Various human GC cell lines, including SGC-7901 (moderately differentiated), BGC-823 (poorly differentiated) and immortalized normal gastric epithelium cells (GES-1), and the human cervical cancer HeLa cell line were provided by the Department of Oncology, The Affiliated Drum Tower Hospital of Nanjing University, Medical School, (Nanjing City, Jiangsu Province). HeLa cell lines were used as a positive control for the present study. Cells were cultured in RPMI1640 (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified air with 5% CO₂ at 37°C (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

A pU6 plasmid, synthesized by Shanghai GeneChem Co., Ltd., (Shanghai, China), containing siRNA targeting PKM2 mRNA and empty plasmids as the negative control were transfected into the SGC-7901 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were cultured and selected in medium containing 400 mg/ml G418 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Cell survival rate was assessed by using a Cell Counting Kit-8 (CCK-8) assay (KeyGen Biotech Co., Ltd., Jiangsu Province, P.R. China), according to the manufacturer's protocol. BGC-823 cells were plated at a density of 1×10⁴ cells/well in 100 µl RPMI-1640 (Hyclone) into 96-well plates and cultured for 24 h (~80% confluent). Cells were then treated with LY294002 at various concentrations (0, 10, 20, 50, 100 µmol/l), or with DMSO (0.2%) as a control, for 24 and 48 h. SGC-7901 cells from the non-transfected, negative control (empty plasmid) and PKM2-siRNA groups were plated at a density of 1×10⁴ cells/well in 96-well plates and cultured at 37°C for 24–48 h. The absorbance was measured at 450 nm using a Cell Counting Kit-8 assay (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China).

BGC-823 cells were plated into 6-well plates at a density of 1×10⁶ cells/well. Following treatment with the indicated concentrations of LY294002 (0, 10, 20, 50, 100 µmol/l), or DMSO (0.2%) for 48 h, the cells were dual-stained using an Annexin V-FITC kit (Nanjing KeyGen Biotech Co., Ltd.). SGC-7901 cells from the non-transfected, negative control and PKM2-siRNA groups were seeded at a density of 1×10⁶ cells/well in 6-well plates and cultured to 80% confluence for 48 h, and then the aforementioned dual staining was performed. Apoptotic cells were detected by flow cytometry using BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, U.S.A.). BD FACSDiva software was used to analyze data (version 7.0, BD Biosciences, U.S.A.).

The protein expression level of PKM2 was evaluated in each cell line (SGC-7901, BGC-823, GES-1 and HeLa cells) by western blot analysis. The protein levels of p-Akt, p-mTOR, hypoxia inducible factor-1α (HIF-1α) and PKM2 were assessed in BGC-823 cells, and PKM2, Glut-1 and LDHA expression levels in SGC-7901 cells of the non-transfected, negative control and PKM2-siRNA groups were evaluated by western blot analysis. Total cell extracts were prepared on ice for 30 min in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% SDS, 0.2% EDTA, 1% Triton X-100, 1% sodium deoxycholate and 0.01% PMSF) and supplemented with protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride, sodium orthovanadate; Roche Diagnostics, Basel, Switzerland), and centrifuged at 9,660 × g in 4°C (cat. no. 5804R; Eppendorf, Hamburg, Germany) for 15 min to remove nuclei and cell debris. Additionally, tumor samples were subjected to homogenate ahead of total cell extracts. Protein concentration was quantified using the bicinchoninic assay kit (Nanjing KeyGen Biotech Co., Ltd., China), according to the manufacturer's protocol. In total, 50 µg of each protein sample loaded onto SDS-PAGE (10%) and transferred on to polyvinylidene fluoride membranes (Immobilon^®-P; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) using a semi-dry transfer system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Non-specific binding was blocked by incubating the membranes in 1X TBS containing 0.05% Tween-20) supplemented with 5% non-fat dry milk for 1 h. Blots were incubated at 4°C overnight with monoclonal rabbit antibodies against PKM2 (cat. no. 4053, dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, U.S.A.), p-Akt (cat. no. 13038, dilution, 1:1,000; Cell Signaling Technology, Inc.), p-mTOR (cat. no. 5536, dilution, 1:1,000; Cell Signaling Technology, Inc.), HIF-1α (cat. no. 36169, dilution, 1:1,000; Cell Signaling Technology, Inc.) glucose transporter-1 (cat. no. 12939, dilution, 1:1,000; Cell Signaling Technology, Inc.) and lactate dehydrogenase A (cat. no. 3582, dilution, 1:1,000; Cell Signaling Technology, Inc.) and a monoclonal mouse antibody to β-actin (cat. no. sc-130300, 1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.) was used as an internal control. Blots were then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (No. 074-1806, dilution, 1:1,000; KPL, Inc., Gaithersburg, MD, USA) and/or goat anti-rabbit antibody (cat. no. L3012, dilution, 1:1,000; KPL, Inc.). Antibody staining was visualized using enhanced chemiluminescence Western Blot Substrate (Pierce; Thermo Fisher Scientific, Inc.) The images of western blot products were collected and analyzed using Quantity One V4.31 (Bio-Rad Laboratories, Inc.).

