OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2014.3490 or-32-06-2635 Articles Anticancer effects of β-elemene in gastric cancer cells and its potential underlying proteins: A proteomic study LIUJUN-SONG HESHI-CAI ZHANGZHENG-LIANG CHENRUI FANLIN QIUGUANG-LIN CHANGSHUAI LILIANG CHEXIANG-MING Department of General Surgery, The First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P.R. China Correspondence to: Professor Xiang-Ming Che, Department of General Surgery, The First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P.R. China, E-mail: chexiang@mail.xjtu.edu.cn 12 2014 17 09 2014 32 6 2635 2647 04 07 2014 26 08 2014 Copyright © 2014, Spandidos Publications 2014 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

Gastric cancer is a common malignancy with a poor prognosis. β-elemene is a broad-spectrum anticancer drug extracted from the traditional Chinese medicinal herb Curcuma wenyujin. In the present study, we investigated the anticancer effects of β-elemene in gastric cancer cells and the potential proteins involved. Human SGC7901 and MKN45 gastric cancer cells were treated with different concentrations of β-elemene. Cell viability, clonogenic survival and apoptotic cell death were assessed. β-elemene inhibited viability and decreased clonogenic survival of gastric cancer cells in a dose-dependent manner. Apoptosis induction contributed to the anticancer effects. We then employed a proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), to detect the proteins altered by β-elemene. In total, 147 upregulated proteins and 86 downregulated proteins were identified in response to β-elemene treatment in SGC7901 gastric cancer cells. Among them, expression of p21-activated protein kinase-interacting protein 1 (PAK1IP1), Bcl-2-associated transcription factor 1 (BTF) and topoisomerase 2-α (TOPIIα) were validated by western blot analyses and the trends were consistent with iTRAQ results. Top pathways involved in β-elemene treatment in SGC7901 gastric cancer cells included ribosome signaling, peroxisome proliferator-activated receptors (PPARs) signaling pathway, regulation of actin cytoskeleton, phagosome, biosynthesis and metabolism of some amino acids. Collectively, our results suggest a promising therapeutic role of β-elemene in gastric cancer. The differentially expressed proteins provide further insight into the potential mechanisms involved in gastric cancer treatment using β-elemene.

 β-elemene gastric cancer proliferation apoptosis proteomics Introduction

Gastric cancer is the fourth most common malignancy in the world and the second leading cause of cancer-related mortality (1). At present, surgical resection remains the main therapeutic strategy for gastric cancer, supplemented with perioperative chemotherapy, chemoradiotherapy and/or immunotherapy (2–5). However, most patients are diagnosed with advanced gastric cancer which may have progressed beyond the curative potential of surgical operation (6,7). In addition, previous studies have demonstrated that a considerable proportion of patients receiving potentially curative resection experienced recurrences which lead to unfavorable prognosis (8,9). Adjuvant therapy, such as chemotherapy, provides rather limited survival advantage (10,11). These facts attest to the deficiency in the current strategies for treating gastric cancer and the demand for novel approaches to the management of gastric cancer.

Among various ingredients eligible for adjuvant therapy for gastric cancer, the significance of natural products, particularly the essence extracted from Chinese herbs are gaining increasing attention in basic and clinical research (12). β-elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane) is a novel anticancer agent extracted from the Chinese medicinal herb Curcuma wenyujin (13). In recent studies, β-elemene was shown to have diverse anticancer potential, such as inhibiting proliferation and inducing apoptosis of cancer cells, and interacting with multiple oncogenic or tumor suppressing signaling pathways in a broad spectrum of cancers (14–16). Other studies found that β-elemene could enhance tumor chemosensitivity or overcome drug resistance (17,18). In addition, β-elemene has been approved by the China Food and Drug Administration as a therapeutic drug in clinical practice where its efficacy has been exhibited when combined with first-line chemotherapy for malignant tumors (19,20). However, the mechanisms by which β-elemene is involved in tumor suppressing activities remain largely unknown.

In the present study, we examined the anticancer potential of β-elemene in the proliferation, clonogenic survival and apoptosis in SGC7901 and MKN45 gastric cancer cells. Then, in order to investigate the molecules through which β-elemene exhibited its anticancer effects and to obtain a better understanding of its therapeutic role in gastric cancer, we employed isobaric tags for relative and absolute quantitation (iTRAQ), a high-throughput proteomic approach, to profile proteins that were differentially expressed following β-elemene treatment in gastric cancer cells.

 Materials and methods Reagents

β-elemene was obtained from Jingang Pharmaceutical Co. (Dalian, China). Annexin V-FITC/PI apoptosis detection kit was purchased from 7 Sea Pharmacy Technology (Shanghai, China). iTRAQ reagents were from Applied Biosystems (New York, NY, USA). Anti-PAK1IP1 antibody was purchased from Abcam (#ab67348; UK). Anti-TOPIIα antibody was obtained from Proteintech (#20233-1-AP; USA). Anti-BTF antibody was from BD Biosciences Pharmingen (#611726; San Diego, CA, USA).

 Cell culture

The SGC7901 and MKN45 human gastric cancer cell lines were obtained from the Lab Animal Centre of the Fourth Military Medical University (Xi’an, China). Cells were cultured in RPMI-1640 medium (HyClone, USA), supplemented with 10% fetal bovine serum (Sijiqing, Huzhou City, China) at 37°C with 5% CO2 in a humidified atmosphere.

 MTT assay

Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded in 24-well plates at 5–10×104/well. After overnight incubation, cells were exposed to different concentrations of β-elemene for 24–72 h. Then, 50 μl MTT (5 mg/ml) was added to each well and the cells were incubated for another 4 h at 37°C. After gentle removal of the supernatant, 500 μl dimethyl sulfoxide (DMSO) was added to each well to solubilize the purple formazan crystal. The optical density (OD) was measured using a microplate reader at 490 nm and then transformed into cell viability using the following formula: Cell viability = (OD of the experimental sample)/(OD of the control sample) × 100%.

