 — Down 158 tr|H7BXZ6 RHOT1 Mitochondrial Rho GTPase 142 81600 5.9 0.77 GTPase SAR1 and related small G proteins Down 159 sp|Q14151 SAFB2 Scaffold attachment factor B2 461 129824 13 0.83 — Down 160 sp|Q96LD4 TRIM47 Tripartite motif-containing protein 47 138 75838 7.8 0.81 — Down 161 tr|A8K2K2 — cDNA FLJ76494, highly similar to Homo sapiens GTPBP2 GTP-binding like protein 2 137 64767 11.7 0.83 GTPase


## 3.2. Classification of Differentially Expressed Proteins

Firstly, screened proteins were functionally catalogued with GO and WEGO to three different groups (Figures2 and 3(a)): biological process (BP), cellular component (CC), and molecular function (MF). As shown in Figure 2, the proteins are involved in BP including cellular process (13.44%), metabolic process (11.16%), single-organism process (10.36%), biological regulation (8.06%), and regulation of biological process (7.59%). The identified proteins separated according to CC include cell (19.40%), cell part (19.40%), organelle (16.68%), organelle part (12.46%), membrane (7.97%), and macromolecular complex (7.94%). MF of the proteins was classified and large groups were found to be binding (50.59%), catalytic activity (27.97%), enzyme regulator activity (3.94%), transporter activity (3.84%), and structural molecular activity (3.43%).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.
(a) (b) (c)Figure 3
WEGO (a) and COG (b) assay of the 205 differentially expressed proteins.
(a) (b)Further COG function classification revealed that posttranslational modification, protein turnover, and ribosomal structure biogenesis were major function of the screened 205 proteins (Figure3(b)). In each category of BP, CC, and MF, top twenty proteins which generated bigger difference in response to ginsenoside F2 treatment are listed in Figure 4.Figure 4
GO annotation of the final selected differentially expressed proteins. The top 20 components for BP (a), CC (b), and MF (c) of the selected differentially expressed proteins are shown along with their enrichment score, represented as ap value.
(a) (b) (c)KEGG is a publicly available pathway database and could provide biologists excellent resources to attain a deeper understanding of biological mechanisms in response to different treatments. Protein analysis through KEGG indicated that 205 differentially expressed proteins were involved in 128 different pathways (data not shown). The connection degree between proteins is calculated by protein-protein interaction network analysis and the results are shown in Figure5. Among these proteins, PRR5, RPS15, and RPL26 were 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 NLRX1 were selected for further validation and study in order to provide a comprehensive perspective for elucidating underlying molecular mechanisms of ginsenoside F2.Figure 5
The protein-protein interaction network of the differentially expressed proteins identified. Red triangle denotes upregulated proteins; green triangle denotes downregulated protein.


## 3.3. Western Blot Analysis

### 3.3.1. For Verification

To validate the information obtained from the iTRAQ-based quantitative proteomics study and bioinformatics analysis, the screened proteins with strong response to ginsenoside F2 treatment 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 the expression of the RPL26 (p<0.01) in SGC7901 cells in comparison with the treatment with vehicle control.Figure 6
Western blot validations of RPS15, RPL26, PRR5, CISD2, NLRX1, p53, PUMA, mTOR, and Bcl-xl in SGC7901 cells with different concentrations of ginsenoside F2. 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: accumulated results show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as p∗∗<0.01, p∗<0.05.
(a) (b)


