- Research article
- Open Access
Integrating transcriptomics and proteomics to show that tanshinone IIA suppresses cell growth by blocking glucose metabolism in gastric cancer cells
© Lin et al.; licensee BioMed Central. 2015
- Received: 2 July 2014
- Accepted: 9 January 2015
- Published: 5 February 2015
Tanshinone IIA (TIIA) is a diterpene quinone extracted from the plant Danshen (Salvia miltiorrhiza) used in traditional Chinese herbal medicine. It has been reported to have anti-tumor potential against several kinds of cancer, including gastric cancer. In most solid tumors, a metabolic switch to glucose is a hallmark of cancer cells, which do this to provide nutrients for cell proliferation. However, the mechanism associated with glucose metabolism by which TIIA acts on gastric cancer cells remains to be elucidated.
We found that TIIA treatment is able to significantly inhibit cell growth and the proliferation of gastric cancer in a dose-dependent manner. Using next-generation sequencing-based RNA-seq transcriptomics and quantitative proteomics-isobaric tags for relative and absolute quantification (iTRAQ), we characterized the mechanism of TIIA regulation in gastric cancer cell line AGS. In total, 16,603 unique transcripts and 102 proteins were identified. After enrichment analysis, we found that TIIA regulated genes are involved in carbohydrate metabolism, the cell cycle, apoptosis, DNA damage and cytoskeleton reorganization. Our proteomics data revealed the downregulation of intracellular ATP levels, glucose-6-phosphate isomerase and L-lactate dehydrogenase B chains by TIIA, which might work with disorders of glucose metabolism and extracellular lactate levels to suppress cell proliferation. The up-regulation of p53 and down-regulation of AKT was shown in TIIA- treated cells, which indicates the transformation of oncogenes. Severe DNA damage, cell cycle arrest at the G2/M transition and apoptosis with cytoskeleton reorganization were detected in TIIA-treated gastric cancer cells.
Combining transcriptomics and proteomics results, we propose that TIIA treatment could lead cell stresses, including nutrient deficiency and DNA damage, by inhibiting the glucose metabolism of cancer cells. This study provides an insight into how the TIIA regulatory metabolism in gastric cancer cells suppresses cell growth, and may help improve the development of cancer therapy.
- Tanshinone IIA
- Gastric cancer
- Isobaric tags for relative and absolute quantification
- Next generation sequencing
Gastric cancer is one of the most notorious cancers worldwide. It is the fourth most frequently occurring cancer, and the second most common cause of death for both sexes among all cancers, claiming over 736,000 lives worldwide in 2008 . However, anti-cancer drug development of gastric cancer today remains slow and costly, and drug-resistance remains a potential obstacle .
Tanshinone IIA (TIIA) is a diterpene quinone extracted from the plant Danshen (Salvia miltiorrhiza), which is used in traditional Chinese medicine . The first usage of Danshen extractions as herbal medicine can be traced back to more than 1800 years ago . Its applications include prevention of cardiac diseases , protection of the nervous system  and hepatocytes , and inhibition of osteoporosis . Drug repositioning is considered to be a promising and valuable method for the reduction of the side effects and cost of anti-cancer drug research and development. The functions of many chemicals extracted from Danshen are therefore explored for their anti-cancer potential, and TIIA is the most abundant and structurally representative of these . TIIA has been recently reported to have anti-cancer potential against several cancers including breast cancer , prostate cancer , colorectal cancer , lung cancer , liver cancer , leukemia , and gastric cancer . However, the regulatory mechanism of TIIA in gastric cancer cells remains unclear.
Metabolic transformation accompanying nutritional imbalance is one of the leading causes of cancer progression. Glucose is a primary source for the pentose phosphate pathway, which makes RNA and DNA. Glycolytic intermediates can be used to assist lipid biosynthesis to produce ATP and non-essential amino acids, such as alanine, for growth . Proliferating cells are commonly maintained by enhancing aerobic glycolysis, also called the Warburg effect, which causes lactate accumulation and contributes to the development of malignancies. In oncogenic pathways, phosphatidylinositide 3-kinases (PI3K)/protein kinase B (AKT) has been shown to enhance glycolysis, while the tumor suppressor p53 inhibits it, suggesting a glycolytic switch intrinsically associated with oncogenic transformation . Through the inference of oncogene expression and glucose metabolism, the supply of nutrients to cancer cells could be blocked, which would stunt their proliferative potential. This may be an important finding for drug discovery.
