Activation Of Autophagic Flux Research Articles

Modulating autophagy to boost the antitumor efficacy of TROP2-directed antibody-drug conjugate in pancreatic cancer

Pancreatic cancer, characterized by a dismal prognosis and limited treatment options, persists as a formidable challenge in oncology. Trophoblast cell surface antigen 2 (TROP2)-directed antibody-drug conjugates have achieved great success in solid tumors such as breast cancer and uroepithelial carcinoma. However, their efficacy against pancreatic cancer was insufficient in clinical trials, necessitating an imperative exploration of underlying mechanisms and new therapeutic strategies. In this study, we indicated that αTROP2-MMAE, an antibody-drug conjugate targeting TROP2, induced apoptosis through the caspase-9/PARP pathway and exerted potent antitumor effects against TROP2-positive pancreatic cancer. Simultaneously, RNA sequencing suggested significant changes in autophagy after αTROP2-MMAE treatment. The formation of autophagosomes and activation of autophagic flux were markedly induced through mechanisms associated with suppressing the activation of the Akt/mTOR pathway. The addition of pharmacological inhibitors of autophagy enhanced the cytotoxicity and apoptosis caused by αTROP2-MMAE, revealing the cytoprotective role of autophagy in TROP2-positive pancreatic cancer. In the subcutaneous xenograft model using BxPC3 cells, the combined administration of αTROP2-MMAE and an autophagy inhibitor elevated the tumor inhibition rate of αTROP2-MMAE from 71.6 % to 99.0 %, resulting in the eradication of tumors in half of the mice. Collectively, our research demonstrated for the first time the cytoprotective role of autophagy in TROP2-targeted antibody-drug conjugate therapy for pancreatic cancer, providing new perspectives for mechanistic exploration and therapeutic strategies in the treatment of pancreatic cancer.

Natural Autophagy Activators to Fight Age-Related Diseases.

The constant increase in the elderly population presents significant challenges in addressing new social, economic, and health problems concerning this population. With respect to health, aging is a primary risk factor for age-related diseases, which are driven by interconnected molecular hallmarks that influence the development of these diseases. One of the main mechanisms that has attracted more attention to aging is autophagy, a catabolic process that removes and recycles damaged or dysfunctional cell components to preserve cell viability. The autophagy process can be induced or deregulated in response to a wide range of internal or external stimuli, such as starvation, oxidative stress, hypoxia, damaged organelles, infectious pathogens, and aging. Natural compounds that promote the stimulation of autophagy regulatory pathways, such as mTOR, FoxO1/3, AMPK, and Sirt1, lead to increased levels of essential proteins such as Beclin-1 and LC3, as well as a decrease in p62. These changes indicate the activation of autophagic flux, which is known to be decreased in cardiovascular diseases, neurodegeneration, and cataracts. The regulated administration of natural compounds offers an adjuvant therapeutic alternative in age-related diseases; however, more experimental evidence is needed to support and confirm these health benefits. Hence, this review aims to highlight the potential benefits of natural compounds in regulating autophagy pathways as an alternative approach to combating age-related diseases.

High glucose-induced p66Shc mitochondrial translocation regulates autophagy initiation and autophagosome formation in syncytiotrophoblast and extravillous trophoblast.

p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.

Two birds with one stone: natural polyphenols boosted periodontitis treatment of chlorhexidine via reducing toxicity and regulating microenvironments.

Chlorhexidine (CHX) is considered the gold standard for controlling periodontal plaque and has been extensively used as a topical agent in treating periodontitis. Nevertheless, the practical clinical application of CHX is still constrained by the inherent limitations of its properties, including toxicity, inadequate biofilm scavenging capacity, and single biological effect. In this study, polyphenolic epigallocatechin gallate (EGCG) has been employed to integrate with CHX to form an EGCG-CHX nanoplatform via a facile one-pot method. Due to the dynamic bonding between EGCG and CHX, the EGCG-CHX nanoparticles (NPs) show reduced toxicity and excellent response release behavior. Moreover, a series of in vitro and in vivo studies demonstrated that the EGCG-CHX NPs significantly enhanced the antibiofilm, antioxidative, anti-inflammatory, and autophagic flux activation effects of CHX, ultimately achieving an improved therapeutic effect on periodontitis. This study successfully developed a strategy boosting the efficiency of CHX for periodontitis treatment.

