Juglanin

Cell experiment:

Breast cancer cells are planted in 96-well plates with a density of 5×103 cells/well. After 12 h, the cells are treated with different concentrations of Juglanin (0 to 40 μM) for different periods of time (24 h and 48 h). Then the fresh mixture of MTS and PMS is added and incubated for 4 h at 37°C according to the manufacturer's protocol. A microplate reader is conducted to determine the absorbance at 500 nm, and the IC50 values are assessed with the probit model. Each one is performed in triplicate[1].

Animal experiment:

Male BALB/c-nude mice, aged 5 weeks, are used. They are maintained under specific pathogenfree conditions and supplied with sterilized food and water. On day 0, about 5×106 MCF-7 cells suspended in 0.1 mL PBS are inoculated subcutaneously in the right flank of each mouse (six mice each group). On day 9, when the tumors reach palpable size of around 200 mm3, mice are randomly assigned to three groups and receive daily intraperitoneal injection with 100 μL of vehicle (10% DMSO, 70% Cremophor/ethanol (3: 1), and 20% PBS), and 5 or 10 Juglanin mg/kg of celastrol. Tumor sizes are measured daily to observe dynamic changes in tumor growth. Body weights are also measured daily. After 7 days of drug administration, when the tumors of control group reach around 1600 mm3, all mice are killed. Tumors are dissected and stored in liquid nitrogen or fixed in formalin for further analysis[1].

References:

[1]. Sun ZL, et al. Juglanin induces apoptosis and autophagy in human breast cancer progression via ROS/JNK promotion. Biomed Pharmacother. 2017 Jan;85:303-312.