High-throughput data can be used to provide a comprehensive inventory of all the biological processes of cells, display their complexity, and increase data accuracy. An accurate picture of the differential expression of experimental samples is important for defining precise targets and networks. Here we use two types of high-throughput data to uncover the regulatory mechanism of TIIA in gastric cancer cells: transcription levels from next-generation sequencing (NGS) data, and isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics analysis. NGS is one of newer transcriptome sequencing approaches and can perform high-throughput sequencing by generating thousands or millions of sequences in parallel . It has better sensitivity and lower background noise than microarray analysis, and more researchers are using it to investigate the mechanisms of anti-cancer drugs [19,20]. iTRAQ is a promising new technique for quantitative proteomics which can quantify proteins from different sources using their different tags . Because of its sensitivity in labeling peptides, it is considered a more powerful quantitative proteomic technique than others, including the two frequently used quantitative proteomics techniques 2D-DIGE (difference gel electrophoresis) and cICAT (cleavable isotope-coded affinity tags) [22,23].
In this study, we identified a total of 16,603 unique transcripts and 102 TIIA-regulated proteins that are involved in glucose metabolic process. We further investigated and found that TIIA blocks glycolysis and gluconeogenesis in cancer cells by altering protein expression, and causes DNA damage, cell cycle arrest, cytoskeleton reorganization and apoptosis.
TIIA reduces the growth rate and suppresses proliferation of gastric cancer AGS cells
To explore whether TIIA could affect gastric cancer cell proliferation rates, we treated AGS cells at different concentrations of TIIA and counted the number of cell colonies stained with crystal violet. The results show that 0.53 μM (1/10 IC50) TIIA reduces colonies to about 60%, whereas 5.3 μM (IC50) TIIA results in almost no colonies. It indicates that the proliferation rate of AGS cells was significantly suppressed by TIIA in a dose-dependent manner (*p < 0.05, **p < 0.01, Student’s t-test) (Figure 1B and C).
Functional annotation enrichment of TIIA -regulated genes
Enrichment analysis of RNA-seq-identified DEGs by MetaCore software
Function description (number of genes)
Differentially expressed genes (DEGs)
Cell cycle_G2-M (75)
Cyclin G, DNMT3B, Ubiquitin, TOP2 beta, ANAPC4, Condensin, UBC, Brca1, Cyclin B, PDGF-B, Cyclin B2, Brca2, Histone H3, HUS1, Miz-1, CDC25C, PTCH1, TRF1, Cyclin A2, BLM, CAP-G, PLK4 (STK18), FANCD2, CAP-G/G2, 14-3-3 sigma, EGFR, GTSE1, ATM, MAD2a, FOXM1, Lamin B1, GADD45 beta, 14-3-3, BUBR1, TOP2 alpha, CAP-H/H2, PDGF-A, LATS2, Lamin B, ETS2, GADD45 alpha, CAP-C, Cyclin A, CNAP1, Rad50, Cyclin G2, DNMT1, Skp2/TrCP/FBXW, CAP-D2/D3, KNSL1, Aurora-B, CDC25A, SKP2, DNMT3A, TOP2, PDGF-R-alpha, Dynamin, p21, BUB1, BRRN1, PDGF receptor, ATR, Cyclin B1, Emi1, c-Myc, Claspin, Aurora-A, CDC25, ATRIP, PKA-cat (cAMP-dependent), CAP-E, Histone H1, CDK1 (p34), CDK7, CDC27
DNA damage_DBS repair (44)
Ubiquitin, TOP2 beta, Tip60, Sirtuin, MRE11, Brca1, BRIP1, Brca2, MeCP2, Histone H3, MRN complex, C1D, HMGB1, BLM, FANCD2, RAD54B, RAD54L, SMC6, ATM, DNA ligase IV, DNA-PK, NEK1, TOP2 alpha, FANCA, Histone H2AX, SMC5, PP2A regulatory, Rad50, ChAF1 subunit B, XRCC2, FANCL, TOP2, Sirtuin6, ATR, PSF, DNA polymerase eta, FANCM, PIR51, Nibrin, HMG1,2, WRN, ATRIP, Histone H4, p53BP1
Apoptosis_Apoptotic nucleus (49)
Tip60, NRIF3, NF-kB2 (p100), DNA polymerase kappa, RelA (p65 NF-kB subunit), Brca1, PARP-1, Tubulin alpha, TIA-1, Separase, Histone H3, ROCK2, Caspase-6, BLM, FANCD2, iASPP, C/EBPbeta, Granzyme B, IEX1, ATM, Histone H2B, IRF5, DOCK1, DNA ligase IV, FOXO3A, DNA-PK, c-Myb, c-Jun, Protein p8, Lamin B1, GADD45 beta, Clusterin, DFF40 (CAD), PHAP1 (pp32), Lamin B, UBE1C, GADD45 alpha, Axin1, NF-kB, p21, ATR, P53DINP1a, Bcl-6, Bard1, APP-BP1, Nibrin, XPD, Perforin, Histone H1
