Anisomycin (Flagecidin) is a bacterial antibiotic, an inhibitor of protein synthesis and a JNK activator. Anisomycin interferes with the synthesis of proteins and DNA by inhibiting the peptidyl transferase or 80S ribosomal system.

方法：黑色素瘤细胞系 FEMX-1 和 WM239 用 Anisomycin (40 nM) 和 lexatumumab (0.75 ng/mL) 处理 48 h，使用 tetrazolium salt reagent 检测细胞活力。
结果：HepG2、Huh7 和 SNU449 细胞都对 Anisomycin 敏感，IC50 值分别为 82.2、118 和 138 nM。[1]
方法：HCC 细胞 HepG2、Huh7 和 SNU449 用 Anisomycin (0-1000 nM) 处理 48 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：Cryptotanshinone 处理后，G2/M 期相关蛋白细胞周期蛋白 B1、CDK1 和 CDC25C 的表达下调，而 p21 (CIP1/WAF1) 上调。关于凋亡相关蛋白，Cryptotanshinone 处理后，Bcl-2 的表达下调，而 p53 和 Bax 的表达上调。[2]

方法：为检测体内抗肿瘤活性，将 Anisomycin (10 mg/kg) 腹腔注射给携带 HepG2 异种移植物的 NOD-SCID 小鼠，每天一次，在治疗开始后第 0 至 5 天和第 15 至 20 天给药。NK 细胞在给药暂停期的第 6 天和第 11 天两次转移到小鼠中。
结果：Anisomycin 显著降低了小鼠的 HepG2 肿瘤大小。在人类原代 NK 细胞存在的情况下，Anisomycin 对肿瘤的抑制协同增强。NK 细胞在 Anisomycin 对 HCC 的抗肿瘤作用中起着关键作用。[3]

JNK phosphorylation: 500,000 cells/well are seeded in 6-well plates and incubated overnight. Cells are then incubated for 1 h with test compounds or DMSO as vehicle control (?nal concentration 1% v/v). Puromycin is added (?nal concentration of 18 μM) and cells incubated for a further 10 min to label nascent polypeptide chains. Background labelling is determined by incubating cells without puromycin. Cells are then washed in HBSS, harvested by scraping and centrifuged (300 g, 5 min). Cells are resuspended in 0.5 mL 50 mM DTT containing phosphatase inhibitors and incubated at 95℃ for 10 min. Samples are then snap frozen in liquid nitrogen and stored at -20℃ until blotted. Samples (20–30 μg protein/sample) are blotted onto a PVDF membrane. The membrane is blocked and incubated with anti-phospho-Thr183/Tyr185-JNK antibody overnight at 4℃. Secondary antibodies are used to label the primary antibody and detected using an infrared scanner. The intensity of the ?uorescence signal for anti-phospho-JNK antibody is background corrected and normalized for loading.

For the assay, EAC cells are plated in 96-well plates at a density of 10,000 cells/well/200 μL of medium. The cells are treated with the different concentrations of anisomycin for 48 h. Adriamycin (500 ng/mL) is used as a positive control. 0.5 mg/mL of MTT is added to each well. 4 h later, the formazan product of MTT reduction is dissolved in DMSO, and absorbance is measured at 570 nm using a Model 680 microplate reader.(Only for Reference)