Influence of Stress Resistance on Myocardial Expression of the Pro-Autophagic Protein Beclin-1 After Cardiac Contusion in Experimental Setting

Objective. Evaluation of myocardial expression of the pro-autophagic protein Beclin-1 after cardiac contusion in experimental animals with different stress resistance.Materials and methods. The study included 68 white mongrel male rats weighing 250–300 g. After ranking for extreme variants of stress resistance, moderately stress-resistant rats (N=36) were excluded from the study. The remaining animals were split into the control (N=16) and study (N=16) groups, each group composed of 8 high stress resistant and 8 low stress resistant rats. In the study group, 24 hours after inflicted cardiac contusion, 5×5 mm myocardial tissue specimens were sampled from the intraventricular septum, anterior walls of the left and right ventricles, histological sections were made, and a reaction with primary polyclonal Anti-Beclin-1 antibodies was performed. Beclin-1 expression was evaluated under the microscope.Results. Immunohistochemical evaluation revealed a statistically significant increase in Beclin-1 protein expression (P=0.0002) in the cytoplasm of cardiomyocytes in the study group vs the control group, regardless of animals’ baseline stress resistance. However, expression of Beclin-1 protein in the myocardium of highly stress-resistant rats (Me=4.3; LQ=4.0; HQ=4.3) was significantly higher versus low-resistant animals (Me=3.6; LQ=3.3; HQ=3.6) (P=0.0009).Conclusion. Increased expression of Beclin-1 protein in the post-traumatic period of experimental cardiac contusion indicates autophagic flux activation. Intensity of autophagy varied depending on the animal’s stress resistance.

KPT330 promotes the sensitivity of glioblastoma to olaparib by retaining SQSTM1 in the nucleus and disrupting lysosomal function

ABSTRACT PARP (poly(ADP-ribose) polymerase) inhibitors have demonstrated promising clinical activity in multiple homologous recombination (HR) deficiency tumors. However, glioblastoma (GBM) patients have obtained little benefit from PARP inhibitors alone. PARP inhibition shows considerable promise when used together with other therapeutic agents. Thus, novel combination therapies may enhance PARP inhibitor efficacy and overcome resistance mechanisms in GBM. Herein, we report that concurrent treatment with the PARP inhibitor olaparib and XPO1 (exportin 1) inhibitor KPT330 showed synergetic anticancer effects on GBM cells. Mechanistically, in the nucleus, we show that KPT330 induced the nuclear retention of SQSTM1 (sequestosome 1) and further inhibited the ubiquitination of the DNA repair signal H2AX (H2A.X variant histone) mediated by olaparib, thus inhibiting DNA damage response and repair in GBM. Moreover, in the cytoplasm, KPT330 blocked the activation of autophagic flux caused by olaparib reagent, downregulated the expression of LAPTM4B (lysosomal protein transmembrane 4 beta) and induced the dysfunction of lysosomes, thereby preventing the degradation of autophagosome, and ultimately promoted cell death. Furthermore, in the LN229-luc mouse orthotopic xenograft model, combination treatment showed significantly increased antitumor efficacy compared to each monotherapy. These data illustrate the application prospects of combined oral administration of olaparib and KPT330 for the treatment of glioblastoma. Abbreviations AO: acridine orange; ATM: ATM serine/threonine kinase; CHEK1: checkpoint kinase 1; CHEK2: checkpoint kinase 2; CI: combination index; DMSO: dimethyl sulfoxide; DSBs: double-strand breaks; GBM: glioblastoma; HR: homologous recombination; H2AX: H2A.X variant histone; IHC: immunohistochemistry; LAPTM4B: lysosomal protein transmembrane 4 beta; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PARP: poly(ADP-ribose) polymerase; RAD51: RAD51 recombinase; SQSTM1: sequestosome 1; SSBs: single-strand breaks; RNF168: ring finger protein 168; XPO1: exportin 1.

