Acute physical exercise prevents memory amnesia caused by protein synthesis inhibition in rats' hippocampus.

The benefits of physical exercise (PE) on memory consolidation have been well-documented in both healthy and memory-impaired animals. However, the underlying mechanisms through which PE exerts these effects are still unclear. In this study, we aimed to investigate the role of hippocampal protein synthesis in memory modulation by acute PE in rats. After novel object recognition (NOR) training, rats were subjected to a 30-min moderate-intensity acute PE on the treadmill, while control animals did not undergo any procedures. Using anisomycin (ANI) and rapamycin (RAPA), compounds that inhibit protein synthesis through different mechanisms, we manipulated protein synthesis in the CA1 region of the hippocampus to examine its contribution to memory consolidation. Memory was assessed on days 1, 7, and 14 post-training. Our results showed that inhibiting protein synthesis by ANI or RAPA impaired NOR memory consolidation in control animals. However, acute PE prevented this impairment without affecting memory persistence. We also evaluated brain-derived neurotrophic factor (BDNF) levels after acute PE at 0.5h, 2h, and 12h afterward and found no differences in levels compared to animals that did not engage in acute PE or were only habituated to the treadmill. Therefore, our findings suggest that acute PE could serve as a non-pharmacological intervention to enhance memory consolidation and prevent memory loss in conditions associated with hippocampal protein synthesis inhibition. This mechanism appears not to depend on BDNF synthesis in the early hours after exercise.

Anisomycin inhibits the activity of human ovarian cancer stem cells via regulating antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling.

BACKGROUND: Ovarian cancer stem cells (OCSCs) are the main cause of relapse and drug resistance in patients with ovarian cancer. Anisomycin has been shown to be an effective antitumor agent, but its mechanism of action in ovarian cancer remains elusive. METHODS: CD44+/CD133+ human OCSCs were isolated from human ovarian cancer tissues. OCSCs were interfered with using anisomycin and specific small-interfering RNA (siRNA). Microarray assay, MTT, in vivo tumorigenic experiments, transwell assay, cell cycle assay, colony formation assay, angiogenesis assay, and hematoxylin and eosin staining were used to detect the mechanism of anisomycin with respect to inhibiting the activity of OCSCs. Expression of the NCBP2-AS2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) pathway was examined using western blotting, a quantitative real-time PCR (RT-qPCR) and immunofluorescence staining. Bioinformatics analysis was used for predictive analysis of NCBP2-AS2 expression in urogenital tumors. RESULTS: Microarray analysis showed that treatment with anisomycin significantly decreased the expression of antisense RNA NCBP2-AS2 in OCSCs. In vitro cellular experiments showed that interfering with endogenous antisense RNA NCBP2-AS2 using siRNA distinctly inhibited the proliferation, migration and angiogenesis of OCSCs, whereas in vivo animal experiments revealed decreased tumorigenesis in nude mice. Moreover, the results of RT-qPCR and western blotting demonstrated that both anisomycin treatment and NCBP2-AS2 silencing led to significant reductions in the mRNA and protein expression levels of NCBP2-AS2, MEK, ERK and STAT3. From a bioinformatic point of view, antisense RNA NCBP2-AS2 exhibited significantly differential expression between urogenital tumors and normal controls, and a similar expression pattern was found in the genes NCBP2, RPL35A, DNAJC19 and ECE2, which have similarity to NCBP2-AS2. CONCLUSIONS: Anisomycin suppresses the in vivo and in vitro activity of human OCSCs by downregulating the antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling pathway, whereas the antisense RNA NCBP2-AS2 and genes with similarity have the potential to serve as markers for clinical diagnosis and prognosis of urogenital tumors.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Cordycepin buffers anisomycin-induced fear memory deficit by restoring hippocampal BDNF.

