Anticancer effects of β-elemene in gastric cancer cells and its potential underlying proteins: A proteomic study

  • Authors:
    • Jun-Song Liu
    • Shi-Cai He
    • Zheng-Liang Zhang
    • Rui Chen
    • Lin Fan
    • Guang‑Lin Qiu
    • Shuai Chang
    • Liang Li
    • Xiang-Ming Che
  • View Affiliations

  • Published online on: September 17, 2014     https://doi.org/10.3892/or.2014.3490
  • Pages: 2635-2647
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

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.

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 (25). 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 (1416). 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.

Table I

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

Table I

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

No.AccessionGene symbolProtein nameFold-ratio (115:114)
1IPI00549540PAK1IP1p21-activated protein kinase-interacting protein 13.967
2IPI00152429SPNS1Isoform 3 of protein spinster homolog 12.979
3IPI00021885FGAIsoform 1 of fibrinogen α chain2.397
4IPI00011200PHGDH D-3-phosphoglycerate dehydrogenase2.319
5IPI00006213PCM1Isoform 1 of pericentriolar material 1 protein2.316
6IPI00418426CNNM4Metal transporter CNNM42.313
7IPI00013809GSTZ1Isoform 1 of maleylacetoacetate isomerase2.292
8IPI00012433F8AFactor VIII intron 22 protein2.172
9IPI00015856DNPEPAspartyl aminopeptidase2.144
10IPI00002564XRCC1DNA repair protein XRCC11.981
11IPI00909584HARS2Histidyl-tRNA synthetase homolog1.922
12IPI00218116OASLIsoform p30 of 59 kDa 2′–5′-oligoadenylate synthase-like protein1.915
13IPI00216138TAGLNTransgelin1.904
14IPI00829741ANKLE2Isoform 1 of ankyrin repeat and LEM domain-containing protein 21.872
15IPI00022145NUCKS1Isoform 1 of nuclear ubiquitous casein and cyclin-dependent kinases substrate1.771
16IPI00290979ABHD5 1-acylglycerol-3-phosphate O-acyltransferase ABHD51.761
17IPI00554777ASNSAsparagine synthetase (glutamine-hydrolyzing)1.664
18IPI00021978PEX11BPeroxisomal membrane protein 11B1.661
19IPI00328737ZNF598Isoform 1 of Zinc finger protein 5981.644
20IPI00101782GMPPAIsoform 1 of mannose-1-phosphate guanyltransferase α1.641
21IPI00007320TCF25Transcription factor 251.627
22IPI00031651C7orf50Uncharacterized protein C7orf501.623
23IPI00247583RPL2160S ribosomal protein L211.622
24IPI00329596TMX2Uncharacterized protein1.613
25IPI00740961INTS1DKFZP586J0619 protein1.568
26IPI00102752RBM15Isoform 1 of putative RNA-binding protein 151.544
27IPI00024650SLC16A1Monocarboxylate transporter 11.539
28IPI00026848LRPAP1α-2-macroglobulin receptor-associated protein1.534
29IPI00019976CCDC85BCoiled-coil domain-containing protein 85B1.52
30IPI00021831PRKAR1AcAMP-dependent protein kinase type I-α regulatory subunit1.485
31IPI00145260IBA57Putative transferase CAF17, mitochondrial1.474
32IPI00550021RPL360S ribosomal protein L31.467
33IPI00853369PLXNB2Plexin-B21.465
34IPI00001734PSAT1Phosphoserine aminotransferase1.463
35IPI00412607RPL3560S ribosomal protein L351.449
36IPI00027463S100A6Protein S100-A61.447
37IPI00012772RPL860S ribosomal protein L81.431
38IPI00299219CYR61Protein CYR611.425
39IPI00006362EDF1Isoform 2 of endothelial differentiation-related factor 11.423
40IPI00007309TIMM23Mitochondrial import inner membrane translocase subunit Tim231.422