Cytoskeleton_Intermediate filaments (30)
Plectin 1, Tubulin beta, Keratin 17, BPAG2, Desmin, PPL(periplakin), ROCK, Tubulin alpha, SYNE2, ROCK2, Keratin 18, Keratin 19, Keratin 8/18, Lamin B1, 14-3-3, Lamin B, Keratin 8, Nestin, Keratin 6A, BPAG1, TMPOA, TMPOB, Kinesin light chain, Plakophilin 2, Actin, Fimbrin, Peripherin, CDK1 (p34), Tubulin (in microtubules), Nesprin 1
Proteomic expression profiling of TIIA-treated gastric cells
List of iTRAQ-identified proteins regulated by TIIA
No. of quantified peptides
Solute carrier family 2, facilitated glucose transporter member 1
Probable phosphoglycerate mutase 4
L-lactate dehydrogenase A chain
L-lactate dehydrogenase B chain
ATP metabolic process
Transitional endoplasmic reticulum ATPase
ATP metabolic process
Fructose-bisphosphate aldolase A
Actin, cytoplasmic 1
Peptidyl-prolyl cis-trans isomerase A
Peptidyl-prolyl cis-trans isomerase B
60 kDa heat shock protein, mitochondrial
Heat shock protein HSP 90-alpha
Stress-70 protein, mitochondrial
Heat shock cognate 71 kDa protein
T-complex protein 1 subunit delta
T-complex protein 1 subunit gamma
T-complex protein 1 subunit eta
Protein disulfide-isomerase A6
Heat shock protein HSP 90-beta
DNA damage response
ATPase family AAA domain-containing protein
DNA damage response
Proteasome subunit beta type-3
DNA damage response
Ubiquitin-40S ribosomal protein S27a
DNA damage response
Histone H2A type 1-A
Nucleoside diphosphate kinase B
Proline-rich protein 5
G2/M transition of mitotic cell cycle
Tubulin beta chain
Keratin, type I cytoskeletal 18
Keratin, type II cytoskeletal 1
Heterogeneous nuclear ribonucleoprotein K
Protein disulfide-isomerase A3
78 kDa glucose-regulated protein
Keratin, type I cytoskeletal 19
Keratin, type II cytoskeletal 8
Actin crosslink formation
Microtubule cytoskeleton organization
Tubulin alpha-1C chain
X-ray repair cross-complementing protein 6
Transcription intermediary factor 1-beta
Nucleosome assembly protein 1-like 1
Histone H2A type 1-C
Histone H2B type 1-M
Histone H3.1 t
40S ribosomal protein S10
60S ribosomal protein L5
40S ribosomal protein SA
40S ribosomal protein S13
60S ribosomal protein L15
60S ribosomal protein L9
40S ribosomal protein S3
Elongation factor 2
40S ribosomal protein S16
60S ribosomal protein L14
40S ribosomal protein S19
60S ribosomal protein L18a
60S ribosomal protein L21
60S ribosomal protein L7
40S ribosomal protein S2
RNA metabolic process
Eukaryotic translation initiation factor 5
RNA metabolic process
Heterogeneous nuclear ribonucleoproteins C1/C2
RNA metabolic process
Heterogeneous nuclear ribonucleoprotein D-like
RNA metabolic process
Heterogeneous nuclear ribonucleoprotein A1-like 2
RNA metabolic process
Eukaryotic initiation factor 4A-I
RNA metabolic process
Heterogeneous nuclear ribonucleoprotein F
Keratin, type I cytoskeletal 14
Keratin, type I cytoskeletal 9
Keratin, type II cytoskeletal 6B
Sulfur amino acid metabolic process
Epithelial to mesenchymal transition
Heterogeneous nuclear ribonucleoprotein A/B
Energy reserve metabolic process
ADP/ATP translocase 2
NADH metabolic process
Malate dehydrogenase, mitochondrial
GTP catabolic process
Tubulin beta-4B chain
Cellular membrane organization
14-3-3 protein zeta/delta
Tropomyosin beta chain
Activation of MAPKK activity
14-3-3 protein beta/alpha
Protein export from nucleus
Eukaryotic translation initiation factor 5A-1
Keratin, type II cytoskeletal 2 epidermal
Cellular response to calcium ion
Keratin, type I cytoskeletal 10
Response to hypoxia
Pyruvate kinase PKM
Heterogeneous nuclear ribonucleoproteins A2/B1
Histone mRNA metabolic process
Small nuclear ribonucleoprotein Sm D3
Positive regulation of protein phosphorylation