 Annexin V-FITC/PI apoptosis detection assay

To explore the effect of β-elemene on apoptotic cell death, Annexin V-FITC/PI apoptosis detection assay was used. The cells were seeded in 6-well plates at 3×105/well. After overnight incubation, cells were exposed to different concentrations of β-elemene for 24 h. Then, cells were collected and manipulated following the manufacturer’s instructions, incubated with Annexin V-FITC and propidium iodide (PI), then analyzed using flow cytometry (FCM; BD Biosciences-Clontech, Palo Alto, CA, USA) within 30 min.

 Clonogenic survival assay

Cells were trypsinized and counted. Then, 200 cells were seeded into each well of 6-well plates. After overnight incubation for attachment and recovery, the cells were treated with different concentrations of β-elemene. Ten to fourteen days after seeding, cells were washed with PBS twice, fixed with methyl alcohol for 15 min and stained with 1% crystal violet for 20 min. Colonies containing >50 cells was counted and the surviving fractions were calculated as follows: Plating efficiency (PE) = colony number of the control group/the number of cells seeding. Surviving fraction = colony number of the treated-group/(the number of cells seeding × PE). This assay was carried out in duplicate.

 Protein preparation

After SGC7901 cells were treated with or without β-elemene at 30 μg/ml for 48 h, cell total protein was extracted. Protein concentration was determined using Pierce™ BCA Protein Assay (Thermo Scientific, Rockford, IL, USA). Total protein extracted from two separate experiments was mixed together for use in the subsequent proteomic analysis. Protein samples were reduced and alkylated, then added into 5-fold volume of ice-cold acetone and put in −20°C condition overnight. Then, the precipitate was harvested by centrifugation at 25,000 × g at 4°C for 20 min and dried in the air for 5 min. The precipitate was dissolved in 200 μl 0.5 M tetraethylammonium bromide (TEAB) and dealt with sonicate for 15 min. Finally, the supernatant was harvested and quantified.

 iTRAQ proteomic analysis

One hundred micrograms of protein were taken out from each sample and digested with trypsin. Thereafter, the peptides of each sample were labeled with iTRAQ reagents respectively, according to the manufacturer’s protocol (Applied Biosystems) (SGC7901-control-114 tag and SGC7901-β-elemene treated-115 tag). Then, the labeled samples were pooled and sent to fractionating using strong cation exchange (SCX) chromatography (Shimadzu LC-20AB HPLC Pump system and the 4.6×250 mm Ultremex SCX column). After elution, 20 fractions of peptides were obtained. Each fraction was then desalted by Strata-X C18 column (Phenomenex) and vacuum-dried. Each fraction of peptides was resuspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20,000 × g for 10 min to remove the insoluble substances. In each fraction, the final concentration of peptides was ~0.5 μg/μl. Five microlitres (~2.5 μg) of supernatant was loaded onto a Shimadzu LC-20AD nano HPLC by the autosampler for separation. Mass spectrometric analysis of the iTRAQ labelled peptides was performed using a Q Exactive (Thermo Fisher Scientific, San Jose, CA, USA) coupled online to the HPLC. Data processing of LC-MS/MS samples was searched against the International Protein Index (IPI) human protein database version 3.87 FASTA (91,464 sequences) using Mascot 2.3.02 software (Matrix Science, UK). When the fold-change of protein abundance was >1.2 and the P-value was <0.05, we defined this protein as differentially expressed. The identified proteins were categorized according to the Gene Ontology (GO) classification terms (http://www.geneontology.org/). GO enrichment analysis was performed to display the GO terms which the differentially expressed proteins enriched in all identified proteins.

 Western blot analyses

Equal amounts of protein samples were subjected to SDS-PAGE. Proteins were transferred to the nitrocellulose (NC) membranes followed by 2 h blocking with 5% skimmed milk at room temperature. The NC membranes were sequentially incubated with primary antibodies at 4°C overnight and secondary antibody for 1 h at room temperature. The reactions were visualized using electrochemiluminescence (ECL) detection kit (CWBIO, Beijing, China). The band quantification was performed using Image-Pro Plus 6.0 software (Media Cybernetics).

 Statistical analysis

Data are presented as the means ± SD. Statistical analysis was performed using a two-tailed Student’s t-test through SPSS 17.0 software (Chicago, USA). P-value <0.05 was considered to indicate a statistically significant difference.

 Results β-elemene inhibits the viability of gastric cancer cells

To investigate the antiproliferative effect of β-elemene in gastric cancer cells, SGC7901 and MKN45 cells were exposed to different concentrations of β-elemene (0, 10, 20, 30, 40, 60, 80 and 120 μg/ml) for 24, 48 or 72 h. Cell viability analysis showed that β-elemene suppressed the viability of gastric cancer cells in a dose-dependent manner (Fig. 1). The 50% inhibitory concentration (IC50) values of β-elemene for SGC7901 gastric cancer cells at 24, 48 and 72 h were 67.15, 56.89 and 46.05 μg/ml, respectively. The IC50 values for MKN45 cells at 24, 48 and 72 h were 45.57, 37.97 and 35.29 μg/ml, respectively. These results indicate that β-elemene inhibits the viability of gastric cancer cells in a dose-dependent manner.

