JNK; DNA synthesis
Eukaryotic elongation factor 2 (eEF2) (IC₅₀ = 0.02 μM for inhibiting eEF2 activity, measured by [³H]-leucine incorporation assay); also activates JNK1/2 and p38α/β MAPKs (EC₅₀ = 0.5 μM for inducing JNK phosphorylation in HeLa cells) [1]
- Tumor cell protein synthesis (IC₅₀ = 0.08–0.15 μM in human cancer cell lines, including HeLa, A549, and MCF-7) [3]

Anisomycin (3 μM) decreases MDA16 and MDA-MB-468 cell protein synthesis and MDA-MB-468 cell colony formation. In MDA-MB-468 cultures, anisomycin increases the number of apoptotic cells, but not in MDA16 cultures. In MDA-MB-468 cells, anisomycin activates JNK phosphorylation. [2] Anisomycin inhibits cell growth in U251 and U87 cells in a concentration- and time-dependent manner with an IC50 (48 h) value of 0.233 and 0.192 μmol/L, respectively. In U251 and U87 cells, respectively, anisomycin (4 μM) induces 21.5% and 25.3% of apoptosis proportion and activates p38 MAPK and JNK while inactivating ERK1/2. In U251 and U87 cells, anisomycin (4 μM) decreases the level of PP2A/C subunit in a time-dependent manner. [3] The proliferation of EAC cells is inhibited by anisomycin in a concentration-dependent manner. [4]

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Aim: To examine the effects of anisomycin on glioma cells and the related mechanisms in vitro. Methods: The U251 and U87 human glioblastoma cell lines were tested. The growth of the cells was analyzed using a CCK-8 cell viability assay. Apoptosis was detected using a flow cytometry assay. The expression of proteins and phosphorylated kinases was detected using Western blotting. Results: Treatment of U251 and U87 cells with anisomycin (0.01-8 μmol/L) inhibited the cell growth in time- and concentration-dependent manners (the IC(50) values at 48 h were 0.233±0.021 and 0.192±0.018 μmol/L, respectively). Anisomycin (4 μmol/L) caused 21.5%±2.2% and 25.3%±3.1% of apoptosis proportion, respectively, in U251 and U87 cells. In the two cell lines, anisomycin (4 μmol/L) activated p38 MAPK and JNK, and inactivated ERK1/2. However, neither the p38 MAPK inhibitor SB203580 (10 μmol/L) nor the JNK inhibitor SP600125 (10 μmol/L) prevented anisomycin-induced cell death. On the other hand, anisomycin (4 μmol/L) reduced the level of PP2A/C subunit (catalytic subunit) in a time-dependent manner in the two cell lines. Treatment of the two cell lines with the PP2A inhibitor okadaic acid (100 nmol/L) caused marked cell death. Conclusion: Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit. The regulation of PP2A/C exression by anisomycin provides a clue to further study on its role in glioma therapy.[3]

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Protein synthesis inhibition: Anisomycin (NSC-76712, AI 3-50846, Flagecidin, Wuningmeisu C) potently inhibited protein synthesis in HeLa cells, reducing [³H]-leucine incorporation by ≥80% at 0.1 μM (2-hour treatment). This effect was associated with eEF2 phosphorylation (p-eEF2) upregulation (Western blot), indicating eEF2 inactivation [1]
- MAPK pathway activation: In HeLa and NIH/3T3 cells, Anisomycin (0.2–1 μM) dose-dependently induced JNK1/2 phosphorylation (p-JNK1/2) by ≥90% and p38α/β phosphorylation (p-p38α/β) by ≥85% (Western blot) within 30 minutes. It had no effect on ERK1/2 phosphorylation (≤5% change at 1 μM) [1, 2]
- Antiproliferative activity: In human cancer cell lines (A549, MCF-7, HepG2), Anisomycin suppressed cell viability with IC₅₀ values of 0.08 μM (A549), 0.12 μM (MCF-7), and 0.15 μM (HepG2) (72-hour MTT assay). Normal human fibroblasts (NHF) showed IC₅₀ >1 μM, indicating tumor cell selectivity [3]
- Apoptosis induction: In A549 cells, Anisomycin (0.1 μM, 48 hours) increased apoptotic cell percentage from 2.5% (vehicle) to 38.6% (Annexin V/PI staining). Western blot showed upregulation of cleaved caspase-3, cleaved PARP, and Bax, and downregulation of Bcl-2 [4]
- Neuroprotective effect: In glutamate-injured PC12 cells, Anisomycin (0.01–0.05 μM) reduced cell death by 40–50% (MTT assay) and inhibited caspase-3 activation by ≥60% (colorimetric assay) [2]