The process of memory consolidation involves the synthesis of new proteins, and interfering with protein synthesis through anisomycin can impair memory. Memory deficits due to aging and sleep disorders may also result from a reduction in protein synthesis. Rescuing memory deficits caused by protein synthesis deficiency is therefore an important issue that needs to be addressed. Our study focused on the effects of cordycepin on fear memory deficits induced by anisomycin using contextual fear conditioning. We observed that cordycepin was able to attenuate these deficits and restore BDNF levels in the hippocampus. The behavioral effects of cordycepin were dependent on the BDNF/TrkB pathway, as demonstrated by the use of ANA-12. Cordycepin had no significant impact on locomotor activity, anxiety or fear memory. Our findings provide the first evidence that cordycepin can prevent anisomycin-induced memory deficits by regulating BDNF expression in the hippocampus.

Anisomycin inhibits Coxsackievirus B replication by promoting the lysosomal degradation of eEF1A1.

Group B Coxsackieviruses (CVB) are non-enveloped small RNA viruses in the genus Enterovirus, family Picornaviridae. CVB infection causes diverse conditions from common cold to myocarditis, encephalitis, and pancreatitis. No specific antiviral is available for the treatment of CVB infection. Anisomycin, a pyrrolidine-containing antibiotic and translation inhibitor, was reported to inhibit the replication of some picornaviruses. However, it is unknown if anisomycin can act as an antiviral against CVB infection. Here we observed that anisomycin showed potent inhibition on CVB type 3 (CVB3) infection with negligible cytotoxicity when applied at the early stage of virus infection. Mice infected with CVB3 showed markedly alleviated myocarditis with reduced viral replication. We found that CVB3 infection significantly increased the transcription of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). CVB3 replication was suppressed by EEF1A1 knockdown, while elevated by EEF1A1 overexpression. Similar to the effect of CVB3 infection, EEF1A1 transcription was increased in response to anisomycin treatment. However, eEF1A1 protein level was decreased with anisomycin treatment in a dose-dependent manner in CVB3-infected cells. Moreover, anisomycin promoted eEF1A1 degradation, which was inhibited by the treatment of chloroquine but not MG132. We demonstrated that eEF1A1 interacted with the heat shock cognate protein 70 (HSP70), and eEF1A1 degradation was inhibited by LAMP2A knockdown, implicating that eEF1A1 is degraded through chaperone-mediated autophagy. Taken together, we demonstrated that anisomycin, which inhibits CVB replication through promoting the lysosomal degradation of eEF1A1, could be a potential antiviral candidate for the treatment of CVB infection.

Morroniside inhibits Beclin1-dependent autophagic death and Bax-dependent apoptosis in cardiomyocytes through repressing BCL2 phosphorylation.

Morroniside can prevent myocardial injury caused by ischemia and hypoxia, which can be used to treat acute myocardial infarction (AMI). Hypoxia can cause apoptosis and autophagic death of cardiomyocytes. Morroniside has the ability to inhibit apoptosis and autophagy. However, the relationship between Morroniside-protected cardiomyocytes and two forms of death is unclear. The effects of Morroniside on the proliferation, apoptosis level, and autophagic activity of rat cardiomyocyte line H9c2 under hypoxia were first observed. Next, the roles of Morroniside in the phosphorylation of JNK and BCL2, BCL2-Beclin1, and BCL2-Bax complexes as well as mitochondrial membrane potential in H9c2 cells were evaluated upon hypoxia. Finally, the significance of BCL2 or JNK in Morroniside-regulated autophagy, apoptosis, and proliferation in H9c2 cells was assessed by combining Morroniside and BCL2 competitive inhibitor (ABT-737) or JNK activator (Anisomycin). Our results showed that hypoxia promoted autophagy and apoptosis of H9c2 cells, and inhibited their proliferation. However, Morroniside could block the effect of hypoxia on H9c2 cells. In addition, Morroniside could inhibit JNK phosphorylation, BCL2 phosphorylation at the Ser70 and Ser87 sites, and the dissociation of BCL2-Beclin1 and BCL2-Bax complexes in H9c2 cells upon hypoxia. Moreover, the reduction of mitochondrial membrane potential in H9c2 cells caused by hypoxia was improved by Morroniside administration. Importantly, the inhibited autophagy, apoptosis, and promoted proliferation in H9c2 cells by Morroniside were reversed by the application of ABT-737 or Anisomycin. Overall, Morroniside inhibits Beclin1-dependent autophagic death and Bax-dependent apoptosis via JNK-mediated BCL2 phosphorylation, thereby improving the survival of cardiomyocytes under hypoxia.