### 3.3.2. For Determining the Expression of Apoptosis and Autophagic Proteins

As shown in Figure6, ginsenoside F2 suppressed the expression of mTOR and upregulated the expression of p53 in a dose-dependent manner. Atg5, Atg7, Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1 are known to be modulated by p53 or Bcl-xl signaling, which may trigger apoptosis or autophagy. Therefore, we proceeded to check the expressions of Atg5, Atg7, Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1. As shown in Figure 7, ginsenoside F2 upregulated the expressions of these proteins in a dose-dependent manner. LC3 is now widely used to monitor autophagy. During autophagy, the cytoplasmic form LC3-I is processed and recruited to phagophores, where LC3-II is generated by site-specific proteolysis and lipidation at the C-terminus. Thus, the amount of LC3-II positively correlates with the number of autophagosomes [10]. We examined the effect of F2 on LC3 conversion in SGC7901 cells. Western blot analysis showed that F2 treatment resulted in dose-dependent accumulation of LC3-II and reduction of LC3-I (Figure 7). The conversion of LC3-I to LC3-II suggested F2 treatment induces autophagy.Figure 7
Effect of ginsenoside F2 on 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 representative western blot analysis. β-actin was used as the loading control. Right panel: accumulated results show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as p∗∗<0.01, p∗<0.05.
(a) (b)In the present study, combination of iTRAQ-based proteomics method with bioinformatics was used to identify critical molecules in SGC7901 cancer cells in response to ginsenoside F2 treatment. Ginsenoside F2 generated significant change of protein profile in SGC7901 cells. Some of them have been demonstrated to participate in either apoptosis or autophagy responses, suggesting that the antitumor mechanisms of ginsenoside F2 in SGC7901 cells are involved in both apoptosis and autophagy.The current findings demonstrate that ginsenoside F2 impacts distinct signaling pathways and induces broad change in the protein profile of SGC7901 cells. Overall, 205 differentially expressed proteins were identified with ≥95% confidence in ginsenoside F2 treated group. Application of a ratio of 1.2-fold change as criteria resulted in 44 and 161 differentially abundant proteins in SGC7901 cells.In our study, some proteins that were significantly altered by ginsenoside F2 show close relationship of protein-protein interaction (Figure 5). Ribosomal proteins, such as RPS15 and 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 of autophagy or apoptosis. The changes of these six potential proteins were verified by western blot analysis.Ribosomal proteins (RPs) are considered to have diverse extra ribosomal functions, ranging from cell cycle progression to cell death and to malignant transformation and cellular metabolism [11]. Relevantly, a number of RPs have been shown to bind to MDM2, the inhibitor of p53 (murine double minute 2, and also HDM2 for its human ortholog), and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and activation, then triggering apoptosis or autophagy [11]. Following the treatment of ginsenoside F2 in SGC7901 cells, the levels of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14, RPL24, RPL7A, and RPL26 were increased, whereas that of RPS15 reduced. Although the functions of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14, RPL24, and RPL7A have not been well studied, RPL26, a positive regulator of p53, was found to increase the translational rate of p53 mRNA by binding to its 50 untranslated region [12] and, in this case, MDM2 acts as an ubiquitin E3 ligase for ubiquitylation and degradation of RPL26 [18]. Thus, under the treatment of ginsenoside F2, the increased level of RPL26 indicated that RPL26 may inhibit MDM2 and subsequently activate p53. RPS15, identified as a direct p53 transcriptional target, was thought to activate p53 by repressing MDM2 activity [19]. Interestingly, in our study, the level of RPS15 reduced in SGC7901 followed by ginsenoside F2 treatment, suggesting that the roles of RPS15 and RPL26 involved in the anticancer mechanism of ginsenoside F2 are different, which warrant further investigation.mTOR, existing in two multiprotein complexes, mTORC1 and mTORC2, regulates cell growth in response to a variety of cellular signals derived from growth factors and environmental stress [20]. mTORC2 is a kinase complex comprised of mTOR, PRR5, Rictor, mSin1, and mLST8/GbL. The expression level of PRR5 is correlated with that of mTORC2. Recent study showed