Fig. 5.

HIF-1α is a key regulator of drug-induced metabolic reprogramming. a and b Western blot demonstrated the time-course (a) and dose-dependent (b) induction of HIF1-α by etoposide. c A549 and H1299 cells were transfected with a HRE-luc plasmid containing five synthetic responsive elements of HIF1-α for 24 h and then treated with etoposide for 5 h. Then luciferase activity was immediately measured and normalized using the dual luciferase reporter system. The bars represent the mean ± S.D. of triplicates (*p < 0.05 for difference from untreated cells by ANOVA for multiple comparison). d A549 Cells were treated with etoposide for 5 h before determination of ROS. e A549 and H1299 cells were pretreated with increasing concentrations of N-acetylcysteine (NAC) for 30 min before 25 μM etoposide was added. Western blot was conducted 5 h later to determine levels of HIF-1α, HK2, MCT4, Glut1. f A549 cells were transfected with HIF-1α siRNA for 24 h and further treated with 25 μM etoposide for 36 h. Western blot was performed to examine the expression of HK2, MCT4, Glut1. g The cells were first stimulated either with different concentrations of LA or different degree of acidification by adding HCl into medium for 3 h followed by further treatment with 25 μM etoposide for additional 36 h and then lysed for western blot analysis of MRP1, cleaved-PARP and Snail expression. h Gradual titration of NaHCO₃ to LA-rich medium dramatically sensitize A549 cells to etoposide treatment as indicated by Cleaved-PARP levels. i Cell apoptosis assay. Addition of NaHCO₃ rescued the etoposide-induced apoptosis inhibited by lactate treatment by Annexin-PI dual staining. The quantification was presented in right panel (**p < 0.01, ***p < 0.001, for difference from the untreated cells, ^##p<0.01 for difference from etoposide-transfected cells, ^$$p < 0.01 for difference from the combined etoposide and LA treated cells by ANOVA with Dunnett’s correction for multiple comparisons) j Cell viability assay showed titration of lactic acid with NaNCO₃ rescued the etoposide-induced inhibition of cell viability. Corresponding EC₅₀ values for etoposide, Etoposide+LA and Etoposide+LA + NaNCO₃ are 20.11 μM, 27.99 μM and 19.71 μM respectively