Juglanin is a JNK activator. Juglanin with inflammation and anti-tumor activities. It can induce apoptosis and autophagy on human breast cancer cells.

Juglanin以剂量依赖和时间依赖的方式抑制乳腺癌细胞的增殖，并对正常细胞的毒性较小。Juglanin导致癌细胞在G2/M期的累积，并相应地降低G0/G1和S期的数量，在MCF-7和SKBR3细胞中均是这种情况。Juglanin上调磷酸化的Chk2、Chk2、磷酸化的Cdc25C、磷酸化的Cdc2、p27、cyclin D的表达，并下调Cdc25C以及Cdc2的水平。对照细胞的凋亡比例可以忽略不计，而暴露于Juglanin 24小时后，会导致MCF-7和SKBR3细胞中凋亡细胞的剂量依赖性增加。Juglanin显著抑制抗凋亡因子Bcl-2的表达，相反，Bad和Bax的表达在Juglanin处理下被上调。Juglanin显著激活caspases，并导致Caspase-9、Caspase-8和Caspase-3的切割。Juglanin启动ROS的生成，并且在高浓度下显著增加。Juglanin还增加了MCF-7和SKBR3细胞中JNK磷酸化的水平。

Breast cancer cells are plated in 96-well plates with a density of 5×10^3 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.

Male BALB/c-nude mice, aged 5 weeks, are used. They are maintained under specific pathogen-free conditions and supplied with sterilized food and water. On day 0, about 5×10^6 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 the 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.