BGC-823 cells were seeded at a density of 1×10⁶ cells/well into 6-well plates, prior to treatment with LY294002 (0, 50 or 100 µmol/l) or with DMSO (0.2%) (2 µl/well) for 48 h at 37°C. Subsequent to washing with PBS 3 times, the cells were fixed with cold acetone for 10 min at 4°C. Next, the cells were blocked with 10% normal goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 30 min and probed with an antibody against PKM2 (cat. no. 4053, dilution, 1:100, Cell Signaling Technology, Inc.) at 4°C overnight. Alexa Fluor dye conjugated secondary antibodies (2 mg/ml goat anti-rabbit IgG (H+L) highly cross-absorbed) (cat. no. R37117, Alexa Fluor 594; dilution, 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) were incubated with cells for 1 h at 20°C to enable the samples to be visualized under a fluorescent microscope (Axio Imager A1; Carl Zeiss AG, Oberkochen, Germany). The nuclei were stained using DAPI (2 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min under dark conditions at 20°C.

Total RNA from SGC-7901 cells of the non-transfected, negative control and PKM2-siRNA groups were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacture's protocol. The first-strand cDNA was synthesized using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-primers of LAT-1 mRNAs were designed and synthesized as follows: Forward, 5′-AGTACCATGCGGGACCATC-3′ and reverse, 5′-GCGTTATCCAGCGTGATTTT-3′ (Invitrogen; Thermo Fisher Scientific, Inc.). Real-time quantitative polymerase chain reaction (qRT-PCR) was performed according to the TaqMan Gene Expression Assays protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.) (28). The PCR program was as follows: 95°C for 10 min, followed by 32 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 45 sec. Fold-induction was calculated using the formula 2^−ΔΔCt (29).

BGC-823 cells were seeded at a density of 1×10⁴ cells/well into 96-well plates and subsequently treated with 0 or 50 µmol/l LY294002 for 48 h. SGC-7901 cells of the non-transfected, negative control and PKM2-siRNA groups were cultured for 48 h at 37°C. LDH activity was determined using an LDH Cytotoxicity Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Lactate in the culture medium was determined using a Lactate Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The data are expressed as the mean ± standard deviation, and analyzed by using Least Significant Difference and Student Newman-Keuls methods following analysis of variance and the Student's t-test method. The data were analyzed using SPSS v.19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Western blot analysis demonstrated that, PKM2 expression was significantly increased in GC cell lines (SGC-7901, BGC-823) and HeLa cells, compared with in the GES-1 cells (Fig. 1A; P<0.05).

Cell viability of BGC-823 cells, following LY294002 inhibitor treatment was assessed using a CCK-8 assay. As illustrated in Fig. 1B, LY294002 was able to inhibit the proliferation of BGC-823 cells in vitro. Cell viability in the 20 µmol/l LY294002 group was significantly decreased (91.64±3.06% vs. 100% for the control group; P<0.05), compared with the control group. This was observed as a dose- and time-dependent decrease in the viability of BGC-823 cells following LY294002 treatment. Furthermore, it was demonstrated that, in response to LY294002 treatment, the early apoptotic rate was significantly increased in the BGC-823 cells compared with the control group, in a dose-dependent manner (50 and 100 µmol/l LY294002; Fig. 1C; P<0.05).

To evaluate whether LY294002 can inhibit p-AKT, p-mTOR, HIF-1α and PKM2 expression, BGC-823 cells treated with 0, 10, 20, 50 or 100 µmol/l LY294002 and DMSO for 48 h were assessed via western blotting and immunofluorescence. Following 10 µmol/l LY294002 treatment for 48 h, p-AKT and p-mTOR expression were decreased when compared with in the control group (P<0.05). Furthermore, HIF-1α expression was reduced following treatment with 20 µmol/l LY294002 for 48 h; and PKM2 expression decreased following treatment with 50 µmol/l LY294002 at 48 h. A dose-dependent association was revealed between the LY294002 concentration and the expression levels of p-AKT, p-mTOR, HIF-1α and PKM2 (Fig. 2A). In addition, the intracellular distribution of PKM2 in BGC-823 cells was detected, revealing that PKM2 was distributed around the nucleus and the expression decreased following treatment with LY294002 (Fig. 2B).

To determine the effects of LY294002 on LDH activity and lactate production in BGC-823 cells, LDH activity and lactate production were measured. LDH activity (Fig. 2C) and lactate production (Fig. 2D) in the 50 µmol/l LY294002 group were significantly decreased compared with in the control group (P<0.05).

PKM2 vectors downregulating the plasmid or control plasmid (non-transfected group and empty plasmid group) were transfected into the SGC-7901 cells according to the protocol aforementioned. The expression levels of PKM2 were analyzed by RT-qPCR and immunoblotting. PKM2-siRNA decreased the mRNA (Fig. 3A) and protein (Fig. 3B) levels of PKM2 (P<0.05). A CCK-8 assay demonstrated significant time-dependent inhibition of SGC-7901 cell proliferation following the knockdown of PKM2 (Fig. 4A; P<0.05). It was further revealed that the early apoptosis rate of SGC-7901/PKM2-siRNA cells was increased compared with that of the control cells (Fig. 4B; P<0.05). These results indicated that PKM2 was able to promote the proliferation and suppress the apoptosis of GC cells. To determine whether PKM2 knockdown exerts any influence on the Warburg effect in SGC7901 cells, LDH activity and lactate production were measured. Compared with the control groups, LDH activity (Fig. 4C) and lactate production (Fig. 4D) in SGC7901 cells of the siRNA-PKM2 group was significantly decreased (P<0.05). Western blotting demonstrated that the knockdown of PKM2 significantly decreased Glut-1 and LDHA protein expression levels (Fig. 4E), compared with in the non-transfected or empty plasmid groups. In combination, these outcomes indicate that PKM2-knockdown in SGC-7901 cells was associated with the Warburg effect.