3,6-Dihydroxyflavone induced apoptosis of breast cancer cells in vitro and in vivo. (A) After being treated with 3,6-dihydroxyflavone (3,6-DHF) for 24 hours at increasing concentrations (5, 10, and 20 μM), apoptosis of breast cancer MDA-MB-453 cells increased in a dose-dependent manner. (B) Treatment with 10 μM 3,6-DHF caused the loss of mitochondrial membrane potential in a time-dependent manner. (C) Treatment with 10 μM 3,6-DHF induced cytochrome C release in a time-dependent manner. (D) Detection of tumor xenograft apoptosis by TUNEL assay. (E) Detection of 1-methyl-1-nitrosourea-induced tumor apoptosis by TUNEL assay. Stained nuclei dark brown in color represent apoptotic cells. Arrows indicate apoptotic nuclei. Data presented as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01 compared with control. JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide.