sirolimus and 3-methyladenine

Octreotide is a synthetic octapeptide of natural somatostatin. We aimed to investigate the influence of Octreotide on lipopolysaccharide (LPS)-stimulated human pulmonary epithelial cell damage. After stimulated by LPS, BEAS-2B cells were treated with various concentrations of Octreotide. CCK-8 assay and LDH kits were to evaluate cell cytotoxicity. ELISA kits were to analyze the levels of inflammatory factors. TUNEL staining was to measure cell apoptosis. Western blot assay was used to assess the expression of apoptosis-related proteins, autophagy-related proteins and AKT/mTOR signaling-related proteins. Then, 3-methyladenine (3-MA) was adopted for treating BEAS-2B cells to determine its effects on inflammation and apoptosis. Afterward, adding AKT agonist (SC79) or mTOR antagonist (rapamycin) to explore the impact of Octreotide on autophagy. Results revealed that Octreotide notably enhanced cell viability and reduced LDH activity. The levels of inflammatory factors were significantly decreased following Octreotide treatment. Additionally, Octreotide attenuated the apoptotic capacity of LPS-induced BEAS-2B cells, led to the up-regulation of Bcl-2 protein level while cut down the protein levels of Bax and cleaved caspase3. Remarkably, the expression of autophagy-related protein LC3II/I and Beclin1 was elevated after Octreotide administration. Importantly, the suppressive effects of Octreotide on the inflammation and apoptosis of LPS-induced BEAS-2B cells was abrogated by 3-MA. Further experiments suggested that Octreotide downregulated p-AKT and mTOR expression in LPS-stimulated BEAS-2B cells. SC79 addition inhibited autophagy, evidenced by downregulated LC3II/I and Beclin1 expression while rapamycin presented the opposite effects. To conclude, Octreotide activates autophagy to alleviate LPS-induced pulmonary epithelial cell injury by inhibiting the AKT/mTOR signaling.

Topics: Acetates; Adenine; Autophagy; Benzopyrans; Cell Line; Cell Survival; Humans; Lipopolysaccharides; Lung Injury; Models, Biological; Octreotide; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Adenine; Adipogenesis; Apoptosis; Autophagy; Cells, Cultured; Humans; Sirolimus; Stem Cells

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice.

Topics: Adenine; Animals; Autophagy; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Transgenic; MTOR Inhibitors; Neuroinflammatory Diseases; Neuroprotective Agents; Parkinson Disease; Parkinson Disease, Secondary; Signal Transduction; Sirolimus; Substantia Nigra; TOR Serine-Threonine Kinases; Trehalose

Autophagy is a self-phagocytic and highly evolutionarily conserved intracellular lysosomal catabolic system, which plays a vital role in a variety of trauma models, including skin wound healing (SWH). However, the roles and potential mechanisms of autophagy in SWH are still controversial. We firstly investigated the role of autophagy in SWH-induced wound closure rate, inflammatory response, and histopathology, utilizing an inhibitor of autophagy 3-methyladenine (3-MA) and its agonist rapamycin (RAP). As expected, we found 3-MA treatment remarkably increased the wound closure rate, combated inflammation response, and mitigated histopathological changes, while RAP delivery aggravated SWH-induced pathological damage. To further exploit the underlying mechanism of autophagy regulating inflammation, the specific inhibitors of yes-associated protein (YAP), Verteporfin, and Anti-IL-33 were applied. Herein, treating with 3-MA markedly suppressed the expression of tumor necrosis factor-α (TNF-α), IL-1β, and IL-6, promoted that of IL-10, IL-33, and ST2, while RAP administration reverted SWH-induced the up-regulation of these inflammatory cytokines mentioned above. Importantly, Verteporfin administration not only down-regulated the expression levels of YAP, TNF-α, and IL-6 but also up-regulated that of IL-33 and IL-10. Unexpectedly, 3-MA or RAP retreatment did not have any impact on the changes in IL-33 among these inflammatory indicators. Furthermore, elevated expression of IL-33 promoted wound closure and alleviated the pathological damage, whereas, its antagonist Anti-IL-33 treatment overtly reversed the above-mentioned effects of IL-33. Moreover, 3-MA in combination with anti-IL-33 treatment reversed the role of 3-MA alone in mitigated pathological changes, but they failed to revert the effect of anti-IL-33 alone on worsening pathological damage. In sum, emerging data support the novel contribution of the YAP/IL-33 pathway in autophagy inhibition against SWH-induced pathological damage, and highlight that the autophagy/YAP/IL-33 signal axis is expected to become a new therapeutic target for SWH.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; Disease Models, Animal; Inflammation; Interleukin-33; Male; Mice; Mice, Inbred ICR; Signal Transduction; Sirolimus; Skin; Wound Healing; YAP-Signaling Proteins

Mosquito-borne dengue virus (DENV) causes major disease worldwide, impacting 50-100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology, including antiviral mechanisms, and developing new control strategies have become an important step towards the elimination of DENV disease. In the study reported here, we focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential antiviral target in the mosquito.. To understand the role played by autophagy in Ae. aegypti, we examined the activation of this pathway in Aag-2 cells, an Ae. aegypti-derived cell line, infected with DENV. Rapamycin and 3-methyladenine, two small molecules that have been shown to affect the function of the autophagy pathway, were used to activate or suppress, respectively, the autophagy pathway.. At 1-day post-DENV infection in Aag-2 cells, transcript levels of both the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) and autophagy-related protein 1 (ATG1) increased. Rapamycin treatment activated the autophagy pathway as early as 1-h post-treatment, and the virus titer had decreased in the Aag-2 cells at 2 days post-infection; in contrast, the 3-methyladenine treatment did not significantly affect the DENV titer. Treatment with these small molecules also impacted the ATG12 transcript levels in DENV-infected cells.. Our studies revealed that activation of the autophagy pathway through rapamycin treatment altered DENV infection in the mosquito cells, suggesting that this pathway could be a possible antiviral mechanism in the mosquito system. Here we provide fundamental information needed to proceed with future experiments and to improve our understanding of the mosquito's immune response against DENV.

Topics: Adenine; Aedes; Animals; Autophagy; Cell Line; Dengue; Dengue Virus; Mosquito Vectors; Sirolimus; Virus Replication

Acute lung injury (ALI) is characterized by alveolar macrophage overactivation and uncontrolled pulmonary inflammation. Mitochondrial damage-associated molecular patterns (MTDs), one type of damage-associated molecular patterns (DAMPs) released from ruptured mitochondrial, can induce inflammation which participates in the pathogenesis of ALI. Despite the critical role of autophagy in inflammatory response, little is known about its function in MTDs-induced ALI. Herein we have studied how autophagy attenuates MTDs-induced ALI in vitro and in vivo.. Exogenous MTDs were injected into mice through tail vein injection or directly treated with cultured alveolar macrophage cell lines to construct MTDs-induced ALI models. Rapamycin and 3-MA were used to regulate autophagy in vivo and in vitro. The expressions of Caspase-1, IL-1β, and their precursor were measured. Inhibition the activation of NLRP3 inflammasome to discover the candidate targets and potential molecular pathways involved in autophagy mitigating the MTDs-induced ALI.. After treatment with MTDs the expression levels of inflammatory cytokines and NLRP3 inflammasome-associated proteins were gradually increased in vitro and in vivo. Most importantly, with autophagy enhanced by rapamycin, all the secretion of inflammation cytokine, the level of lung injury, and the expression level of NLRP3 inflammasome-associated proteins were greatly decreased in MTDs-induced mouse model. MTDs-induced inflammation and lung injury were alleviated by autophagy enhancement. Autophagy can function as an effective way to alleviate inflammation in MTDs-induced ALI by inhibiting NLRP3 inflammasome and may represent a therapeutic target in modulating MTDs-induced inflammatory response.

Topics: Acute Lung Injury; Adenine; Alarmins; Animals; Autophagy; Cytokines; Disease Models, Animal; Inflammasomes; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Pneumonia; Sirolimus

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Arsenic Trioxide; Autophagy; Cell Line; Cell Survival; Mice; Microtubule-Associated Proteins; Sirolimus

We aimed to clarify the changes in apoptosis, proliferation, senescence, and adipogenesis after promoting and inhibiting autophagy in adipose-derived stem cells (ADSCs) by rapamycin and 3-methyladenine in vitro and in vivo.. After rapamycin and 3-methyladenine pretreatment, ADSC autophagy was detected by immunofluorescence for LC3, RT-PCR for ATG genes, and western blotting (WB) for the LC3 II/I and p62 proteins. TUNEL staining, PCR of BAX, and WB of Caspase-3 were preformed to assess ADSC apoptosis. The adipogenesis of ADSCs was evaluated by Oil red O staining and PCR of PPAR-γ. CCK8 assays were conducted to detect proliferation. Senescence was tested by Sa-β-gal staining and PCR of the P16/ 19/21 genes. Moreover, the mass and volume retention rate were determined, and perilipin and CD31 staining were performed in vivo.. Rapamycin and 3-methyladenine pretreatment increased and decreased autophagy of ADSCs, respectively, under normal and oxygen-glucose deprivation conditions. Apoptosis and senescence of ADSCs were decreased, and adipogenesis was increased along with the upregulation of autophagy. However, the proliferation of ADSCs was inhibited after either rapamycin or 3-methyladenine pretreatment. In vivo, the volume and mass retention rate and the angiogenesis of the grafts were also improved after rapamycin pretreatment.. Rapamycin pretreatment reduced apoptosis, delayed senescence, and promoted adipogenesis of ADSCs. These effects were inhibited by 3-methyladenine, indicating that the changes may be mediated by autophagy. Moreover, the survival rate and angiogenesis of the grafts were increased after upregulation of ADSC autophagy in vivo, which may help improve the efficiency of clinical fat transplantation.. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Topics: Adenine; Adipogenesis; Adipose Tissue; Apoptosis; Autophagy; Humans; Sirolimus; Stem Cells

As the skin is the largest organ of the human body, it is aging inevitably and produces cosmetic and psychological problems, and even disease. Therefore, the molecular mechanisms related to the prevention of skin aging need to be further explored.. Aging models were constructed by D-galactose. Mice were administrated with polygoni multiflori radix preparat (PMRP), PMRP and 3-methyladenine, or PMRP and rapamycin intragastrically. The apparent and viscera index of aged rats was measured. Then, the physicochemical property, antioxidant ability, histological structure, mitochondrial membrane potential, ATP and ROS levels, and mitophagy of aged skins were determined. Finally, the expression of PINK1, Parkin, P62, and LC3II/I; apoptosis-related proteins; and the percentage of apoptotic cells were measured.. PMRP relieved skin aging with reducing of thymus index, improvement of pathological damage and histological structure, increase of the expression area of fibrous tissue, the ratio of type I to type III collagen, and antioxidant ability of aged skins. Importantly, PMRP also improved mitochondrial dysfunction with an increase in the content of mitochondrial membrane potential and ATP and a decrease of ROS levels. Moreover, mitophagy was enhanced with the treatment of PMRP when observed using electron microscopy, and the expression of PINK1, Parkin, and LC3I/II was increased with PMRP treatment but P62 expression was decreased. Meanwhile, PMRP alleviated apoptosis with a decrease of apoptotic cell and the expression of Cleaved-cas3, Bax, Cyt-c, AIF, and Smac as well as an increase of Bcl-2 expression.. The results demonstrated that the polygoni multiflori radix preparata may delay skin aging by inducing mitophagy.

Topics: Adenine; Aging; Animals; Antioxidants; Apoptosis; Drugs, Chinese Herbal; Fallopia multiflora; Male; Membrane Potential, Mitochondrial; Mitophagy; Rats; Sirolimus; Skin; Skin Aging

Signal transduction of Angiotensin II (Ang II) induced autophagy and its role in Ang II-induced dysfunction of HUVECs are still unclear.. HUVECs are stimulated with different doses of Ang II (10-9-10-5 mol/L) for different time (6-48 hours). Autophagy-related protein markers: LC3, Beclin-1 and SQSTM1/p62 are measured by western blot.. Incubation with Ang II increases autophagic flux (Beclin-1, autophagosomes formation, and degradation of SQSTM1/p62, LC3-I). Increased autophagic levels are inhibited by pretreatment with Ang II type 1 receptor (AT1) blocker (Candesartan), NADPH Oxidase inhibitor (apocycin), mitochondrial K. Our results demonstrate that Ang II stimulation increases autophagy levels via AT1 receptor, NADPH oxidase, mitochondrial K

Topics: Acetophenones; Adenine; Angiotensin II; Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Benzimidazoles; Biphenyl Compounds; Decanoic Acids; Human Umbilical Vein Endothelial Cells; Humans; Hydroxy Acids; Models, Biological; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; Signal Transduction; Sirolimus; Tetrazoles; Time Factors

The role of autophagic mechanisms in the protective effect of berberine (BBR) on lipopolysaccharide (LPS)-induced injury in the endothelial cells human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) was investigated. Cell viability, proliferation, and apoptosis were detected by the CCK-8 assay, the EdU kit, and flow cytometry, respectively, and autophagy-related protein expression, the number of autophagic vacuoles, and LC3 double-fluorescence were examined using western blot analysis, transmission electron microscopy, and confocal microscopy, respectively. LPS resulted in a decrease in the cell viability and proliferation of HUVECs and HPMECs and an increase in the number of apoptotic cells, while BBR treatment resulted in an increase in cell viability and proliferation, as well as a decrease in cell apoptosis. Furthermore, BBR could inhibit LPS-induced autophagy, as demonstrated by its inhibitory effects on the LC3-II/LC3-I ratio and Beclin-1 levels and its promotive effect on p62 expression. Addition of the autophagy inducer rapamycin (RAPA) aggravated LPS-induced injury, while treatment with the autophagy blocker 3-methyladenine (3-MA) attenuated the injury. Further, the protective effect of BBR was inhibited by rapamycin. JNK inhibition by SP600125 inhibited LPS-induced autophagy, and BBR could not alter the LPS-induced autophagy in HUVECs and HPMECs that were pretreated with SP600125. The present data indicate that BBR attenuated LPS-induced cell apoptosis by blocking JNK-mediated autophagy in HUVECs and HPMECs. Therefore, the JNK-mediated autophagy pathway could be a potential target for the prevention and treatment of cardiovascular disease.