Liraglutide ameliorates hepatic steatosis via retinoic acid receptor-related orphan receptor α-mediated autophagy pathway.

Liraglutide, an analog of human glucagon-like peptide-1 (GLP-1), has been found to improve hepatic steatosis in clinical practice. However, the underlying mechanism remains to be fully defined. Increasing evidence suggests that retinoic acid receptor-related orphan receptor α (RORα) is involved in hepatic lipid accumulation. In the current study, we investigated whether the ameliorating impact of liraglutide on lipid-induced hepatic steatosis is dependent on RORα activity and examined the underlying mechanisms. Cre-loxP-mediated, liver-specific Rorα knockout (Rora LKO) mice, and littermate controls with a Roraloxp/loxp genotype were established. The effects of liraglutide on lipid accumulation were evaluated in mice challenged with a high-fat diet (HFD) for 12 weeks. Moreover, mouse AML12 hepatocytes expressing small interfering RNA (siRNA) of Rora were exposed to palmitic acid to explore the pharmacological mechanism of liraglutide. The results showed that liraglutide treatment significantly alleviated HFD-induced liver steatosis, marked by reduced liver weight and triglyceride accumulation, improved glucose tolerance and serum levels of lipid profiles and aminotransferase. Consistently, liraglutide also ameliorated lipid deposits in a steatotic hepatocyte model in vitro. In addition, liraglutide treatment reversed the HFD-induced downregulation of Rora expression and autophagic activity in mouse liver tissues. However, the beneficial effect of liraglutide on hepatic steatosis was not observed in Rora LKO mice. Mechanistically, the ablation of Rorα in hepatocytes diminished liraglutide-induced autophagosome formation and the fusion of autophagosomes and lysosomes, resulting in weakened autophagic flux activation. Thus, our findings suggest that RORα is essential for the beneficial impact of liraglutide on lipid deposition in hepatocytes and regulates autophagic activity in the underlying mechanism.

A prolyl oligopeptidase inhibitor reduces tau pathology in cellular models and in mice with tauopathy.

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

Orf virus induces complete autophagy to promote viral replication via inhibition of AKT/mTOR and activation of the ERK1/2/mTOR signalling pathway in OFTu cells

Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.

Functional TFEB activation characterizes multiple models of renal cystic disease and loss of polycystin-1.

Polycystic kidney disease is a disorder of renal epithelial growth and differentiation. Transcription factor EB (TFEB), a master regulator of lysosome biogenesis and function, was studied for a potential role in this disorder. Nuclear translocation and functional responses to TFEB activation were studied in three murine models of renal cystic disease, including knockouts of folliculin, folliculin interacting proteins 1 and 2, and polycystin-1 (Pkd1) as well as in mouse embryonic fibroblasts lacking Pkd1 and three-dimensional cultures of Madin-Darby canine kidney cells. Nuclear translocation of Tfeb characterized cystic but not noncystic renal tubular epithelia in all three murine models as both an early and sustained response to cyst formation. Epithelia expressed elevated levels of Tfeb-dependent gene products, including cathepsin B and glycoprotein nonmetastatic melanoma protein B. Nuclear Tfeb translocation was observed in mouse embryonic fibroblasts lacking Pkd1 but not wild-type fibroblasts. Pkd1 knockout fibroblasts were characterized by increased Tfeb-dependent transcripts, lysosomal biogenesis and repositioning, and increased autophagy. The growth of Madin-Darby canine kidney cell cysts was markedly increased following exposure to the TFEB agonist compound C1, and nuclear Tfeb translocation was observed in response to both forskolin and compound C1 treatment. Nuclear TFEB also characterized cystic epithelia but not noncystic tubular epithelia in human patients with autosomal dominant polycystic kidney disease. Noncanonical activation of TFEB is characteristic of cystic epithelia in multiple models of renal cystic disease including those associated with loss of Pkd1. Nuclear TFEB translocation is functionally active in these models and may be a component of a general pathway contributing to cystogenesis and growth.NEW & NOTEWORTHY Changes in epithelial cell metabolism are important in renal cyst development. The role of TFEB, a transcriptional regulator of lysosomal function, was explored in several models of renal cystic disease and human ADPKD tissue sections. Nuclear TFEB translocation was uniformly observed in cystic epithelia in each model of renal cystic disease examined. TFEB translocation was functionally active and associated with lysosomal biogenesis and perinuclear repositioning, increased TFEB-associated protein expression, and activation of autophagic flux. Compound C1, a TFEB agonist, promoted cyst growth in 3-D cultures of MDCK cells. Nuclear TFEB translocation is an underappreciated signaling pathway for cystogenesis that may represent a new paradigm for cystic kidney disease.

