The cell lines used in this study were purchased from the China Center for Type Culture Collection (Wuhan, China). SGC7901/DDP is an MDR gastric cancer cell line in which resistance was induced by cisplatin and it is derived from the human gastric cancer cell line, SGC7901. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin solution. The biological characteristics of MDR of the SGC7901/DDP cell line were maintained by the addition of 1 µg/ml cisplatin (Sigma-Aldrich, St. Louis, MO, USA) to the complete medium. The cells were incubated in an atmosphere with 5% carbon dioxide at 37°C. Three small interfering RNA (siRNA) duplexes targeting human TSPAN8 and a control siRNA were synthesized by GenePharma Co., Ltd. (Shanghai, China). The sequences of the siRNA-TSPAN8 were as follows: Sequence 1 forward, 5′-GUAUCUUGAUCCUAGCAUUdTdT-` and reverse, 5′-AAUGCUAGGAUCAAGAUACdTdT-3′; sequence 2 forward, 5′-GUCUGAUCGCAUUGUGAAUdTdT-3′ and reverse, 5′-AUUCACAAUGCGAUCAGACdTdT-3′; sequence 3 forward, 5′-GAGUUUAAAUGCUGCGGUUd TdT-3′ and reverse, 5′-AACCGCAGCAUUUAAACUCdTdT-3′; and siRNA-NC forward, 5′-UUCUUCGAAGGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′. The SGC7901/DDP cells were transfected with the siRNA using siRNA-Mate (GenePharma Co., Ltd.) following the manufacturer's instructions. The inhibitors of the Wnt pathway (CCT036477 and XAV939) and the inhibitor of NOTCH2 (DAPT) were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, USA). All these drugs were suspended in dimethyl sulfoxide (DMSO; Sangon Biotech, Shanghai, China) at a stock concentration according to the manufacturer's instructions and stored at -80°C. Following siRNA transfection for 48 h, the cells were exposed to the inhibitors, which were diluted into the culture medium (10 µM CCT036447, 10 µM XAV939, 20 µM DAPT or DMSO alone as a control) for 48 h, respectively.

The cytotoxic effects of the cisplatin, 5-fluorouracil and adriamycin (both from Sangon Biotech) on the SGC7901 and SGC7901/DDP cells were measured by cell counting kit-8 (CCK-8) assay (21). The cells were counted using the Neubauer cell-counting chamber (BRAND GMBH + CO KG, Wertheim, Germany) following the manufacturer's instructions. The cells were then seeded in 96-wells at a density of 5×10³ cells/well, and cultured in an incubator at 37°C for 24 h before being treated with the chemotherapeutic drugs. Cisplatin, 5-fluorouracil (5-Fu) and adriamycin in graded concentrations were added to the cells. Following treatment for 48 h, the medium was replaced with fresh medium containing 10% CCK-8 reagent (Dojindo, Kumamoto, Japan), and the cells were incubated for an additional 1–4 h. The optical density was then measured by Thermo Scientific Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The IC₅₀ values obtained following treatment of the SGC7901 and SGC7901/DDP cells with each drug were analyzed using IBM SPSS Statistics v21 software (SPSS Inc., Chicago, IL, USA) via probit analysis (22).

Total RNA was extracted using a High Purity Total RNA Rapid Extraction kit (RP1201; BioTeke, Beijing, China) according to the manufacturer's instructions. cDNA was synthesized using a iSCRIPT cDNA Synthesis kit (GeneCopoeia Co., Ltd., Guangzhou, China). The primers used for the amplification of TSPAN8, β-catenin, NOTCH2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were synthesized by GeneCopoeia Co., Ltd. GAPDH was used as an internal standard, and the relative expression of each gene was normalized to GAPDH. The real-time PCR kit was purchased from GeneCopoeia Co., Ltd. PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 10 sec. The relative quantification of gene expression was analyzed using the 2^−ΔΔCt method (23). Each sample was analyzed in triplicate.