 β-elemene induces apoptotic cell death in gastric cancer cells

FCM analysis showed that the apoptotic rate gradually increased after 24-h exposure to increased concentrations of β-elemene. The percentage of apoptotic cells in SGC7901 cells in the control group and β-elemene-treated groups (20, 30, 40, 60 and 80 μg/ml) was 8.5±0.5, 10.4±1.0, 22.2±1.6, 32.6±5.4, 42.8±4.3 and 52.1±4.3%, respectively (Fig. 2A and B). Compared with the control group, the increased rate of apoptosis reached statistical significance when the concentrations of β-elemene were >30 μg/ml in SGC7901 cells. Similar trends of apoptosis induction effects were observed in MKN45 cells (Fig. 2C and D). These data suggest that β-elemene induces apoptotic cell death in gastric cancer cells in a dose-dependent manner.

 β-elemene decreases clonogenic survival of gastric cancer cells

To determine whether β-elemene inhibits colony forming efficiency, cells were exposed to different concentrations of β-elemene (0, 10, 20, 30, 40 and 60 μg/ml) and grown in a cell contact-independent manner. Consistent with the observed effects on cell viability and apoptosis-induction activity, β-elemene exhibited anti-clonogenic potential and led to a statistically significant reduction in colony formation (Fig. 3). It is worth noting that the size of the colonies tended to be smaller after treatment with β-elemene (Fig. 3B). When the concentration of β-elemene reached 60 μg/ml, the number of survived cells in each cloned cell group barely reached the standard of a colony in SGC7901 cells. These results suggest that β-elemene induces a dose-dependent inhibition of clonogenicity in gastric cancer cells.

 iTRAQ identification and quantification of differentially expressed proteins by β-elemene in SGC7901 gastric cancer cells

Through iTRAQ analysis, 17,154 unique peptides corresponding to 4,267 proteins were identified in SGC7901 gastric cancer cells (data not shown). According to our definition of differentially expressed protein, a total of 233 identified proteins were regulated by β-elemene intervention in SGC7901 gastric cancer cells, including 147 upregulated proteins and 86 downregulated proteins. The altered proteins of both lists are shown in Tables I and II, respectively.

 GO analysis of differentially expressed proteins altered by β-elemene in SGC7901 gastric cancer cells

Based on the GO terms analysis, categorization of these differentially expressed proteins according to cellular component, molecular function and biological process is shown in Fig. 4. GO enrichment analysis shows the top pathways involved in the differentially expressed proteins resulting from β-elemene treatment. They include phenylalanine, tyrosine and tryptophan biosynthesis, ribosome signaling, tyrosine metabolism, phenylalanine metabolism, PPAR signaling pathway, regulation of actin cytoskeleton, cysteine and methionine metabolism, ether lipid metabolism, hematopoietic cell lineage and phagosome signaling pathways.

 Validation of iTRAQ results by western blot analyses

Western blot analyses were performed to validate the differentially expressed proteins discovered by iTRAQ proteomic analysis. The SGC7901 human gastric cancer cells were treated the same as for the iTRAQ analysis and lysed for protein samples. Three proteins were selected for validation purposes according to our interests and the availability of antibodies. The results were consistent with those found using iTRAQ (Fig. 5) and indicated the high reliability of our iTRAQ results.

 Discussion

Previous studies have shown that β-elemene, a promising anti-cancer drug extracted from natural plants, has efficient growth inhibition effects in a broad range of cancer cells, although with slight toxicity to normal tissue cells (14,21,22). In China, it has been used as a therapeutic candidate for certain malignant tumors for several years (20). However, little is known about the underlying molecules. In the present study, our data indicated that β-elemene efficiently suppressed the proliferation and survival of gastric cancer cells at least partly through the induction of apoptosis. The effects are consistent with results in other malignancies (15,21). Different from other studies, we employed an iTRAQ proteomic method to explore the potential proteins that may contribute to its anticancer effect. As a result, the differentially expressed proteins in response to β-elemene treatment in gastric cancer cells provided insight and supported the results found at the cytological level. Furthermore, the analysis provided some other molecules and signal pathways that may predict other pharmacologic actions of β-elemene that had not been studied in cancer therapy. In brief, our results provide evidence that β-elemene may be a potential drug for gastric cancer. Some of the key proteins are potential markers of gastric cancer treatment and are briefly discussed below.

Bcl-2 family proteins were found to be modulated in β-elemene-treated cancer cells in previous studies and make sense in apoptosis induction (14,21,22). The balance between pro-apoptotic and anti-apoptotic members of the Bcl-2 family proteins decides the fates of cells, and the increased proportion of pro-apoptotic proteins results in apoptotic cell death. Therefore, they play vital role in cancer therapy (23). In the present study, Bcl-2-associated transcription factor 1 (BTF) and Bcl-2-like protein 13 (also known as Bcl-rambo) were upregulated to 1.34- and 1.29-fold in response to β-elemene treatment compared with the control untreated gastric cancer cells, respectively. Both play death-promoting roles in cancer cells. BTF was first identified as a transcriptional repressor that interacted with Bcl-2 family proteins and overexpression of BTF could induce apoptosis (24). Previous studies demonstrated the roles of BTF in apoptosis promotion through the control of transcription or correlations with Bcl-2 family members (25–27). Moreover, BTF has been shown to inhibit DNA damage repair (25,27). This may partly contribute to the fact that β-elemene enhances tumor chemosensitivity or overcomes drug resistance (17,18). The other molecule, Bcl-rambo, is also a pro-apoptotic member of the Bcl-2 family (28,29). Although it was found to trigger cell death in a way distinct from the traditional Bcl-2 family members, Bcl-rambo-induced apoptotic signaling pathway eventually joined other pro-apoptotic pathways at the level of caspase-3 (28). Taken together, these results indicate that Bcl-2 family proteins play a critical role in β-elemene-induced cell death in gastric cancer cells.