Reduced secretion of LCN2 (lipocalin 2) from reactive astrocytes through autophagic and proteasomal regulation alleviates inflammatory stress and neuronal damage

ABSTRACT LCN2/neutrophil gelatinase-associated lipocalin/24p3 (lipocalin 2) is a secretory protein that acts as a mammalian bacteriostatic molecule. Under neuroinflammatory stress conditions, LCN2 is produced and secreted by activated microglia and reactive astrocytes, resulting in neuronal apoptosis. However, it remains largely unknown whether inflammatory stress and neuronal loss can be minimized by modulating LCN2 production and secretion. Here, we first demonstrated that LCN2 was secreted from reactive astrocytes, which were stimulated by treatment with lipopolysaccharide (LPS) as an inflammatory stressor. Notably, we found two effective conditions that led to the reduction of induced LCN2 levels in reactive astrocytes: proteasome inhibition and macroautophagic/autophagic flux activation. Mechanistically, proteasome inhibition suppresses NFKB/NF-κB activation through NFKBIA/IκBα stabilization in primary astrocytes, even under inflammatory stress conditions, resulting in the downregulation of Lcn2 expression. In contrast, autophagic flux activation via MTOR inhibition reduced the intracellular levels of LCN2 through its pre-secretory degradation. In addition, we demonstrated that the N-terminal signal peptide of LCN2 is critical for its secretion and degradation, suggesting that these two pathways may be mechanistically coupled. Finally, we observed that LPS-induced and secreted LCN2 levels were reduced in the astrocyte-cultured medium under the above-mentioned conditions, resulting in increased neuronal viability, even under inflammatory stress. Abbreviations: ACM, astrocyte-conditioned medium; ALP, autophagy-lysosome pathway; BAF, bafilomycin A1; BTZ, bortezomib; CHX, cycloheximide; CNS, central nervous system; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; JAK, Janus kinase; KD, knockdown; LCN2, lipocalin 2; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin kinase; NFKB/NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; NFKBIA/IκBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; OVEX, overexpression; SLC22A17, solute carrier family 22 member 17; SP, signal peptide; SQSTM1, sequestosome 1; STAT3, signal transducer and activator of transcription 3; TNF/TNF-α, tumor necrosis factor; TUBA, tubulin, alpha; TUBB3/β3-TUB, tubulin, beta 3 class III; UB, ubiquitin; UPS, ubiquitin-proteasome system

HO-1/autophagic flux axis alleviated sepsis-induced acute lung injury via inhibiting NLRP3 inflammasome