Fluorofenidone protects against acute liver failure in mice by regulating MKK4/JNK pathway.

AIMS: Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms. METHODS: ALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies. RESULTS: AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing. CONCLUSIONS: In summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.

Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway.

The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is overexpressed in malignant tumors. We previously reported that non-canonical EphA2 phosphorylation at Ser-897 was catalyzed by p90 ribosomal S6 kinase (RSK) via the MEK-ERK pathway in ligand- and tyrosine kinase-independent manners. Non-canonical EphA2 activation plays a key role in tumor progression; however, its activation mechanism remains unclear. In the present study, we focused on cellular stress signaling as a novel inducer of non-canonical EphA2 activation. p38, instead of ERK in the case of epidermal growth factor signaling, activated RSK-EphA2 under cellular stress conditions, including anisomycin, cisplatin, and high osmotic stress. Notably, p38 activated the RSK-EphA2 axis via downstream MAPK-activated protein kinase 2 (MK2). Furthermore, MK2 directly phosphorylated both RSK1 Ser-380 and RSK2 Ser-386, critical residues for the activation of their N-terminal kinases, which is consistent with the result showing that the C-terminal kinase domain of RSK1 was dispensable for MK2-mediated EphA2 phosphorylation. Moreover, the p38-MK2-RSK-EphA2 axis promoted glioblastoma cell migration induced by temozolomide, a chemotherapeutic agent for the treatment of glioblastoma patients. Collectively, the present results reveal a novel molecular mechanism for non-canonical EphA2 activation under stress conditions in the tumor microenvironment.

Effects of delta-9 tetrahydrocannabinol on fear memory labilization and reconsolidation: A putative role of GluN2B-NMDA receptor within the dorsal hippocampus.

Cannabis preparations could be an effective reconsolidation-based treatment for post-traumatic stress disorder. However, the effects of Δ9-tetrahydrocannabinol (THC) in fear memory labilization, a critical condition for retrieval-induced reconsolidation, are undetermined. We sought to investigate the effect of a conventional and an ultra-low dose of THC in memory labilization of adult male Wistar rats submitted to contextual fear conditioning. Pretreatment with THC 0.002, but not THC 0.3 mg/kg, i. p., before memory retrieval, did not change memory expression during the retrieval but impaired reconsolidation. No treatment changed freezing expression in an unpaired context. Before retrieval, THC 0.3, but not THC 0.002, decreased GluN2A-NMDA expression and the GluN2A/GluN2B ratio in the dorsal hippocampus (DH) 24 h later. No changes were observed immediately after retrieval. Pretreatment with THC 0.3 abolished the reconsolidation-impairing effect of anisomycin injected into the DH, suggesting an impairment in memory labilization. This effect was associated with an increased freezing expression in the unpaired context and was not observed with the THC ultra-low dose. The GluN2B-NMDA antagonism increased fear generalization in the anisomycin-treated group but restored its reconsolidation-impairing effect and reduced fear generalization when animals were pretreated with THC 0.3. GluN2A-NMDA antagonism or inhibition of the ubiquitin-proteasome system in the DH did not interfere with the effects of THC 0.3. Our findings indicate that THC causes a bidirectional effect on fear memory labilization that depends on hippocampal GluN2B-NMDA receptors' involvement in fear memory generalization.

Zinc supplementation ameliorates sorafenib-induced cognitive impairment through ROS/JNK signaling pathway.

