Fig. 1

Induction of lactate metabolism inhibits genotoxic and apoptotic effects of etoposide in NSCLC. a Treatment of cells with etoposide for 5 h resulted in an increase in lactate secretion (*p<0.05, **P<0.01, ***p<0.001 for difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). b The cells were treated with different concentrations of etoposide or 1% Triton X-100 as positive control for 5 h. Culture medium was collected for determination of released lactate dehydrogenase (LDH) reflecting the membrane rapture. No significant difference of LDH release was detected after etoposide treatment (difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). c The extracellular acidification rate (ECAR) was measured in cells exposed to 25 μM etoposide for 5 h. The concentrations of oligomycin, FCCP were 10 μM and the bar is representative of mean ± S.D. of three independent experiments (*p<0.05, student t-test). d Measurement of 2-DG uptake in cells after exposed etoposide for 5 h. (*p<0.05, **P<0.01, for difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). e Western blot analysis was performed to examine glycolysis enzymes, HK2, MCT4, LDHA, MCT1 and Glut1, in different histological lung cancer cell lines A549, H1299 and H446 treated for indicated time (upper panel, 25 μM) or for various concentrations (lower panel) of etoposide. f The cells were first stimulated either with LA (lactic acid) or NaL (sodium lactate) for 3 h followed by further treatment with 25 μM etoposide for additional 36 h and then lysed for western blot analysis using antibodies against Cleaved-PARP and γH2AX. It is lactic acid, but not its sodium salt, suppressed the levels of γH2AX in both A549 and H1299 cell lines. g Comet assay reveal that 20 mM LA (lactic acid) inhibited DNA damage induced by etoposide. The quantification was present in right panel. The bars represent the mean ± S.D. of triplicates (**p<0.01 for difference from control cells, ^##p<0.01 for difference from etoposide-treated cells by ANOVA with Dunnett’s correction for multiple comparisons). h A549 and H1299 were incubated first with etoposide for 5 h and then subjected to LA (lactic acid) exposure in the presence of etoposide for additional 36 h. The results revealed that etoposide induced γ-H2AX was not inhibited by lagging treatment with LA. i A549 and H1299 cells were pre-treated with LA (20 mM) for 3 h before etoposide treatment for 36 h. Flow cytometric analysis of cell death showed that lactate significantly reduced etoposide-induced apoptosis in A549 but not in H1299 cells. The quantification was present in right panel. The bars represent the mean ± S.D. of triplicates (**p<0.01 for difference from control cells, ^##p<0.01 for difference from etoposide-treated cells by ANOVA with Dunnett’s correction for multiple comparisons, ns means no statistical difference). j Cell viability assay showed that LA significantly increased A549 rather than H1299 cell viability. In A549 cells, the EC₅₀ (concentration for 50% of maximal effect) of etoposide was 19.87 μM, the cell survival was increased in the presence of LA with EC₅₀ of 28.07 μM. However, the LA stimulation had not effect of EC₅₀ values in H1299 cells. (*p<0.05, for difference from untreated control by ANOVA with Dunnett’s correction for multiple comparisons). k Cells were treated with different concentrations of LA for 3 h in the absence and presence of 25 μM etoposide. ATM and p-ATM expression were examined by western blot. l 24 h after transfection with wild-type p53 plasmids, cells were treated with etoposide in the absence and presence of 20 mM LA. The expression levels of Cleaved-PARP, γ-H2AX, ATM, p-ATM were analyzed by western blot