Guanine nucleotide-binding protein subunit beta-2-like 1
GTP catabolic process
Elongation factor 1-alpha 1
Response to virus
Elongation factor 1-gamma
IQ calmodulin-binding motif-containing protein 1
Cell redox homeostasis
TATA-binding protein-associated factor 2 N
TIIA suppresses glucose metabolism of gastric cancer cells
TIIA arrests the cell cycle at the G2/M phase transition
As indicated in Table 1, CDK1, cyclin B1 and Cdc25C are associated with the cell cycle at the G2/M phase, and were identified in our transcriptomics data. CDK1 activation can regulate the progression of the cell cycle from the G2 to the M phase, which is dependent on coordination with cyclin B [24,25]. The activation of the CDK1/cyclin-B complex is maintained through phosphorylation of Thr161 and dephosphorylation of Thr14 and Tyr15 in CDK1. Dephosphorylation of Thr14 and Tyr15 in CDK1 is catalyzed by phosphatase Cdc25C, which is considered a rate-limiting step for the G2 to M phase transition [24,25]. Previous reports suggest monitoring the alteration of CDK1, cyclin B1, Cdc25C, and phospho-CDK1 (CDK1-Thr161) protein expressions is a useful way to validate the occurrence of cell cycle arrest at the G2/M transition . For these reasons, to confirm whether TIIA induces cell cycle arrest at G2/M in gastric cancer cells, we treated AGS cells with TIIA at a concentration of 5.3 μM (IC50), and then measured protein expression levels using western blotting analysis. Levels of phospho-CDK1 (CDK1-Thr161), cyclin B1, and Cdc25C were all reduced in cells treated with TIIA (Figure 5B). Our results indicate that TIIA treatment induced characteristic cell cycle arrest at G2/M in AGS cells by altering cyclin B1 and Cdc25C expression as well as the phosphorylation of CDK1.
TIIA treatment causes apoptosis and reorganization of actin filaments and microtubules
It is widely understood that reorganization of the cytoskeleton, including actin filaments and microtubules, plays a crucial role in apoptosis [28,29]. Links between this process and TIIA treatment can be seen in our transcriptomics and proteomics data (Figure 2A and Table 1). To detect whether the cytoskeletons of AGS cells undergo reorganization after TIIA exposure, we treated AGS cells with TIIA at different concentrations, then examined the consequent distribution of actin filaments and microtubules using immunofluorescence staining. Many cells were seen to manifest shrinking morphology after TIIA treatment (Figure 6B). Actin filaments under TIIA treatment became more condensed, especially at the cell periphery, and underwent crumbling. On the other hand, microtubules aggregated to become thick bundles, and were distributed along nuclear fragmentation sites with condensed chromatin (Figure 6B). These kinds of cytoskeletal reorganizations, combined with nuclear fragmentation, are all characteristic of apoptosis [2,28-30], showing that TIIA induced cytoskeleton reorganization arising from apoptosis in AGS cells.
TIIA triggers γ-H2AX nuclear foci in response to DNA double strand breaks
Botanical herbs have been used for disease treatment and prevention, and as alternative and complementary therapies . For instance, paclitaxel isolated from one kind of botanical herb, Taxus brevifolia, has been used as second-line chemotherapy for advanced or recurrent gastric cancer . In this study, we treated gastric cancer with TIIA, which can be used to improve heart function by limiting apoptosis or oxidative damage . Recently, many studies have shown that TIIA exhibits anti-tumor potential [10-16], and that on cancers such as breast cancer, prostate cancer, and leukemia, it can induce the mitochondria-dependent apoptosis pathway [38-40]. In MKN45 gastric cancer cells, TIIA was reported to inhibit cell growth, induce G2/M cell cycle arrest and apoptosis [16,41]. However, few of them actually constructed possible pathways or mechanisms in gastric cancer.