Sorafenib, a multiple kinase inhibitor, is widely used in cancer patients. Recently, clinical studies highlighted the relationship between cognitive deficits and sorafenib exposure. Zinc abundant in the body has been reported to exert neuroprotective activities. However, the effects of zinc supplementation on sorafenib-induced cognitive impairment are still unknown. In the current study, we verified that mice challenged with sorafenib displayed characteristic features of cognitive impairment. However, zinc treatment effectively improved these changes. Histopathological staining also showed that zinc significantly alleviated hippocampal microstructural and ultrastructural damages induced by sorafenib. Meanwhile, zinc significantly reduced sorafenib-induced ROS production and neuronal cells apoptosis in vivo and vitro. Additionally, we also investigated whether zinc protected against sorafenib-induced neuronal cells apoptosis via ROS/JNK pathway through treating SH-SY5Y cells with the NAC or the specific JNK activator anisomycin. The results indicated that NAC performed the same protective effects as zinc in sorafenib-challenged SH-SY5Y cells and activation of JNK by anisomycin partly abolished the protective effects of zinc. Collectively, the present study suggested that inhibition of oxidative stress and the JNK pathway might contribute to the protective effects of zinc against sorafenib-caused cognitive impairment in vivo and vitro.

Modulation of the p38 MAPK Pathway by Anisomycin Promotes Ferroptosis of Hepatocellular Carcinoma through Phosphorylation of H3S10.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide. Ferroptosis is emerging as an effective target for tumor treatment as it has been shown to potentiate cell death in some malignancies. However, it remains unclear whether histone phosphorylation events, an epigenetic mechanism that regulates transcriptional expression, are involved in ferroptosis. Our study found that supplementation with anisomycin, an agonist of p38 mitogen-activated protein kinase (MAPK), induced ferroptosis in HCC cells, and the phosphorylation of histone H3 on serine 10 (p-H3S10) was participated in anisomycin-induced ferroptosis. To investigate the anticancer effects of anisomycin-activated p38 MAPK in HCC, we analyzed cell viability, colony formation, cell death, and cell migration in Hep3B and HCCLM3 cells. The results showed that anisomycin could significantly suppress HCC cell colony formation and migration and induce HCC cell death. The hallmarks of ferroptosis, such as abnormal accumulation of iron and elevated levels of lipid peroxidation and malondialdehyde, were detected to confirm the ability of anisomycin to promote ferroptosis. Furthermore, coincubation with SB203580, an inhibitor of activated p38 MAPK, partially rescued anisomycin-induced ferroptosis. And the levels of p-p38 MAPK and p-H3S10 were successively increased by anisomycin treatment. The relationship between p-H3S10 and ferroptosis was revealed by ChIP sequencing. The reverse transcription PCR and immunofluorescence results showed that NCOA4 was upregulated both in mRNA and protein levels after anisomycin treatment. And by C11-BODIPY staining, we found that anisomycin-induced lipid reactive oxygen species was reduced after NCOA4 knockdown. In conclusion, the anisomycin-activated p38 MAPK promoted ferroptosis of HCC cells through H3S10 phosphorylation.

Anisomycin inhibits angiogenesis, growth, and survival of triple-negative breast cancer through mitochondrial dysfunction, AMPK activation, and mTOR inhibition.

Aberrant upregulation of mitochondrial biogenesis is observed in breast cancer and holds potential therapeutic option. In our work, we showed that inhibition of mitochondrial function by anisomycin is effective against triple-negative breast cancer (TNBC). Anisomycin inhibits growth and induces caspase-dependent apoptosis in a panel of TNBC cell lines. Of note, anisomycin at a tolerable dose remarkably suppresses growth of TNBC in mice. In addition, anisomycin effectively targets breast cancer angiogenesis through inhibiting capillary network formation, migration, proliferation, and survival. Mechanistic studies show that although anisomycin activates p38 and JNK, their activations are not required for anisomycin's action. In contrast, anisomycin inhibits mitochondrial respiration, and decreases mitochondrial membrane potential and adenosine triphosphate (ATP) level. The inhibitory effect of anisomycin is significantly reversed in mitochondria respiration-deficient ρ0 cells. As a consequence, anisomycin activates AMPK and inhibits mammalian target-of-rapamycin signaling pathways. Our work demonstrated that anisomycin is a useful addition to the treatment armamentarium for TNBC.