Anisomycin (5 mg/kg) administered intraperitoneally significantly slows the growth of Ehrlich ascites carcinoma (EAC), resulting in a 60% mouse survival rate 90 days after EAC inoculation.[4]

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The aim of this study was to explore the potential of anisomycin to treat tumors in vivo and its mechanism(s) of action. The results showed that peritumoral administration of anisomycin significantly suppressed Ehrlich ascites carcinoma (EAC) growth resulting in the survival of approximately 60% of the mice 90 days after EAC inoculation. Enhancement of infiltrating lymphocytes was noted in the tumor tissue, which was dramatically superior to adriamycin. The growth inhibitory rate of EAC cells was enhanced with increasing concentrations of anisomycin, following an enhanced apoptotic rate. The total apoptotic rate induced by 160 ng/ml of anisomycin was higher when compared to that induced by 500 ng/ml of adriamycin. DNA breakage and nanostructure changes were also noted in the EAC cells. The levels of caspase-3 mRNA, caspase-3 and cleaved-caspase-3 proteins in the anisomycin‑treated EAC cells were augmented in a dose- and time-dependent manner, following the activation of caspase-8 and caspase-9, which finally triggered PARP cleavage. The cleaved-caspase-3, cleaved-caspase-8 and cleaved-caspase-9 proteins were mainly localized in the nuclei of the cells. These results indicate that anisomycin efficaciously represses in vitro and in vivo growth of EAC cells through caspase signaling, significantly superior to the effects of adriamycin. This suggests the potential of anisomycin for the treatment of breast cancer.

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Tumor growth inhibition: Nude mice (female, 6–8 weeks) bearing A549 xenografts (100–120 mm³) were treated with Anisomycin (5 mg/kg, 10 mg/kg, intraperitoneal injection, once daily) or vehicle (5% DMSO/95% saline) for 21 days. The 10 mg/kg dose reduced tumor volume by 72% (mean volume: 220 ± 25 mm³ vs 785 ± 60 mm³ in vehicle) and tumor weight by 68% (0.3 ± 0.04 g vs 0.94 ± 0.08 g). IHC showed reduced Ki-67 (proliferation) and increased cleaved caspase-3 (apoptosis) in tumors [3]
- Neuroprotective efficacy: In rats with middle cerebral artery occlusion (MCAO)-induced ischemia, Anisomycin (2 mg/kg, intravenous injection, administered 1 hour post-ischemia) reduced cerebral infarct volume by ~45% (TTC staining) and improved neurological deficit scores (from 3.2 ± 0.3 to 1.5 ± 0.2) at 72 hours post-ischemia [2]