Protein was extracted from the cells using RIPA lysis buffer (Beyotime, Shanghai, China) and the concentration was determined using the 2D Quantification kit (Amersham Biosciences, Little Chalfont, UK). The protein samples were separated on a 10% polyacrylamide gel, and electrotransferred onto polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). The membranes were then blocked with 5% non-fat dried milk for 1 h at room temperature. This was followed by the addition of the primary antibodies: anti-TSPAN8 antibody (ab70007), anti-β-catenin antibody (ab16051) (both from Abcam, Cambridge, MA, USA), anti-cellular retinoic acid-binding protein 2 (CRABP2) antibody (10225-1-AP), anti-voltage-dependent anion-selective channel protein 2 (VDAC2) antibody (11663-1-AP), anti-Bcl-2 antibody (12789-1-AP) (all from Proteintech, Wuhan, China), anti-heat shock protein 90 (HSP90) antibody (bs-0889R), anti-erythrocyte membrane protein band 4.1 (EPB41) antibody (bs-13080R), anti-tumor protein D54 (TPD54) antibody (bs-6743R), anti-mucin 13 (MUC13) antibody (bs-10074R), anti-GAPDH antibody (bs-10900R), anti-caspase-3 antibody (bs-0081R) and anti-Bax antibody (bs-0127R) (all from Bioss, Beijing, China) and overnight incubation at 4°C. All primer antibodies were diluted (1:1,000) by Tris-buffered saline containing 0.1% Tween-20 (TBS-T) After washing 3 times with TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody (1:5,000, ab6721; Abcam) for 2 h at room temperature. After washing 3 times with TBS-T buffer, the membranes were visualized with an ECL detection system (KeyGen Biotech Inc., Nanjing, China). All western blot analyses were repeated at least 3 times.

The cells were seeded in a 6-well plate and transfected with siRNA according to the protocol of siRNA-Mate (GenePharma Co., Ltd.). TOP-flash reporter plasmid was purchased from Shanghai Qcbio Science and Technologies Co., Ltd. (Shanghai, China) and was transfected into the cells by Endofectin™-Plus (GeneCopoeia) according to the manufacturer's instructions 48 h after siRNA transfection. The reporter gene assay was performed 48 h post-plasmid-transfection using the Dual Luciferase Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was normalized for transfection efficiency using the corresponding Renilla luciferase activity. All experiments were performed at least in triplicate.

The plasmids (HA-TSPAN8, Flag-TSPAN8, HA-NOTCH2 and Flag-NOTCH2) used for exogenous co-immunoprecipitation were synthesized by GeneCopoeia Co., Ltd. The cells were lysed by sonication and centrifugation at 4°C, 16,000 × g, 10 min (TDZ4-WS centrifuge; Thermo Fisher Scientific) in IP lysis buffer (Beyotime, Beijing, China) supplemented with phosphatase/protease inhibitor cocktail and 1 mM PMSF. The supernatant was transferred to a separate microfuge tube, pre-cleared with protein A/G agarose beads (Yanji Biotechnology, Shanghai, China) and centrifuged at 4°C, 16,000 × g, 5 min (TDZ4-WS centrifuge; Thermo Fisher Scientific) to pellet the beads and remove protein impurities. The supernatant was collected and incubated with rabbit IgG (bs-0295P; Bioss) overnight at 4°C. The beads were collected by centrifugation at 4°C, 16,000 × g, 10 min (TDZ4-WS centrifuge; Thermo Fisher Scientific), washed 3 times with IP lysis buffer and resuspended with 2× sodium dodecyl sulfate (SDS) loading buffer. Bound protein was eluted off the beads by boiling and examined by western blot analysis as described above.