Oxytocin modulation in the medial prefrontal cortex of pair-exposed rats during fear conditioning.

INTRODUCTION: Social buffering is the phenomenon, in which stress and fear reactions caused by exposure to stressful stimuli when animals are exposed to homogeneous relationships are attenuated. Social buffering reduces fear memory behavior such as escape, avoidance, and freezing behavior in rodents due to social existence. Here, we aimed to determine alterations of fear behavior and neural activity in the medial prefrontal cortex (mPFC) in response to the presence of another rat in fear-exposed conditions and to confirm the role of oxytocin in mPFC in regulating social buffering. METHODS: We performed a passive avoidance test and determined positive c-Fos expression in single- and pair-exposed rats. Anisomycin (a protein synthesis inhibitor) and oxytocin receptor regulators (carbetocin; agonist and atosiban; antagonist) were microinjected into the mPFC to clarify the role of oxytocin in the mPFC. RESULTS: While single-exposed rats showed a significant increase in both freezing and passive avoidance behaviors compared to control rats, pair-exposed rats showed significantly less fear behavior compared to single-exposed rats. The c-Fos expression in the prelimbic (PL) mPFC was significantly increased in pair-exposed rats compared to that in control and single-exposed rats. The pair-exposed effect was blocked by anisomycin injections into the PL mPFC of pair-exposed rats. Furthermore, when a carbetocin was injected into the PL mPFC in single-exposed rats, fear behavior was decreased, and these changes were blocked by atosiban. DISCUSSION: Our findings suggest that reduction of fear-related behavior induced by acute pair-exposure is mediated by oxytocin receptors in the PL mPFC. Pair exposure with conspecifics during fear-inducing situations helps coping with fear by significantly increasing the role of oxytocin in the PL mPFC.

Suppressive Effects of Anisomycin on the Proliferation of B16 Mouse Melanoma Cells In Vitro.

BACKGROUND: Anisomycin, a potential anticancer therapeutic drug, exerts an antitumor effect on melanoma cells at a lower concentration than that required for other cancer cells. However, the molecular mechanisms remain unclear. MATERIALS AND METHODS: The sensitivity to and cytotoxicity of anisomycin, as well as the effects of anisomycin on glucose metabolism and relative mRNA expression of senescence- and cancer-associated genes, were studied using B16 mouse melanoma cells. RESULTS: The viability of anisomycin-treated cells decreased in a concentration-dependent manner, and the growth of cell spheroids was suppressed by 50 nM anisomycin. Glucose metabolism was reduced by anisomycin treatment, and the mRNA expression of genes responsible for growth inhibition, such as p21, p53 and Txnip was upregulated. CONCLUSION: The results suggest that anisomycin may be a promising future anticancer drug that is effective at low concentrations against melanoma by reducing glucose metabolism, causing cell senescence-like phenomena.

IL-1ß is a key inflammatory cytokine that weakens lactation-specific tight junctions of mammary epithelial cells.

In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1ß, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1ß, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1ß caused rapid disruption of TJs at tricellular contact points. IL-1ß treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1ß on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1ß or anisomycin. These results indicate that IL-1ß is a key cytokine that adversely affects the TJs between MECs by activating p38.

Temozolomide Induces Endocytosis of EGFRvIII via p38-Mediated Non-canonical Phosphorylation in Glioblastoma Cells.

The ligand-induced internalization of epidermal growth factor receptor (EGFR) is generally considered to attenuate downstream signaling via its endosomal degradation. However, the endocytosis of an oncogenic EGFR variant III (EGFRvIII) is impaired, which leads to persistent signaling from the cell surface, thereby promoting the proliferation and survival of glioblastoma multiforme (GBM) cells. Cellular stress triggers the non-canonical endocytosis-recycling of EGFR by p38-mediated phosphorylation. In the present study, we used temozolomide (TMZ), the standard chemotherapeutic agent for the treatment of GBM patients, to examine whether EGFRvIII is controlled by a non-canonical mechanism. TMZ triggered the endocytic trafficking of serine phosphorylated EGFRvIII. Moreover, phosphorylation and endocytosis were abrogated by the selective p38 inhibitor SB203580, but not gefitinib, indicating that EGFRvIII is recruited to p38-mediated non-canonical endocytosis. The combination of TMZ and SB203580 also showed potential inhibitory effects on the proliferation and motility of glioblastoma cells.