Topics: Adenine; Anthracenes; Autophagy; Berberine; Cell Proliferation; Cell Survival; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; MAP Kinase Signaling System; Protective Agents; Sirolimus

Methylmercury (MeHg) is a environmental contaminant, which can induce neurotoxic effects. So far, the exact molecular mechanisms of autophagy and its effect on apoptosis in MeHg-induced neurotoxicity have not been elucidated. Here, rats were exposed to MeHg (4, 8, or 12 μmol/kg) for 4 weeks to evaluate the dose-effect relationship between MeHg and apoptosis, or autophagy in cerebral cortex. On this basis, rapamycin (Rapa) or 3-methyladenine (3-MA) was administrated to further explore the regulatory mechanisms of autophagy on MeHg-induced neuronal apoptosis. The pathological changes, autophagy or apoptosis levels, expression of autophagic or apoptotic-associated factors such as mTOR, S6K1, 4EBP1, Vps34, Beclin1, p62, LC3, Bcl-2/Bax, caspase, or MAPKs were investigated. Results showed that MeHg dose-dependently induced pathological changes in cerebral cortex, and the levels of autophagy and apoptosis were increased. Furthermore, Rapa pretreatment antagonized MeHg-induced apoptosis, whereas 3-MA further aggravated apoptosis, which were supported by findings that Rapa activated mTOR-mediated autophagy while 3-MA inhibited Vps34-related autophagy, further affect neuronal apoptosis through regulation of apoptotic factors mentioned above. In conclusion, the findings indicated that MeHg dose-dependently induced autophagy or apoptosis, and mTOR or Vps34 may play important roles in mediating autophagy, which further regulated apoptosis through MAPKs or mitochondrial apoptosis pathways.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cerebral Cortex; Class III Phosphatidylinositol 3-Kinases; Dose-Response Relationship, Drug; Female; Male; Methylmercury Compounds; Neurons; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases

Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy.. Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres.. All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research.. The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells.

Topics: Adenine; Autophagy; Autophagy-Related Proteins; Cell Differentiation; Cytokines; Dendritic Cells; Flow Cytometry; Genotype; HL-60 Cells; Humans; Macrolides; Microscopy, Fluorescence; Monocytes; Phenotype; Polymorphism, Single Nucleotide; Sirolimus; THP-1 Cells; U937 Cells

The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson's disease (PD). In this study, we investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, we found that Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. Our results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models. Our findings not only provide a novel insight into the mechanisms of Nrf2-antioxidant-autophagy signaling, but also provide potential targets for PD interventions.

Topics: Adenine; Animals; Animals, Genetically Modified; Antioxidants; Antiparkinson Agents; Astrocytes; Autophagy; Behavior, Animal; Dihydroxyphenylalanine; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Male; Motor Activity; Nerve Degeneration; NF-E2-Related Factor 2; Oleanolic Acid; Parkinsonian Disorders; Phenotype; Rats, Sprague-Dawley; Repressor Proteins; Rotenone; Signal Transduction; Sirolimus

Autophagy is a self-degrading process. Previously, we showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel transforming growth factor β1 (TGFβ1)-interacting factor in liver fibrosis; the role of TGFβ1-mediated autophagy in hepatic stellate cells (HSCs) activation has been investigated. However, whether autophagy is regulated by IGFBPrP1 remains unknown.. We investigated the interactions among IGFBPrP1, autophagy, and activation of primary rat HSCs.. Primary HSCs were separated from Sprague Dawley rats by two-step enzymatic digestion, and then, we overexpressed or inhibited IGFBPrP1 expression in HSCs under serum-starved condition. Autophagy inducer rapamycin or inhibitor 3-methyladenine (3MA) was used to assess the relationship between autophagy and HSCs activation.. We observed the expression of activation marker α-SMA and autophagy markers such as LC3B and Beclin1, which were significantly increased in HSCs treated with adenovirus vector harboring the IGFBPrP1 gene (AdIGFBPrP1) compared to cells cultured under serum-starved. In comparison, HSCs treated with shIGFBPrP1 showed opposite results. Furthermore, HSCs activation and autophagy increased when cells were treated with rapamycin, whereas opposite results were obtained when cells were treated with 3MA. AdIGFBPrP1 treatment downregulated the phosphorylation of Akt and mTOR.. Autophagy was induced in IGFBPrP1-treated primary HSCs, and IGFBPrP1-induced autophagy promoted the activation of HSCs and extracellular matrix expression, the underlying mechanism of which may involve the phosphatidylinositide 3-kinase/Akt/mTOR signaling pathway.

Topics: Actins; Adenine; Animals; Autophagy; Beclin-1; Hepatic Stellate Cells; Insulin-Like Growth Factor Binding Proteins; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinase; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Transforming Growth Factor beta1

Autophagy is a cellular process activated in response to various stresses such as starvation, hypoxia, and oxidative stress. Autophagy was reported to modulate the inflammatory pathways. However, whether autophagy is involved in regulation of palmitate-induced inflammation of skeletal muscle C2C12 cells is still unknown. The present study aimed to investigate the autophagic pathway in C2C12 cells treated with 0.5 mM palmitate. The results showed that the protein levels of LC3BII and P62 were increased in C2C12 cells after 12 h palmitate treatment. Besides, inhibition of autophagy by chloroquine or 3-methyladenin and its activation by rapamycin were associated with elevated mRNA and protein levels of IL-6 and TNF-α inflammatory cytokines in C2C12 cells. To study the mechanism by which autophagy impairment leads to activation of inflammatory responses, reactive oxygen species (ROS) levels in palmitate-treated cells were measured. The results showed that while palmitate stimulates ROS production, pretreatment of the cells with N-acetyl cysteine (NAC), a ROS scavenger, reduced inflammatory responses and also improved LC3-BII and P62 protein in the C2C12 cells exposed to palmitate. These findings suggest that palmitate-induced defect of autophagic flux leads to elevated inflammatory cytokine expression in the skeletal muscle cells by regulating the oxidative stress process.

Topics: Acetylcysteine; Adenine; Animals; Autophagy; Cell Line; Chloroquine; Cytokines; Free Radical Scavengers; Inflammation; Interleukin-6; Mice; Microtubule-Associated Proteins; Muscle, Skeletal; Oxidative Stress; Palmitates; Reactive Oxygen Species; Sequestosome-1 Protein; Sirolimus; Tumor Necrosis Factor-alpha

Although there is an increment in stroke burden in the world, stroke therapeutic strategies are still extremely limited to a minority of patients. We previously demonstrated that dexmedetomidine (DEX) protects against focal cerebral ischemia via inhibiting neurons autophagy. Nevertheless, the role of DEX in regulating astrocytes autophagic status in oxygen-glucose deprivation, a condition that mimics cerebral ischemia, is still unknown. In this study, we have shown that DEX and DEX + RAPA (autophagy inducer) increased viability and reduced apoptosis of primary astrocytes in oxygen-glucose deprivation (OGD) model compared with DEX + 3-methyladenine (3-MA) (autophagy inhibitor). DEX induced the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1, while reduced the expression of p62 in primary cultured astrocytes through induction of autophagy. In addition, DEX enhanced the expression of tuberous sclerosis complex 2 (TSC2) in primary cultured astrocytes, while reduced the expression of mammalian target of rapamycin (mTOR). In conclusion, our study suggests that DEX exerts a neuroprotection against OGD-induced astrocytes injury via activation of astrocytes autophagy by regulating the TSC2/mTOR signaling pathway, which provides a new insight into the mechanisms of DEX treatment for acute ischemic injury.

Topics: Adenine; Animals; Astrocytes; Autophagy; Cell Hypoxia; Cells, Cultured; Dexmedetomidine; Drug Evaluation, Preclinical; Glucose; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neuroprotective Agents; Random Allocation; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein

The molecular mechanism of autophagy in Lactoferrin (LF) induced osteoblast differentiation is not fully demonstrated. In this study, alkaline phosphatase (ALP) activity, alizarin red S staining and ELISA were used to study N-terminal propeptide of type I procollagen (PINP) expression. mRFP-GFP-LC3 adenoviruses, mono-dansylcadaverine (MDC) staining, scanning electron microscopy, and western blot analysis was employed to probe the LF induced autophagy. The interaction between autophagy receptor Neighbor of Brca1 gene (Nbr1) and pp38 was studied. 3-methyladenine (3-MA) and chloroquine (CQ) could inhibit the activity of ALP, PINP and the autophagy in LF group. LF treatment could up-regulate and down-regulate the expressions of pp38 and Nbr1with a dose-dependent manner, respectively. LF could inhibit the recognition of pp38 and Nbr1. In addition, LF can prompt Nbr1-medicated autophagy and prevent pp38 degradation by autophagy. LF can induce Nbr1-mediated autophagy and inhibit pp38 entering into autophagy flux in the physiological process of osteoblast differentiation.

Topics: Adenine; Animals; Autophagy; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Chloroquine; Intracellular Signaling Peptides and Proteins; Lactoferrin; Mice; Osteoblasts; Osteogenesis; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Sirolimus

Penehyclidine hydrochloride (PHC) suppresses renal ischemia and reperfusion (I/R) injury (IRI); however, the underlying mechanism of action that achieves this function remains largely unknown. The present study aimed to investigate the potential role of autophagy in PHC‑induced suppression of renal IRI, as well as the involvement of cell proliferation and apoptosis. A rat IRI model and a cellular hypoxia/oxygenation (H/R) model were established; PHC, 3‑methyladenine (3‑MA) and rapamycin (Rapa) were administered to the IRI model rats prior to I/R induction and to H/R cells following reperfusion. Serum creatinine was measured using a biochemistry analyzer, whereas aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) expression levels were detected using ELISA kits. Renal tissue injury was evaluated by histological examination. In addition, microtubule‑associated protein light chain 3B (LC3B) expression, autophagosome formation, cell proliferation and apoptosis were detected in the cellular H/R model. The results demonstrated that I/R induced renal injury in IRI model rats, upregulated serum creatinine, ALAT and ASAT expression levels, and increased autophagic processes. In contrast, pretreatment with PHC or Rapa significantly prevented these I/R‑induced changes, whereas the administration of 3‑MA enhanced I/R‑induced injuries through suppressing autophagy. PHC and Rapa increased LC3B and Beclin‑1 expression levels, but decreased sequestome 1 (p62) expression in the cellular H/R model, whereas 3‑MA prevented these PHC‑induced changes. PHC and Rapa promoted proliferation and autophagy in the cellular H/R model; these effects were accompanied by increased expression levels of LC3B and Beclin‑1, and reduced p62 expression levels, whereas these levels were inhibited by 3‑MA. Furthermore, PHC and Rapa inhibited apoptosis in the cellular H/R model through increasing Bcl‑2 expression levels, and suppressing Bax and caspase‑3 expression levels; the opposite effect was induced by 3‑MA. In conclusion, PHC suppressed renal IRI through the induction of autophagy, which in turn promoted proliferation and suppressed apoptosis in renal cells.

Topics: Adenine; Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Autophagy; Beclin-1; Caspase 3; Cell Proliferation; Disease Models, Animal; Ischemia; Kidney; Male; Quinuclidines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Microvascular changes are the earliest adverse events in diabetic retinopathy, but recent studies have shown that oxidative stress induced by photoreceptors is associated with the development of the retinopathy. The purpose of this study was to determine the roles played by superoxides formed by photoreceptors under hyperglycemic conditions on autophagy. To accomplish this, we cultured 661 W cells, a transformed murine cone cell line, with 5.5 or 25 mM glucose in the presence or absence of 3 methyl adenine (3MA) or rapamycin. The superoxides were determined by flow cytometry using hydroethidine as a fluorescence probe. The autophagy activity was determined by changes in the expression of LC3B2 and P62 by immunoblotting. The degree of mitophagy was determined by the accumulation of mitochondria and lysosomes. Apoptotic changes of 661 W cells were determined by the caspase 3/7 activities. Our results showed higher levels of P62 and superoxides in cells cultured in 25 mM glucose than in 5.5 mM glucose. Addition of 3MA caused a significant increase of P62, superoxides, and caspase 3/7 activities in the 661 W cells cultured in high glucose but not in low glucose. These findings suggest that autophagy is important for the functioning and survival of 661 W cells under hyperglycemic conditions.

Topics: Adenine; Animals; Autophagy; Cell Line; Cell Survival; Diabetic Retinopathy; Dose-Response Relationship, Drug; Gene Expression Regulation; Glucose; Mice; Microtubule-Associated Proteins; Models, Biological; Oxidative Stress; Retinal Cone Photoreceptor Cells; Sequestosome-1 Protein; Sirolimus; Superoxides

Autophagy is involved in the modulation of nutrition, immunity, and disease in humans and animals. To understand the impact of autophagy modulation on a rainbow trout gill cell line, RTgill-W1, treatments including reduced nutrition (2% fetal bovine serum compared with 10% control), rapamycin, 3-methyladenine, deoxynivalenol, and chloroquine were tested. Western blot and immunofluorescence were used to detect microtubule-associated protein 1A/1B-light chain protein and quantitative polymerase chain reaction was used to detect the expression of 10 autophagy-related genes. At 3-d post-treatment, reduced nutrition significantly (p < 0.05) increased autophagy while deoxynivalenol significantly (p < 0.01) suppressed it compared to controls. These phenomena were confirmed by using immunofluorescence to detect the number of autophagosomes in RTgill-W1. Chloroquine is critical to allow observation of microtubule-associated protein 1A/1B-light chain protein in this model. The commonly used autophagy-modulating chemicals rapamycin and 3-methyladenine either activated or suppressed microtubule-associated protein 1A/1B-light chain protein, respectively, as expected from the literature, but did not act in a consistently significant manner. Expression of five of the 10 Atg genes, including lc3, gabarap, atg4, atg7, and atg12, were altered in a similar pattern to microtubule-associated protein 1A/1B-light chain protein. The consistent trend of autophagy-related gene upregulation including becn1, lc3, gabarap, and atg9 following treatment with 3-methyladenine and chloroquine is suggestive of a novel feedback regulation in the autophagy machinery.

