Figure 4. Glucose deprivation potentiates the anticancer activity of 64B.

MDA-MB-231 and A549 cells were cultured in normoglycemic or hyperglycemic DMEM, and were treated with 64B (5 μM) and/or 2-DG (2 mM) for 24 h unless indicated otherwise. A, Knockdown of Glut-1 expression by siGlut-1 was verified by immunofluorescent staining in tumor cells after 48 h. The nuclei of tumor cells were counter-stained with DAPI. B, C, Effect of the co-treatment with siGlut-1 and 64B on the cytotoxicity IC₅₀ of 64B in tumor cells (B) and intracellular ATP level (C). Equal amount of scrambled siRNA (SCR) was used as a control. D, Western blot analysis showing effect of co-treatment with siGlut-1 and 64B on levels of indicated proteins. E, F, Effect of co-treatment with 64B and 2-DG on the cytotoxicity IC₅₀ of 64B in tumor cells (E) and intracellular ATP level (F). G, Western blot analysis showing the effect of combined treatment with 64B and 2-DG on levels of indicated proteins. H, Effect of co-treatment with 64B and 2-DG on the cytotoxicity IC₅₀ of 64B under hypoxia (1% O₂) for 72 h. I, Western blot analysis showing effect of co-reatment with 64B and 2-DG on levels of indicated proteins under hypoxia. All data show representative results obtained from three independent experiments, and the results are reported as the mean ± SD (n = 3). *, p < 0.05. **, p < 0.01.