Among the multiple organ injuries induced by sepsis, acute lung injury (ALI) triggered by an excessive inflammatory response is one of the main causes contributing to patient death, and inhibition of the inflammation cascade is the key therapeutic strategy to improve prognosis. The NLRP3 inflammasome complex is considered an intracellular signaling molecule closely associated with the uncontrolled inflammatory response in sepsis-induced ALI. Therefore, exploring new targets to repress its activation is regarded as a potential therapeutic strategy. Growing evidence demonstrated that heme oxygenase-1 (HO-1) contributed to general anti-inflammation and exerted a protective role in ALI, but its underlying mechanisms have not been clarified completely. Herein, we investigated HO-1 was elevated in alveolar macrophages isolated from bronchoalveolar lavage fluid (BALF) of sepsis mice. HO-1 abundance suppressed NLRP3 inflammasome complex activation and attenuated pro-inflammatory cytokines release, thereby alleviating sepsis-induced ALI. Whereas inhibition of HO-1 reached the opposite effect. Meanwhile, HO-1 is an effective and functionally relevant regulator of autophagic flux. HO-1 activator decreased the expression of P62 and enhanced the LC3 II/LC3 I ratio, resulting in autophagic flux activation. In addition, the protective effects HO-1 exerted in sepsis-induced ALI could be abolished by autophagic flux inhibitor. Autophagic flux activator could suppress NLRP3 inflammasome activation and attenuate ALI, while autophagic flux inhibitor had the opposite effect. In conclusion, our study revealed increased HO-1 expression inhibited the level of NLRP3 inflammasome via regulating the activation of autophagic flux, thus attenuating inflammatory response and alleviating sepsis-induced ALI.

Activation of Autophagic Flux Maintains Mitochondrial Homeostasis during Cardiac Ischemia/Reperfusion Injury.

Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Integrated Metabolomics and Transcriptomics Analyses Reveal Metabolic Mechanisms in Porcine Intestinal Epithelial Cells under Zearalenone Stress.

Zearalenone (ZEA) is a mycotoxin that frequently occurs in agricultural crops and related products and seriously threatens both animal feed and human food safety. To identify key metabolites and regulators involved in ZEA toxicological processes, we performed metabolomic and transcriptomic analyses of porcine IPEC-J2 intestinal epithelial cells upon ZEA exposure using liquid chromatography-mass spectrometry (LC-MS)/MS and RNA-seq techniques. A total of 325 differential metabolites and 5646 differentially expressed genes were detected. Integrated analyses of metabolomic and transcriptomic data indicated that metabolic processes including lipid metabolism, amino acid metabolism, and carbohydrate metabolism were most affected. Exogenous addition of the key metabolite l-arginine significantly facilitated ZEA metabolism and ameliorated ZEA-induced reactive oxygen species levels and cell apoptosis. Furthermore, l -arginine contributed to the expression of phase II detoxification genes (SULT2B1, GSTA1, GSTM3, and GPX4). l-Arginine addition also increased the protein levels of LC3-II and Beclin 1, and downregulated p62/SQSTM1 levels, indicating its regulatory roles in autophagic flux activation upon ZEA exposure. This study provided global insights into metabolic and transcriptional changes as well as key metabolites and regulators underlying the cellular response to ZEA exposure, and paved the way for the identification of metabolic and molecular targets for biomonitoring and controlling contamination by ZEA.

Distinct ginseng extracts produce disparate effects on proteostatic support through the autophagy‐lysosomal pathway that is linked to synaptic resilience and cognitive health