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Cell Survival; Chloroquine; Fish Proteins; Gene Expression Regulation; Gills; Microtubule-Associated Proteins; Nutrients; Oncorhynchus mykiss; Pharmaceutical Preparations; Serum; Sirolimus; Time Factors; Trichothecenes

Rabies is an important zoonotic disease in Iran. Autophagy is a process that maintains homeostasis and can be used as an innate defense mechanism against viruses. Apoptosis is the process of programmed cell death induced by physiological and pathological conditions. The crosstalk of autophagy and apoptosis plays a key role in rabies virus infection. In the current study, NMRI mice intra-cranially received 3-Methyl Adenine (3-MA), rapamycin, street rabies virus (SRABV) and drugs plus SRABV. SRABV and Map1lc3, Beclin-1, Atg5 gene expression were assayed by real-time PCR. Immunohistochemistry was carried out via LC3 protein staining as an autophagy marker, and apoptotic cell death was measured using a TUNEL assay. Map1lc3, Beclin-1 and Atg5 genes expression was significantly increased in drug-plus-SRBV-treated tissues compared to control at 24 hpi. Map1lc3 and Atg5 gene expression showed a slight change in the drugs-plus-virus group compared with the control at 72 hpi. The presence of LC3 in the tissues of the group treated with rapamycin plus SRBV confirmed induction of autophagy, but it was not present in the tissues treated with 3-MA plus SRBV. Our data revealed that apoptosis was induced only in the groups receiving the SRBV or rapamycin or both at 24 hpi. Apoptosis was observed after 72 hours, when the drugs' effect had disappeared in all but the autophagy inhibitor group. Understanding the interaction of SRABV with autophagy pathway genes and its effect on host cell apoptosis may open a new horizon for human intervention and allow a deeper understanding of rabies infections.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain; Disease Models, Animal; Fluorescent Antibody Technique, Direct; Mice; Neurons; Rabies; Rabies virus; Sirolimus; Viral Proteins; Virus Replication

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is identified as the main risk factor of severe dengue diseases. The underlying mechanisms leading to severe dengue fever remain unclear.. THP-1 cells were treated with an autophagy inducer (rapamycin) or inhibitor (3-methyladenine [3-MA]) and infected with DENV and DENV-ADE. In order to investigate the expression profile of autophagy-related genes in DENV-ADE and DENV direct infection of THP-1 cells, the PCR array including 84 autophagy-related genes was selected to detect the expression of related genes, and then heat map and clustergram were established by analysis software to compare the expression differences of these genes between the DENV-ADE and DENV direct infection.. Autophagy-inducing complex related genes ATG5 and ATG12 were upregulated, and autophagosomes were also observed by transmission electron microscopy among DENV-ADE- and DENV-infected THP-1 cells, which indicated that autophagy was involved in dengue infection. The results show that 3-MA has a significant inhibitory effect on ATG12 in THP-1 cells; on the contrary, the expression of ATG12 was upreg-ulated in THP-1 cells that were treated with rapamycin. The autophagy-related genes ESR1, INS, BNIP3, FAS, TGM2, ATG9B, and DAPK1 exhibited significant differences between DENV-ADE and DENV direct infection groups.. In the present study, an additional mechanism of autophagy was inhibited by the autophagy inhibitor (3-MA) in DENV- and DENV-ADE-infected THP-1 cells. Our finding provided a clear link between autophagy and antibody-enhanced infection of DENV.

Topics: Adenine; Antibodies, Viral; Antibody-Dependent Enhancement; Autophagosomes; Autophagy; Autophagy-Related Protein 12; Autophagy-Related Proteins; Dengue; Dengue Virus; Humans; Sirolimus; THP-1 Cells; Transcriptome

NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation contributes to the progression of atherosclerosis, and autophagy inhibits inflammasome activation by targeting macrophages. We investigated whether fucoidan, a marine sulfated polysaccharide derived from brown seaweeds, could reduce NLRP3 inflammasome activation by enhancing sequestosome 1 (p62/SQSTM1)-dependent selective autophagy to alleviate atherosclerosis in high-fat-fed ApoE-/- mice with partial carotid ligation and differentiated THP-1 cells incubated with oxidized low-density lipoprotein (oxLDL). Fucoidan significantly ameliorated lipid accumulation, attenuated progression of carotid atherosclerotic plaques, deregulated the expression of NLRP3 inflammasome, autophagy receptor p62, and upregulated microtubule-associated protein light chain 3 (LC3)-II/I levels. Transmission electron microscopy and GFP-RFP-LC3 lentivirus transfection further demonstrated that fucoidan could activate autophagy. Mechanistically, fucoidan remarkably inhibited NLRP3 inflammasome activation, which was mostly dependent on autophagy. The inhibitory effects of fucoidan on NLRP3 inflammasome were enhanced by autophagy activator rapamycin (Rapa) and alleviated by autophagy inhibitor 3-methyladenine (3-MA). Fucoidan promoted the colocalization of NLRP3 and p62. Knockdown of p62 and ATG5 by small interfering RNA significantly reduced the inhibitory effects of fucoidan treatment on NLRP3 inflammasome. The data suggest that fucoidan can inhibit NLRP3 inflammasome activation by enhancing p62/SQSTM1-dependent selective autophagy to alleviate atherosclerosis.

Topics: Adenine; Animals; Atherosclerosis; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Gene Knockdown Techniques; Humans; Inflammasomes; Lipid Metabolism; Lipoproteins, LDL; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; NLR Family, Pyrin Domain-Containing 3 Protein; Plaque, Atherosclerotic; Polysaccharides; Sequestosome-1 Protein; Sirolimus

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that eventually leads to joint deformities and loss of joint function. Previous studies have demonstrated a close relationship between autophagy and the development of RA. Although autophagy and apoptosis are two different forms of programmed death, the relationship between them in relation to RA remains unclear. In this study, we explored the effect of autophagy on apoptosis of articular chondrocytes in vivo and in vitro. Adjuvant arthritis (AA) and acid-induced primary articular chondrocyte apoptosis were used as in vivo and in vitro models, respectively. Articular chondrocyte autophagy and apoptosis were both observed dynamically in AA rat articular cartilage at different stages (15 days, 25 days and 35 days). Moreover, chondrocyte apoptosis and articular cartilage injury in AA rats were increased by the autophagy inhibitor 3-methyladenine (3-MA) and decreased by the autophagy activator rapamycin. In addition, pre-treatment with 3-MA increased acid-induced chondrocyte apoptosis, while pre-treatment with rapamycin reduced acid-induced chondrocyte apoptosis in vitro. These results suggest that autophagy might be a potential target for the treatment of RA.

Topics: Adenine; Animals; Apoptosis; Arthritis, Experimental; Autophagy; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; Male; Rats, Sprague-Dawley; Sirolimus

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

Topics: Adenine; Animals; Autophagy; Cell Size; Cells, Cultured; Cumulus Cells; Embryonic Development; Female; In Vitro Oocyte Maturation Techniques; Meiosis; Oocytes; Oogenesis; Ovarian Follicle; Parthenogenesis; Sirolimus; Swine

To investigate the process by which quercetin suppresses atherosclerosis by upregulating MST1-mediated autophagy in RAW264.7 macrophages.. An in vitro foam cell model was established by culturing RAW264.7 macrophages with oxidized low-density lipoprotein (ox-LDL). The cells were treated with quercetin alone or in combination with the autophagy inhibitor, 3-methyladenine, and autophagy agonist, rapamycin. Cell viability was detected with a CCK-8 kit. Lipid accumulation was detected by oil red O staining, senescence was detected by SA-β-gal (senescence-associated β-galactosidase) staining, reactive oxygen species were detected by ROS assay kit. Autophagosomes and mitochondria were detected by transmission electron microscope (TEM), and expression of MST1, LC3-II/I, Beclin1, Bcl-2, P21, and P16 were detected by immunofluorescence and Western blot.. Ox-LDL induced RAW264.7 macrophage-derived foam cell formation, reduced survival, aggravated cell lipid accumulation, and induced a senescence phenotype. This was accompanied by decreased formation of autophagosome; increased expression of P53, P21, and P16; and decreased expression of LC3-II/I and Beclin1. After intervention with quercetin, the cell survival rate was increased, and lipid accumulation and senescence phenotype were reduced. Furthermore, the expression of LC3-II/I and Beclin1 were increased, which was consistent with the ability of quercetin to promote autophagy. Ox-LDL also increased the expression of MST1, and this increase was blocked by quercetin, which provided a potential mechanism by which quercetin may protect foam cells against age-related detrimental effects.. Quercetin can inhibit the formation of foam cells induced by ox-LDL and delay senescence. The mechanism may be related to the regulation of MST1-mediated autophagy of RAW264.7 cells.

Topics: Adenine; Animals; Atherosclerosis; Autophagy; Cell Survival; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Foam Cells; Hepatocyte Growth Factor; Lipid Metabolism; Lipoproteins, LDL; Mice; Proto-Oncogene Proteins; Quercetin; RAW 264.7 Cells; Sirolimus; Up-Regulation

Sepsis-induced myopathy is a heavy burden for patients during respiratory failure as well as after discharge, which could be characterized with qualitative changes to nAChR in a rat model of sepsis, regulated by NRG-1. Autophagy is an innate immune defense mechanism against microbial challenges. We found neuromuscular dysfunction in anterior tibial muscle of male Sprague-Dawley rats 24 h after cecal ligation and puncture (CLP). CLP resulted in increased systemic and local inflammation in anterior tibial muscle tissue. The start-up phase of autophagy, as measured by LC3II, was activated immediately after CLP and continued until 24 h; the degradation phase was suppressed until 24 h, after a brief increase at 4 h (revealed by p62). NRG-1 first increased, and then decreased to a level lower than that in the sham group. Meanwhile, expression of γ- and α7- acetylcholine receptors was detected at 8 and 16 h after CLP; levels increased continuously until 24 h. Subsequently, we investigated the significance of autophagy in CLP-induced neuromuscular dysfunction by treatment with rapamycin or 3-methyladenine, which were classical pharmaceuticals for enhancing or suppressing autophagy. Rapamycin activated autophagy, limited the CLP-induced systemic pro-inflammatory response and blood bacterial load without affecting local inflammatory response, upregulated NRG-1, downregulated γ- and α7-acetylcholine receptors, and improved 7-day neuromuscular function and survival rate. In contrast, 3-methyladenine enhanced local inflammatory response, suppressed autophagy, worsened 7-day neuromuscular function. We conclude that impaired autophagy may contribute to sepsis-induced neuromuscular dysfunction in young male rats. Enhancing autophagy with rapamycin alleviated qualitative changes to acetylcholine receptors without triggering local anti-inflammatory response and improved anterior tibial muscle function in septic early phase (24 h) as well as in septic chronic phase (7d). Enhancing autophagy soon after sepsis is a potential strategy for treatment of sepsis-induced myopathy.

Topics: Adenine; Animals; Autophagy; Cecum; Ligation; Male; Punctures; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Sepsis; Sirolimus; Sphingosine-1-Phosphate Receptors

Selenium deficiency and T-2 toxin exposure may contribute to the development of Keshan disease characterized by congestive cardiomyopathy. The aim of this study was to explore the role of autophagy in the aggravation of selenium deficiency on T-2 toxin-induced damages on primary cardiomyocyte. Our present study demonstrated that 0.25-1 μM T-2 toxin damaged primary cardiomyocytes and selenium deficiency exacerbated T-2 toxin-induced damages by measuring the levels of MTT, lactate dehydrogenase and cleaved-caspase 3. T-2 toxin triggered autophagy in primary cardiomyocytes, as indicated by markedly increased expressions of LC3-Ⅱ and Beclin-1 mRNA levels. Rapamycin (autophagy agonist) treatment increased autophagy levels and decreased the cytotoxicity caused by T-2 toxin while 3-methyladenine (autophagy inhibitor) treatment reduced autophagy levels and enhanced the cytotoxicity of T-2 toxin, suggesting that autophagy protect primary cardiomyocytes from the cytotoxicity of T-2 toxin. Selenium deficiency lowered cytoprotective autophagy in the primary cardiomyocytes treated by T-2 toxin. It can be concluded that autophagy induced by T-2 toxin plays a role in protecting primary cardiomyocyte, but selenium deficiency decreases the protective autophagy and then exacerbate T-2 toxin-induced damages. Our finding may partly interpret the combination effects of selenium deficiency and T-2 toxin on the development of Keshan disease.

Topics: Adenine; Animals; Autophagy; Beclin-1; Caspase 3; Cell Survival; Microtubule-Associated Proteins; Myocytes, Cardiac; Rats; Rats, Wistar; Selenium; Sirolimus; T-2 Toxin

T cells have emerged as a therapeutically-relevant target for ex vivo gene delivery and editing. However, most commercially available reagents cannot transfect T cells and designing cationic polymers for non-viral gene delivery to T cells has resulted in moderate success. Here, we assess various barriers to successful gene transfer in the Jurkat human T cell line and primary human T cells. Using two polymers previously developed by our group, we show that uptake is one barrier to gene delivery in primary human T cells but is not predictive of successful gene delivery. We then probe intracellular pathways for barriers to gene transfer including endosomal acidification, autophagy, and immune sensing pathways. We find that endosomal acidification is slower and not as robust in human T cells compared to the model HeLa human cell line commonly used to evaluate cationic polymers for gene delivery. These studies inform the future design of cationic polymers for non-viral gene delivery to T cells, specifically, to rely on alternative endosomal release mechanisms rather than on pH-triggered release.