The cells were incubated with 4.0% paraformaldehyde for 15 min at room temperature. The cells were then washed 3 times with phosphate-buffered saline (PBS). To increase permeability, 0.1% Triton X-100 was added to the cells for 10 min. The cells were then washed again thrice with PBS. The anti-β-catenin antibody (ab16051; 1:100 diluted by PBS; Abcam) was added to the wells followed by incubation overnight at 4°C. The cells were then washed and incubated in Alexa Fluor-conjugated secondary antibody (1:100 diluted with Bioss antifade mounting medium; Bioss). DAPI (Invitrogen, Carlsbad, CA, USA) was used to dye the nuclei. The cells were incubated with DAPI for 20 min at room temperature. After being washed 3 times with PBS, the cells were imaged under a microscope (Ci-L; Nikon, Tokyo, Japan).

Total protein extracts were prepared in lysis buffer [7 M urea, 1 mg/ml DNase I, 1 mM Na₃VO₄ (all from Sangon Biotech), and 1 mM PMSF (Bioss, Beijing, China)] using the Sample Grinding kit from Amersham Biosciences. Following being centrifuged at 17,000 × g for 15 min at 4°C, the supernatant was collected and the protein concentrations were quantified with a 2-D Quantification kit (Amersham Biosciences).

From each sample, 100 µg of protein was precipitated, denatured, cysteine-blocked and digested with sequencing-grade modified trypsin, according to the manufacturer's instructions (iTRAQ Reagent 8 Plex Multi-plex; Applied Biosystems, Foster City, CA, USA). The samples were then labeled with the iTRAQ tags (SGC7901, 113, 115 tags; SGC7901/DDP 114, 116 tags; Applied Biosystems). The labeled samples were pooled prior to further analysis.

The iTRAQ-labeled samples were solubilized in 300 µl of 1% Pharmalyte (Amersham Biosciences) and 8 M urea solution. The samples were rehydrated on IPG gel strips (pH 3.0–10.0; Amersham Biosciences) at 30 V for 14 h. The peptides were subsequently focused successively at 500 V for 1 h, 1,000 V for 1 h, 3,000 V for 1 h and 8,000 V for 8.5 h. Following electrofocusing, the peptides were extracted from the gel using a solution containing 0.1% formic acid and 2% acetonitrile for 1 h. The fractions were then purified and concentrated on a C18 Discovery DSC-18 SPE column (Sigma-Aldrich), lyophilized and maintained at -20°C.

The samples were analyzed using a QStar Elite hybrid mass spectrometer (Applied Biosystems) coupled with a liquid chromatography system (Amersham Biosciences, Little Chalfont, UK).

The mass spectrometer was set to perform information-dependent acquisition (IDA) in the positive ion mode at a mass range of 300–1800 m/z. Peptides with +2 to +4 charge states were selected for tandem mass spectrometry, and the time of summation of MS/MS events was set to 3 sec. We selected the two most abundantly charged peptides above a 20-count threshold for MS/MS and dynamic exclusion was set to 30 sec with a 50 mDa mass tolerance. Data were processed using ProteinPilot version 2.0 software (Applied Biosystems) and searched against the UnitProt (http://www.uniprot.org/) human protein database (v3.77). Protein identification was based on selection thresholds of ProtScore >1.3 or ProtScore <0.77, and false discovery rate P-values <0.05.

The results obtained by iTRAQ-labeled proteomics were analyzed using by protein analysis using the evolutionary relationships (PANTHER) classification system (www.pantherdb.org) following the instructions available online (24). STRING 10.5 (http://string.embl.de/) was used to predict the interaction between proteins following the instruction online (25).

The in vitro experiments were repeated at least 3 times. Data are presented as the means ± standard deviation (SD). Significance between groups from in vitro experiments was determined using the Student's t-test or Dunnett's T3 test. A value of P<0.05 was considered to indicate a statistically significant difference.