The growing incidence of age‐related proteinopathies like Alzheimer’s disease (AD) warrants high‐level understanding of protein quality control pathways and their regulatory factors that influence protein clearance (Farizatto et al. 2017 PLoS ONE 12:e0182895; Boland et al. 2018 Nat Rev Drug Discov 17:660). Alterations to proteostasis mechanisms occur with age leading to the associated gradual disruption of synaptic integrity, the best correlate of cognitive decline during aging and dementia. Poor nutrition is believed to contribute to cognitive aging and many reports suggest natural products, including different types of ginsengs, promote cognitive health. Thus, this study assessed two plant extracts for their ability to amplify a protein clearing enzyme of the autophagy‐lysosomal pathway. Note, we focused on American ginseng (P. quinquefolius) and Asian ginseng (P. ginseng) since ginseng root has a long history as a traditional medicine for many diseases and was recently associated with anti‐brain aging effects by protecting neurons against oxidative stress, inflammation and stimulating autophagy (Ong et al. 2015 Front Aging Neurosci 16;7:129; Choi et al. 2021 Integr Med Res 10:100450). Long‐term hippocampal explants with native circuitries and synaptic density were treated daily with the specific extracts for 3 days. Asian ginseng caused a 50% increase while American ginseng caused a 250‐350% increase in the active form of cathepsin B (CatB), a lysosomal protease found to improve clearance of pathogenic proteins and preserve synaptic and cognitive measures in models of proteinopathy (Mueller‐Steiner et al. 2006 ‐ Neuron 51:703; Butler et al. 2011 ‐ PLoS ONE 6:e20501; Hwang et al. 2019 ‐ Inter J Mol Sci 20:4432). Of the two treatments, only American ginseng produced a significant correlation between improved levels of the CatB active form and the postsynaptic protein GluA1, and corresponding protection was evident towards both pre‐ and postsynaptic markers. The LC3I to LC3II conversion was also enhanced in the American ginseng‐treated explants, and such was confirmed to occur in the presence of the lysosomal inhibitor chloroquine, thus indicating activation of autophagic flux. Chemical assessment of the two ginsengs by UPLC‐Q‐ToF‐MS suggests the extracts have different profiles of ginsenosides and other molecules, differences that may explain their distinct effects on the hippocampal tissue. The extract‐treated hippocampal slice cultures were also subjected to chloroquine infusion that results in a synaptopathogenic cascade. However, American ginseng treatment prevented the synaptic decline due to chloroquine‐mediated protein accumulation stress. These findings indicate that ginseng type and/or extraction method are critical for the ability of ginseng extracts to improve protein quality control in the hippocampus, a critical issue as disrupted hippocampal performance underlies the cognitive impairment of several neurodegenerative disorders. The protective influence by a specific natural product points to the protein clearance capacity of neurons as having a mechanistic role for healthy cognitive aging.

Neuroprotective effect of liraglutide in an experimental mouse model of multiple sclerosis: role of AMPK/SIRT1 signaling and NLRP3 inflammasome

The heterogeneous nature of multiple sclerosis (MS) and the unavailability of treatments addressing its intricate network and reversing the disease state is yet an area that needs to be elucidated. Liraglutide, a glucagon-like peptide-1 analogue, recently exhibited intriguing potential neuroprotective effects. The currents study investigated its potential effect against mouse model of MS and the possible underlying mechanisms. Demyelination was induced in C57Bl/6 mice by cuprizone (400 mg/kg/day p.o.) for 5 weeks. Animals received either liraglutide (25 nmol/kg/day i.p.) or dorsomorphin, an AMPK inhibitor, (2.5 mg/Kg i.p.) 30 min before the liraglutide dose, for 4 weeks (starting from the second week). Liraglutide improved the behavioral profile in cuprizone-treated mice. Furthermore, it induced the re-myelination process through stimulating oligodendrocyte progenitor cells differentiation via Olig2 transcription activation, reflected by increased myelin basic protein and myelinated nerve fiber percentage. Liraglutide elevated the protein content of p-AMPK and SIRT1, in addition to the autophagy proteins Beclin-1 and LC3B. Liraglutide halted cellular damage as manifested by reduced HMGB1 protein and consequently TLR-4 downregulation, coupled with a decrease in NF-κB. Liraglutide also suppressed NLRP3 transcription. Dorsomorphin pre-administration indicated a possible interplay between AMPK/SIRT1 and NLRP3 inflammasome activation as it partially reversed liraglutide’s effects. Immunohistochemical examination of Iba+ microglia emphasized these findings. In conclusion, liraglutide exerts neuroprotection against cuprizone-induced demyelination via anti-inflammatory, autophagic flux activation, NLRP3 inflammasome suppression, and anti-apoptotic mechanisms, possibly mediated, at least in part, via AMPK/SIRT1, autophagy, TLR-4/ NF-κB/NLRP3 signaling.Graphical abstractThe potential mechanistic insight of Lira in alleviating Cup-induced neurotoxicity via: (1) AMPK/SIRT1 pathways activation resulting in the stimulation of brain autophagy flux (confirmed by lowering Beclin-1 and LC3-B protein expression). (2) Inhibition of NLRP3 inflammasome activation, as evidenced by reduced HMGB1, TLR-4, NF-κB and NLRP3 protein expression, alongside diminishing the activation of its downstream cascade as reflected by reduced levels of caspase-1 and IL-1β protein expression. (3) A possible modulating interplay between the previously mentioned two pathways.