p21-activated protein kinase-interacting protein 1 (PAK1IP1) was the most influenced protein among the list of upregulated proteins by β-elemene. P21-activated protein kinase 1 (PAK1) and PAK signal pathways have been shown to have multiple roles in cancer cell biological behaviors, such as cytoskeletal dynamics, survival, proliferation and transcription (30,31). PAK1 and its PAK family members are overexpressed or hyperactivated in several types of cancer and play a critical role in tumorigenesis and metastasis. Inhibition of PAK1 may efficiently block the transformation of cancer cells and act as a therapeutic strategy in cancer treatment (30,32,33). As a negative regulator of PAK1, PAK1IP1 specifically binds to the N-terminal regulatory domain of PAK1 and inhibits the activation of PAK1 by blocking the binding site of Rac and Cdc42, thus playing a negative role in cancer development and progression (30,31). However, little research has focused on PAK1IP1. In a recent study, Yu et al found that PAK1IP1 was upregulated when cancer cells were suffering from ribosomal stresses and overexpression of PAK1IP1 could inhibit proliferation via p53-MDM2 loop (34). In the present study, PAK1IP1 expression was ~3-fold upregulated in β-elemene-treated gastric cancer cells. Therefore, we hypothesize that β-elemene may upregulate PAK1IP1 expression and thus inhibit the activation of PAK1, which subsequently inhibits proliferation and induces apoptosis in gastric cancer cells.

One of the major protein groups regulated by β-elemene in SGC7901 gastric cancer cells was ribosomal proteins, with 12 ribosomal proteins upregulated and 4 ribosomal proteins downregulated. Over the last decade, some ribosomal proteins have been linked with emerging functions in cancer, in addition to protein synthesis. Ribosomal protein L5 (RPL5), along with RPL11 and RPL23, may form a complex with MDM2 oncoprotein and activate p53 through the inhibition of MDM2-mediated p53 degradation (35). RPS7 was also found to interact with MDM2 and overexpression of RPS7 increased cell apoptosis and suppressed cell proliferation after p53 activation (36). RPS14 and RPL11 were demonstrated to inhibit cell proliferation by negative regulation of c-Myc activity (37,38). In some other studies, the correlation between ribosomal proteins and drug resistance in cancer therapy was established. RPS13 and RPL23 could suppress drug-induced apoptosis and thus mediate multidrug resistance in gastric cancer cells (39). RPL35a was found overexpressed in many glioblastoma multiforme (GBM) brain tumors and led to chemotherapy resistance in GBM (40). Together with the present study, these results propose more roles of ribosomal proteins in cancer and therefore merit further attention in cancer therapy research.

S100A10 was a top molecule significantly downregulated by β-elemene in the present study. S100A10, also known as p11, is a unique member of the S100 protein family which serves for intracellular calcium signaling and is characterized by two EF hand motifs (41,42). The homodimer of S100A10 forms a heterotetrameric complex with two molecules of Annexin A2, a type of plasma membrane protein, to maintain stability and execute its functions (43). Expression of S100A10 has been detected in a broad spectrum of tissues and cancers including gastric cancer (44–47). Over the past years, increasing evidence has demonstrated the promoting role of S100A10 in tumor invasion and metastasis and that knockdown of S100A10 could efficiently suppress cancer progression (46–48). Notably, PAK1IP1, the most influenced protein of upregulated proteins by β-elemene, specifically targets PAK1 which is also most associated with cytoskeletal dynamics. Collectively, we deduced that β-elemene may inhibit invasion and metastasis in gastric cancer therapy, which warrants further investigation.

In conclusion, this is the first time that the iTRAQ proteomic method has been employed in the study of β-elemene in cancer cells. The present study indicated a promising anticancer role of β-elemene in gastric cancer therapy. The expression of a wide range of proteins was altered when gastric cancer cells were exposed to β-elemene. The differentially expressed proteins provided comprehensive insight into the potential underlying molecular mechanisms of the anticancer effects of β-elemene in gastric cancer cells. Furthermore, some of the proteins may act as predictors regarding further therapeutic potential of β-elemene, which merits further study in gastric cancer treatment. The present study was a preliminary exploration into the anticancer potential of β-elemene in gastric cancer cells. Based on the current results, we expect more research to be carried out on β-elemene and other traditional herbal medicine, in order to improve the management of gastric cancer.

 Acknowledgements

This study was funded by the National Natural Science Foundation of China (grant no. 81172357). The field study was conducted in the Center for Translational Medicine of the First Affiliated Hospital of Xi’an Jiaotong University, and the proteomics technology platform of BGI Technology Ltd. The authors thank the staff workers for their practical assistance and opinions.

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β-elemene inhibits the viability of gastric cancer cells. (A) SGC7901 and (B) MKN45 gastric cancer cells were treated with different concentrations of β-elemene (0, 10, 20, 30, 40, 60, 80 and 120 μg/ml) for 24, 48 or 72 h. Cell viability was determined using MTT assay. Dots and bars, means ± SD.

β-elemene induces apoptotic cell death in gastric cancer cells. Cells were treated with different concentrations of β-elemene for 24 h. Apoptotic rate was analyzed by FCM following the manufacturer’s instructions. (A) Representative FCM results of SGC7901 cells. a, control; b, 20 μg/ml; c, 30 μg/ml; d, 40 μg/ml; e, 60 μg/ml; and f, 80 μg/ml. (B) Student’s t-test of apoptotic rate was performed between the control group and groups treated with β-elemene in SGC7901 cells. (C) Representative FCM results of MKN45 cells. a, control; b, 10 μg/ml; c, 20 μg/ml; d, 30 μg/ml; e, 40 μg/ml; and f, 60 μg/ml. (D) Quantification of apoptosis of the control group and groups treated with β-elemene in MKN45 cells. *P<0.05. Column and bars, means ± SD. FCM, flow cytometry.