Topics: Adenine; Autophagy; Carbocyanines; Cations; Cell Survival; HeLa Cells; Humans; Hydrogen-Ion Concentration; Jurkat Cells; Microscopy, Confocal; Plasmids; Polymers; Sirolimus; T-Lymphocytes; Transfection

Ischemia-reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI). Numerous therapeutic approaches for I/R injury have been studied, including autophagy, particularly in animal models of renal I/R injury derived from young or adult animals. However, the precise role of autophagy in renal ischemia-reperfusion in the aged animal model remains unclear. The purpose of this study was to demonstrate whether autophagy has similar effects on renal I/R injury in young and aged rats.. All rats were divided into two age groups (3 months and 24 months) with each group being further divided into four subgroups (sham, I/R, I/R+Rap (rapamycin, an activator of autophagy), I/R+3-MA (3-methyladenine, an inhibitor of autophagy)). The I/R+Rap and I/R+3-MA groups were intraperitoneally injected with rapamycin and 3-MA prior to ischemia. We then measured serum levels of urea nitrogen, creatinine and assessed damage in the renal tissue. Immunohistochemistry was used to assess LC3-II and caspase-3, and Western blotting was used to evaluate the autophagy-related proteins LC3-II, Beclin-1 and P62. Apoptosis and autophagosomes were evaluated by TUNEL and transmission electron microscopy, respectively.. Autophagy was activated in both young and aged rats by I/R and enhanced by rapamycin, although the level of autophagy was lower in the aged groups. In young rats, the activation of autophagy markedly improved renal function, reduced apoptosis in the renal tubular epithelial cells and the injury score in the renal tissue, thereby exerting protective effects on renal I/R injury. However, this level of protection was not present in aged rats.. Our data indicated that the activation of autophagy was ineffective in aged rat kidneys. These discoveries may have major implications in that severe apoptosis in aged kidneys might be refractory to antiapoptotic effect induced by the activation of autophagy.

Topics: Acute Kidney Injury; Adenine; Age Factors; Animals; Apoptosis; Autophagosomes; Autophagy; Beclin-1; Blood Urea Nitrogen; Caspase 3; Creatinine; Disease Models, Animal; Kidney; Male; Microtubule-Associated Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Impaired mitochondrial function is a key factor attributing to lung ischaemia-reperfusion (IR) injury, which contributes to major post-transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3-MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia-reoxygenation (H/R) were monitored after 3-MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3-MA inhibited cell apoptosis of donor lung to alleviate I/R-induced lung injury as well as inhibited H/R-induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R- and H/R-mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R-induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury.

Topics: Adenine; Animals; Autophagy; Endothelial Cells; Humans; Lung; Lung Transplantation; Mitochondria; Rats; Reperfusion Injury; Signal Transduction; Sirolimus; Tissue Donors; TOR Serine-Threonine Kinases

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.

Topics: Adenine; Adenosine Triphosphate; Autophagosomes; Autophagy; Eimeria tenella; Sirolimus; Sporozoites

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV-inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3-MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.

Topics: Adenine; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Autophagy; Cell Line; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Haemophilus parasuis; Host-Pathogen Interactions; Luminescent Proteins; Macrophages, Alveolar; Microtubule-Associated Proteins; Oxazines; Protein Isoforms; Red Fluorescent Protein; Ribonucleotides; Signal Transduction; Sirolimus; Swine; Virulence

The cardioprotective effect of FSTL1 has been extensively studied in recent years, but its role in myocardial ischemia/reperfusion injury (IRI) is unclear. In this study, we investigated the effect of FSTL1 pretreatment on myocardial IRI as well as the possible involvement of autophagic pathways in its effects.. The effects of FSTL1 on the viability and apoptosis of rat cardiomyocytes were investigated after exposure of cardiomyocytes to hypoxia/ischemia by using the CCK-8 assay and Annexin V/PI staining. Further, western blot analysis was used to detect the effects of FSTL1 pretreatment on autophagy-associated proteins, and confocal microscopy was used to observe autophagic flux. To confirm the role of autophagy, the cells were treated with the autophagy promoter rapamycin or the autophagy inhibitor 3-methyladenine, and cell viability and apoptosis during IRI were observed. These effects were also observed after treatment with rapamycin or 3-methyladenine followed by FSTL1 administration and IRI.. FSTL1 pretreatment significantly increased viability and reduced apoptosis in cardiomyocytes exposed to hypoxia/ischemia conditions. Further, FSTL1 pretreatment affected the levels of the autophagy-related proteins and enhanced autophagic flux during IRI. In addition, cell viability was enhanced and apoptosis was decreased by rapamycin treatment, while these effects were reversed by 3-MA treatment. However, when the myocardial cells were pretreated with rapamycin or 3-methyladenine, there was no significant change in their viability or apoptosis with FSTL1 treatment during IRI.. FSTL1 plays a protective role in myocardial IRI by regulating autophagy.

Topics: Adenine; Apoptosis; Autophagy; Cell Line; Cell Proliferation; Cell Survival; Follistatin-Related Proteins; Models, Biological; Myocardial Reperfusion Injury; Myocytes, Cardiac; Sirolimus

Intestinal ischemia/reperfusion (I/R) is a grave condition related to high morbidity and mortality. Autophagy, which can induce a new cell death named type II programmed cell death, has been reported in some intestinal diseases, but little is known in I/R-induced intestinal injury. In this study, we aimed to explore the role of autophagy in intestinal injury induced by I/R and its potential mechanisms.. The rats pretreated with rapamycin or 3-methyladenine had intestinal I/R injury. After reperfusion, intestinal injury was measured by Chiu's score, intestinal mucosal wet-to-dry ratio, and lactic acid level. Intestinal mucosal oxidative stress level was measured by malondialdehyde and superoxide dismutase. Autophagosome, LC3, and p62 were detected to evaluate autophagy level. Mammalian target of rapamycin (mTOR) was detected to explore potential mechanism.. Chiu's score, intestinal mucosal wet-to-dry ratio, lactic acid level, malondialdehyde level, autophagosomes, and LC3-II/LC3-I were significantly increased, and superoxide dismutase level and expression of p62 were significantly decreased in intestinal mucosa after intestinal ischemia/reperfusion. Pretreatment with rapamycin significantly aggravated intestinal injury evidenced by increased Chiu's score, intestinal mucosal wet-to-dry ratio and lactic acid level, increased autophagy level evidenced by increased autophagosomes and LC3-II/LC3-I and decreased expression of p62, and downregulated expression of p-mTOR/mTOR. On the contrary, pretreatment with 3-methyladenine significantly attenuated intestinal injury and autophagy level and upregulated expression of p-mTOR/mTOR.. In summary, autophagy was significantly enhanced in intestinal mucosa after intestinal ischemia/reperfusion, and inhibition of autophagy attenuated intestinal injury induced by I/R through activating mTOR signaling.

Topics: Adenine; Animals; Autophagy; Drug Evaluation, Preclinical; Intestinal Diseases; Intestinal Mucosa; Male; Malondialdehyde; Random Allocation; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Superoxide Dismutase; TOR Serine-Threonine Kinases

Topics: Adenine; Ammonium Chloride; Cell Line; Chaperone-Mediated Autophagy; Exosomes; Humans; Lysosomal-Associated Membrane Protein 2; Macrolides; Microautophagy; Mycophenolic Acid; RNA, Small Interfering; Sirolimus

Autophagy alterations have been observed in a variety of neurological disorders, however, very few studies have focused on autophagy alterations in epilepsy. The ketogenic diet (KD) likely ameliorates neuronal loss in several seizure models. However, whether this neuroprotective function occurs via starvation-induced autophagy and its prevalence in chronic kindled seizures remains unknown. The aim of this study was to determine the role of autophagy following seizure under KD, and the potential mechanism involved. Pentylenetetrazol (PTZ)-kindled rats, which were fed a Normal diet (ND) or KD, were pretreated with intraventricular infusions of saline, autophagy inducer rapamycin (RAP), or inhibitor 3-methyladenine (3-MA). KD alleviated seizure severity, decreased the number of Fluoro-jade B (FJB)-positive cells in the hippocampus of kindled rats. These effects were abolished by 3-MA pretreatment. RAP pretreatment did not affect seizure severity, but decreased the number of FJB-positive cells in ND group. KD decreased the percentage of damaged mitochondria in kindled group. Hippocampal Beclin-1 was increased by KD in vehicle group. The autophagy proteins Atg5, Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3-I in kindled KD-fed rats were higher, and the autophagy substrate P62 was lower than those in the kindled ND-fed rats, indicating an increase in autophagy following KD. Pretreatment with RAP increased the level of LC3-II/LC3-I, and pretreatment with 3-MA increased the level of P62 in KD-fed rats. To further clarify the mechanism of autophagy protection, the levels of key mitochondria related molecules were examed. The results showed that mitochondrial cytochrome c was up-regulated, cytosolic cytochrome c and the downstream cleaved caspase-3 was down-regulated in KD-fed rats, indicating a decrease in mitochondrial apoptosis. Taken together, our results indicated that KD activates autophagic pathways and reduces brain injury during PTZ-kindled seizures. The neuroprotective effect of KD is likely exerted via a reduction of mitochondrial cytochrome c release.

Topics: Adenine; Animals; Apoptosis; Autophagy; Caspase 3; Cytochromes c; Diet, Ketogenic; Epilepsy; Hippocampus; Male; Mitochondria; Neurons; Neuroprotective Agents; Pentylenetetrazole; Rats; Rats, Sprague-Dawley; Seizures; Sirolimus

Autophagy is a homeostatic process that has been shown to be vital in the innate immune defense against pathogens. However, little is known about the regulatory role of autophagy in porcine teschovirus 2 (PTV-2) replication. In this study, we found that PTV-2 infection induces a strong increase in GFP-LC3 punctae and endogenous LC3 lipidation. However, PTV-2 infection did not enhance autophagic protein degradation. When cellular autophagy was pharmacologically inhibited by wortmannin or 3-methyladenine, PTV-2 replication increased. The increase in virus yield via autophagy inhibition was further confirmed by silencing atg5, which is required for autophagy. Furthermore, PTV-2 replication was suppressed when autophagy was activated by rapamycin. Together, the results suggest that PTV-2 infection activates incomplete autophagy and that autophagy then inhibits further PTV-2 replication.

Topics: Adenine; Androstadienes; Animals; Autophagy; Autophagy-Related Protein 5; Cell Line; Epithelial Cells; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Host-Pathogen Interactions; Kidney; Microtubule-Associated Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; Swine; Teschovirus; Virus Replication; Wortmannin

Overexposure to manganese (Mn) has been known to induce alpha-synuclein (α-Syn) oligomerization, which is degraded mainly depending on endoplasmic reticulum stress (ER stress) and autophagy pathways. However, little data reported the cross-talk between ER stress and autophagy on Mn-induced α-Syn oligomerization. To explore the relationship between ER stress and autophagy, we used 4-phenylbutyric acid (4-PBA, the ER stress inhibitor), rapamycin (Rap, autophagy activator) and 3-methyladenine (3-MA, autophagy inhibitor) in mice model of manganism. After 4 weeks of treatment with Mn, both ER stress and autophagy were activated. Exposed to Mn also resulted in α-Syn oligomerization and neuronal cell damage in the brain tissue of mice, which could be relieved by 4-PBA pretreatment. Moreover, when the ER stress was inhibited, the activation of autophagy was also inhibited. Rap pretreatment significantly activated autophagy and decreased α-Syn oligomers. However, 3-MA pretreatment inhibited autophagy resulting in increase of α-Syn oligomers, and compensatorily activated PERK signaling pathway. Our results also demonstrated that the inhibition of autophagy by 3-MA aggravated neuronal cell damage. The findings clearly demonstrated that the cross-talking between autophagy and ER stress might play an important role in the α-Syn oligomerization and neurotoxicity by Mn.

Topics: Adenine; alpha-Synuclein; Animals; Apoptosis; Autophagy; Brain; Butylamines; Chlorides; Endoplasmic Reticulum Stress; Environmental Pollutants; Manganese; Manganese Compounds; Mice, Inbred C57BL; Neurons; Phenylbutyrates; Polymerization; Signal Transduction; Sirolimus

Hyperthermia causes oxidative stress in testes, which triggers antioxidant signals including autophagy and nuclear factor erythroid 2-related factor 2 (Nrf2). However, their relationship in testes under oxidative stress is unclear. In this study, we conducted testes injection for autophagy alteration and heat exposure to reveal the interaction between autophagy and the Nrf2-antioxidant system. Male mice were injected once with normal saline as control (Cont group), autophagy inhibitor 3-methyladenine (3-MA group) or autophagy inducer rapamycin (Rapa group). Then, each group was divided into two parts: one received a 2-h 42°C heat treatment for eight days (HT groups), and the other was kept thermal neutral (NT groups). Heat-exposed mice showed significantly increased rectal, scrotal surface and body surface temperatures. Histology of the testes revealed many vacuoles inserted in the seminiferous tubules in the HT Cont group and two 3-MA groups. Ultrastructural changes in germ cells revealed autophagosomes in two 3-MA groups. Immunohistochemical detection of Nrf2 and p62/SQSTM1 proteins showed prominent expression in Leydig cells. Heat exposure increased Nrf2 protein and mRNA levels. 3-MA and Rapa testes injection also resulted in Nrf2 cytoplasm accumulation. Massive conversion of LC3 (microtubule-associated protein light chain 3)Ⅰ to LC3Ⅱ was detected in two 3-MA groups, accompanied by decreased ATG5 (autophagy related gene 5) mRNA levels in the HT 3-MA group. These results indicated autophagy alteration triggered the Nrf2 signaling pathway with consequences such that the autophagy inducer protected the testes and the autophagy inhibitor enhanced the detrimental effects caused by heat exposure.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Proteins; Heat-Shock Response; Hot Temperature; Male; Mice; NF-E2-Related Factor 2; Seminiferous Tubules; Sequestosome-1 Protein; Signal Transduction; Sirolimus

Glucocorticoids (GCs) are closely associated with the progression of GC‑induced osteoporosis (GIOP) by inhibiting osteoblast viability. However, endogenous GCs are important for bone development. In addition, previous studies have demonstrated that GCs could induce autophagy, a cytoprotective process that is protective against various stressors. In the present study, the aim is to explore whether osteoblasts exhibited dose‑dependent viability in the presence of GCs due to autophagy. hFOB 1.19 osteoblasts were treated with various doses of dexamethasone (DEX; 10‑8‑10‑4 M) for 0, 24, 48 and 72 h. The results revealed a biphasic effect of DEX on the viability of hFOB 1.19 cells; a high dose of DEX (≥10‑6 M) accelerated cell apoptosis, while a low dose of DEX (10‑8 M) increased cell viability. Furthermore, significantly increased autophagy was observed in the low dose DEX treatment group, as indicated by the expression of the autophagy‑associated proteins beclin 1 and microtubule‑associated protein light chain 3, and the detection of autophagosomes. Another finding was that DEX upregulated intracellular reactive oxygen species (ROS), which was decreased by the autophagy agonist rapamycin. The increase in autophagy and cell viability associated with low‑dose DEX (10‑8 M) was suppressed by the ROS scavenger catalase and the autophagy inhibitor 3‑methyladenine. In conclusion, the results revealed that GCs affected osteoblast viability in a dose‑dependent manner. A low dose of GCs increased osteoblast viability by inducing autophagy via intracellular ROS. The results indicate that autophagy may be a novel mechanism by which osteoblasts survive GC exposure and provide a potential therapeutic target for treating GIOP.