Beclin 1, LC3 and P62 Expression in Equine Sarcoids.

Simple SummaryEquine sarcoids, caused by bovine papillomaviruses, are equine skin tumors of fibroblastic origin. It is well known that bovine papillomaviruses are able to interfere with the survival and proliferation of cells by regulating autophagy, a mechanism implicated in the breakdown and reuse of old and damaged cellular material. The present study focused on the evaluation in equine sarcoids and normal skins of the expression level of some of the main proteins involved in the autophagic pathway, such as Beclin 1, LC3 and P62, by immunohistochemical and biochemical techniques. Results obtained in equine sarcoids suggested an alteration of the autophagic process which could lead to a predominance of a particular population of fibroblast. Those fibroblasts could survive longer in a hypoxic microenvironment and produce more and/or altered collagen, giving an origin to the equine sarcoid. Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

Autophagy Blockade Disrupts Myeloma Cell Recovery from Proteasome Inhibition and Enhances Apoptosis

BackgroundExtensive protein synthesis in multiple myeloma (MM) cells renders them vulnerable to proteasome inhibitors (PI), a cornerstone of anti-myeloma therapy. However, relapse is inevitable and PI resistance remains a major barrier to improving outcomes. Adaptive responses to PI include upregulation of autophagy, another major degradation pathway in cells. We hypothesised that autophagy inhibition increases cell death and impedes recovery of MM cells exposed to Carfilzomib (K), a second generation PI effective in newly diagnosed and relapsed/refractory MM.Methods:Human myeloma cell lines (HMCL) and patient samples were exposed to a 1-hour pulse of K to mimic pharmacokinetics, ± autophagy inhibitors (AI) vacuolar protein sorting kinase 34 inhibitor (VPS34i) or unc-51 like autophagy initiating kinase inhibitor (ULKi). Apoptosis was assessed by Annexin V/Propidium iodide (AnV/PI) flow cytometry (FC) and autophagic flux by western blotting (WB) and FC for light chain 3B (LC3B) ± bafilomycin (B). Cell cycle was examined by FC using KI67/PI.Results:Basal autophagic flux was seen in all HMCL (MM1s, H929, KMS12BM) tested (±B). Autophagic flux increased in response to K: MM1s mean fluorescence intensity (MFI) control (CTL) 318±28 vs K 567±72, (mean±SEM),p=0.01, n=6). Autophagy was inhibited in all 3 HMCL with AI. Combined exposure to K and AI (VPS34i or ULKi) increased time dependent apoptosis in all HMCL tested: MM1s 31±8% (K) vs 58±9% (K+VPS34i), p=0.03, (n=3), and 13±0.4% (K) vs 63±16% (K+ULKi), p=0.03 (n=3), (K 10nM, AI 1uM) 48 hours(h) post K exposure.Basal autophagic flux was detected in all CD138+ primary MM patient samples tested by LC3B FC assay (MFI CTL B- 407±57 vs B+ 1081±125, p<0.0001, n=14). This increased in response to K: CTL B- 407±57 vs K B- 862±224, p=0.03, n=14. VPS34i alone reduced viable cell numbers in 26/33 primary samples (% viable cells relative to starting viable MM cell number at day 0: CTL 99±14% vs VPS34i 71±12%, p<0.0001 at 48h), and cell loss correlated with basal autophagic flux (r=0.9, Pearson's correlation coefficient, p=0.0003, n=10). Addition of VPS34i to K increased loss of viable CD138+ cells (n=33) (Fig 1). Both apoptosis with K and enhanced cell death with K+VPS34i correlated with basal autophagic flux (r=0.8, p=0.0005 and r=0.8, p<0.0001 respectively), but no correlation was seen with previous therapy, disease stage, or adverse risk by cytogenetics or ISS stage.K treated MM1s cells were examined for kinetics of ubiquitinated protein build up and autophagic induction by WB. Increased ubiquitinated proteins were detectable up to 24h post K pulse, whilst autophagic flux increased up to 72h but returned to baseline thereafter (n=3). MM1s cells were treated with K±VPS34i for up to 7 days and apoptosis, absolute cell number and cell cycle assessed. In K treated cells, apoptosis was maximal at 72h but declined thereafter. Viable cell numbers fell initially but increased after 96h, indicating cell recovery. However, in MM1s cells treated with K+VPS34i/ULKi, apoptosis was enhanced with a rapid loss of viable cells (Fig 2) and viable cell numbers remained low at all timepoints (Fig 3). Cell cycle assays confirmed persistent G0/G1 arrest in MM1s cells treated with K+VPS34i or ULKi at all timepoints, whilst cell proliferation recovered in K treated cells on day 3, (15±3% cells in S/G2M in K treated cells vs 2±0.4% with K+VPS34i, p=0.02, n=4, 11±3% in S/G2M with K vs 1± 0.7% with K+ULKi, p=0.04, n=3, day 4)(Fig 4). To confirm that effects of AI were mediated via autophagy, KMS28PE (ATG12 null HMCL) were treated with K±VPS34i. These autophagy depleted cells showed limited basal or K induced autophagic flux and addition of VPS34i to K had no effect on apoptosis or cell cycle.ConclusionMM cells displayed active autophagic flux that increased with K treatment. Inhibition of autophagy increased K induced apoptosis, prolonged K-induced cell cycle arrest and prevented MM cell recovery. Addition of AI to K increased cytotoxicity in patient samples and many patients showed sensitivity to AI alone; these effects correlated with basal autophagic flux. These data suggest that autophagy plays a vital role in cell recovery from proteasome inhibition independent of proteotoxic stress. Dual inhibition of the proteasome and autophagosome may therefore help overcome PI resistance, which remains an unmet need in MM. [Display omitted] DisclosuresAuner: Takeda, Karyopharm: Other: Advisory role; Amgen: Research Funding; Janssen: Speakers Bureau. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yong: Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding.