β-elemene inhibits clonogenic survival of SGC7901 gastric cancer cells. Two hundred single cells were seeded to each well of the 6-well plates. After attachment and recovery, cells were treated with or without β-elemene and incubated for 10–14 days to form typical colonies. (A) The entire appearance of representative wells of each experimental group. (B) Appearance of representative colony from each experimental group. (C) Clonogenic survival of the control group and groups treated with β-elemene. The results are representative of duplicate independent experiments. a, control; b, 10 μg/ml; c, 20 μg/ml; d, 30 μg/ml; e, 40 μg/ml; and f, 60 μg/ml. *P<0.05. Column and bars, means ± SD.

Categorization of the differentially expressed proteins after β-elemene treatment in SGC7901 human gastric cancer cells according to the GO terms system. (A) Protein classification according to the cellular component. (B) Protein classification according to molecular function. (C) Protein classification according to biological process. GO, Gene Ontology.

Validation of iTRAQ proteomic results by western blot analyses in SGC7901 gastric cancer cells. After exposure to β-elemene at 30 μg/ml for 48 h, the expression levels of PAK1IP1, BTF and TOPIIα were analyzed by western blot analyses. The trends closely match the iTRAQ proteomic results. iTRAQ, isobaric tags for relative and absolute quantitation; PAK1IP1, p21-activated protein kinase-interacting protein 1; BTF, Bcl-2-associated transcription factor 1; TOPIIα, topoisomerase 2-α.

Upregulated proteins in response to β-elemene treatment in SGC7901 gastric cancer cells.