Genes for glycolysis are overexpressed in 70% of all human cancers worldwide, including gastric cancer . By comparing our transcriptomics and proteomics data, we are the first to propose that the transformation of glucose metabolism, glycolysis and gluconeogenesis in gastric cancer cells is a major biological process that is regulated by TIIA treatment. An enzyme involved in glycolysis, G6PI, catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, and is downregulated by TIIA . Increased synthesis of G6PI, known as autocrine motility factor (AMF), is considered to be a unique feature of cancer cells, which stimulates cell growth and contributes to cancer metastasis and malignancy [44-47]. AMF can also down-regulate caspase-9 and Apaf-1, making cancer cells more resistant to mitochondria-dependent apoptosis . Previous studies have reported that TIIA can induce mitochondria-dependent apoptosis by regulating caspase-9 and Apaf-1 in several types of cancer [38-40]. In gastric cancer, we also found that TIIA may induce apoptosis (Figure 6A). Based on our results, we suggest that TIIA might induce the occurrence of apoptosis by suppressing G6PI expression, which decreases glucose consumption and inhibits glycolysis in cancer cells.
In gluconeogenesis, expression of LDHB, one of the subunits of lactate dehydrogenase (LDH), which converts lactate to pyruvate, was shown to decrease in AGS cells after TIIA treatment. Pyruvate is the end product of glycolysis and contributes to gluconeogenesis, acetyl-CoA which enters the Krebs (TCA) cycle, lipid synthesis and nonessential amino acid synthesis for proliferative responses in tumor cells . Decreased LDHB expression might enhance negative regulation of the Warburg effect, which mediates pyruvate into lactate and back in tumor cells . In mouse immortalized cell lines, LDHB is critical in the mechanistic target of rapamycin (mTOR) pathway to induce tumor formation . LDHB is also considered as a tumor marker that increases in many cancers because it facilitates tumor growth and cell proliferation [51-53]. Serum LDH is also considered to be predictors for overall survival of advanced nasopharyngeal carcinoma patients . In gastric cancer cells, we suggest that TIIA treatment might block nutrient supply, which reduces cell survival and proliferation by causing lactate dehydrogenase deficiency.
The glucose metabolic switch is associated with the cell cycle, apoptosis and DNA repair in tumor cells . Our transcriptomics analyses and proteomics data also showed that TIIA can regulate these functions and their related proteins, such as PSMB3 and RS2. Previous studies have shown that the expression of PSMB3 and RS2 is involved the development of cancer [60,61]. PSMB3, which belongs to the proteasome B-type family, was up-regulated by TIIA. Proteasomes are necessary for the DNA damage response, and accumulate at double strand break sites to recruit other repair proteins . TIIA treatment increased the likelihood of the occurrence of DNA damage in gastric cancer cells (Figure 7), which might stimulate cancer cells to increase PSMB3 expression.
Moreover, RS2, as a cancer-related ribosomal protein, was down-regulated by TIIA. Increased RS2 is present when p53 mutates, which occurs in cell cycle progression or apoptosis in response to DNA damage . Elevated expression of p53, cell cycle arrest in the G2/M phase, apoptosis and cytoskeleton reorganization were observed in AGS cells following TIIA treatment. DNA damage is one of the major causes of cell cycle arrest in the G2/M phase . Cytoskeleton reorganization, including the redistribution of actin and microtubules, is a characteristic of chromatin remodeling during apoptosis . This has also been observed in cancer cells treated with clinical anti-cancer drugs, such as etoposide, doxorubicin and taxol .
We integrate transcriptomics and proteomics data to uncover TIIA-regulated mechanisms in gastric cancer. In glycolysis and gluconeogenesis, TIIA reduces glucose consumption and the production of pyruvate by regulating G6PI, LDHB, MDH1, PCK2 and PGK1 expression. AKT decreases and p53 increases in response to apoptosis and DNA damage in gastric cancer cells following TIIA treatment, while the glucose metabolism switch induced by the transformation of oncogenes is destroyed (Figure 8). We suggest that TIIA treatment could cause cell stress, nutrient deficiency and DNA damage, to induce cycle arrest at the G2/M transition, apoptosis and cytoskeleton reorganization, all of which would inhibit cell proliferation. In this study, we provide new insight into the TIIA regulatory mechanism in gastric cancer cells, so that future cancer research can improve therapeutic strategies.
Human stomach adenocarcinoma AGS (CRL-1739; ATCC) cells were grown in 90% RPMI 1640 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (Biological Industries). Cells were cultured at 37°C in an incubator with controlled humidified atmosphere containing 5% CO2. Cells were disaggregated for subculturing with trypsin plus ethylenediaminetetraacetic acid (EDTA), then spun down by centrifugation at 1200 rpm for 5 min, after which the pellet was re-suspended in culture medium.
Tanshinone IIA powder (T4952; Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) (BioShop), which was diluted to applicable concentrations for treatment.