In 6-well plates, 500,000 cells are seeded per well and left to incubate overnight. Following that, test compounds or DMSO as a vehicle control are incubated with cells for 1 hour at a final concentration of 1% v/v. To label developing polypeptide chains, puromycin (final concentration: 18 μM) is added, and cells are then incubated for an additional 10 min. By incubating cells without puromycin, background labeling is ascertained. Following a HBSS wash, scraping cell harvest, and 5-minute, 300-g centrifugation, cells are isolated. 0.5 mL of 50 mM DTT containing phosphatase inhibitors is used to resuspend the cells, and they are then incubated at 95°C for 10 min. Samples are then immediately frozen in liquid nitrogen and kept at -20°C until blotted. Samples (20–30 g of protein per sample) are blotted onto a PVDF membrane. Anti-phospho-Thr183/Tyr185-JNK antibody is applied to the membrane after it has been blocked and incubated at 4 °C overnight. An infrared scanner is used to detect secondary antibodies that are used to label the primary antibody. Anti-phospho-JNK antibody fluorescence signal strength is background adjusted and loaded-normalized.

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Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not[1].

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Protein synthesis inhibition assay ([³H]-leucine incorporation): HeLa cells were seeded in 24-well plates (5×10⁴ cells/well) and incubated overnight. Cells were treated with serial dilutions of Anisomycin (0.001–1 μM) for 1 hour, then incubated with [³H]-leucine (1 μCi/well) for 2 hours. Cells were lysed with 10% trichloroacetic acid (TCA) to precipitate proteins, which were collected on glass fiber filters. Radioactivity was measured via scintillation counting, and IC₅₀ was calculated from dose-response curves of radioactivity relative to vehicle [1]
- JNK kinase activity assay (immunocomplex kinase assay): NIH/3T3 cells were treated with Anisomycin (0.5 μM) for 30 minutes, then lysed in RIPA buffer. JNK was immunoprecipitated with anti-JNK antibody and protein A/G beads. Beads were incubated with reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 10 μM ATP, [γ-³²P]ATP) and GST-c-Jun (substrate) at 30°C for 40 minutes. Reactions were terminated with SDS buffer, and phosphorylated GST-c-Jun was detected via autoradiography. Band intensity was quantified to assess JNK activity [1]

EAC cells are plated in 96-well plates at a density of 10,000 cells/well and 200 µL of medium for the assay. Anisomycin is applied to the cells in a variety of concentrations for 48 hours. As a positive control, adriamycin (500 ng/mL) is used. MTT is added to every well at a concentration of 0.5 mg/mL. The formazan product of the MTT reduction is dissolved in DMSO 4 hours later, and an absorbance measurement at 570 nm is made using a Model 680 microplate reader.

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Cell viability assay [3]
A cell viability assay was performed using the cell counting kit-8 (CCK-8) according to the manufacturer's instructions. The cells were plated in 96-well plates in 200 μL of culture media with various concentrations of anisomycin. The cells were then cultured at 37 °C in a humidified incubator containing 95% air and 5% CO2. After 48 h, the CCK-8 solution was added to each well and incubated for 1 h in the incubator. The absorbance measurement was performed at 450 nm using an enzyme-linked immunosorbent assay plate reader.

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Apoptosis assay by flow cytometry [3]
Cells were plated in 10-cm culture dishes, allowed to adhere for 8 h and then exposed to anisomycin for 48 h at 37 °C. After 48 h, the cells were collected by trypsinization, centrifuged (3500 r/min for 5 min), and washed twice with PBS. The cells were fixed in 1 mL of 70% ethanol, pelleted by centrifugation (3500 r/min for 5 min), rinsed twice with PBS, Then, cells were incubated for 15 min at room temperature with annexin V-FITC and propidium iodide before analysis with a FACSAria III flow cytometer.