41IPI00030362PLP2Isoform 1 of proteolipid protein 21.421
42IPI00219840AP2S1Isoform 1 of AP-2 complex subunit sigma1.42
43IPI00980827UnknownUncharacterized protein1.419
44IPI00296526NAGK N-acetyl-D-glucosamine kinase1.417
45IPI00465361RPL1360S ribosomal protein L131.416
46IPI00306749SLC4A1APKanadaptin1.409
47IPI00168262GLT25D1Procollagen galactosyltransferase 11.404
48IPI00410067ZC3HAV1Isoform 1 of Zinc finger CCCH-type antiviral protein 11.397
49IPI00789805DIAPH3Isoform 3 of protein diaphanous homolog 31.395
50IPI00293425FXNIsoform 1 of frataxin, mitochondrial1.394
51IPI00021389CCSCopper chaperone for superoxide dismutase1.376
52IPI00027096MRPL1939S ribosomal protein L19, mitochondrial1.376
53IPI00005040ACADMIsoform 1 of medium-chain specific acyl-CoA dehydrogenase, mitochondrial1.376
54IPI00556655LAMP1LAMP1 protein variant (fragment)1.375
55IPI00101968DBNLIsoform 3 of drebrin-like protein1.37
56IPI00008418DIABLODiablo homolog, mitochondrial precursor1.36
57IPI00396174CCDC25Coiled-coil domain-containing protein 251.357
58IPI00010404SF3B5Splicing factor 3B subunit 51.356
59IPI00216999C14orf21Pumilio domain-containing protein C14orf211.354
60IPI00293276MIFMacrophage migration inhibitory factor1.352
61IPI00297241URB1Nucleolar pre-ribosomal-associated protein 11.351
62IPI00004406UPP1Isoform 1 of uridine phosphorylase 11.347
63IPI00023122PDLIM7Isoform 1 of PDZ and LIM domain protein 71.346
64IPI00375731RBM10Isoform 1 of RNA-binding protein 101.345
65IPI00170786WBP11WW domain-binding protein 111.345
66IPI00010740SFPQIsoform long of splicing factor, proline- and glutamine-rich1.343
67IPI00329321LYRM7LYR motif-containing protein 71.341
68IPI00290857KRT3Keratin, type II cytoskeletal 31.34
69IPI00456758RPL27A60S ribosomal protein L27a1.339
70IPI00063673ISY1Isoform 1 of pre-mRNA-splicing factor ISY1 homolog1.339
71IPI00217236TBCATubulin-specific chaperone A1.339
72IPI00301280TMEM43Transmembrane protein 431.337
73IPI00006079BCLAF1Isoform 1 of Bcl-2-associated transcription factor 11.336
74IPI00746351DIS3Isoform 1 of exosome complex exonuclease RRP441.33
75IPI00180781MLKLIsoform 1 of mixed lineage kinase domain-like protein1.328
76IPI00012493RPS2040S ribosomal protein S201.321
77IPI00008922IFITM2Interferon-induced transmembrane protein 21.32
78IPI00647650EIF3HEukaryotic translation initiation factor 3 subunit 31.317
79IPI00299573RPL7A60S ribosomal protein L7a1.317
80IPI01012991LUC7L2Isoform 1 of putative RNA-binding protein Luc7-like 21.315
81IPI00300078PWP2Periodic tryptophan protein 2 homolog1.311
82IPI00003016STRN4Striatin-41.31
83IPI00745955EBNA1BP2Probable rRNA-processing protein EBP21.307
84IPI00220527SNX1Isoform 1A of sorting nexin-11.305
85IPI00017448RPS2140S ribosomal protein S211.3
86IPI00002135TACC3Transforming acidic coiled-coil-containing protein 31.296
87IPI00011635BCL2L13Isoform 2 of Bcl-2-like protein 131.291
88IPI00157176MEA1Male-enhanced antigen 11.288
89IPI00012756IFIT5Interferon-induced protein with tetratricopeptide repeats 51.287
90IPI00022202SLC25A3Isoform A of phosphate carrier protein, mitochondrial1.287
91IPI00064767ARHGAP17Isoform 1 of Rho GTPase-activating protein 171.284
92IPI00063827ABHD14BIsoform 1 of abhydrolase domain-containing protein 14B1.282
93IPI00243221NRD1Nardilysin isoform a1.274
94IPI00297160CD44Isoform 12 of CD44 antigen1.272
