The GC cell lines (AGS, BGC-823, MGC-803 and SGC-7901) were purchased from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). The normal gastric mucosa endothelial cell line GES-1 was purchased from Beijing Cancer Institute (Beijing, China). All primary antibodies were purchased from Bioworld Technology (St. Louis Park, MN, USA). The secondary antibodies were purchased from Beijing Zhongshan Biotechnology Co., Ltd. (Beijing, China). RIPA lysis buffer and protease phenylmethanesulfonyl fluoride (PMSF) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). The Super ECL kit was purchased from Applygen Technologies Inc. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) without glucose and pyruvate was purchased from Gibco-BRL (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). L(+)-Lactic Acid assay kit and D-galactose and glucose powder were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neutral red staining solution was purchased from Beyotime Institute of Biotechnology. Transwell migration chamber, Annexin V-FITC and propidium iodide (PI) were purchased from Invitrogen Corporation (Carlsbad, CA, USA). The PCR primers, reverse transcription kit, and qPCR reaction kits were purchased from Takara Biotechnology Co. Ltd. (Dalian, China). The primer sequences used were: LDH-A forward, 5′-GGTTGGTGCTGTTGGCAT GG-3′ and reverse, 5′-TGCCCCAGCCGTGATAATGA-3′; β-actin forward, 5′-TGGCACCCAGCACAATGAA-3′ and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′.

Two different culture media were used: DMEM (containing 25 mM glucose) and glucose-free DMEM (supplemented with 10 mM galactose, for the neutral red staining assay). Both media were deprived of sodium pyruvate, supplemented with 10% FBS, 100 IU/l penicillin and 100 IU/l streptomycin. All cells were cultured under standard conditions in humidified 5% CO₂ at 37°C.

The effect of LDH-A inhibitor oxamate on GC cells was tested in glucose-free condition in which 10 mM of galactose was added to the glucose-free DMEM medium.

Total RNA was extracted with TRIzol reagent according to the manufacturer’s instructions. Approximately 30 ng of total RNA was reverse-transcribed into cDNA. The synthesized cDNA samples were subjected to qPCR using a SYBR-Green quantitative PCR kit. Amplification was carried out in a total volume of 20 μl for 40 cycles of 15 sec at 95°C, 20 sec at 60°C and 30 sec at 72°C. Samples were run in triplicate and the relative expression of target genes was determined by normalizing expression of each target to that of β-actin. The amplification was monitored on a Rotor-Gene real-time PCR apparatus (Rotor-Gene, Australia).

The effect of modulating LDH-A activity on cell proliferation was tested in AGS and SGC-7901 cells by neutral red staining assay (16). Briefly, ~5×10⁴ cells suspended in 200 μl of DMEM containing 25 mM glucose or 10 mM galactose were seeded into each well of a 96-well plate and incubated under standard growth conditions overnight. Cells were treated with various concentrations of oxamate (0–100 mM) for 24 h, incubated with neutral red, and the absorbance was read at 490 nm in a microplate reader. The IC₅₀ value (dose needed for 50% growth inhibition) was determined using CalcuSyn software (17).

For morphological analysis, ~1×10⁶ cells/well were grown in a 6-well plate and were treated with oxamate (0, 20, 40, 60, 80 and 100 mM) for 24 h. Cells were observed under an inverted phase contrast microscope (CHK-213; Olympus, Japan).

Approximately 5×10⁴ cells in 200 μl culture medium containing 25 mM glucose were seeded into each well of a 96-well plate and incubated overnight. Cells were then treated with various concentrations of oxamate (0, 20, 40, 60 and 80 mM). Lactic acid secreted into the culture medium was measured at the start of the experiment and 4 h after oxamate treatment. The concentration of lactic acid was determined by an enzyme assay kit, and the absorbance of NADH formation was measured in a microplate reader at 450 nm. The amounts of lactic acid were calculated by substracting the value measured at the start time from that of the 4-h treatment and the final concentration of lactic acid was normalized against the cell number. Inhibition rate (%) of lactic acid production was defined as (control group − experimental group)/control group × 100%.