that mTORC2 is implicated in actin cytoskeleton regulation, as well as phosphorylation of Akt [13]. Although TOR kinase has been largely attributed as a negative regulator of autophagy through TORC1, resent study indicated that mTORC2 was an independent positive regulator of autophagy during amino acid starvation [21]. In the present study, ginsenoside F2 decreased level of PPR5, indicated that ginsenoside F2 may 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 may activate the AMPK pathway and inhibit the mTOR pathway, subsequently triggering autophagy. p53 may also transactivate multiple genes with proautophagic roles, including proapoptotic Bcl-2 proteins (Bax, PUMA) [22, 23]. In this network, PUMA induces the noncanonical autophagy pathway regulated via Atg5, Atg7, and Atg10. PUMA’s initiation of autophagy promotes cytochrome c release, which then leads to apoptosis [22]. Interestingly, in our previous work, increasing level of cytochrome c and decreased mitochondrial transmembrane potential (MTP) were observed [6]. In present study, decreased expressions of PRR5 and RPL26 were found, which implied that ginsenoside F2 might trigger p53 signal pathway. It was reported that western blot analyses tended to show greater differential abundance compared with iTRAQ analyses [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 and reduced level of P53 and mTORC2 suggested that ginsenoside F2 may 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]. Our results showed that CISD2 was significantly decreased in ginsenoside F2 treated group, confirmed by western blot analysis. Recent work identified CISD2 as a Bcl-xl binding partner at a branch point between autophagy and apoptosis, life and death, under nutrient-deprived and oxidative stress conditionsin vivo cells [25, 26]. Bcl-xl, also called Bcl-2L, is known to function through inhibition of the autophagy effector and tumor suppressor Beclin-1 [15]. CISD2 is required in this pathway for Bcl-xl to functionally antagonize Beclin-1-dependent autophagy. In our study, the expression of Bcl-xl decreased, confirmed by western blot analysis. Thus, CISD2 may be a Bcl-xl-associated cofactor that targets Bcl-2 for the autophagy pathway.During initiation of autophagosome formation, after release from Bcl-xl, Beclin-1 functions as a platform by binding to class III PI3K/vacuolar protein sorting-34 (Vps34), UV-resistance-associated gene (UVRAG), activating molecule in Beclin-1-regulated autophagy (AMBRA-1) [15, 26, 27]. Previous studies have shown that binding of Beclin-1 to Bcl-2/Bcl-xl inhibits the autophagic function of Beclin-1, suggesting that Beclin-1 might have a role in the convergence between autophagy and apoptotic cell death [22]. For confirming the Beclin-1/Bcl-xl pathway, western blot was employed. The expressions of Beclin-1, UVRAG, and AMBRA-1 were increased, while Bcl-xl was decreased, which suggested that ginsenoside F2 may induce autophagy via Bcl-xl/Beclin-1 pathway.NLRX1, a mitochondrial NOD-like receptor that amplifies apoptosis by inducing reactive oxygen species production, is an important component of TLR mediated inflammatory pathways [13, 16]. Recent evidence suggested that upregulated expression of NLRX1 may synergistically regulate metabolism and autophagy for highly invasive growth of 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. In our iTRAQ results, expression of NLRX1 was significantly decreased in SGC7901 cells treated with ginsenoside F2. The phenomenon suggested different role of NLRX1 involved in the ginsenoside F2 treatment that may be different from that of published reports [16, 17], though the mechanism needs further research.Mai et al. reported that F2 induces apoptotic cell death accompanied by protective autophagy in breast cancer stem cells [28]. In one of our previous studies, we found that F2 induces apoptosis by causing an accumulation of ROS and activating the apoptosis signaling pathway [6]. However, there was no report systemically comparing differently regulated proteins and building a network of F2-treated cancer cells at proteome level. In the current study, by the close look at cellular mechanisms at proteome level, we clearly identified the distinct pattern of cellular responses for the F2-treated cells, and 6 differentially regulated proteins were identified, which provide useful information on elucidating the anticancer mechanism of F2 to SGC7901 cells. Moreover, the integration of networks and pathway with the proteomic data enhanced our understanding of the functional relationship of proteome changes caused by the compound.