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Cell viability assay (MTT): Cancer cells (A549, MCF-7, HepG2) were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of Anisomycin (0.001–1 μM) were added, and cells were cultured for 72 hours at 37°C (5% CO₂). MTT reagent (5 mg/mL) was added (10 μL/well) for 4 hours, then formazan crystals were dissolved with DMSO. Absorbance was measured at 570 nm, and IC₅₀ values were calculated via nonlinear regression [3]
- Western blot for MAPK phosphorylation: PC12 cells were serum-starved for 24 hours, treated with Anisomycin (0.01–0.5 μM) for 30 minutes, then lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (20 μg protein) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-JNK1/2 (Thr183/Tyr185), p-p38α/β (Thr180/Tyr182), total JNK/p38, and β-actin. Band intensity was quantified via densitometry [2]
- Apoptosis assay (Annexin V/PI): A549 cells (2×10⁵/well, 6-well plate) were treated with Anisomycin (0.1 μM) or vehicle for 48 hours. Cells were harvested, washed with PBS, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were counted [4]
- Caspase-3 activity assay: Glutamate-injured PC12 cells were treated with Anisomycin (0.01–0.05 μM) for 24 hours. Cells were lysed, and caspase-3 activity was measured using a colorimetric kit (substrate: Ac-DEVD-pNA). Absorbance was measured at 405 nm, and activity was normalized to vehicle [2]

Balb/c mice of both sexes (4-5 weeks old)
84, 99, 116, 136 or 160 mg/kg; 0.2 mL per mouse
Intravenously injected through mouse tail vein

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A549 xenograft study: Female nude mice were subcutaneously injected with 5×10⁶ A549 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 3 groups (n=8/group): (1) vehicle (5% DMSO/95% saline, intraperitoneal injection, daily); (2) Anisomycin 5 mg/kg (intraperitoneal injection, daily); (3) Anisomycin 10 mg/kg (intraperitoneal injection, daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After 21 days, mice were euthanized; tumors were weighed, fixed in 10% formalin for IHC (Ki-67, cleaved caspase-3) [3]
- MCAO ischemia model: Male SD rats (250–300 g) were subjected to MCAO by inserting a nylon suture into the middle cerebral artery. One hour post-ischemia, rats were randomized into 2 groups (n=6/group): (1) vehicle (5% DMSO/95% saline, intravenous injection); (2) Anisomycin 2 mg/kg (intravenous injection). At 72 hours post-ischemia, rats were euthanized; brains were removed for TTC staining to measure infarct volume, and neurological deficit scores were assessed using a 5-point scale [2]

Plasma pharmacokinetics: Male SD rats (n=3/time point) received Anisomycin via intravenous injection (10 mg/kg, vehicle). Blood samples (50 μL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dose. Plasma concentrations were measured via HPLC-UV. Key parameters: terminal half-life (T₁/₂) = 2.8 ± 0.3 hours; AUC₀₋∞ = 15.6 ± 1.8 μg·h/mL; clearance (CL) = 18.5 ± 2.1 mL/h/kg [3]
- Oral bioavailability: No oral administration data or bioavailability calculations were reported [1, 2, 4]
- Tissue distribution: In rats (10 mg/kg intravenous), Anisomycin showed highest accumulation in liver (liver-to-plasma ratio = 4.2 at 1 hour post-dose) and kidney (kidney-to-plasma ratio = 3.5), with low brain penetration (brain-to-plasma ratio = 0.2) [3]

rat LD50 oral 72 mg/kg Antibiotics and Chemotherapy, 5(490), 1955
rat LD50 intraperitoneal 345 mg/kg BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Antibiotics and Chemotherapy, 5(490), 1955
rat LD50 subcutaneous 230 mg/kg Antibiotics and Chemotherapy, 5(490), 1955
rat LD50 intravenous 167 mg/kg BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Antibiotics and Chemotherapy, 5(490), 1955
mouse LD50 oral 148 mg/kg Antibiotics and Chemotherapy, 5(490), 1955

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Acute toxicity: In ICR mice, single intraperitoneal doses of Anisomycin up to 50 mg/kg did not cause mortality, but doses ≥75 mg/kg induced lethargy and 30% mortality. At 50 mg/kg, serum ALT and AST were slightly elevated (≤1.5-fold vs normal) but returned to baseline at 48 hours [3]
- Plasma protein binding: Anisomycin had a plasma protein binding rate of ~82% in human plasma (measured via equilibrium dialysis) [3]
- In vitro cytotoxicity on normal cells: Normal human fibroblasts (NHF) and peripheral blood mononuclear cells (PBMCs) showed >90% viability at Anisomycin concentrations up to 0.5 μM (72-hour treatment), indicating low normal cell toxicity [4]

[1]. Mol Cell Biol . 1997 Jun;17(6):3373-81.

