FL-411 (BRD4-IN-1)

Cell experiment:

The MCF-7 and MDA-MB-231 cells are dispensed in 96-well flat bottom microtiter plates at a density of 5×104 cells/mL. After 24 h incubation, MCF-7 or MDA-MB-231 cells are treated with 1.5 and 3 μM FL-411, respectively. 3-MA (1 mM) is added 1 h before treated with FL-411. After treatment, cell viability is measured by the MTT assay. 5 mg/mL MTT is added to each well. After 4 h incubation, the medium is removed and 150 μL of DMSO is added to each well to dissolve the crystal formazan dye. Absorbance is measured at 570 nm on an enzyme-linked immunosorbent assay reader[1].

Animal experiment:

Mice[1]52 female nude mice (BALB/c, 6-8 weeks, 20-22 g) are injected subcutaneously with MCF-7 cells or MDA-MB-231 cells (5×106 cells/mouse), respectively. When the tumors reach 100 mm3 in volume, the mice are divided into four groups for each cell line. Three groups are treated with different doses of FL-411 (low dose, 25 mg/kg; median dose, 50 mg/kg; high dose, 100 mg/kg) once a day by intragastric administration for 24 or 27 days, whereas the control group is treated with vehicle control. During the treatment, the tumor volumes and body weight are measured every 3 days until the end of the study. At the end of treatment, all mice are sacrificed. The tumor tissues are harvested, weighed, and photographed. Then, the tumor tissues are frozen in liquid nitrogen or fixed in formalin immediately[1].

References:

[1]. Ouyang L, et al. Discovery of a Small-Molecule Bromodomain-Containing Protein 4 (BRD4) Inhibitor That InducesAMP-Activated Protein Kinase-Modulated Autophagy-Associated Cell Death in Breast Cancer. J Med Chem. 2017 Dec 28;60(24):9990-10012.