Nanosize aminated fullerene for autophagic flux activation and G0/G1 phase arrest in cancer cells via post-transcriptional regulation

Nanosize aminated fullerene for autophagic flux activation and G0/G1 phase arrest in cancer cells via post-transcriptional regulation

The NUPR1/p73 axis contributes to sorafenib resistance in hepatocellular carcinoma.

The multikinase inhibitor sorafenib was the first drug approved by the FDA for treating patients with advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a major challenge for improving the effectiveness of HCC treatment. Previously, we identified several genes modulated after sorafenib treatment of human HCC cells, including the stress-inducible nuclear protein 1 (NUPR1) gene. Multiple studies have shown that NUPR1 regulates autophagy, apoptosis, and chemoresistance. Here, we demonstrate that treatment of HCC cells with sorafenib resulted in the activation of autophagic flux. NUPR1 knock-down (KD) in HCC cells was associated with increased p62 expression, suggesting an impairment of autophagic flux, and with a significant increase of cell sensitivity to sorafenib. In NUPR1 KD cells, reduced levels of NUPR1 were associated with the increased expression of p73 as well as its downstream transcription targets PUMA, NOXA, and p21. Simultaneous silencing of p73 and NUPR1 in HCC cells resulted in increased resistance to sorafenib, as compared to the single KD of either gene. Conversely, pharmacological activation of p73, via the novel p73 small molecule activator NSC59984, determined synergistic anti-tumor effects in sorafenib-treated HCC cells. The combination of NSC59984 and sorafenib, when compared to either treatment alone, synergistically suppressed tumor growth of HCC cells in vivo. Our data suggest that the activation of the p73 pathway achieved by NUPR1 KD potentiates sorafenib-induced anti-tumor effects in HCC cells. Moreover, combined pharmacological therapy with the p73 activator NSC59984 and sorafenib could represent a novel approach for HCC treatment.