## 3.3.1. For Verification

To validate the information obtained from the iTRAQ-based quantitative proteomics study and bioinformatics analysis, the screened proteins with strong response to ginsenoside F2 treatment 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 the expression of the RPL26 (p<0.01) in SGC7901 cells in comparison with the treatment with vehicle control.Figure 6
Western blot validations of RPS15, RPL26, PRR5, CISD2, NLRX1, p53, PUMA, mTOR, and Bcl-xl in SGC7901 cells with different concentrations of ginsenoside F2. 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: accumulated results show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as p∗∗<0.01, p∗<0.05.
(a) (b)


## 3.3.2. For Determining the Expression of Apoptosis and Autophagic Proteins

As shown in Figure6, ginsenoside F2 suppressed the expression of mTOR and upregulated the expression of p53 in a dose-dependent manner. Atg5, Atg7, Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1 are known to be modulated by p53 or Bcl-xl signaling, which may trigger apoptosis or autophagy. Therefore, we proceeded to check the expressions of Atg5, Atg7, Atg10, PUMA, Beclin-1, UVRAG, and AMBRA-1. As shown in Figure 7, ginsenoside F2 upregulated the expressions of these proteins in a dose-dependent manner. LC3 is now widely used to monitor autophagy. During autophagy, the cytoplasmic form LC3-I is processed and recruited to phagophores, where LC3-II is generated by site-specific proteolysis and lipidation at the C-terminus. Thus, the amount of LC3-II positively correlates with the number of autophagosomes [10]. We examined the effect of F2 on LC3 conversion in SGC7901 cells. Western blot analysis showed that F2 treatment resulted in dose-dependent accumulation of LC3-II and reduction of LC3-I (Figure 7). The conversion of LC3-I to LC3-II suggested F2 treatment induces autophagy.Figure 7
Effect of ginsenoside F2 on 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 representative western blot analysis. β-actin was used as the loading control. Right panel: accumulated results show the relative protein density. Error bars represent means ± SEMs. Significant difference is expressed as p∗∗<0.01, p∗<0.05.
(a) (b)In the present study, combination of iTRAQ-based proteomics method with bioinformatics was used to identify critical molecules in SGC7901 cancer cells in response to ginsenoside F2 treatment. Ginsenoside F2 generated significant change of protein profile in SGC7901 cells. Some of them have been demonstrated to participate in either apoptosis or autophagy responses, suggesting that the antitumor mechanisms of ginsenoside F2 in SGC7901 cells are involved in both apoptosis and autophagy.The current findings demonstrate that ginsenoside F2 impacts distinct signaling pathways and induces broad change in the protein profile of SGC7901 cells. Overall, 205 differentially expressed proteins were identified with ≥95% confidence in ginsenoside F2 treated group. Application of a ratio of 1.2-fold change as criteria resulted in 44 and 161 differentially abundant proteins in SGC7901 cells.In our study, some proteins that were significantly altered by ginsenoside F2 show close relationship of protein-protein interaction (Figure 5). Ribosomal proteins, such as RPS15 and 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 of autophagy or apoptosis. The changes of these six potential proteins were verified by western blot analysis.Ribosomal proteins (RPs) are considered to have diverse extra ribosomal functions, ranging from cell cycle progression to cell death and to malignant transformation and cellular metabolism [11]. Relevantly, a number of RPs have been shown to bind to MDM2, the inhibitor of p53 (murine double minute 2, and also HDM2 for its human ortholog), and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and activation, then triggering apoptosis or autophagy [11]. Following the treatment of ginsenoside F2 in SGC7901 cells, the levels of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14, RPL24, RPL7A, and RPL26 were increased, whereas that of RPS15 reduced. Although the functions of RPL28, RPL34, RPL35, RPS16, RPL17, RPL14, RPL24, and RPL7A have not been well studied, RPL26, a positive regulator of p53, was found to increase the translational rate of p53 mRNA by binding to its 50 untranslated region [12] and, in this case, MDM2 acts as an ubiquitin E3 ligase for ubiquitylation and degradation of RPL26 [18]. Thus, under the treatment of ginsenoside F2, the increased level of RPL26 indicated that RPL26 may inhibit MDM2 and subsequently activate p53. RPS15, identified as a direct p53 transcriptional target, was thought to activate p53 by repressing MDM2 activity [19]. Interestingly, in our study, the level of RPS15 reduced in SGC7901 followed by ginsenoside F2 treatment, suggesting that the roles of RPS15 and RPL26 involved in the anticancer mechanism of ginsenoside F2 are different, which warrant further investigation.mTOR, existing in two multiprotein complexes, mTORC1 and mTORC2, regulates cell growth in response to a variety of cellular signals derived from growth factors and environmental stress [20]. mTORC2 is a kinase complex comprised of mTOR, PRR5, Rictor, mSin1, and mLST8/GbL. The expression