Anisomycin is an antibiotic and protein synthesis inhibitor produced by Streptomyces griseolus. It is also a classic activator of p38 MAPK and JNK. By inhibiting protein synthesis, anisomycin induces cellular stress, which activates upstream kinases and subsequently leads to the phosphorylation and activation of p38 MAPK and JNK.

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)