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

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.


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][5][6][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][5][6][7]. Structuralfunction 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 F 2 (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, our in vitro and in vivo studies demonstrated that ginsenoside F 2 possesses anticancer effects in human gastric carcinoma cells SGC7901 [6]. However, the involved exact mechanisms of ginsenoside F 2 on SGC7901 cancer cells at proteome level have not been systemically investigated.
Advancements in the field of proteomics have made it possible to accurately monitor and quantitatively detect 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 iTRAQtagged 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 iTRAQbased 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 F 2 in gastric SGC7901 cells. General molecular biological techniques such as western blot were utilized for validation.

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), 100 g/mL streptomycin, and 100 g/mL penicillin and grown at 37 ∘ C in 5% carbon dioxide.

Protein Preparation.
In one of our recent reports [6], we have shown that the IC 50 of ginsenoside F 2 is in <50 M in 24 hours. In order to characterize ginsenoside F 2 -related mechanism it is imperative to use samples that are at the early stages of ginsenoside F 2 treatment. So, a lower dose than the IC 50 (20 M) and a shorter duration (12 hours in the study) were chosen in the study. The treated (20 M) and untreated SGC7901 cells were suspended in the lysis buffer and sonicated in ice. The proteins were reduced with 10 M 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.

iTRAQ Labeling and SCX Fractionation.
Total protein (100 g) 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 L isopropanol. iTRAQ labels 113 were used to label control sample separately, and 115 and 117 were used to label twice F 2 -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 NaH 2 PO 4 in 25% acetonitrile, pH 2.7) and loaded onto a 4.6 × Evidence-Based Complementary and Alternative Medicine 3 250 mm Ul tremex SCX column containing 5 m 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 NaH 2 PO 4 , 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.

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 0.5 g/ L. 10 L 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 m) packed inhouse. The samples were loaded at 8 L/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 highenergy 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 × 10 5 by pAGC and fragmented HCD. A datadependent 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 3e 6  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 of N-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 value must be below 0.05 to identify significant changes.

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). Approximately 30 g 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 M 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 -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).

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's t-test or ANOVA test by using GraphPad Prism v4.0 software (GraphPad Software, San Diego, CA, USA). Statistical significance is expressed as * * * < 0.001; * * < 0.01; * < 0.05.

Proteome Analysis.
Human gastric carcinoma cells (SGC7901) are treated with ginsenoside F 2 at a dose of 20 M 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 < 0.05 with relative expression levels at least >1.2-fold (Table 1) 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%).
Further COG function classification revealed that posttranslational modification, protein turnover, and ribosomal structure biogenesis were major function of the screened 205 proteins (Figure 3(b)). In each category of BP, CC, and MF, top twenty proteins which generated bigger difference in response to ginsenoside F 2 treatment are listed in Figure 4.
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 Figure 5. 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 F 2 .

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 F 2 treatment were further confirmed by western blot. As shown in Figure 6, ginsenoside F 2 significantly reduced protein expressions of PRR5, CISD2, Bcl-xl, NLRX1, and RPS15 ( < 0.01) and enhanced the expression of the RPL26 ( < 0.01) in SGC7901 cells in comparison with the treatment with vehicle control. Figure 6, ginsenoside F 2 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 F 2 upregulated the expressions of these proteins in a dosedependent 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 Cterminus. Thus, the amount of LC3-II positively correlates with the number of autophagosomes [10]. We examined the effect of F 2 on LC3 conversion in SGC7901 cells. Western blot analysis showed that F 2 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 F 2 treatment induces autophagy.

For Determining the Expression of Apoptosis and Autophagic Proteins. As shown in
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 F 2 treatment. Ginsenoside F 2 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 F 2 in SGC7901 cells are involved in both apoptosis and autophagy.
The current findings demonstrate that ginsenoside F 2 impacts distinct signaling pathways and induces broad change in the protein profile of SGC7901 cells. Overall, 205 Evidence-Based Complementary and Alternative Medicine 5              differentially expressed proteins were identified with ≥95% confidence in ginsenoside F 2 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 F 2 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 F 2 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 F 2 , the increased level of RPL26 indicated that RPL26 may inhibit MDM2 and subsequently activate p53.  Figure 5: The protein-protein interaction network of the differentially expressed proteins identified. Red triangle denotes upregulated proteins; green triangle denotes downregulated protein.
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 F 2 treatment, suggesting that the roles of RPS15 and RPL26 involved in the anticancer mechanism of ginsenoside F 2 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 F 2 decreased level of PPR5, indicated that ginsenoside F 2 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 F 2 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 F 2 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 F 2 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 conditions in 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-1dependent 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 F 2 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 TNFinduced apoptosis and metabolism in cancer cells. In our iTRAQ results, expression of NLRX1 was significantly decreased in SGC7901 cells treated with ginsenoside F 2 . The phenomenon suggested different role of NLRX1 involved in the ginsenoside F 2 treatment that may be different from that of published reports [16,17], though the mechanism needs further research.
Mai et al. reported that F 2 induces apoptotic cell death accompanied by protective autophagy in breast cancer stem cells [28]. In one of our previous studies, we found that F 2 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 F 2 -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 F 2 -treated cells, and 6 differentially regulated proteins were identified, which provide useful information on elucidating the anticancer mechanism of F 2 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.

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 F 2 , compared with our previous study. 6 differentially abundant common proteins, PRR5, CISD2, Bcl-xl, NLRX1, RPS15, and RPL26, were confirmed by western blot analysis. Ribosomal protein-p53 signaling pathway and Bcl-xl/Beclin-1 pathway might be significantly regulated biological process by ginsenoside F 2 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 F 2 for gastric cancer treatment.