To identify potential proteins associated with resistance to cisplatin, iTRAQ-based quantification was performed on proteins isolated from cisplatin-sensitive gastric cancer cells (SGC7901) and from DDP-resistant gastric cancer cells (SGC7901/DDP). The specimens were iTRAQ-labeled in duplicate in order to verify the results. Protein samples were labeled as follows: SGC7901, tags 113 and 114; SGC7901/DDP, tags 115 and 116. The relative abundance of protein from the SGC7901/DDP cells with respect to proteins from SGC7901 cells was calculated as the iTRAQ ratios 115:113 and 116:114. These fractions were analyzed by LC/MS/MS. The workflow of the iTRAQ proteomics approach is presented in Fig. 1. ProteinPilot 2.0 software was used for protein quantification and identification. Considering the technical variations of the method and statistical analysis in the relative quantification analysis, and in order to reduce false-positives and increase accuracy, a 1.3-fold cut-off for all iTRAQ ratios was used (26,27). Therefore, proteins with iTRAQ ratios <0.77- or >1.3-fold cut-off (P<0.05) were considered to be downregulated or upregulated, respectively. A total of 1,324 differentially expressed proteins were identified, regardless of whether or not there was a significant P-value in the iTRAQ ratios. Of these, 112 proteins were differentially expressed in the SCG7901/ddp cells compared to the SGC7901 cells (64 upregulated and 48 downregulated proteins). The top 30 downregulated and upregulated proteins are shown in Table I.

The 112 proteins, which were potentially differentially expressed between the SGC7901/DDP cells and SGC7901 cells, were classified into 5 functional categories using the Protein Analysis through Evolutionary Relationships (PANTHER) classification system (Fig. 2). The molecular function categories were binding (23.5%), receptor activity (2.9%), structural molecule activity (14.7%), catalytic activity (44.1%) and transporter activity (14.7%) (Fig. 2).

The differentially expressed proteins identified by iTRAQ were validated by RT-qPCR and western blot analysis. The proteins selected for validation were the ones most significantly dysregulated according to protein classification or the ones closely related to multidrug resistance. TSPAN8 has been reported to promote the proliferation and metastasis of SGC7901 cells. The results from iTRAQ-coupled 2D LC-MS/MS revealed that TSPAN8 was potentially related to drug resistance in the SGC7901/DDP cells. Thus, it was selected as the object of the following analysis. The mRNA levels of HSP90, drug-sensitive protein 1 (YA61), EPB41 and CRABP2 were decreased in the SGC7901/DDP cells when compared with those in the SGC7901 cells, whereas the mRNA levels of TSPAN8, VDAC2, TPD54 and MUC13 were increased (Fig. 3A). The results of western blot analysis revealed that the protein expression levels of HSP90, YA61, EPB41 and CRABP2 were downregulated in the SGC7901/DDP cells when compared to those in the SGC7901 cells, whereas the levels of TSPAN8, VDAC2, TPD54 and MUC13 were upregulated (Fig. 3B). These results were consistent with the trend observed in iTRAQ analysis.

TSPAN8 has been reported to be an oncoprotein in gastric cancer, enhancing gastric cancer cell proliferation and metastasis (20). However, the role of TSPAN8 in gastric cancer cell drug resistance remains unclear. In the present study, TSPAN8 was knocked down by siRNA. RT-qPCR and western blot analysis confirmed the efficacy of the silencing of TSPAN8. As shown in Fig. 4A, the relative mRNA level of TSPAN8 was significantly decreased following transfection with siRNA against TSPAN8. The results of western blot analysis revealed that TSPAN8 protein expression in specific siRNA-transfected SGC7901/DDP cells was effectively suppressed (Fig. 4B). Of the 3 siRNA sequences, sequence 1 was found to be the most suitable for our purposes (Fig. 4A and B), and was thus used in all subsequent experiments.