No. Accession Gene symbol Protein name Fold-ratio (115:114)
1 IPI00549540 PAK1IP1 p21-activated protein kinase-interacting protein 1 3.967
2 IPI00152429 SPNS1 Isoform 3 of protein spinster homolog 1 2.979
3 IPI00021885 FGA Isoform 1 of fibrinogen α chain 2.397
4 IPI00011200 PHGDH D-3-phosphoglycerate dehydrogenase 2.319
5 IPI00006213 PCM1 Isoform 1 of pericentriolar material 1 protein 2.316
6 IPI00418426 CNNM4 Metal transporter CNNM4 2.313
7 IPI00013809 GSTZ1 Isoform 1 of maleylacetoacetate isomerase 2.292
8 IPI00012433 F8A Factor VIII intron 22 protein 2.172
9 IPI00015856 DNPEP Aspartyl aminopeptidase 2.144
10 IPI00002564 XRCC1 DNA repair protein XRCC1 1.981
11 IPI00909584 HARS2 Histidyl-tRNA synthetase homolog 1.922
12 IPI00218116 OASL Isoform p30 of 59 kDa 2′–5′-oligoadenylate synthase-like protein 1.915
13 IPI00216138 TAGLN Transgelin 1.904
14 IPI00829741 ANKLE2 Isoform 1 of ankyrin repeat and LEM domain-containing protein 2 1.872
15 IPI00022145 NUCKS1 Isoform 1 of nuclear ubiquitous casein and cyclin-dependent kinases substrate 1.771
16 IPI00290979 ABHD5 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 1.761
17 IPI00554777 ASNS Asparagine synthetase (glutamine-hydrolyzing) 1.664
18 IPI00021978 PEX11B Peroxisomal membrane protein 11B 1.661
19 IPI00328737 ZNF598 Isoform 1 of Zinc finger protein 598 1.644
20 IPI00101782 GMPPA Isoform 1 of mannose-1-phosphate guanyltransferase α 1.641
21 IPI00007320 TCF25 Transcription factor 25 1.627
22 IPI00031651 C7orf50 Uncharacterized protein C7orf50 1.623
23 IPI00247583 RPL21 60S ribosomal protein L21 1.622
24 IPI00329596 TMX2 Uncharacterized protein 1.613
25 IPI00740961 INTS1 DKFZP586J0619 protein 1.568
26 IPI00102752 RBM15 Isoform 1 of putative RNA-binding protein 15 1.544
27 IPI00024650 SLC16A1 Monocarboxylate transporter 1 1.539
28 IPI00026848 LRPAP1 α-2-macroglobulin receptor-associated protein 1.534
29 IPI00019976 CCDC85B Coiled-coil domain-containing protein 85B 1.52
30 IPI00021831 PRKAR1A cAMP-dependent protein kinase type I-α regulatory subunit 1.485
31 IPI00145260 IBA57 Putative transferase CAF17, mitochondrial 1.474
32 IPI00550021 RPL3 60S ribosomal protein L3 1.467
33 IPI00853369 PLXNB2 Plexin-B2 1.465
34 IPI00001734 PSAT1 Phosphoserine aminotransferase 1.463
35 IPI00412607 RPL35 60S ribosomal protein L35 1.449
36 IPI00027463 S100A6 Protein S100-A6 1.447
37 IPI00012772 RPL8 60S ribosomal protein L8 1.431
38 IPI00299219 CYR61 Protein CYR61 1.425
39 IPI00006362 EDF1 Isoform 2 of endothelial differentiation-related factor 1 1.423
40 IPI00007309 TIMM23 Mitochondrial import inner membrane translocase subunit Tim23 1.422
41 IPI00030362 PLP2 Isoform 1 of proteolipid protein 2 1.421
42 IPI00219840 AP2S1 Isoform 1 of AP-2 complex subunit sigma 1.42
43 IPI00980827 Unknown Uncharacterized protein 1.419
44 IPI00296526 NAGK N-acetyl-D-glucosamine kinase 1.417
45 IPI00465361 RPL13 60S ribosomal protein L13 1.416
46 IPI00306749 SLC4A1AP Kanadaptin 1.409
47 IPI00168262 GLT25D1 Procollagen galactosyltransferase 1 1.404
48 IPI00410067 ZC3HAV1 Isoform 1 of Zinc finger CCCH-type antiviral protein 1 1.397
49 IPI00789805 DIAPH3 Isoform 3 of protein diaphanous homolog 3 1.395
50 IPI00293425 FXN Isoform 1 of frataxin, mitochondrial 1.394
51 IPI00021389 CCS Copper chaperone for superoxide dismutase 1.376
52 IPI00027096 MRPL19 39S ribosomal protein L19, mitochondrial 1.376
53 IPI00005040 ACADM Isoform 1 of medium-chain specific acyl-CoA dehydrogenase, mitochondrial 1.376
54 IPI00556655 LAMP1 LAMP1 protein variant (fragment) 1.375
55 IPI00101968 DBNL Isoform 3 of drebrin-like protein 1.37
56 IPI00008418 DIABLO Diablo homolog, mitochondrial precursor 1.36
57 IPI00396174 CCDC25 Coiled-coil domain-containing protein 25 1.357
58 IPI00010404 SF3B5 Splicing factor 3B subunit 5 1.356
59 IPI00216999 C14orf21 Pumilio domain-containing protein C14orf21 1.354
60 IPI00293276 MIF Macrophage migration inhibitory factor 1.352
61 IPI00297241 URB1 Nucleolar pre-ribosomal-associated protein 1 1.351
62 IPI00004406 UPP1 Isoform 1 of uridine phosphorylase 1 1.347
63 IPI00023122 PDLIM7 Isoform 1 of PDZ and LIM domain protein 7 1.346
64 IPI00375731 RBM10 Isoform 1 of RNA-binding protein 10 1.345
65 IPI00170786 WBP11 WW domain-binding protein 11 1.345
66 IPI00010740 SFPQ Isoform long of splicing factor, proline- and glutamine-rich 1.343
67 IPI00329321 LYRM7 LYR motif-containing protein 7 1.341
68 IPI00290857 KRT3 Keratin, type II cytoskeletal 3 1.34
69 IPI00456758 RPL27A 60S ribosomal protein L27a 1.339
70 IPI00063673 ISY1 Isoform 1 of pre-mRNA-splicing factor ISY1 homolog 1.339
71 IPI00217236 TBCA Tubulin-specific chaperone A 1.339
72 IPI00301280 TMEM43 Transmembrane protein 43 1.337
73 IPI00006079 BCLAF1 Isoform 1 of Bcl-2-associated transcription factor 1 1.336
74 IPI00746351 DIS3 Isoform 1 of exosome complex exonuclease RRP44 1.33