95IPI00014808PAFAH1B3Platelet-activating factor acetylhydrolase IB subunit γ1.269
96IPI00022694PSMD4Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 41.264
97IPI00011229CTSDCathepsin D1.261
98IPI00003856ATP6V1E1V-type proton ATPase subunit E 11.255
99IPI00291669UBLCP1Ubiquitin-like domain-containing CTD phosphatase 11.252
100IPI00029081LIG3Isoform α of DNA ligase 31.248
101IPI00007797FABP5Fatty acid-binding protein, epidermal1.248
102IPI00028387DDRGK1Isoform 1 of DDRGK domain-containing protein 11.245
103IPI00007694PPME1Isoform 1 of protein phosphatase methylesterase 11.243
104IPI00031519DNMT1Isoform 1 of DNA (cytosine-5)-methyltransferase 11.241
105IPI00219684FABP3Fatty acid-binding protein, heart1.24
106IPI00219153RPL2260S ribosomal protein L221.237
107IPI00219029GOT1Aspartate aminotransferase, cytoplasmic1.236
108IPI00297178DHX16119 kDa protein1.234
109IPI00305267GOLGA3Isoform 1 of golgin subfamily A member 31.234
110IPI00010697ITGA6Isoform α-6X1X2B of integrin α-61.233
111IPI00396387GNL1Isoform 1 of guanine nucleotide-binding protein-like 11.233
112IPI00000030PPP2R5DIsoform δ-1 of serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit δ isoform1.233
113IPI00007118SERPINE1Plasminogen activator inhibitor 11.23
114IPI00021057SLC12A4Isoform 1 of solute carrier family 12 member 41.229
115IPI00018206GOT2Aspartate aminotransferase, mitochondrial1.229
116IPI00019472SLC1A5Neutral amino acid transporter B(0)1.229
117IPI00306353DUSP23Dual specificity protein phosphatase 231.227
118IPI00032825TMED7Transmembrane emp24 domain-containing protein 71.227
119IPI01010270WIZWIZ protein1.225
120IPI00006980C14orf166UPF0568 protein C14orf1661.224
121IPI00298625LYNIsoform LYN A of tyrosine-protein kinase Lyn1.223
122IPI00550523ATL3Atlastin-31.222
123IPI00333215TCEA1Isoform 1 of transcription elongation factor A protein 11.222
124IPI00032038CPT1AIsoform 1 of carnitine O-palmitoyltransferase 1, liver isoform1.221
125IPI00025512HSPB1Heat shock protein β-11.221
126IPI00013468BUB3Isoform 1 of mitotic checkpoint protein BUB31.221
127IPI00307165TRIM47Tripartite motif-containing protein 471.22
128IPI00181396VPRBPIsoform 3 of protein VPRBP1.217
129IPI00216237RPL3660S ribosomal protein L361.217
130IPI00550069RNH1Ribonuclease inhibitor1.217
131IPI00215995ITGA3Isoform 1 of integrin α-31.216
132IPI00902512PPP1CA Serine/threonine-protein phosphatase1.215
133IPI00003923UMPSIsoform 1 of uridine 5′-monophosphate synthase1.21
134IPI00010414PDLIM1PDZ and LIM domain protein 11.21
135IPI01026021SHMT2SHMT2 protein1.21
136IPI00021828CSTBCystatin-B1.21
137IPI00060627CCDC124Coiled-coil domain-containing protein 1241.209
138IPI00218466SEC61A1Protein transport protein Sec61 subunit α isoform 11.207
139IPI00026546PAFAH1B2Platelet-activating factor acetylhydrolase IB subunit β1.207
140IPI00011592DYNC1LI2Cytoplasmic dynein 1 light intermediate chain 21.206
141IPI00216682CNN3Calponin-31.206
142IPI00297900DDX10Probable ATP-dependent RNA helicase DDX101.206
143IPI00292657PTGR1Prostaglandin reductase 11.204
144IPI00925046QARSGlutaminyl-tRNA synthetase1.204
145IPI00339379ARHGEF1Isoform 2 of Rho guanine nucleotide exchange factor 11.202
146IPI00294486DUSP9Dual specificity protein phosphatase 91.201
147IPI00159899ANKFY1Isoform 1 of ankyrin repeat and FYVE domain-containing protein 11.201

