Anticancer effects of β-elemene in gastric cancer cells and its potential underlying proteins: A proteomic study
- Authors:
- Published online on: September 17, 2014 https://doi.org/10.3892/or.2014.3490
- Pages: 2635-2647
Abstract
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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