level of PRR5 is correlated with that of mTORC2. Recent study showed that mTORC2 is implicated in actin cytoskeleton regulation, as well as phosphorylation of Akt [13]. Although TOR kinase has been largely attributed as a negative regulator of autophagy through TORC1, resent study indicated that mTORC2 was an independent positive regulator of autophagy during amino acid starvation [21]. In the present study, ginsenoside F2 decreased level of PPR5, indicated that ginsenoside F2 may 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 may activate the AMPK pathway and inhibit the mTOR pathway, subsequently triggering autophagy. p53 may also transactivate multiple genes with proautophagic roles, including proapoptotic Bcl-2 proteins (Bax, PUMA) [22, 23]. In this network, PUMA induces the noncanonical autophagy pathway regulated via Atg5, Atg7, and Atg10. PUMA’s initiation of autophagy promotes cytochrome c release, which then leads to apoptosis [22]. Interestingly, in our previous work, increasing level of cytochrome c and decreased mitochondrial transmembrane potential (MTP) were observed [6]. In present study, decreased expressions of PRR5 and RPL26 were found, which implied that ginsenoside F2 might trigger p53 signal pathway. It was reported that western blot analyses tended to show greater differential abundance compared with iTRAQ analyses [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 and reduced level of P53 and mTORC2 suggested that ginsenoside F2 may 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]. Our results showed that CISD2 was significantly decreased in ginsenoside F2 treated group, confirmed by western blot analysis. Recent work identified CISD2 as a Bcl-xl binding partner at a branch point between autophagy and apoptosis, life and death, under nutrient-deprived and oxidative stress conditionsin vivo cells [25, 26]. Bcl-xl, also called Bcl-2L, is known to function through inhibition of the autophagy effector and tumor suppressor Beclin-1 [15]. CISD2 is required in this pathway for Bcl-xl to functionally antagonize Beclin-1-dependent autophagy. In our study, the expression of Bcl-xl decreased, confirmed by western blot analysis. Thus, CISD2 may be a Bcl-xl-associated cofactor that targets Bcl-2 for the autophagy pathway.During initiation of autophagosome formation, after release from Bcl-xl, Beclin-1 functions as a platform by binding to class III PI3K/vacuolar protein sorting-34 (Vps34), UV-resistance-associated gene (UVRAG), activating molecule in Beclin-1-regulated autophagy (AMBRA-1) [15, 26, 27]. Previous studies have shown that binding of Beclin-1 to Bcl-2/Bcl-xl inhibits the autophagic function of Beclin-1, suggesting that Beclin-1 might have a role in the convergence between autophagy and apoptotic cell death [22]. For confirming the Beclin-1/Bcl-xl pathway, western blot was employed. The expressions of Beclin-1, UVRAG, and AMBRA-1 were increased, while Bcl-xl was decreased, which suggested that ginsenoside F2 may induce autophagy via Bcl-xl/Beclin-1 pathway.NLRX1, a mitochondrial NOD-like receptor that amplifies apoptosis by inducing reactive oxygen species production, is an important component of TLR mediated inflammatory pathways [13, 16]. Recent evidence suggested that upregulated expression of NLRX1 may synergistically regulate metabolism and autophagy for highly invasive growth of 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. In our iTRAQ results, expression of NLRX1 was significantly decreased in SGC7901 cells treated with ginsenoside F2. The phenomenon suggested different role of NLRX1 involved in the ginsenoside F2 treatment that may be different from that of published reports [16, 17], though the mechanism needs further research.Mai et al. reported that F2 induces apoptotic cell death accompanied by protective autophagy in breast cancer stem cells [28]. In one of our previous studies, we found that F2 induces apoptosis by causing an accumulation of ROS and activating the apoptosis signaling pathway [6]. However, there was no report systemically comparing differently regulated proteins and building a network of F2-treated cancer cells at proteome level. In the current study, by the close look at cellular mechanisms at proteome level, we clearly identified the distinct pattern of cellular responses for the F2-treated cells, and 6 differentially regulated proteins were identified, which provide useful information on elucidating the anticancer mechanism of F2 to SGC7901 cells. Moreover, the integration of networks and pathway with the proteomic data enhanced our understanding of the functional relationship of proteome changes caused by the compound.


## 4. Conclusions

In conclusion, 44 upregulated proteins and 161 downregulated proteins were discovered by iTRAQ analysis in SGC7901 cells treated with lower dose and shorter duration of ginsenoside F2, 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 process by ginsenoside F2 treatment in SGC7901 cells. Although more work is required to find out the precise role of targeted proteins, our data lead to a better understanding of the molecular mechanisms of ginsenoside F2 for gastric cancer treatment.



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*Source: 2635483-2016-10-18.xml*