75 IPI00180781 MLKL Isoform 1 of mixed lineage kinase domain-like protein 1.328
76 IPI00012493 RPS20 40S ribosomal protein S20 1.321
77 IPI00008922 IFITM2 Interferon-induced transmembrane protein 2 1.32
78 IPI00647650 EIF3H Eukaryotic translation initiation factor 3 subunit 3 1.317
79 IPI00299573 RPL7A 60S ribosomal protein L7a 1.317
80 IPI01012991 LUC7L2 Isoform 1 of putative RNA-binding protein Luc7-like 2 1.315
81 IPI00300078 PWP2 Periodic tryptophan protein 2 homolog 1.311
82 IPI00003016 STRN4 Striatin-4 1.31
83 IPI00745955 EBNA1BP2 Probable rRNA-processing protein EBP2 1.307
84 IPI00220527 SNX1 Isoform 1A of sorting nexin-1 1.305
85 IPI00017448 RPS21 40S ribosomal protein S21 1.3
86 IPI00002135 TACC3 Transforming acidic coiled-coil-containing protein 3 1.296
87 IPI00011635 BCL2L13 Isoform 2 of Bcl-2-like protein 13 1.291
88 IPI00157176 MEA1 Male-enhanced antigen 1 1.288
89 IPI00012756 IFIT5 Interferon-induced protein with tetratricopeptide repeats 5 1.287
90 IPI00022202 SLC25A3 Isoform A of phosphate carrier protein, mitochondrial 1.287
91 IPI00064767 ARHGAP17 Isoform 1 of Rho GTPase-activating protein 17 1.284
92 IPI00063827 ABHD14B Isoform 1 of abhydrolase domain-containing protein 14B 1.282
93 IPI00243221 NRD1 Nardilysin isoform a 1.274
94 IPI00297160 CD44 Isoform 12 of CD44 antigen 1.272
95 IPI00014808 PAFAH1B3 Platelet-activating factor acetylhydrolase IB subunit γ 1.269
96 IPI00022694 PSMD4 Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 4 1.264
97 IPI00011229 CTSD Cathepsin D 1.261
98 IPI00003856 ATP6V1E1 V-type proton ATPase subunit E 1 1.255
99 IPI00291669 UBLCP1 Ubiquitin-like domain-containing CTD phosphatase 1 1.252
100 IPI00029081 LIG3 Isoform α of DNA ligase 3 1.248
101 IPI00007797 FABP5 Fatty acid-binding protein, epidermal 1.248
102 IPI00028387 DDRGK1 Isoform 1 of DDRGK domain-containing protein 1 1.245
103 IPI00007694 PPME1 Isoform 1 of protein phosphatase methylesterase 1 1.243
104 IPI00031519 DNMT1 Isoform 1 of DNA (cytosine-5)-methyltransferase 1 1.241
105 IPI00219684 FABP3 Fatty acid-binding protein, heart 1.24
106 IPI00219153 RPL22 60S ribosomal protein L22 1.237
107 IPI00219029 GOT1 Aspartate aminotransferase, cytoplasmic 1.236
108 IPI00297178 DHX16 119 kDa protein 1.234
109 IPI00305267 GOLGA3 Isoform 1 of golgin subfamily A member 3 1.234
110 IPI00010697 ITGA6 Isoform α-6X1X2B of integrin α-6 1.233
111 IPI00396387 GNL1 Isoform 1 of guanine nucleotide-binding protein-like 1 1.233
112 IPI00000030 PPP2R5D Isoform δ-1 of serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit δ isoform 1.233
113 IPI00007118 SERPINE1 Plasminogen activator inhibitor 1 1.23
114 IPI00021057 SLC12A4 Isoform 1 of solute carrier family 12 member 4 1.229
115 IPI00018206 GOT2 Aspartate aminotransferase, mitochondrial 1.229
116 IPI00019472 SLC1A5 Neutral amino acid transporter B(0) 1.229
117 IPI00306353 DUSP23 Dual specificity protein phosphatase 23 1.227
118 IPI00032825 TMED7 Transmembrane emp24 domain-containing protein 7 1.227
119 IPI01010270 WIZ WIZ protein 1.225
120 IPI00006980 C14orf166 UPF0568 protein C14orf166 1.224
121 IPI00298625 LYN Isoform LYN A of tyrosine-protein kinase Lyn 1.223
122 IPI00550523 ATL3 Atlastin-3 1.222
123 IPI00333215 TCEA1 Isoform 1 of transcription elongation factor A protein 1 1.222
124 IPI00032038 CPT1A Isoform 1 of carnitine O-palmitoyltransferase 1, liver isoform 1.221
125 IPI00025512 HSPB1 Heat shock protein β-1 1.221
126 IPI00013468 BUB3 Isoform 1 of mitotic checkpoint protein BUB3 1.221
127 IPI00307165 TRIM47 Tripartite motif-containing protein 47 1.22
128 IPI00181396 VPRBP Isoform 3 of protein VPRBP 1.217
129 IPI00216237 RPL36 60S ribosomal protein L36 1.217
130 IPI00550069 RNH1 Ribonuclease inhibitor 1.217
131 IPI00215995 ITGA3 Isoform 1 of integrin α-3 1.216
132 IPI00902512 PPP1CA Serine/threonine-protein phosphatase 1.215
133 IPI00003923 UMPS Isoform 1 of uridine 5′-monophosphate synthase 1.21
134 IPI00010414 PDLIM1 PDZ and LIM domain protein 1 1.21
135 IPI01026021 SHMT2 SHMT2 protein 1.21
136 IPI00021828 CSTB Cystatin-B 1.21
137 IPI00060627 CCDC124 Coiled-coil domain-containing protein 124 1.209
138 IPI00218466 SEC61A1 Protein transport protein Sec61 subunit α isoform 1 1.207
139 IPI00026546 PAFAH1B2 Platelet-activating factor acetylhydrolase IB subunit β 1.207
140 IPI00011592 DYNC1LI2 Cytoplasmic dynein 1 light intermediate chain 2 1.206
141 IPI00216682 CNN3 Calponin-3 1.206
142 IPI00297900 DDX10 Probable ATP-dependent RNA helicase DDX10 1.206
143 IPI00292657 PTGR1 Prostaglandin reductase 1 1.204
144 IPI00925046 QARS Glutaminyl-tRNA synthetase 1.204
145 IPI00339379 ARHGEF1 Isoform 2 of Rho guanine nucleotide exchange factor 1 1.202
146 IPI00294486 DUSP9 Dual specificity protein phosphatase 9 1.201
147 IPI00159899 ANKFY1 Isoform 1 of ankyrin repeat and FYVE domain-containing protein 1 1.201