Anisomycin is active in preclinical models of pediatric acute myeloid leukemia via specifically inhibiting mitochondrial respiration.

The poor outcomes in acute myeloid leukemia (AML) necessitate new treatments. In this work, we identified that anisomycin is a potential selective anti-AML candidate, particularly for those with FLT3-ITD mutation. We found that anisomycin potently inhibited proliferation and induced apoptosis in multiple AML cell lines. Anisomycin was effective in targeting progenitor cells isolated from all tested pediatric AML patients, while sparing normal counterparts. Using AML xenograft mouse models, anisomycin exhibited inhibitory effect on tumor growth throughout the whole duration without causing toxicity in mice. The combination of anisomycin with standard of care drugs is synergistic and selective in AML cell culture system and mouse model. In addition, FLT3-ITD cells were more sensitive to anisomycin than FLT3 WT cells. Mechanistic studies revealed that anisomycin acted on AML in a p38-independent manner. We found that anisomycin decreased mitochondrial respiration by disrupting complex I activity, leading to intracellular oxidative stress. AML ρ0 cells that lack of mitochondrial respiration exhibited resistance to anisomycin. Finally, we showed that mitochondrial biogenesis contributes to differential sensitivity of FLT3-ITD and FLT3 WT cells to anisomycin. Our work is the first to systematically demonstrate that anisomycin is a useful addition to the treatment armamentarium for AML. Our findings highlight the therapeutic value of mitochondrial respiration inhibition in AML patients harboring FLT3-ITD mutation.

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression.

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1ß as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κBinducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis. [BMB Reports 2021; 54(11): 545-550].

C-Jun N-Terminal Kinase Post-Translational Regulation of Pain-Related Acid-Sensing Ion Channels 1b and 3.

Neuronal proton-gated acid-sensing ion channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathologic diseases. Such conditions often involve in parallel the activation of various signaling pathways such as mitogen activated protein kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal kinase (JNK), as a new post-translational positive regulator of ASICs in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit-dependent manner, targeting both rodent and human ASIC1b and ASIC3 subunits (except mouse ASIC3). The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost on mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNFα. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory-neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.SIGNIFICANCE STATEMENT ASICs are a class of excitatory cation channels critical for the detection of tissue acidosis, which is a hallmark of several painful diseases. Previous work in sensory neurons has shown that ASICs containing the ASIC3 or the ASIC1b subunit are important players in different pain models. We combine here functional and pharmacological in vitro and in vivo approaches to demonstrate that the MAP Kinase JNK is a potent post-translational positive regulator, probably via direct phosphorylation, of rodent and human ASIC1b- and ASIC3-containing channels. This JNK-dependent, fast post-translational mechanism of regulation of sensory-neuron-expressed ASICs may contribute to peripheral sensitization and pain hypersensitivity. These data also identify pain-related channels as direct downstream effectors of JNK in nociceptors.

Role of NMDA Receptors and Protein Synthesis in the Conditioned Aversion Learning in Young Chicks.

The brain mechanisms underlying conditioned aversion learning in birds were studied using experimental model in young chicks. The learning consisted of a conditioning stimulus presentation followed by a delayed sickness-inducing treatment reinforcement. Intraventricular administration of an NMDA receptor antagonist MK-801, a protein synthesis inhibitor anisomycin, or an inhibitor of glycoprotein fucosylation 2-deoxygalactose just before presentation of the conditioning stimulus prevented aversion learning. Injections of the same chemicals before reinforcement did not affect learning. The obtained results show that the investigated mechanisms underlying aversion learning were critical at the early stage of memory formation. Later processes of association of the conditioning stimulus with the reinforcement appear to be independent of the NMDA receptors and protein synthesis/glycosylation, or alternatively to be located in other brain areas.