Topics: Adenine; Apoptosis; Autophagy; Beclin-1; Biomarkers; Catalase; Cell Line; Cell Survival; Dexamethasone; Fetus; Gene Expression; Glucocorticoids; Hormesis; Humans; Microtubule-Associated Proteins; Osteoblasts; Reactive Oxygen Species; Sirolimus

Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down-regulation of Cdk2 and Cdk4 and the up-regulation of cell cycle suppressors, p21 and p27. In addition, the caspase-dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3-MA or bafilomycin, potentiated the honokiol-mediated anti-OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5-FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5-FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC-xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.

Topics: Adenine; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Biphenyl Compounds; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Fluorouracil; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lignans; Macrolides; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Lupus nephritis (LN) is one of the most common and severe complications in Systemic lupus erythematosus patients, and the mechanism underlining the pathogenesis of LN is still unknown. Autophagy plays vital roles in maintaining cell homeostasis and is involved in the pathogenesis of many diseases. In this study, we investigated the role of autophagy in the progression of LN.. Autophagic activities in podocytes of both LN patients (Class IV and V) and mice were evaluated. Podocytes were observed by electron microscopy, and autophagic activity was evaluated by immunofluorescence staining and western blot analysis. Apoptotic activity was evaluated by immunohistochemistry, TUNEL assays and flow cytometric analysis.. Significantly greater podocyte injury and discrepant autophagic levels were observed in LN patients. Differentiated mouse podocytes in the LN group showed reduced nephrin expression and increased apoptosis, as well as significantly higher levels of apoptosis-related proteins (cleaved caspase-3 and Bax). In the mice LN group, the increased number of autophagosomes was accompanied by increased LC3-II/LC3-I ratios and decreased p62, suggesting increased autophagic and apoptotic activity in podocytes. Blockade of autophagic activity by 3-MA or siRNA-mediated silencing of Atg5 resulted in decreases in LC3-II/LC3-I ratios, podocyte apoptosis and damage in the mice LN group. Futhermore, Rapamycin treatment increased LC3-II/LC3-I ratios, and enhanced LN-induced apoptosis in podocyte from modal animal.. This study demonstrates that autophagic activity of podocytes is a crucial factor in renal injury by directly affecting the function of podocyte; thus, inhibiting this activity during the early stages of LN is implicated as a potential therapeutic strategy for delaying the progression of LN. Also, clinical application in LN needs to consider patients' pathological type and drugs' comprehensive effectiveness.

Topics: Adenine; Adolescent; Adult; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Disease Progression; Female; Humans; Lupus Nephritis; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Podocytes; Sirolimus; Young Adult

Autophagy and apoptosis are two different biological processes that determine cell fates. We previously reported that autophagy inhibition and apoptosis induction are involved in lead(II)-induced cytotoxicity in primary rat proximal tubular (rPT) cells, but the interplay between them remains to be elucidated. Firstly, data showed that lead(II)-induced elevation of LC3-II protein levels can be significantly modulated by 3-methyladenine or rapamycin; moreover, protein levels of Autophagy-related protein 5 (Atg5) and Beclin-1 were markedly up-regulated by lead(II) treatment, demonstrating that lead(II) could promote the autophagosomes formation in rPT cells. Next, we applied three pharmacological agents and genetic method targeting the early stage of autophagy to validate that enhancement of autophagosomes formation can inhibit lead(II)-induced apoptotic cell death in rPT cells. Simultaneously, lead(II) inhibited the autophagic degradation of rPT cells, while the addition of autophagic degradation inhibitor bafilomycin A1 aggravated lead(II)-induced apoptotic death in rPT cells. Collectively, this study provided us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells, and the interplay between autophagy and apoptosis highlights a new sight into the mechanism of lead(II)-induced nephrotoxicity.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Beclin-1; Cells, Cultured; Kidney Tubules, Proximal; Lead; Microtubule-Associated Proteins; Rats; Sirolimus

The functional outcome after peripheral nerve repair is often unpredictable for many reasons, e.g., the severity of neuronal death and scarring. Axonal degeneration significantly affects outcomes. Post-injury axonal degeneration in peripheral nerves is accompanied by myelin degradation initiated by Schwann cells (SCs), which activate autophagy, a ubiquitous cytoprotective process essential for degrading and recycling cellular constituents. Scar formation occurs concomitantly with nerve insult and axonal degeneration. The association between SC autophagy and the mechanisms of nerve scar formation is still unknown. A rat model of peripheral nerve lesions induced by sciatic nerve transection injuries was used to examine the function of autophagy in fibrosis reduction during the early phase of nerve repair. Rats were treated with rapamycin (autophagy inducer) or 3-methyladenine (autophagy inhibitor). One week after the nerve damage, fibrosis was potently inhibited in rapamycin-treated rats and, based on gait analysis, yielded a better functional outcome. Immunohistochemistry showed that the autophagic activity of SCs and the accumulation of neurofilaments were upregulated in rapamycin-treated rats. A deficiency of SC autophagic activity might be an early event in nerve scar formation, and modulating autophagy might be a powerful pharmacological approach for improving functional outcomes.

Topics: Adenine; Animals; Autophagy; Cicatrix; Male; Nerve Regeneration; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; Schwann Cells; Sciatic Nerve; Sirolimus

Here, the regulatory role of autophagy is examined in both an LPS-induced lethal endotoxic shock mouse model and cecal ligation and puncture (CLP) mouse model. Autophagy-inhibitor 3-methyladenine (3-MA) and autophagy-enhancer rapamycin were administrated to mice challenged with LPS or CLP. Animals challenged with LPS or CLP combined with 3-MA displayed increased survival after endotoxemia, but LPS combined with rapamycin worsened the endotoxic shock of the mice. Among the different organs studied, the lungs and intestines exhibited significant differences among LPS alone, LPS combined with 3-MA and LPS combined with rapamycin. LPS combined with 3-MA attenuated the inflammatory damages of these organs as compared with LPS alone. In contrast, LPS combined with rapamycin increased damage in these organs. Consistently, serum inflammatory mediators TNF-α and IL-6 were decreased by the treatment of LPS combined with 3-MA as compared with LPS alone, while administration of LPS combined with rapamycin increased the serum TNF-α and IL-6 levels. Similar results were found in mouse bone marrow-derived macrophages exposed to LPS. Moreover, the regulatory effect of autophagy to endotoxic shock is dependent on the TLR4 signaling pathway. Our results demonstrate the central role of autophagy in the regulation of endotoxic shock and its potential modulation for endotoxic shock treatment.

Topics: Adenine; Animals; Autophagy; Carrier Proteins; Endotoxemia; Interleukin-6; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Sepsis; Shock, Septic; Signal Transduction; Sirolimus; Tumor Necrosis Factor-alpha

Contrast induced-acute kidney injury (CI-AKI) is one of the most common causes of acute kidney injury (AKI) in hospitalized patients. Mitophagy, the selective elimination of mitochondria via autophagy, is an important mechanism of mitochondrial quality control in physiological and pathological conditions. In this study, we aimed to determine effects of iohexol and iodixanol on mitochondrial reactive oxygen species (ROS), mitophagy and the potential role of mitophagy in CI-AKI cell models.. Cell viability was measured by cell counting kit-8. Cell apoptosis, mitochondrial ROS and mitochondrial membrane potential were detected by western blot, MitoSOX fluorescence and TMRE staining respectively. Mitophagy was detected by the colocalization of LC3-FITC with MitoTracker Red, western blot and electronic microscope.. The results showed that mitophagy was induced in human renal tubular cells (HK-2 cells) under different concentrations of iodinated contrast media. Mitochondrial ROS displayed increased expression after the treatment. Rapamycin (Rap) enhanced mitophagy and alleviated contrast media induced HK-2 cells injury. In contrast, autophagy inhibitor 3-methyladenine (3-MA) down-regulated mitophagy and aggravated cells injury.. Together, our finding indicates that iohexol and iodixanol contribute to the generation of mitochondrial ROS and mitophagy. The enhancement of mitophagy can effectively protect the kidney from iodinated contrast (iohexol)-induced renal tubular epithelial cells injury.

Topics: Acute Kidney Injury; Adenine; Apoptosis; Autophagy; Cell Line; Contrast Media; Epithelial Cells; Humans; Iodine; Iohexol; Kidney Tubules; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Mitochondria; Mitophagy; Reactive Oxygen Species; Sirolimus; Triiodobenzoic Acids

BACKGROUND HIF-1α plays an important role in hypoxia-ischemia brain damage. Accumulating evidences demonstrates that HIF-1α can contribute to cell autophagy. Oxygen-glucose deprivation (OGD) is a commonly used ischemic model in vitro. Our study was performed to investigate the influences of HIF-1α on autophagy in SH-SY5Y cells under OGD treatment. MATERIAL AND METHODS An OGD model was constructed in SH-SY5Y cells. PI method and MTT assay were used to test cell death and viability, respectively. Western blot assay was used to estimate the protein levels of HIF-1α and LC3. Quantitative GFP-LC3 light microscopy autophagy assay was performed for SH-SY5Y cells. 2ME2 and siRNA-HIF-1α were applied to investigate the effects of HIF-1α-knockdown on LC3 expression. Additionally, 3-MA (autophagy inhibitor) and autophagy inducer rapamycin (Rapa) were used to investigate the effects of autophagy on cell survival under OGD condition. RESULTS Under OGD, the apoptosis of SH-SY5Ycells was increased while cell viability rate was decreased. The expression of HIF-1α was increased along with the advancement of OGD treatment and achieved the highest level at 24 h. However, inhibiting HIF-1α expression decreased the cell apoptosis and increased cell viability. LC3-II expression was gradually increased with the duration of OGD condition and knockdown of HIF-1α resulted in decreased expression of LC3. Inhibiting autophagy significantly enhanced the viability and reduced the apoptosis of SH-SY5Y cells, while enhancing autophagy showed the opposite effects. CONCLUSIONS Enhanced expression of HIF-1α may be related to autophagy activation in SH-SY5Y cells, thus contributing to ischemic/hypoxic brain damage.

Topics: Adenine; Apoptosis; Autophagy; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Gene Knockdown Techniques; Glucose; Green Fluorescent Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Microtubule-Associated Proteins; Models, Biological; Oxygen; RNA, Small Interfering; Sirolimus

Previous studies by our group have demonstrated that extract of clove exhibits potent anticancer effects in vitro and in vivo. In the present study, the effect of an extracted and isolated active fraction of clove (AFC) on induction of cellular apoptosis in human colorectal cancer HCT-116 cells was investigated by morphological observation, flow cytometry, and western blotting analysis. The results revealed that AFC induced apoptosis of HCT-116 cells. AFC also induced autophagy, demonstrated by increased punctuate microtubule-associated protein 1A/1B-light chain 3 (LC3) staining, and LC3-II and Beclin-1 protein expression levels. Furthermore, the autophagy inhibitors 3-MA and baflomycin A1 potentiated the pro-apoptotic activity of AFC in HCT-116 cells. AFC also inhibited the phosphorylation of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signaling pathway. The present study may improve the existing understanding of the anticancer mechanisms of clove and provide a scientific rationale for AFC to be further developed as a promising novel anticancer agent for the treatment of colorectal cancer.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Macrolides; Phosphatidylinositol 3-Kinases; Plant Extracts; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Syzygium; TOR Serine-Threonine Kinases

Paracoccidioides spp. is a thermally dimorphic fungus endemic to Latin America and the etiological agent of paracoccidioidomycosis (PCM), a granulomatous disease acquired through fungal propagule inhalation by its mammalian host. The infection is established after successful mycelia to yeast transition in the host pulmonary alveoli. The challenging environment inside the host exposes the fungus to the need of adaptation in order to circumvent nutritional, thermal, oxidative, immunological and other stresses that can directly affect their survival. Considering that autophagy is a response to abrupt environmental changes and is induced by stress conditions, this study hypothesizes that this process might be crucially involved in the adaptation of Paracoccidioides spp. to the host and, therefore, it is essential for the proper establishment of the disease. By labelling autophagous vesicles with monodansylcadaverine, autophagy was observed as an early event in cells during the normal mycelium to yeast transition, as well as in yeast cells of P. brasiliensis under glucose deprivation, and under either rapamycin or 3-methyladenine (3-MA). Findings in this study demonstrated that autophagy is triggered in P. brasiliensis during the thermal-induced mycelium to yeast transition and by glucose-limited conditions in yeasts, both of which modulated by rapamycin or 3-MA. Certainly, further genetic and in vivo analyses are needed in order to finally address the contribution of autophagy for adaptation. Yet, our data propose that autophagy possibly plays an important role in Paracoccidioides brasiliensis virulence and pathogenicity.