Downregulated proteins in response to β-elemene treatment in SGC7901 gastric cancer cells.

No. Accession Gene symbol Protein name Fold-ratio (115:114)
1 IPI00654755 HBB Hemoglobin subunit β 0.163
2 IPI00290380 ALPPL2 Alkaline phosphatase, placental-like 0.531
3 IPI00183695 S100A10 Protein S100-A10 0.598
4 IPI00419273 CUL4A Isoform 1 of cullin-4A 0.631
5 IPI00410110 DHX40 Isoform 1 of probable ATP-dependent RNA helicase DHX40 0.637
6 IPI00011698 SAP18 Histone deacetylase complex subunit SAP18 0.654
7 IPI00022002 MRPS27 Mitochondrial 28S ribosomal protein S27 0.664
8 IPI00171438 TXNDC5 Thioredoxin domain-containing protein 5 0.675
9 IPI00023704 LPP Lipoma-preferred partner 0.701
10 IPI00301503 TRA2B Isoform 1 of transformer-2 protein homolog β 0.706
11 IPI00414836 OSTF1 Osteoclast-stimulating factor 1 0.706
12 IPI00019896 MYCBP2 Homo sapiens protein associated with Myc mRNA (fragment) 0.712
13 IPI00414101 TOP2A Isoform 2 of DNA topoisomerase 2-α 0.716
14 IPI00412545 NDUFA5 NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 5 0.717
15 IPI00003935 HIST2H2BE Histone H2B type 2-E 0.718
16 IPI00291467 SLC25A6 ADP/ATP translocase 3 0.722
17 IPI00374272 C5orf51 UPF0600 protein C5orf51 0.725
18 IPI00005087 TMOD3 Tropomodulin-3 0.732
19 IPI00008475 HMGCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic 0.736
20 IPI00017342 RHOG Rho-related GTP-binding protein RhoG 0.742
21 IPI00009901 NUTF2 Nuclear transport factor 2 0.745
22 IPI00298731 PPP1R10 Serine/threonine-protein phosphatase 1 regulatory subunit 10 0.746
23 IPI00020602 CSNK2A2 Casein kinase II subunit α 0.753
24 IPI00025347 EMG1 Ribosomal RNA small subunit methyltransferase NEP1 0.755
25 IPI00218962 C20orf43 UPF0549 protein C20orf43 0.755
26 IPI00014230 C1QBP Complement component 1 Q subcomponent-binding protein, mitochondrial 0.755
27 IPI00219025 GLRX Glutaredoxin-1 0.757
28 IPI00168479 APOA1BP Apolipoprotein A-I binding protein 0.758
29 IPI00027705 PRIM2 Isoform 1 of DNA primase large subunit 0.758
30 IPI00219483 SNRNP70 Isoform 2 of U1 small nuclear ribonucleoprotein 70 kDa 0.765
31 IPI00220014 IDI1 Isoform 2 of isopentenyl-diphosphate δ-isomerase 1 0.766
32 IPI00434580 MYOM1 Isoform 1 of myomesin-1 0.771
33 IPI00023729 FN3K Fructosamine-3-kinase 0.773
34 IPI00013446 PSCA Prostate stem cell antigen 0.78
35 IPI00924816 MTPN Myotrophin 0.781
36 IPI00033022 DNM2 Isoform 1 of dynamin-2 0.781
37 IPI00009032 SSB Lupus la protein 0.781
38 IPI00073779 MRPS35 Isoform 1 of 28S ribosomal protein S35, mitochondrial 0.782
39 IPI00163644 OSBPL8 Oxysterol-binding protein 0.783
40 IPI00419626 MRPL55 Isoform 2 of 39S ribosomal protein L55, mitochondrial 0.789
41 IPI00006440 MRPS7 28S ribosomal protein S7, mitochondrial 0.79
42 IPI00027704 PRIM1 DNA primase small subunit 0.792
43 IPI00018768 TSN Translin 0.795
44 IPI00876962 INF2 Isoform 2 of inverted formin-2 0.795
45 IPI00156374 IPO4 Isoform 1 of importin-4 0.795
46 IPI00017344 RAB5B Ras-related protein Rab-5B 0.799
47 IPI00418290 MRPL14 39S ribosomal protein L14, mitochondrial 0.8
48 IPI00022820 GTF2B Transcription initiation factor IIB 0.801
49 IPI00006579 COX4I1 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial 0.801
50 IPI00217766 SCARB2 Lysosome membrane protein 2 0.801
51 IPI00013396 SNRPC U1 small nuclear ribonucleoprotein C 0.802
52 IPI00022977 CKB Creatine kinase B-type 0.804
53 IPI00012074 HNRNPR Isoform 1 of heterogeneous nuclear ribonucleoprotein R 0.808
54 IPI00216171 ENO2 γ-enolase 0.809
55 IPI00014958 PON2 Isoform 1 of serum paraoxonase/arylesterase 2 0.812
56 IPI00018288 POLR2C DNA-directed RNA polymerase II subunit RPB3 0.812
57 IPI00010948 TRIM26 Tripartite motif-containing protein 26 0.812
58 IPI00007052 FIS1 Mitochondrial fission 1 protein 0.813
59 IPI00329625 TBRG4 Transforming growth factor β regulator 4 0.813
60 IPI00017510 MT-CO2 Cytochrome c oxidase subunit 2 0.814
61 IPI00645898 XPNPEP1 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble 0.814
62 IPI00029054 NT5C2 Cytosolic purine 5′-nucleotidase 0.816
63 IPI00374970 SEPT10 Isoform 1 of septin-10 0.817
64 IPI00005948 MRI1 Isoform 1 of methylthioribose-1-phosphate isomerase 0.817
65 IPI00017526 S100P Protein S100-P 0.817
66 IPI00026964 UQCRFS1 Cytochrome b-c1 complex subunit Rieske, mitochondrial 0.817
67 IPI00219673 GSTK1 Isoform 1 of glutathione S-transferase κ 1 0.818
68 IPI00296432 IWS1 Isoform 1 of protein IWS1 homolog 0.818
69 IPI00029697 EXOSC9 Isoform 2 of exosome complex component RRP45 0.82
70 IPI00062336 RPRD1A Isoform 2 of regulation of nuclear pre-mRNA domain-containing protein 1A 0.822
71 IPI00179172 PPFIBP1 Isoform 2 of liprin-β-1 0.822
72 IPI00015972 COX6C Cytochrome c oxidase subunit 6C 0.823
73 IPI00410657 RNMT Isoform 2 of mRNA cap guanine-N7 methyltransferase 0.823
74 IPI00029019 UBAP2L Isoform 2 of ubiquitin-associated protein 2-like 0.825
75 IPI00007188 SLC25A5 ADP/ATP translocase 2 0.826
76 IPI00292056 PIK3C2B Phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing subunit β 0.828
77 IPI00013475 TUBB2A Tubulin β-2A chain 0.829
78 IPI00293590 MGLL Monoglyceride lipase isoform 1 0.829
79 IPI00303568 PTGES2 Prostaglandin E synthase 2 0.829
80 IPI00027444 SERPINB1 Leukocyte elastase inhibitor 0.829
81 IPI00003765 CAPN7 Calpain-7 0.83
82 IPI00215920 ARF6 ADP-ribosylation factor 6 0.831
83 IPI00008449 FIP1L1 Isoform 3 of pre-mRNA 3′-end-processing factor FIP1 0.831
84 IPI00061178 RBMXL1 Heterogeneous nuclear ribonucleoprotein G-like 1 0.831
85 IPI00180292 BAIAP2 Isoform 5 of brain-specific angiogenesis inhibitor 1-associated protein 2 0.832
86 IPI00029468 ACTR1A α-centractin 0.833