Table II

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

Table II

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

No.AccessionGene symbolProtein nameFold-ratio (115:114)
1IPI00654755HBBHemoglobin subunit β0.163
2IPI00290380ALPPL2Alkaline phosphatase, placental-like0.531
3IPI00183695S100A10Protein S100-A100.598
4IPI00419273CUL4AIsoform 1 of cullin-4A0.631
5IPI00410110DHX40Isoform 1 of probable ATP-dependent RNA helicase DHX400.637
6IPI00011698SAP18Histone deacetylase complex subunit SAP180.654
7IPI00022002MRPS27Mitochondrial 28S ribosomal protein S270.664
8IPI00171438TXNDC5Thioredoxin domain-containing protein 50.675
9IPI00023704LPPLipoma-preferred partner0.701
10IPI00301503TRA2BIsoform 1 of transformer-2 protein homolog β0.706
11IPI00414836OSTF1 Osteoclast-stimulating factor 10.706
12IPI00019896MYCBP2Homo sapiens protein associated with Myc mRNA (fragment)0.712
13IPI00414101TOP2AIsoform 2 of DNA topoisomerase 2-α0.716
14IPI00412545NDUFA5NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 50.717
15IPI00003935HIST2H2BEHistone H2B type 2-E0.718
16IPI00291467SLC25A6ADP/ATP translocase 30.722
17IPI00374272C5orf51UPF0600 protein C5orf510.725
18IPI00005087TMOD3Tropomodulin-30.732
19IPI00008475HMGCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic0.736
20IPI00017342RHOGRho-related GTP-binding protein RhoG0.742
21IPI00009901NUTF2Nuclear transport factor 20.745
22IPI00298731PPP1R10 Serine/threonine-protein phosphatase 1 regulatory subunit 100.746
23IPI00020602CSNK2A2Casein kinase II subunit α0.753
24IPI00025347EMG1Ribosomal RNA small subunit methyltransferase NEP10.755
25IPI00218962C20orf43UPF0549 protein C20orf430.755
26IPI00014230C1QBPComplement component 1 Q subcomponent-binding protein, mitochondrial0.755
27IPI00219025GLRXGlutaredoxin-10.757
28IPI00168479APOA1BPApolipoprotein A-I binding protein0.758
29IPI00027705PRIM2Isoform 1 of DNA primase large subunit0.758
30IPI00219483SNRNP70Isoform 2 of U1 small nuclear ribonucleoprotein 70 kDa0.765
31IPI00220014IDI1Isoform 2 of isopentenyl-diphosphate δ-isomerase 10.766
32IPI00434580MYOM1Isoform 1 of myomesin-10.771
33IPI00023729FN3K Fructosamine-3-kinase0.773
34IPI00013446PSCAProstate stem cell antigen0.78
35IPI00924816MTPNMyotrophin0.781
36IPI00033022DNM2Isoform 1 of dynamin-20.781
37IPI00009032SSBLupus la protein0.781
38IPI00073779MRPS35Isoform 1 of 28S ribosomal protein S35, mitochondrial0.782
39IPI00163644OSBPL8Oxysterol-binding protein0.783
40IPI00419626MRPL55Isoform 2 of 39S ribosomal protein L55, mitochondrial0.789
41IPI00006440MRPS728S ribosomal protein S7, mitochondrial0.79
42IPI00027704PRIM1DNA primase small subunit0.792
43IPI00018768TSNTranslin0.795
44IPI00876962INF2Isoform 2 of inverted formin-20.795
45IPI00156374IPO4Isoform 1 of importin-40.795
46IPI00017344RAB5BRas-related protein Rab-5B0.799
47IPI00418290MRPL1439S ribosomal protein L14, mitochondrial0.8
48IPI00022820GTF2BTranscription initiation factor IIB0.801
49IPI00006579COX4I1Cytochrome c oxidase subunit 4 isoform 1, mitochondrial0.801
50IPI00217766SCARB2Lysosome membrane protein 20.801
51IPI00013396SNRPCU1 small nuclear ribonucleoprotein C0.802
52IPI00022977CKBCreatine kinase B-type0.804
53IPI00012074HNRNPRIsoform 1 of heterogeneous nuclear ribonucleoprotein R0.808