Topics: Adenine; Autophagy; Gene Expression Regulation, Fungal; Mycelium; Nutrients; Oxidative Stress; Paracoccidioides; Paracoccidioidomycosis; Saccharomyces cerevisiae; Sirolimus

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Chemical and Drug Induced Liver Injury; Chromones; Cobalt; Disease Models, Animal; Erythropoietin; Humans; Liver; Liver Function Tests; Mice; Mice, Inbred C57BL; Morpholines; Peptide Fragments; Proto-Oncogene Proteins c-akt; Random Allocation; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway.. For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins.. Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin.. Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

Topics: Acetylcysteine; Adenine; Autophagy; Beclin-1; Blood Platelets; Cell Adhesion; Humans; Lipoproteins, LDL; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; Platelet Activation; Platelet Aggregation; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA-Binding Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Our previous study demonstrated that angiogenesis increased during the recovery of heat-denatured endothelial cells. However, the mechanism is still unclear. This study aimed to investigate the relation of autophagy and angiogenesis during the recovery of heat-denatured endothelial cells. A rat deep partial-thickness burn model and heat-denatured human umbilical vein endothelial cells (HUVECs) model (52 °C for 35 s) were used. Autophagy increased significantly in the dermis and HUVECs in a time-dependent manner after heat denaturation and recovery for 2-5 days. Rapamycin-mediated autophagy enhanced the pro-angiogenic effect, evidenced by increased proliferation and migration of HUVECs, and formation of tube-like structures. Autophagy inhibition by 3-Methyladenine (3-MA) abolished the angiogenesis in heat-denatured HUVECs after recovery for 3-5 days. Moreover, heat denaturation augmented the phosphorylation of AMP-activated protein kinase (AMPK) but reduced the phosphorylation of Akt and mTOR in HUVECs. Furthermore, autophagy inhibition by antioxidant NAC, compound C or AMPK siRNA impaired cell proliferation, migration and tube formation heat-denatured HUVECs. At last, the in vivo experiments also showed that inhibition of autophagy by bafilomycin A1 could suppress angiogenesis and recovery of heat-denatured dermis.Taken together, we firstly revealed that autophagy promotes angiogenesis via AMPK/Akt/mTOR signaling during the recovery of heat-denatured endothelial cells and may provide a potential therapeutic target for the recovery of heat-denatured dermis.

Topics: Adenine; AMP-Activated Protein Kinase Kinases; Animals; Autophagy; Cell Proliferation; Hot Temperature; Human Umbilical Vein Endothelial Cells; Humans; Macrolides; Neovascularization, Physiologic; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The roles of autophagy performed in chemotherapy-induced cell death or proliferation inhibition were still in debate. In this study, we aimed to disclose the function of autophagy in chemotherapy of HCT-116 colon cells.. Pharmacological and genetic methods were applied to induce and inhibit autophagy and elucidate the roles of autophagy performed in chemotherapy-induced proliferation inhibition and apoptosis. Autophagy was assessed by microtubule-associated protein light chain 3 (LC3) expression and monodansylcadaverine (MDC) staining.. After treatment with 5-fluorouracil (5-FU), HCT-116 cells showed typical autophagy as stained by MDC. Autophagy inhibitor (3-methyladenine [3-MA]) or inducer (rapamycin) was applied in combination with 5-FU, respectively. As evidenced by our data, 3-MA inhibited while rapamycin facilitated 5-FU-induced apoptosis and proliferation inhibition of HCT-116 cells. Consistently, 3-MA inhibited, while rapamycin facilitated 5-FU-induced expressions of Beclin1 and LC3B. Moreover, 3-MA inhibited while rapamycin facilitated 5-FU-induced p53 protein expression. Using genetic method, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced cell proliferation inhibition and apoptosis. Especially, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced p53 expression.. Our study provides both of pharmacological and genetic evidence to support that autophagy facilitates anticancer effect of the chemotherapeutic agent. The associated application of autophagy inducer with 5-FU would be beneficial for the chemotherapy in HCT-116 cancer cells.

Topics: Adenine; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Beclin-1; Cell Proliferation; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Drug Synergism; Fluorouracil; HCT116 Cells; Humans; Microtubule-Associated Proteins; RNA, Small Interfering; Sirolimus

Although liver regeneration has been intensively studied in various ways, the mechanisms underlying liver regeneration remain elusive. Apoptosis-stimulating protein two of p53 (ASPP2) was discovered as a binding partner of p53 and plays an important role in regulating cell apoptosis and growth. However, the role of ASPP2 in hepatocyte proliferation and liver regeneration has not been reported. The expression profile of ASPP2 was measured in a mouse model with 70% partial hepatectomy (PH

Topics: Adenine; Animals; Autophagy; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation; Haploinsufficiency; Hepatocytes; Liver Regeneration; Mechanistic Target of Rapamycin Complex 1; Mice; Sirolimus; Tumor Suppressor Proteins

The administration of cisplatin is limited due to its nephrotoxic side effects, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. In this study, we demonstrated that the pretreatment of human umbilical cord MSC-derived exosomes (hucMSC-Ex) can prevent the development of cisplatin-induced renal toxicity by activation of autophagy in vitro and in vivo.. In vitro, rat renal tubular epithelial (NRK-52E) cells were pre-incubated with exosomes from hucMSC or HFL1 (human lung fibroblast cells; as control) for 30 min, and 3-methyladenine (an autophagic inhibitor) and rapamycin (an autophagic inducer) for 1 h before cisplatin treatment for 8 h, respectively. Cells were harvested for apoptosis assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we constructed cisplatin-induced acute kidney injury rat models. Prior to treatment with cisplatin for 0.5 h, hucMSC-Ex or HFL1-Ex were injected into the kidneys via the renal capsule. 3-methyladenine and rapamycin were injected under the kidney capsule before hucMSC-Ex. All animals were sacrificed at 3 days after cisplatin injection. Renal function, Luminex assay, tubular apoptosis and proliferation, and autophagy response were evaluated.. hucMSC-Ex inhibited cisplatin-induced mitochondrial apoptosis and secretion of inflammatory cytokines in renal tubular epithelial cells in vitro. hucMSC-Ex increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in NRK-52E cells. Rapamycin mimicked the effects of hucMSC-Ex in protecting against cisplatin-induced renal injury, while the effects were abrogated by the autophagy inhibitor 3-methyladenine in the animals.. Our findings indicate that the activation of autophagy induced by hucMSC-Ex can effectively relieve the nephrotoxicity of cisplatin. Therefore, pre-treatment of hucMSC-Ex may be a new method to improve the therapeutic effect of cisplatin.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Biomarkers; Cell Differentiation; Cell Line; Cisplatin; Epithelial Cells; Exosomes; Female; Fetus; Fibroblasts; Gene Expression; Humans; Mesenchymal Stem Cells; Microtubule-Associated Proteins; Nephritis; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Sirolimus; Umbilical Cord

The hypothalamus regulates metabolism and feeding behavior by perceiving the levels of peripheral insulin. However, little is known about the hypothalamic changes after aberrant metabolism. In this study, we investigated the changes of insulin and autophagy relevant signals of hypothalamus under diabetes mellitus.. C57B/L mice were injected with low-dose streptozotocin (STZ) and fed with high-fat diet to induce type 2 diabetes mellitus. In vitro, PC12 cells were treated with oleic acid to mimic lipotoxicity.. Results showed that the cholesterol level in the hypothalamus of the diabetic mice was higher than that of the normal mice. The expression of insulin receptors and insulin receptor substrate-1 were downregulated and the number of Fluoro-Jade C positive cells significantly increased in the hypothalamic arcuate nucleus of the diabetic mice. Furthermore, Upregulation of mammalian target of rapamycin (mTOR) and downregulation of LC 3II were obvious in the hypothalamus of the diabetic mice. In vitro, results showed that high-lipid caused PC12 cell damage and upregulated LC3 II expression. Pretreatment of cells with 3-methyladenine evidently downregulated LC3 II expression and aggravated PC12 cell death under high lipid conditions. By contrast, pretreatment of cells with rapamycin upregulated LC3 II expression and ameliorated PC12 cell death caused by lipotoxicity.. These results demonstrate that autophagy activation confers protection to neurons under aberrant metabolism and that autophagy dysfunction in the hypothalamus occurs in the chronic metabolic disorder such as T2DM.

Topics: Adenine; Animals; Arcuate Nucleus of Hypothalamus; Autophagy; Blotting, Western; Brain Diseases; Cholesterol; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diet, High-Fat; Down-Regulation; Glucose Tolerance Test; Hypothalamus; Immunosuppressive Agents; In Vitro Techniques; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Lipid Metabolism; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Neurons; Oleic Acid; PC12 Cells; Rats; Receptor, Insulin; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation; Ventromedial Hypothalamic Nucleus

Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-β (Aβ) production and deposition, its role in pathogenesis of Alzheimer's disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aβ-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aβ1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aβ1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca

Topics: Adenine; Amyloid beta-Peptides; Apoptosis; Autophagy; Blotting, Western; Cell Line, Tumor; Cell Survival; Cyclic AMP Response Element-Binding Protein; Humans; Membrane Potential, Mitochondrial; Neural Cell Adhesion Molecules; Neurons; Neuroprotection; Oxidative Stress; Peptide Fragments; Real-Time Polymerase Chain Reaction; Sirolimus

The present study was designed to examine the effect of autophagy and apoptosis on intestinal injury in mice after severe burns.. Kunming mice were subjected to third degree burns over 30% of the total body surface area. Damage to the intestine was assessed by examining changes in intestinal mucosal morphology, enzyme-linked immunosorbent assay of serum d-lactate, diamine oxidase, lipopolysaccharide, interleukin-6, and tumor necrosis factor α (marker of intestinal damage), hematoxylin and eosin staining, and Western blotting under 4 experimental conditions: control group, burn only (burn group), burn and administration of rapamycin to stimulate intestinal autophagy (rapamycin group), or burn and administration of 3-methyladenine to inhibit intestinal autophagy (3-methyladenine group).. At day 1 postburn, the expression levels of light chain 3 II, beclin-1, and cleaved caspase-3 were significantly greater in all 3 groups of mice subjected to the burn injury than in the control group 1 day postburn; while the levels of light chain 3 II and beclin-1 were significantly greater and those of cleaved caspase-3 were significantly less in the rapamycin group than in the burn group. In contrast, light chain 3 II and beclin-1 levels were significantly less and those of cleaved caspase-3 significantly greater in the 3-methyladenine group. All 3 groups subjected to burn injury showed significantly increased levels of d-lactate, diamine oxidase, lipopolysaccharide, interleukin-6, and tumor necrosis factor α. Of the 3 groups, the rapamycin group exhibited the least observed levels, the 3-methyladenine group the greatest, and the burn group intermediate. Pathologic sections of the intestinal tissue showed that all 3 burn groups exhibited severe intestinal mucosal damage at 1 day postburn. The condition of the 3-methyladenine treatment group was worse than that of the rapamycin treatment group, but better than that of the burn group.. Intestinal autophagy is activated in response to intestinal apoptosis after severe burns and may alleviate burn-induced intestinal injury.

Topics: Adenine; Animals; Autophagy; Burns; Disease Models, Animal; Female; Interleukin-6; Intestinal Mucosa; Intestines; Male; Mice; Sirolimus; Tumor Necrosis Factor-alpha

Hypoxia may induce apoptosis and autophagy to promote cardiomyocyte injury. The present study investigated the effect of berberine, a natural extract of Rhizoma Coptidis, on hypoxia‑induced autophagy and apoptosis in the H9c2 rat myocardial cell line. Expression levels of apoptosis and autophagy markers were upregulated in H9c2 myocytes during hypoxia and cell viability was reduced. However, berberine significantly reduced hypoxia‑induced autophagy in H9c2 myocytes, as demonstrated by the ratio of microtubule‑associated proteins 1A/1B light chain 3 I/II and the expression levels of B‑cell lymphoma 2 (Bcl‑2)/adenovirus E1B 19 kDa protein‑interacting protein 3, and promoted cell viability. In addition, expression levels of the Bcl‑2 anti‑apoptotic protein were significantly downregulated, and expression levels of pro‑apoptotic proteins Bcl‑2‑associated X protein and cleaved caspase‑3 were upregulated during hypoxia injury in cardiac myocytes. This was reversed by treatment with berberine or the autophagy inhibitor 3‑methyladenine, whereas the autophagy agonist rapamycin had the opposite effects, suggesting that berberine reduces myocyte cell death via inhibition of autophagy and apoptosis during hypoxia. In addition, Compound C, a 5' adenosine monophosphate‑activated protein kinase (AMPK) inhibitor, reduced apoptosis and autophagy in hypoxic myocytes, suggesting that the activation of the AMPK signaling pathway may be involved in this process. These findings suggested that berberine protects cells from hypoxia‑induced apoptosis via inhibition of autophagy and suppression of AMPK activation. Therefore, berberine may be a potential therapeutic agent for the treatment of patients with cardiac myocyte injury and ischemia.