The effect of LDH-A inhibition by oxamate on the migratory effect of SGC-7901 cells was examined by the Transwell migration assay. Briefly, cells were placed onto 8-μm pore sized Transwell filters at a concentration of 2×10⁵ cells/well in 250 μl of FBS-free DMEM medium. To the lower chamber, 1 ml of medium with 10% FBS and various concentrations of oxamate were added. After 24 h, the cells that transmigrated through the membrane to the lower side of the filter were stained and counted under a microscope. Incorporated dye was extracted with cell detachment buffer, and the absorbance of the released dye was read in a micro-plate reader at 550 nm.

The effect of oxamate on apoptosis was detected by flow cytometry following Annexin V-FITC and PI staining. Briefly, cells were incubated with various concentrations of oxamate for 24 h, washed and resuspended in 0.5 ml PBS, and stained in Annexin V-FITC and PI buffer. Cells were then analyzed by flow cytometry.

Whole-cell extracts were prepared from the treated cells with 2 ml of RIPA buffer containing protease inhibitors. Cell lysates were centrifuged at 8,000 rpm for 10 min, and the total protein concentrations in the supernatant were determined. An equal amount of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% fat-free milk at room temperature for 1 h, incubated with the respective primary antibodies against LDH-A and the apoptosis-associated protein, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (all diluted at 1:1,000) for 2 h at room temperature. β-actin was used as a loading control. The membranes were detected using an enhanced chemiluminescence (ECL) system.

All experiments were repeated for three times. Data are presented as means ± standard deviation (SD). Statistical comparisons were performed using analysis of variance (ANOVA) with a post-hoc test. A p-value <0.05 was considered to indicate a statistically significant result. All data were analyzed using SPSS software (version 11.0).

Initially, LDH-A mRNA expression was examined in four GC cell lines (AGS, BGC-823, MGC-803 and SGC-7901) and a normal gastric mucosal cell line (GES-1) by qPCR and western blotting. As shown in Fig. 1, a significantly increased expression of LDH-A mRNA (Fig. 1A) and protein (Fig. 1B) were found in the GC cells compared with these levels in the GES-1 cells, with the highest expression level of LDH-A being found in AGS and SGC-7901 cells. Thus, most of the following experiments were performed in these two cell lines.

To test the effect of LDH-A blockade on cell proliferation, AGS and SGC-7901 cells were treated for 24 h with various concentrations of oxamate in the presence or absence of glucose. As shown in Fig. 2, in the presence of glucose, oxamate inhibited cell proliferation in a dose-dependent manner. The IC₅₀ value of oxamate for AGS cells was 38.11 mM (r=0.9990) (Fig. 2A), and 49.26 mM (r=0.9963) for SGC-7901 (Fig. 2B) cells.

As revealed by phase contrast microscopy, treatment of SGC-7901 cells with 40 mM of oxamate for 24 h led to a significant detachment (Fig. 3B), compared to the control SGC-7901 cells (non-oxamate-exposed cells, Fig. 3A). Cells treated with 20 mM of oxamate only showed a decrease in the cell number without morphological changes (Fig. 3C).

As shown in Fig. 4, treatment of SGC-7901 cells with oxamate led to a reduced production of lactic acid. When exposed to 40 mM (IC₅₀ value) of oxamate for 4 h, the production of lactic acid was reduced by 50%. The effective concentration of oxamate to suppress lactic acid production was similar to that previously reported (18,19). Hence, oxamate inhibits glycolysis in SGC-7901 cells.

Treatment of SGC-7901 cells with 40 mM of oxamate for 24 h resulted in a 50% reduction in cell migration (Fig. 5).

As revealed by flow cytometric analysis (Fig. 6) treatment of SGC-7901 cells with 40 mM of oxamate caused a marked increase in the proportion of early apoptotic cells, compared to the proportion in the control cells (54.29±7.90 vs. 10.95±2.08%, respectively, p<0.001). The pro-apoptotic effect of oxamate was also demonstrated by concomitant changes in the expression of apoptosis-associated proteins, as detected by western blot assay (Fig. 7). As exemplified in SGC-7901 cells, oxamate significantly decreased Bcl-2 expression, yet enhanced the expression of Bax and caspase-3.