Triple-negative breast cancer (TNBC) cells are more sensitive to 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP) than other subtypes of breast cancer cells due to its effect on ATP level and their dependence on glycolysis for survival. (A) 3-BrOP caused more severe depletion of ATP in TNBC and SKBR3 cells (Group I: BT20, MDA468, MDA231, MDA436, SKBR3) compared to estrogen receptor (ER)/progesterone receptor (PR)-positive cells (Group II: BT474, MCF7, T47D, ZR751). Breast cancer cells were treated with 3-BrOP at the indicated concentration for 8 hours. Intracellular ATP level was measured; bar graph represents mean ± SD from three experiments with triplicate wells for each assay. (B) TNBC cells are more dependent on glycolysis for survival. Top panel, confirmation of HKII downregulated expression by western blotting in ER-positive cells (MCF7) and TNBC cells (MDA436) after HKII siRNA transfection (72 hours). Data are representative of at least three independent experiments. Bottom panel, TNBC cells are more dependent on HKII for survival assessed by annexin-V/Pi staining. MCF7 and MDA436 cells were transfected with HKII siRNA or scramble siRNA and 72 hours after transfection cell death was detected by flow cytometric analysis using annexin V/PI double staining (bar graph represents mean ± SD from three experiments). (C) Representative profile of fluorescence-activated cell sorting analysis after Annexin-V/PI staining. 3-BrOP is more toxic to cancer cells than normal cells. MDA436 cells and immortalized human mammary epithelial cells (HMLE) were treated with 50 μM 3-BrOP (24 h). Cell death was detected by flow cytometric analysis using annexin V/PI double staining.