Topics: Adenine; AMP-Activated Protein Kinases; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Berberine; Caspase 3; Cell Hypoxia; Cell Line; Cell Survival; Microtubule-Associated Proteins; Myocytes, Cardiac; Pinellia; Rats; Sirolimus

The objective of this study was to examine the effect of autophagy on stress-induced M2 macrophage polarization in the tumor microenvironment of breast cancer and to determine whether the underlying mechanism was related to the reactive oxygen species (ROS)/ERK and mTOR pathway. In vitro, we found that the basal autophagy level in mouse RAW 264.7 macrophages decreased with the incubation of tumor cell culture supernatant. Similarly, the polarization of RAW 264.7 to M2 macrophages was inhibited by the autophagy inducer rapamycin and increased by the autophagy inhibitor 3-MA or by siBeclin1. In addition, we found that not only was M2 molecule expression down-regulated but intracellular ROS generation was also blocked by autophagy induction. In vivo, we observed that mice that received an isoprenaline injection as a stress agent exhibited augmented implanted breast tumor growth, lung metastasis, intratumoral mRNA expression of M2 molecules and serum ROS generation. In contrast, the intratumoral expression of LC3-II and Beclin1 was decreased. In addition, we observed that isoprenaline induced the up-regulation of the intratumoral expression of phosphorylated mTOR, phosphorylated ERK1/2, phosphorylated Tyr705-STAT3 and HIF-1α, whereas rapamycin induced an opposite effect on the same molecules and could abolish the effects of isoprenaline. These results suggest that autophagy might suppress M2 macrophage polarization induced by isoprenaline via the ROS/ERK and mTOR signaling pathway. Our findings provide a theoretical basis for why high levels of stress hormones accelerate the progression of breast cancer, and autophagy may play a role in determining the outcomes of cancer therapy.

Topics: Adenine; Animals; Antibiotics, Antineoplastic; Autophagy; Cell Line, Tumor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isoproterenol; Lung Neoplasms; Mammary Neoplasms, Experimental; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; RAW 264.7 Cells; Reactive Oxygen Species; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases; Tumor Microenvironment

Mono-(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di-(2-ethylhexyl) phthalate (DEHP). MEHP has toxic effects on cardiovascular system, but the possible molecular mechanisms are not completely elucidated. In our study, 3-methyladenine (3-MA), an autophagosome formation inhibitor, protected the EA.hy926 cells against MEHP cytotoxicity, and rapamycin, an autophagosome formation stimulator, further decreased the cell viability in the MEHP-treated EA.hy926 cells. Thus, autophagy may play an important role in MEHP-induced toxicity. MEHP increased the autophagosome number in EA.hy926 cells detected under transmission electron microscope. Collapses of ΔΨm and reactive oxygen species (ROS) level were increased in a dose-dependent manner under treatment with 0-200μM MEHP for 24h. N-acetyl-l-cysteine (NAC), a ROS inhibitor, protected against MEHP-induced cytotoxicity and decreased the protein expression of LC3-II. These findings suggested that MEHP-induced autophagic cell death was ROS-dependent in EA.hy926 cells. Knockdown of Akt1 with Akt1 siRNA aggravated MEHP-induced cell death, and insulin, an Akt1 activator, alleviated MEHP-induced cell death. These results were consistent with the expression of LC3-II using western blot. The phospho-Akt1

Topics: Acetylcysteine; Adenine; Autophagy; Cell Line; Cell Survival; Diethylhexyl Phthalate; Endothelial Cells; Humans; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA, Small Interfering; Sirolimus

the anti-vascular endothelial growth factors (VEGF), Aflibercept and Ranibizumab, are used for the treatment of macular degeneration. Here we examined the involvement of nitric oxide (NO), mitochondria function and of apoptosis/autophagy in their antioxidant effects in human retinal pigment epithelium cells (RPE).. RPE were exposed to Ranibizumab/Aflibercept in the absence or presence of NO synthase (NOS) inhibitor and of autophagy activator/blocker, rapamicyn/3-methyladenine. Specific kits were used for cell viability, NO and reactive oxygen species detection and mitochondrial membrane potential measurement, whereas Western Blot was performed for apoptosis/ autophagy markers and other kinases detection.. In RPE cultured in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Moreover, both the anti-VEGF agents were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by the NOS inhibitor and 3-methyladenine and were potentiated by rapamycin. Finally, Aflibercept and Ranibizumab counteracted the changes of apoptosis/autophagy markers, NOS, Phosphatidylinositol-3-Kinase/Protein Kinase B and Extracellular signal-regulated kinases 1/2 caused by peroxidation.. Aflibercept and Ranibizumab protect RPE against peroxidation through the modulation of NO release, apoptosis and autophagy.

Topics: Adenine; Angiogenesis Inhibitors; Animals; Apoptosis; Autophagy; Cell Line; Cell Proliferation; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Proto-Oncogene Proteins c-akt; Ranibizumab; Reactive Oxygen Species; Receptors, Vascular Endothelial Growth Factor; Recombinant Fusion Proteins; Retinal Pigment Epithelium; Sirolimus; Swine

Cardiac microvascular endothelial cells (CMECs) dysfunction is an important pathophysiological event in the cardiovascular complications induced by diabetes. However, the underlying mechanism is not fully clarified. Autophagy is involved in programmed cell death. Here we investigated the potential role of autophagy on the CMECs injury induced by high glucose. CMECs were cultured in normal or high glucose medium for 6, 12 and 24 h respectively. The autophagy of CMECs was measured by green fluorescence protein (GFP)-LC3 plasmid transfection. Moreover, the apoptosis of CMEC was determined by flow cytometry. Furthermore, 3-Methyladenine (3MA), ATG7 siRNA and rapamycin were administrated to regulate the autophagy state. Moreover, Western blotting assay was performed to measure the expressions of Akt, mTOR, LC3 and p62. High glucose stress decreased the autophagy, whereas increased the apoptosis in CMECs time dependently. Meanwhile, high glucose stress activated the Akt/mTOR signal pathway. Furthermore, autophagy inhibitor, 3-MA and ATG7 siRNA impaired the autophagy and increased the apoptosis in CMECs induced by high glucose stress. Conversely, rapamycin up-regulated the autophagy and decreased the apoptosis in CMECs under high glucose condition. Our data provide evidence that high glucose directly inhibits autophagy, as a beneficial adaptive response to protect CMECs against apoptosis. Furthermore, the autophagy was mediated, at least in part, by mTOR signaling.

Topics: Adenine; Animals; Apoptosis; Caspases; Cells, Cultured; Endothelial Cells; Glucose; Heart; Male; Myocardium; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.

Topics: Acetylcysteine; Adenine; Animals; Anti-Bacterial Agents; Apoptosis; Autophagy; Autophagy-Related Proteins; Cell Line; Hair Cells, Auditory; Mice; Mitochondria; Neomycin; Reactive Oxygen Species; Sirolimus

Autophagy is a highly conserved process of self-digestion to promote cell survival in response to nutrient starvation and other metabolic stresses. However, whether ischemic-hypoxic (IH) injury-induced autophagy acts as a neuroprotective mechanism or leads to neuroinjury is a subject of debate. It is known that autophagy is regulated by signaling pathways, including the mammalian target of rapamycin pathway. However, in neural IH injury, whether other signaling pathways are involved in the regulation of autophagy remains to be fully elucidated. In the present study, using the autophagy agonist (rampycin), autophagy antagonist [3-methyl adenine (3-MA)] and lysosome antagonist (MHY1485), autophagy was intervened with at oxygen-glucose deprivation (OGD) 6 h, in order to elucidate the regulatory mechanisms of autophagy. Using immunocytochemistry and western blot analysis, the expression levels of stress-related proteins, such as hypoxia-inducible factor-1α (HIF-1α) (a key regulator in hypoxia) and cyclooxygenase 2 (COX2; inflammatory indicator), were analyzed. In addition, the upstream proteins (Wnt1 and Wnt3a), downstream proteins (Dvl2, β-catenin) and target proteins (C-myc and cyclin D) in the Wnt/β-catenin signaling pathway were examined by immunocytochemistry and western blot analysis. The present study revealed that autophagy was activated with the upregulation of autophagic flux in IH injury; it was demonstrated that autophagy had a protective role in IH injury. The Wnt/β-catenin pathway was involved in IH injury regulation, and the upstream proteins in the Wnt/β-catenin signaling pathway were upregulated, whereas downstream proteins were downregulated by the activity of autophagy accordingly.

Topics: Adenine; Animals; Autophagy; beta Catenin; Hypoxia-Ischemia, Brain; Models, Biological; Morpholines; PC12 Cells; Rats; Sirolimus; Triazines; Wnt Signaling Pathway

Gambogic acid (GA) has been shown to inhibit cancer cell proliferation, induce apoptosis, and enhance reactive oxygen species accumulation. However, whether GA could improve multidrug resistance through modulating autophagy has never been explored. We demonstrated that the combination of GA and cisplatin (CDDP) resulted in a stronger growth inhibition effect on A549 and NCI-H460 cells using the MTT assay. Furthermore, treatment with GA significantly increased autophagy in these cells. More importantly, GA-induced cell death could be largely abolished by 3-methyladenine (3-MA) or chloroquine (CQ) treatment, suggesting that GA-induced cell death was dependent on autophagy. Western blot analysis showed that GA treatment suppressed the activation of Akt, mTOR, and S6. In addition, using a GA and rapamycin combination induced more cell death compared to either GA or rapamycin alone. In summary, GA may have utility as an adjunct therapy for non-small cell lung cancer (NSCLC) patients through autophagy-dependent cell death, even when cancer cells have developed resistance to apoptosis.

Topics: A549 Cells; Adenine; Antineoplastic Agents, Phytogenic; Autophagy; Cell Line, Tumor; Cell Proliferation; Chloroquine; Cisplatin; Drug Combinations; Drug Synergism; Garcinia; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Plant Extracts; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xanthones

To investigate the effects of resveratrol on autophagy in the chronically diabetic nephropathy and to study the effects of the different expression of microRNAs after resveratrol (RSV) treated in db/db mice (diabetic mice).. Db/m (non- diabetic) and db/db mice were randomly divided into intra gastric RSV treatment group or control group. Renal tissues were prepared for HE/PAS staining. In vitro, mouse podocytes cell lines were grown in different mediums with different dose of resveratrol treatment. microRNA (miRNA) gene chips assay was performed for differentially expressed miRNAs screening. Western blot was used to detect protein levels.. In vivo, RSV significantly decreased urinary albumin, serum creatinine, mesangial area and glomerular size in db/db mice. After RSV treatment, LC3-II/LC3-I and synaptopodin were increased while cleaved-caspase 3 was decreased in kidney tissues. In vitro, podocytes treated with RSV exhibited significantly increased LC3-II/LC3-I and decreased cleaved caspase 3. Moreover, this effect of RSV can be enhanced by rapamycin (RAPA, an activator of autophagy) but partially reversed by 3-MA (an autophagy inhibitor). Further, we found that miR-18a-5p was significantly upregulated after RSV treatment in db/db mice. Overexpression of miR-18a-5p in podocytes resulted in significant inhibition of cleaved-caspase 3 protein, and increased the ratio of LC3-II/LC3-I. Dual luciferase report assay validated that Atactic telangiectasis mutation (ATM) was a target of miR-18a-5p. In podocytes, downregulation of cleaved caspase 3 and the enhanced ratio of protein LC3-II/LC3-I were detected in cells transfected with ATM siRNA.. Role of miRNA-18a-5p in the regulation of autophagy via targeting ATM may represent a promising therapeutic target for preventing and attenuating diabetic nephropathy.

Topics: Adenine; Animals; Ataxia Telangiectasia Mutated Proteins; Autophagy; Caspase 3; Cell Line; Creatinine; Diabetes Mellitus, Experimental; Down-Regulation; Male; Mice; Mice, Obese; MicroRNAs; Microtubule-Associated Proteins; Resveratrol; RNA Interference; RNA, Small Interfering; Sirolimus; Stilbenes; Up-Regulation

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.

Topics: Adenine; Animals; Apoptosis; Autophagy; Disease Models, Animal; Genotype; In Situ Nick-End Labeling; Mice; Motor Neurons; Muscular Atrophy, Spinal; Sirolimus

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, autophagy, translation, and survival. Dysregulation of mTOR signaling is associated with cancer, diabetes, and autism. However, a role for mTOR signaling in neuronal death is not well delineated. Here we show that global ischemia triggers a transient increase in mTOR phosphorylation at S2448, whereas decreasing p-mTOR and functional activity in selectively vulnerable hippocampal CA1 neurons. The decrease in mTOR coincides with an increase in biochemical markers of autophagy, pS317-ULK-1, pS14-Beclin-1, and LC3-II, a decrease in the cargo adaptor p62, and an increase in autophagic flux, a functional readout of autophagy. This is significant in that autophagy, a catabolic process downstream of mTORC1, promotes the formation of autophagosomes that capture and target cytoplasmic components to lysosomes. Inhibitors of the lysosomal (but not proteasomal) pathway rescued the ischemia-induced decrease in mTOR, consistent with degradation of mTOR via the autophagy/lysosomal pathway. Administration of the mTORC1 inhibitor rapamycin or acute knockdown of mTOR promotes autophagy and attenuates ischemia-induced neuronal death, indicating an inverse causal relation between mTOR, autophagy, and neuronal death. Our findings identify a novel and previously unappreciated mechanism by which mTOR self-regulates its own levels in hippocampal neurons in a clinically relevant model of ischemic stroke.

Topics: Acetylcysteine; Adenine; AMP-Activated Protein Kinases; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Beclin-1; Cells, Cultured; Hippocampus; Ischemia; Leupeptins; Lysosomes; Male; Microtubule-Associated Proteins; Neurons; Phosphorylation; Rats; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases

Neurodegeneration, along with inflammatory demyelination, is an important component of multiple sclerosis (MS) pathogenesis. Autophagy is known to play a pivotal role in neuronal homeostasis and is implicated in several neurodegenerative disorders. However, whether autophagy is involved in the mechanisms of neuronal damage during MS remains to be investigated. Experimental autoimmune encephalomyelitis (EAE), an in vivo model of MS, was induced in female C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein p35-55. After that, autophagic flux in the spinal cord of mice was evaluated by detection of LC3-II and Beclin1 protein expressions. EAE mice were then administered with rapamycin and 3-methyladenine (3-MA) for 10 days. Afterward, the changes in LC3-II, Beclin1, and p62 expression, number of infiltrated inflammatory cells, demyelinated lesion area, and neuronal damage, as well as clinical scores, were assessed. Further, apoptotic cell rate and apoptosis-related protein expressions were monitored. We observed an impaired autophagic flux and increased neuronal damage in the spinal cords of EAE mice. We also found that rapamycin, an autophagy inducer, mitigated EAE-induced autophagy decrease, inflammation, demyelination and neuronal injury, as well as the abnormal clinical score. In addition, rapamycin suppressed cell apoptosis, and decreased Bax/Bcl-2 ratio and cleaved caspase-3 expression. Conversely, the effect of autophagy inhibitor 3-MA on EAE mice resulted in completely opposite results. These results indicated that autophagy deficiency, at least in part, contributed to EAE-induced neuronal injury and that pharmacological modulation of autophagy might be a therapeutic strategy for MS.