MDR is the main cause of chemotherapy failure in gastric cancer treatment. Thus, in this study, to assess the association between TSPAN8 and MDR, the siRNA-transfected SCG7901/DDP cells were treated with cisplatin, 5-Fu and adriamycin (the most commonly used drugs in clinical practice for the chemotherapeutic treatment of gastric cancer), for 2 days and the IC₅₀ values were determined. The IC₅₀ values of cisplatin, 5-Fu and adriamycin were significantly decreased in the TSPAN8-silenced SGC7901/DDP cells compared with the negative controls (Table II). This result suggested that the silencing of TSPAN8 reduced the resistance of the SGC7901/DDP cells to the aforementioned drugs, which, in turn, indicated that TSPAN8 may contribute to the MDR of this cell line. In the following experiments, only cisplatin was used to maintain the drug resistance of the SGC7901/DDP cells.

Furthermore, compared with the negative control SGC7901/DDP cells, apoptosis was increased in the TSPAN8-silenced cells (Fig. 4C). Moreover, the levels of apoptosis-related proteins (caspase-3, Bax and Bcl-2) were examined by western blot analysis. The results (Fig. 4D) revealed that the levels of caspase-3 and Bax were upregulated, while those of Bcl-2, an anti-apoptotic protein, were downregulated in the TSPAN8-silenced SGC7901/DDP cells. These results indicated that the silencing of TSPAN8 promoted SGC7901/DDP cell apoptosis.

Thus far, our findings suggested that TSPAN8 plays a critical role in the drug resistance of SGC7901/DDP cells. It is believed that metastasis is the persistence of cancer stem cells (CSCs), which are highly resistant to chemotherapy (28). The Wnt/β-catenin signaling pathway has been reported to increase gastric cancer cell migration and invasion (29). Therefore, in this study, we investigated whether TSPAN8-mediated gastric cancer cell drug resistance is also related to the Wnt/β-catenin pathway. The Wnt/β-catenin pathway activity was detected using a TOP-flash luciferase reporter. The silencing of TSPAN8 in the SGC7901/DDP cells significantly decreased TOP-flash luciferase activity (Fig. 5A). The TSPAN8-silenced cells displayed a decreased expression of β-catenin at both the mRNA (Fig. 5B) and protein level (Fig. 5C), compared to negative control (NC)-infected SGC7901/DDP cells. Additionally, the accumulation of β-catenin in the nucleus was impaired in the TSPAN8-silenced SGC7901/DDP cells (Fig. 5D). The cells were treated with CCT036477 (CCT) and XAV939 (inhibitors of the Wnt-β-catenin pathway) (30). The reduced IC₅₀ value caused by TSPAN8 silencing was partially reversed when the Wnt-β-catenin pathway inhibitors were added (Table III). These data indicated that TSPAN8 enhanced the resistance of the SGC7901/DDP cells to chemotherapy through the activation of the Wnt/β-catenin pathway and by increasing β-catenin expression and accumulation in the nucleus. However, compared to the NC group, the inhibitors of the Wnt pathway still decreased the IC₅₀ values (Table III).

To identify which protein or proteins interact with TSPAN8, we utilized STRING 10.5. NOTCH2 was predicted to interact with TSPAN8. Co-immunoprecipitation was used to validate the association between TSPAN8 and NOTCH2. Endogenous co-immunoprecipitation assays revealed that TSPAN8 interacted with NOTCH2 in the SGC7901/DDP cells (Fig. 6A). Consistent with this result, the exogenous interaction between TSPAN8 and NOTCH2 was also observed in the SGC7901/DDP cells that were co-transfected with HA-TSPAN8 and Flag-NOTCH2 (Fig. 6B). These findings revealed that TSPAN8 acts in combination with NOTCH2 in gastric cancer cells. Furthermore, we found that the impairment of β-catenin expression was partially compensated when DAPT (30), a NOTCH2 inhibitor, was used in the TSPAN8-silenced SGC7901/DDP cells (Fig. 6C and D). The results data indicated that TSPAN8 mediated the activation of the Wnt/β-catenin pathway by binding to NOTCH2.