Growth curves and IC50 measurement
Growth curves of AGS cells were recorded by an RTCA DP® system (xCELLigence™; Roche Applied Science & ACEA Biosciences). Cells were treated with TIIA or 0.1% DMSO as control for 72 hr after 24 hr of seeding (5,000 cells/well in 96-well E-plates). TIIA treatment concentrations were 0.1 μM, 1 μM, 5.3 μM, and 10 μM; 0.1% DMSO dissolved in culture medium was used as control. Each condition was performed in triplicate. The IC50 of TIIA at 48 hr was calculated with RTCA software 1.2 (xCELLigence™) by using AGS cell growth curves under TIIA treatments of 0.1 μM, 1 μM, 5.3 μM, and 10 μM.
Colony formation assay
AGS cells were treated with TIIA or 0.1% DMSO control medium for 5 days after 24 hr of seeding (2,500 cells/well in 6-well plates). TIIA treatment concentrations were 0.53 μM, 1.06 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were fixed with methanol for 10 min, stained with 200 μg/mL crystal violet (Sigma-Aldrich) for 10 min, and then colonies were counted using AlphaView SA 3.4.0 (ProteinSimple). Each condition was performed in triplicate.
RNA sequencing and data analysis
Total RNA was extracted from AGS cells treated with DMSO (control) or TIIA, using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The quantity and quality of RNA were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and were found to have an RNA Integrity Number (RIN) value of more than 9. Poly(A) mRNA was isolated with oligo(dT)-bound magnetic beads and incubated with fragmentation buffer to form short RNA fragments. Reverse transcriptase and random hexamer primers were used to synthesize the first-strand cDNA, and DNA polymerase I and RNaseH were used to synthesize the second-strand cDNA. Double stranded cDNA was end-repaired with T4 DNA polymerase, Klenow fragments, and T4 Polynucleotide Kinase, after which a single “A” base was added, and the entire sequence was ligated to Illumina sequencing adapters (Illumina, San Diego, CA). PCR was performed to amplify the fragments. Using Illumina HiSeqTM 2000 (Illumina), the cDNA library was sequenced on a flow cell after validation on an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System.
After the base calling, the reads with adaptor sequences and low quality scores were removed. The reads with low quality scores are defined as the reads with greater than 10% of unknown bases (N) or greater than 50% of low quality bases which quality values are less than 5. The reads with high quality scores were mapped to the human reference genome hg19 assembly using SOAPaligner in SOAP2 with 2 mismatch allowance  and annotated based on the GENCODE . Read counts for individual GENCODE genes were subsequently determined using HTSeq-count (http://www-huber.embl.de/users/anders/HTSeq), by considering only uniquely mapped reads. Expression of each individual gene was quantified by using the Reads Per Kilobase per Million mapped reads (RPKM) method. The differentially expressed genes were identified by Poisson distribution model . The sequences reported in this study have been deposited in the Sequence Read Archive database with accession number SRP049450.
Functional enrichment analysis
We used MetaCore version 6.18 (GeneGo, St. Joseph, MI, USA) to perform a gene function analysis and to enrich the functional networks of identified genes collected from RNA-seq. There were 16,110 of 16,603 identified genes eligible for network enrichment via GO Processes and Metabolic Networks analysis. For analysis of differentially expressed genes (|log2(fold–change)| > 1), 2,717 of 2,761 DEGs were eligible for GO Processes enrichment analysis, and displayed classified genes (Table 1). All enrichment analysis was tested using the p-value threshold p < 0.0001 for the data inputs.
iTRAQ labeling and LC-MS/MS analysis
AGS cells were treated with 5.3 μM (IC50) TIIA or 0.1% DMSO control medium for 48 hr after 24 hr of seeding (8 × 104 cells/well in 6-well plates). Cells were harvested with trypsin/EDTA and then total proteins were extracted by using lysis buffer (1% SDS (Bioman), 50 mM Tris–HCl (pH 6.8), 10% glycerol), and 1× protease inhibitor (Bioman) and sonication. Proteins were processed through reduction, alkylation, and gel-assisted trypsin digestion overnight to yield peptides, which were later extracted from the gels as previous methods . Equal amounts of peptides from control and TIIA-treated samples were labeled by different iTRAQ reagents (AB SCIEX; control samples labeled by 114 or 115; TIIA-treated samples labeled by 116 or 117) and incubated at room temperature for 1 hr. Peptides were combined together and dried with a centrifugal evaporator (CVE-2000; EYELA). After iTRAQ-labeling, samples were desalted and analyzed by using a LC-ESI-Q-TOF mass spectrometer (Waters SYNAPT® G2 HDMS; Waters Corp.). Samples were injected into a 180 mm × 2 cm capillary trap column and separated by a 75 mm × 25 cm nanoACQUITY UPLC™ 1.7 mm Ethylene Bridged Hybrid C18 column using a nanoACQUITY Ultra Performance LC™ System (Waters Corp.). The mass spectrometer (MS) was operated in electrospray ionization sensitivity mode, and calibrated with a synthetic human [Glu1]-Fibrinopeptide B solution (1 pmol/ml; Sigma-Aldrich) delivered through a NanoLockSpray™ source, which was used for accurate mass measurements. Data was acquired in the data directed analysis (DDA) mode, which included one full MS scan (m/z 350–1700, 1 s) and three sequential MS/MS scans (m/z 100–1990. 1.5 s for each scan) on the three most intense ions present in the full scan mass spectrum.
