In vitro activity: Anisomycin (3 μM) decreases protein synthesis in MDA16 and MDA-MB-468 cells, and reduces colony formation by MDA-MB-468 cells. Anisomycin causes an increase in the number of apoptotic cells in MDA-MB-468 cultures, but not in MDA16 cultures. Anisomycin actives JNK phosphorylation in MDA-MB-468 cells. In U251 and U87 cells, anisomycin (0.01-8 μM) inhibits the cell growth in time- and concentration-dependent manners with the IC50 (48 h) values of 0.233 and 0.192 μmol/L, respectively. Anisomycin (4 μM) causes 21.5% and 25.3% of apoptosis proportion in U251 and U87 cells, respectively, and activates p38 MAPK and JNK, while inactivated ERK1/2. Anisomycin (4 μM) reduces the level of PP2A/C subunit in a time-dependent manner in U251 and U87 cells. Anisomycin inhibits EAC cell proliferation in concentration-dependent manner.

Kinase Assay: Cells (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 (final concentration 1% v/v). Puromycin is added (final 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 fluorescence signal for anti-phospho-JNK antibody is background corrected and normalized for loading.

Cell Assay: For the assay, Ehrlich ascites carcinoma (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.