54IPI00216171ENO2γ-enolase0.809
55IPI00014958PON2Isoform 1 of serum paraoxonase/arylesterase 20.812
56IPI00018288POLR2CDNA-directed RNA polymerase II subunit RPB30.812
57IPI00010948TRIM26Tripartite motif-containing protein 260.812
58IPI00007052FIS1Mitochondrial fission 1 protein0.813
59IPI00329625TBRG4Transforming growth factor β regulator 40.813
60IPI00017510MT-CO2Cytochrome c oxidase subunit 20.814
61IPI00645898XPNPEP1X-prolyl aminopeptidase (aminopeptidase P) 1, soluble0.814
62IPI00029054NT5C2Cytosolic purine 5′-nucleotidase0.816
63IPI00374970SEPT10Isoform 1 of septin-100.817
64IPI00005948MRI1Isoform 1 of methylthioribose-1-phosphate isomerase0.817
65IPI00017526S100PProtein S100-P0.817
66IPI00026964UQCRFS1Cytochrome b-c1 complex subunit Rieske, mitochondrial0.817
67IPI00219673GSTK1Isoform 1 of glutathione S-transferase κ 10.818
68IPI00296432IWS1Isoform 1 of protein IWS1 homolog0.818
69IPI00029697EXOSC9Isoform 2 of exosome complex component RRP450.82
70IPI00062336RPRD1AIsoform 2 of regulation of nuclear pre-mRNA domain-containing protein 1A0.822
71IPI00179172PPFIBP1Isoform 2 of liprin-β-10.822
72IPI00015972COX6CCytochrome c oxidase subunit 6C0.823
73IPI00410657RNMTIsoform 2 of mRNA cap guanine-N7 methyltransferase0.823
74IPI00029019UBAP2LIsoform 2 of ubiquitin-associated protein 2-like0.825
75IPI00007188SLC25A5ADP/ATP translocase 20.826
76IPI00292056PIK3C2B Phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing subunit β0.828
77IPI00013475TUBB2ATubulin β-2A chain0.829
78IPI00293590MGLLMonoglyceride lipase isoform 10.829
79IPI00303568PTGES2Prostaglandin E synthase 20.829
80IPI00027444SERPINB1Leukocyte elastase inhibitor0.829
81IPI00003765CAPN7Calpain-70.83
82IPI00215920ARF6ADP-ribosylation factor 60.831
83IPI00008449FIP1L1Isoform 3 of pre-mRNA 3′-end-processing factor FIP10.831
84IPI00061178RBMXL1Heterogeneous nuclear ribonucleoprotein G-like 10.831
85IPI00180292BAIAP2Isoform 5 of brain-specific angiogenesis inhibitor 1-associated protein 20.832
86IPI00029468ACTR1Aα-centractin0.833
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 (2527). 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 (4447). 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 (4648). 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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December 2014
Volume 32 Issue 6

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APA
Liu, J., He, S., Zhang, Z., Chen, R., Fan, L., Qiu, G. ... Che, X. (2014). Anticancer effects of β-elemene in gastric cancer cells and its potential underlying proteins: A proteomic study. Oncology Reports, 32, 2635-2647. https://doi.org/10.3892/or.2014.3490
MLA
Liu, J., He, S., Zhang, Z., Chen, R., Fan, L., Qiu, G., Chang, S., Li, L., Che, X."Anticancer effects of β-elemene in gastric cancer cells and its potential underlying proteins: A proteomic study". Oncology Reports 32.6 (2014): 2635-2647.
Chicago
Liu, J., He, S., Zhang, Z., Chen, R., Fan, L., Qiu, G., Chang, S., Li, L., Che, X."Anticancer effects of β-elemene in gastric cancer cells and its potential underlying proteins: A proteomic study". Oncology Reports 32, no. 6 (2014): 2635-2647. https://doi.org/10.3892/or.2014.3490