The peak list resulting from MS/MS spectra was generated by Mascot Distiller v2.3.2 (Matrix Science, London, United Kingdom). Data files were searched against the sequence database (containing 536,789 sequences entries) of the Swiss-Prot human database, using Mascot search engine v2.3.02 (Matrix Science, London, United Kingdom). Both the precursor peptide and fragment ion tolerances were set to ±0.1 Da. The search parameter settings were as follows: allowances for two missed cleavages from trypsin digestion and variable modifications of deamidation (NQ), oxidation (M), iTRAQ (N terminal), iTRAQ (K), and methylthio (C). The peptide charge was set to Mr, the instrument was set to ESI-QUAD-TOF, and the decoy database was searched. Mascot search results were filtered using “Significance threshold” set at p < 0.05 and “Ions score or expect cut-off” set at 0.05. To evaluate the false discovery rate (FDR), we compared a decoy database search against a randomized decoy database created by Mascot using identical search parameters and validation criteria. FDR was calculated as D/R × 100%, where D and R are the number of matches above identity threshold using the decoy and real databases, respectively. The mass spectrometry proteomics data have been uploaded to the ProteomeXchange Consortium  via the PRIDE partner repository with the data set identifier PXD000998 and DOI 10.6019/PXD000998.
For protein quantitation, signature ions (m/z = 114, 115, 116 and 117) and peptides were detected and analyzed using Multi-Q software (v22.214.171.124) . Peptides that satisfied the following four criteria were subjected to further analysis. Firstly, the peptide is labeled with iTRAQ tags; secondly, the peptide has an ion score higher than the Mascot identity score (p < 0.05); thirdly, the peptide is nondegenerate (unique); fourthly, the iTRAQ signature ion peak intensity (ion count) of the peptide is within the dynamic range (the peak intensity of each iTRAQ signature ion must be > 0, and the average of the peak intensities of all iTRAQ signature ions must be ≥ 30). Before quantitation of the expression of each protein, the peak intensity of the iTRAQ signature ion was normalized, as “Method 1” of our previous study . To determine the expression ratio of identified proteins in AGS cells from both the control and the TIIA treatment, the normalized peptide iTRAQ signal of each identified protein was summarized, to calculate protein ratios (TIIA treatment/control).
Western blot analysis
AGS cells were treated with 5.3 μM (IC50) TIIA or 0.1% DMSO control medium for 48 hr after 24 hr of seeding (8 × 104 cells/well in 6-well plates). Cells were harvested with trypsin/EDTA and total proteins were extracted. Then, proteins from control and TIIA-treated samples were separated in 12% SDS-PAGE gels, and transferred onto 0.45 μm PVDF membranes (Millipore) in a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad) for 50 min at 400 mA. The membrane was blocked for 1 hr at room temperature in 5% non-fat milk powder/PBS-T (1× PBS, 0.1% Tween 20 (Sigma-Aldrich)) and incubated overnight at 4°C with blocking buffer containing rabbit monoclonal antibodies to human RS2 (GeneTex; 1:1,000), PSMB3 (GeneTex; 1:1,000), phospho-CDK1 (Santa Cruz; 1:100), CDK1 (Santa Cruz; 1:100), Cyclin B1 (GeneTax; 1:500), Cdc25C (GeneTex; 1:1,000), G6PI (GeneTex; 1:1000), ENO1 (GeneTex; 1:2000), MDH1 (GeneTex; 1:500), PGK1 (GeneTex; 1:500), ALDOC (GeneTex; 1:250), PCK2 (GeneTex; 1:1000), LDH-B (GeneTex; 1:100), p53 (Santa Cruz; 1:500) or AKT (Santa Cruz; 1:1000). The membrane was washed with PBS-T, incubated 1 hr with 5% non-fat milk powder/PBS-T containing anti-rabbit IgG antibodies (1:10,000) (Sigma-Aldrich) or anti-mouse IgG antibodies (Sigma-Aldrich, 1:10,000), washed and imaged with enhanced chemiluminescence (PerkinElmer). The membrane image was then analyzed by an AutoChemi Image System (UVP) or exposed to Fuji medical X-ray film, followed by quantification with AlphaView SA 3.4.0 (ProteinSimple).
