Isorhamnetin

Cell experiment:

MCF7, T47D, BT474, BT-549, MDA-MB-231 and MDA-MB-468 breast cancer cell lines, as well as a MCF10A normal breast epithelial cell line (control) are seeded into 96-well plates at a density of 5×103 cells/well in 100 µL DMEM and placed in cell incubator for 12 h at 37°C in an atmosphere containing 5% CO2. The cells are then treated with various concentrations of Isorhamnetin (100, 33.3, 11.1, 3.7, 1.2, 0.4 and 0 µM) for 48 h, and cell proliferation rates are determined by adding 10 µL CCK-8 solution prior to incubation at 37°C for 2 h. The absorbance is measured at a wavelength of 450 nm using a SpectraMax 190 Microplate Reader. For each assay, four parallel wells are included, and the half maximal inhibitory concentration (IC50) is measured using the inhibition curve and presented as the mean of three independent experiments[2].

Animal experiment:

Mice[1] Female athymic nude mice are injected subcutaneously in the flank with A431 cells (1×106 cells in 50 μL of medium and 50 μL of Matrigel). Cells are allowed to form tumors, and once the tumors reach a size of 40 mm3, the mice are randomly assigned into groups (6 mice/group) and treated with (1 or 5 mg/kg body weight) or without Isorhamnetin in 40% DMSO/PBS buffer, administered intraperitoneally every other day for 28 days. Tumor size is measured every week with calipers, and the tumor volume is calculated. Mice are sacrificed after 28 days of treatment when the control tumors reach approximately 600 mm3. The tumors are harvested, photographed, and weighed. Tumor tissues are used for western blot analysis and immunohistochemical analysis.

References:

[1]. Kim JE, et al. Isorhamnetin suppresses skin cancer through direct inhibition of MEK1 and PI3-K. Cancer Prev Res (Phila). 2011 Apr;4(4):582-91.
[2]. Hu S, et al. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen activated protein kinase kinase signaling pathways. Mol Med Rep. 2015 Nov;12(5):6745-51.