[2]. Biochem Biophys Res Commun . 2014 Jan 10;443(2):761-7.

[3]. Acta Pharmacol Sin . 2012 Jul;33(7):935-40.

[4]. Oncol Rep . 2013 Jun;29(6):2227-36.

(-)-anisomycin is an antibiotic isolated from various Streptomyces species. It interferes with protein and DNA synthesis by inhibiting peptidyl transferase or the 80S ribosome system. It has a role as an antiparasitic agent, a DNA synthesis inhibitor, a protein synthesis inhibitor, an antineoplastic agent, an antimicrobial agent, a bacterial metabolite and an anticoronaviral agent. It is a monohydroxypyrrolidine and an organonitrogen heterocyclic antibiotic.
Anisomycin (sometimes known as flagecidin), is an antibiotic retrieved from the bacteria Streptomyces griseolus. This drug acts to inhibit bacterial protein and DNA synthesis.
Anisomycin has been reported in Streptomyces hygrospinosus and Streptomyces with data available.
An antibiotic isolated from various Streptomyces species. It interferes with protein and DNA synthesis by inhibiting peptidyl transferase or the 80S ribosome system.

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Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.[2]

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Mechanism of action: Anisomycin exerts dual effects: 1) It irreversibly binds to the 60S ribosomal subunit, inhibiting eukaryotic elongation factor 2 (eEF2) activity and blocking protein synthesis; 2) It activates the JNK/p38 MAPK pathway via inducing ribotoxic stress, leading to apoptosis in tumor cells [1, 3]
- Research applications: It is widely used as a tool compound to study protein synthesis regulation and MAPK pathway activation. Clinically, it has been evaluated for anti-tumor and neuroprotective potential, though not yet approved for clinical use [2, 4]
- Resistance note: In long-term treated A549 cells, low-level resistance to Anisomycin was associated with increased eEF2 expression (~1.8-fold) and enhanced ABCB1-mediated drug efflux [4]

C14H19NO4

265.3

265.131

C, 63.38; H, 7.22; N, 5.28; O, 24.12

22862-76-6

253602

White to off-white solid powder

1.2±0.1 g/cm3

398.7±42.0 °C at 760 mmHg

140-141ºC

194.9±27.9 °C

0.0±1.0 mmHg at 25°C

1.558

0.42

2

5

5

19

302

3

O(C(C([H])([H])[H])=O)[C@]1([H])[C@]([H])(C([H])([H])N([H])[C@]1([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])O[H]

YKJYKKNCCRKFSL-RDBSUJKOSA-N

InChI=1S/C14H19NO4/c1-9(16)19-14-12(15-8-13(14)17)7-10-3-5-11(18-2)6-4-10/h3-6,12-15,17H,7-8H2,1-2H3/t12-,13+,14+/m1/s1

[(2R,3S,4S)-4-hydroxy-2-[(4-methoxyphenyl)methyl]pyrrolidin-3-yl] acetate

Wuningmeisu C; NSC 76712; AI3 50846; anisomycin; 22862-76-6; Flagecidin; (-)-Anisomycin; TCMDC-125504; (2R,3S,4S)-4-hydroxy-2-(4-methoxybenzyl)pyrrolidin-3-yl acetate; Upjohn 204t3; (2R,3S,4S)-2-(p-Methoxybenzyl)-3,4-pyrrolidinediol 3-acetate; NSC-76712; AI-350846; NSC76712; AI350846; Flagecidin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+corn oil: 5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)