Intracellular ATP generation assay
Cells were seeded onto 6-well plates at 8 × 104 cells/well, and then incubated for 24 h. For the control, 0.1% DMSO was added to the medium, and for the treatment, 5.3 μM TIIA was added. After 48 h of drug exposure, the medium was removed, and then cells were washed twice with PBS. The levels of intracellular ATP were determined using a bioluminescent somatic cell assay kit (Sigma-Aldrich), according to the manufacturer’s instructions, and normalized to protein concentrations. Luminescence was detected using a FlexStation III (Molecular Devices). The ATP content of each sample was calculated as the average of the relative light readings and based on the ATP standard curve.
For cell cycle analysis, AGS cells were treated with TIIA or DMSO as control for 48 hr. TIIA treatment concentrations were 0.625 μM, 1.25 μM, 2.5 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were harvested with trypsin/EDTA, fixed with 70% ethanol, then spun down, after which ethanol was removed. Then each sample was mixed with RNase A (100 μg/mL), incubated at 37°C for 1 hr, and stained with propidium iodide (PI) (Santa Cruz Biotechnology, Inc.) at a concentration of 100 μg/mL in the dark at room temperature for 15 min. For apoptosis analysis, AGS cells were treated with TIIA or DMSO control medium for 48 hr after 24 hr of seeding (3.5 × 105 cells in 10-cm plates). TIIA treatment concentrations were 1.25 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were harvested with trypsin/EDTA, suspended, and counted. Then each sample was adjusted to a concentration of 106 cells/tube and stained with Annexin V-FITC (Santa Cruz Biotechnology, Inc.) and PI (Santa Cruz Biotechnology, Inc.) dissolved in binding buffer (Santa Cruz Biotechnology, Inc.) in the dark at room temperature for 15 min. Both cell cycle distribution and apoptotic cells proportion were then analyzed with a BD FACSCanto II flow cytometer (BD Biosciences) and FCS Express 4 (BD Biosciences).
AGS cells were treated with TIIA or DMSO control medium for 48 hr after 24 hr of seeding (4 × 104 cells in 6-well plates). TIIA treatment concentrations were 1.25 μM and 5.3 μM, and 0.1% DMSO dissolved in culture medium was used as control. Cells were washed, fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 20 min at 37°C, then permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature. Then cells were incubated with 1% BSA (Bioshop) in PBS as blocking buffer for 30 min at room temperature, and labeled with mouse monoclonal antibodies to human α-tubulin (Millipore; 1:500) or γ-H2AX (Abcam; 1:500) dissolved in blocking buffer at 4°C overnight. After being washed with PBS three times, cells were labeled with anti-mouse FITC-IgG (Millipore; 1:100) and TRITC-conjugated Phalloidin (Millipore; 1:2000) dissolved in blocking buffer for 1 hr in the dark at room temperature. Then cells were washed with PBS three times and mounted with ProLong® Gold reagent with DAPI (Invitrogen). Images were acquired by using a fluorescence microscope with Leica HCX FL PLAN 1006/1.25 oil objective, a SPOT camera (Diagnostic Instruments), and were analyzed with SPOT Advanced software (Diagnostic Instruments).
Data were expressed as mean ± standard deviation (SD) and analyzed using two-tailed Student’s t-tests. In the cell proliferation assays, data were analyzed using the Wilcoxon Signed-Rank test. A P-value of less than 0.05 was taken to indicate statistical significance.
We acknowledged Technology Commons in College of Life Science, National Taiwan University for technical assistance with the Mascot search engine and flow cytometer. This work was supported by the National Science Council of Taiwan (NSC 102-2628-B-002-041-MY3, 102-2627-B-002-002, and 102-2311-B-010-004) and the National Taiwan University Cutting-Edge Steering Research Project (103R7602C3) and Translational Medicine Piloted Model Cooperation Projects of National Taiwan University Medical Campus and College of Life Science (103-L004).
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