生物活性

Anisomycin is a phenylpyrolidine derivative shown to strongly activate the stress-induced MAP kinases JNK and p38. This activity results in the induction of the immediate-early genes: c-Fos, c-Jun, Fos B, Jun B and Jun D, and enhanced phosphorylation of p54/nrb, ATF-2, ERK, Caldesmon, IRS-1 and IRS-2. This compound behaves as a true signaling agonist, and has been implicated to play an important role in hyperosmolarity and UV-stimulated responses. Anisomycin has also been shown to increase the phosphorylation of p70 S6 kinase α. Acts as a potent signaling agonist to selectively elicit homologous desensitization of immediate early gene induction (c-fos, fosB, c-jun, junB and junD).

Cell viability

体外研究

体内研究

激酶实验

SUnSETassay and assay for JNK phosphorylation[2]

5x10⁵ cells/well were seeded in6-well plates and incubated overnight. Cells were then incubated for 1 h withtest compounds or DMSO as vehicle control (final concentration 1% v/v). After 1h puromycin was added (final concentration of 18μM) and cells incubatedfor a further 10 min to label nascent polypeptide chains. Background labellingwas determined by incubating cells without puromycin. Cells were then washed inHBSS, harvested by scraping and centrifuged (300g, 5 min). Cells wereresuspended in 0.5 ml 50mM DTT containing phosphatase inhibitors and incubatedat 95^oC for 10 min. Samples were then snap frozen in liquid nitrogenand stored at -20^oC until blotted. Samples (20–30μgprotein/ sample) were blotted onto a PVDF membrane using a vacuum manifold. Themembrane was blocked and incubated with anti-phospho-Thr183/Tyr185-JNK antibodyovernight at 4^oC. Secondary antibodies conjugated to a fluorophorewere used to label the primary antibody and detected using an infrared scanner.The intensity of the fluorescence signal for anti-phospho-JNK antibody wasbackground corrected and normalized for loading.

Rat-1 cells from a 6-cm-diameter tissueculture dishes were harvested by lysis in a solution containing 20mM HEPES-KOH(pH 7.4), 2mM EGTA, 50mM β-glycerophosphate, 1mM dithiothreitol (DTT), 10% glycerol, 1% TritonX-100, 1mM sodium vanadate, 1μM microcystin, 1mM phenylmethylsulfonyl fluoride, 1μgof aprotinin per ml, and 1μg of leupeptin per ml. SAPK/JNK1 was immunoprecipitated for 3 h at4°C with an anti-JNK1 antibody (sc-474) precoupled to protein A-agarose. Theimmunoprecipitates were washed once with lysis buffer, once with a solution consistingof 100mM Tris-HCl (pH 7.6), 500mM LiCl, 1mM DTT, and 0.1% Triton X-100, andonce with a buffer containing 20mM morpholinepropanesulfonic acid (MOPS; pH7.2), 10mM MgCl₂, 2mM EGTA, 1mM DTT, and 0.1% Triton X-100. For thekinase reaction, theimmunoprecipitates were incubated with 1μgof either glutathione S-transferase (GST)-Elk1 or GST-cJun fusion proteins inthe presence of 10mM MOPS (pH 7.2), 20mM MgCl₂, 1mM EGTA, 0.5mM DTT,0.05% Triton X-100 and 1μCi [γ-³²P]ATP for 20 min at 30°C. After the reactions were stoppedby adding 10μl of 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)loading buffer, the samples were resolved by SDS–13% PAGE. The phosphorylatedGST-Elk1 was quantified from dried gels with a Molecular Dynamics PhosphorImagerand IP Lab Gel software.

细胞实验

Cellculture[4]

Wild-type PC12 (wtPC12) ratpheochromocytoma cells and the p143p53PC12 subclone were cultured in Dulbecco’smodified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS) and 10%heat-inactivated horse serum at 37oC in a humidified atmospherecontaining 5% CO2. Cells were treated with 1μg/mlanisomycin for 2, 4, 8, 12, or 24 h. DMEM and anisomycin were purchased fromSigma-Aldrich; FBS and horse serum were obtained from Invitrogen.

DNAfragmentation assay

5x106cells were cultured in100-mm dishes and treated as described in the legend to Fig. 1. At the end ofthe treatment, cells were collected into their medium; low molecular weight DNAfragments were isolated and analyzed by gel electrophoresis in 1.8% agarosegels. DNA fragments were visualized in ethidium bromide stained gels with anUV-transilluminator. Photographs were taken with a Kodak Image Station 440 geldocumentation system.

Cellviability assay

Cell viability was determined by a WST-1reagent. To detect the effect of anisomycin on cell viability, 103cells were plated in 24-well plates. The cells were treated with 1μg/mlanisomycin for 2, 4, 8, 10, or 24 h. simultaneously; the viability ofnon-treated, control cells was assessed. At the end of the exposure period, themedium was replaced in each well by 200μl of theWST-1 reagent (in 1:10 dilution) in a fresh medium, incubated for 4 h, and 100μl aliquotsfrom each well were transferred to a 96-well plate. Absorbance was measured onan ELISA plate reader. The analysis was performed in three independent experiments.

动物实验

Animals[5]

Male BALB/c mice, 6-8 weeks of age,weighing 20±2g, were held for a quarantine period of 1 week at a temperature of25±2˚C, relative humidity of 55±2% and with a 12-h light/12-h dark cycle.

Invivo therapy

The animals were subcutaneously inoculatedwith EAC cells (0.2 ml of 1x107cells/mouse), and divided randomlyinto three groups (n=10). The experimental treatment started when the tumorvolume reached ~50mm3. Anisomycin (5mg/kg), adriamycin or 100μl ofPBS was peritumorally injected into EAC-bearing mice every other day for 7times. The animals were weighed and inspected daily for survival. The solidtumor sizes were measured every day, and the tumor volumes and tumor growthinhibition rate were calculated by the following formula：

Tumor volume (mm3) = (length xwidth2)/2, where the length and width are given in mm.

Tumor inhibition rate (%) = [(Average tumorvolume of the control group - average tumor volume of the test group)/averagetumor volume of the control group] x 100.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Li JY HJ, Li M, Zhang H, Xing B, Chen G, Wei D, Gu PY, Hu WX. Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro. Acta Pharmacol Sin. . 2012;33(7):935-940.
[2] Monaghan D, O'Connell E, Cruickshank FL, et al. Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues. Biochem Biophys Res Commun. 2014;443(2):761-767.
[more]

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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