Topics: Adenine; Animals; Apoptosis; Autophagy; Beclin-1; Caspase 3; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Inflammation; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Microtubule-Associated Proteins; Multiple Sclerosis; Neurons; Sirolimus; Spinal Cord

Malignant glioma is the most aggressive brain tumor. Hypoxic condition has been explored for killing cancer stem cells or drug-resistant tumor cells. This study investigated the effects of hypoxia on autophagic death and the possible mechanisms. Exposure of human malignant glioma U87-MG cells to cobalt chloride (CoCl2) increased cellular hypoxia-inducible factor-1α levels and concurrently decreased cell viability concentration- and time-dependently. In parallel, treatment with CoCl2 suppressed proliferation of human U87-MG cells. Autophagic cells and levels of LC3-II were concentration- and time-dependently induced in human U87-MG cells after exposure to CoCl2. However, pretreatment with 3-mehyladenine (3-MA) and chloroquine, inhibitors of cell autophagy, caused significant alleviations in CoCl2-induced cell autophagy. In contrast, exposure to rapamycin, an inducer of cell autophagy, synergistically induced hypoxia-induced autophagy of U87-MG cells. Administration of human U87-MG cells with CoCl2 triggered caspase-3 activation and cell apoptosis. Interestingly, pretreatment with 3-MA and chloroquine remarkably suppressed CoCl2-induced caspase-3 activation and cell apoptosis. Application of p53 small interference (si)RNA into human U87-MG cells downregulated levels of this protein and simultaneously lowered hypoxia- and 3-MA-induced alterations in cell autophagy, apoptosis, and death. The hypoxia-induced autophagy and apoptosis of DBTRG-05MG cells were significantly lowered by 3-MA pretreatment and p53 knockdown. Therefore, the present study shows that CoCl2 treatment can induce autophagy of human glioma cells and subsequent autophagic apoptosis via a p53-dependent pathway. Hypoxia-induced autophagic apoptosis may be applied as a therapeutic strategy for treatment of glioma patients.

Topics: Adenine; Antimutagenic Agents; Apoptosis; Autophagy; Brain Neoplasms; Caspase 3; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chloroquine; Cobalt; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Microtubule-Associated Proteins; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Sirolimus; Tumor Suppressor Protein p53

To determine potential effects of autophagy activation on hypoxia-reoxygenation (H/R) induced damage of a rat alveolar epithelial cell line.. CCL149 cells were subjected to autophagy agonist (rapamycin, Rap), autophagy inhibitor (3-methyladenine, 3-MA) or PBS for 1 h before H/R treatment for 2 h, 4 h and 6 h. The optimal concentration of Rap (150 nM, 200 nM and 250 nM) or 3-MA (5 mM, 10 mM and 15 mM) was obtained from MTT assay. Autophagy was determined by fluorescence microscopy of eRFP-LC3 positive cells, transmission electron microscopy of autophagosome, western blot of LC3, AMPK, Beclin-1, HDAC6 and p62 proteins. Endoplasmatic reticulum stress was indicated by detecting expressions of BIP, XBP-1 and CHOP via western blot.. Rap at concentration of 250 nM before H/R increased the autophagy formation with more eRFP-LC3 positive cells and higher expressions of LC3-II, Beclin-1, HDAC6 and p62, but lower expressions of BIP, XBP-1 and CHOP in H/R treated CCL149. This effect seemed to be still obvious after H/R exposure for 6 h. The contrary results were obtained by treatment with 5 mM 3-MA.. Rap might be a promising agent before mechanical ventilation or reperfusion to prevent re-damage in hypoxia related lung diseases.

Topics: Adenine; Alveolar Epithelial Cells; Animals; Autophagy; Beclin-1; Cell Hypoxia; Cell Line; Endoplasmic Reticulum Stress; Immunohistochemistry; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Microtubule-Associated Proteins; Oxygen; Rats; Sirolimus; Transcription Factor CHOP; X-Box Binding Protein 1

This study sought to explore the effect of staphylococcal lipoteichoic acid (LTA) on autophagy in mouse mesenchymal stem cells (MSCs), and then influence osteogenesis through the change of autophagy. C3H10T1/2 cells were induced by osteogenic medium with the treatment of LTA at different concentrations (1, 5, 10 μg/mL); 3-methyladenine (3-MA) were used as the autophagy inhibitor, and rapamycin (rapamycin, Rap) were used to activate autophagy; the effects on osteogenesis were detected by alkaline phosphatase staining, alizarin red staining, real-time quantitative PCR, and western blotting; autophagic activity was investigated by the expression of LC3-Ⅱand p62 proteins. Compared with control group, the expression of osteogenesis markers was significantly up-regulated with the LTA treatment on the mRNA and protein level; the positive rate of alkaline phosphatase was enhanced in the LTA groups; and the formation of calcium nodules was increased simultaneously. The expression of LC3-Ⅱ protein was increased in LTA groups, while the expression of p62 protein was decreased. Inhibition of autophagy significantly reduced the effect of LTA on osteogenesis of MSCs; the promotion of LTA on osteogenic differentiation was further enhanced when adding rapamycin to activate autophagic activity. It provides new insight of prevention and treatment for bone infection.

Topics: Actins; Adenine; Alkaline Phosphatase; Animals; Autophagy; Blotting, Western; Cell Differentiation; Cell Line; Collagen Type I; Core Binding Factor Alpha 1 Subunit; Gene Expression; Immunosuppressive Agents; Lipopolysaccharides; Mesenchymal Stem Cells; Mice, Inbred C3H; Microtubule-Associated Proteins; Osteogenesis; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Staphylococcus aureus; Teichoic Acids

Hydroquinone (HQ), one of the metabolic products of benzene, is a carcinogen. It can induce apoptosis in lymphoma cells. However, whether HQ can induce autophagy and what roles autophagy plays in TK6 cells exposured to HQ remains unclear. In this study, we found that HQ could induce autophagy through techniques of qRT-PCR, Western blot, immunofluorescent assay of LC3 and transmission electron microscope. Furthermore, inhibiting autophagy using 3-methyladenine (3-MA) or chloroquine (CQ) significantly enhanced HQ-induced cell apoptosis, suggesting that autophagy may be a survival mechanism. Our study also showed that HQ activated PARP-1. Moreover, knockdown of PARP-1 strongly exhibited decreased autophagy related genes expression. In contrast, the absence of SIRT1 increased that. Altogether, our data provided evidence that HQ induced autophagy in TK6 cells and autophagy protected TK6 from HQ attack-induced injury in vitro, and the autophagy was partially mediated via activation of the PARP-1-SIRT1 signaling pathway.

Topics: Adenine; Apoptosis; Autophagy; Autophagy-Related Protein 5; Beclin-1; Cell Line; Chloroquine; Gene Knockdown Techniques; Humans; Hydrogen Peroxide; Hydroquinones; Microtubule-Associated Proteins; Poly (ADP-Ribose) Polymerase-1; Sirolimus; Sirtuin 1

Autophagy is an evolutionarily conserved process that plays a crucial role in maintaining a series of cellular functions. It has been found that autophagy is closely involved in the physiological process of spermatogenesis and the regulation of sperm survival and motility. However, the role of autophagy in high-fat diet (HFD)-induced impaired spermatogenesis remains unknown. This study was designed to investigate the role of autophagy in HFD-induced spermatogenesis deficiency and employed chloroquine (CQ) to inhibit autophagy and rapamycin (RAP) to induce autophagy. 3-methyladenine (3-MA) and CQ were administered via intratesticular injection in vivo. The effects of CQ and 3-MA on the parameters of spermatozoa co-cultured with palmitic acid (PA) in vitro were also investigated. Human semen samples from obese, subfertile male patients were also collected to examine the level of autophagy. The results suggested that HFD mice subjected to CQ showed improved spermatogenesis. Inhibiting autophagy with CQ improved the decreased fertility of HFD male mice. Moreover, the in vivo and in vitro results indicated that both CQ and 3-MA could suppress the pathological changes in spermatozoa caused by HFD or PA treatment. Additionally, the excessive activation of autophagy was also observed in sperm samples from obese, subfertile male patients.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cells, Cultured; Chloroquine; Diet, High-Fat; Humans; Infertility, Male; Male; Mice; Mice, Inbred C57BL; Obesity; Palmitic Acid; Semen Analysis; Sirolimus; Spermatogenesis; Spermatozoa; Testis

Autophagy is a basic biological metabolic process involving in intracellular membrane transport pathways that recycle cellular components and eliminate intracellular microorganisms within the lysosome. Autophagy also plays an important part in virus infection and propagation. However, some pathogens, including viruses, have evolved unique trick to escape or exploit autophagy. This study explores the mechanism of autophagy induction by porcine hemagglutinating encephalomyelitis virus (PHEV) in Neuro-2a cells, and examines the role of autophagy in PHEV replication. PHEV triggered autophagy in Neuro-2a cells is dependent on the presence of bulk double- or single-membrane vacuoles, the accumulation of GFP-LC3 fluorescent dots, and the LC3 lipidation. In addition, PHEV induced an incomplete autophagic effect because the degradation level of p62 did not change in PHEV-infected cells. Further validation was captured using LysoTracker and lysosome-associated membrane protein by indirect immunofluorescence labeling in PHEV-infected cells. We also investigated the change in viral replication by pharmacological experiments with the autophagy inducer rapamycin or the autophagy inhibitor 3-MA, and the lysosomal inhibitor chloroquine (CQ). Suppression of autophagy by 3-MA increased viral replication, compared with the mock treatment, while promoting of autophagy by rapamycin reduced PHEV replication. CQ treatment enhanced the LC3 lipidation in PHEV-infected Neuro-2a cells but lowered PHEV replication. These results show that PHEV infection induces atypical autophagy and causes the appearance of autophagosomes but blocks the fusion with lysosomes, which is necessary for the replication of PHEV in nerve cells.

Topics: Adenine; Animals; Autophagosomes; Autophagy; Betacoronavirus 1; Cell Line; Cell Survival; Chloroquine; Coronavirus Infections; Lysosomes; Membrane Proteins; Mice; Sirolimus; Vacuoles; Virus Replication

Since melamine was illegally added to raw milk for increasing the apparent protein content, such a scandal has not been quite blown out. Previous studies showed that melamine induced apoptosis and oxidative damage in both in vivo and in vitro experiments. It is well known that autophagy is closely related to oxidative stress. In the present study, we examined whether autophagy played an important role in protecting PC12 cells, which were damaged by melamine. Immunofluorescence assay showed that melamine enhanced the number of punctuate dot, indicating the increase of autophagosomes. Western blot assay presented that melamine significantly elevated the expression level of autophagy markers including LC3-II/LC3-I ratio, beclin-1, and Atg 7. Rapamycin further enhanced the effect, whereas 3-methyadenine (3-MA) inhibited it. MTT assay exhibited that rapamycin significantly enhanced the cell viability (P < 0.01), while 3-MA considerably reduced it in melamine-treated PC12 cells (P < 0.01). Furthermore, flow cytometry assay showed that rapamycin considerably reduced the reactive oxygen species (ROS) level of the cells (P < 0.01), but 3-MA increased the generation of ROS (P < 0.01). Additionally, the superoxide dismutase (SOD) activity was notably increased by rapamycin in melamine-treated PC12 cells (P < 0.01), while the activity of which was prominently decreased by 3-MA (P < 0.01). Malondialdehyde (MDA) assay showed that rapamycin remarkably decreased the MDA level of the cells (P < 0.05), while 3-MA increased it (P < 0.01). Consequently, this study demonstrated that autophagy protected PC12 cells from melamine-induced cell death via inhibiting the excessive generation of ROS. Regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Protein 7; Beclin-1; Cell Shape; Cell Survival; Fluorescent Antibody Technique; Malondialdehyde; Microtubule-Associated Proteins; PC12 Cells; Rats; Reactive Oxygen Species; Sirolimus; Superoxide Dismutase; Triazines

Acute kidney injury (AKI) is characterized by a rapid loss of kidney function and an antigen-independent inflammatory process that causes tissue damage, which was one of the main manifestations of kidney ischemia/reperfusion (I/R). Recent studies have demonstrated autophagy participated in the pathological process of acute kidney injury. In this study, we discuss how autophagy regulated inflammation response in the kidney I/R. AKI was performed by renal I/R. Autophagy activator rapamycin (Rap) and inhibitor 3-methyladenine (MA) were used to investigate the role of autophagy on kidney function and inflammation response. After the experiment, kidney tissues were obtained for the detection of autophagy-related protein microtubule-associated protein light chain 3(LC3)II, Beclin1, and Rab7 and lysosome-associated membrane protein type (LAMP)2 protein by reverse transcription-polymerase chain reaction (PT-PCR) and Western blotting, and histopathology and tissue injury scores also. The blood was harvested to measure kidney function (creatinine (Cr) and blood urea nitrogen (BUN) levels) after I/R. Cytokines TNF-α, IL-6, HMGB1, and IL-10 were measured after I/R. I/R induced the expression of LC3II, Beclin1, LAMP2, and Rab7. The activation and inhibition of autophagy by rapamycin and 3-MA were promoted and attenuated histological and renal function in renal I/R rats, respectively. Cytokines TNF-α, IL-6, and HMGB1 were decreased, and IL-10 was further increased after activation of autophagy treated in I/R rats, while 3-MA exacerbated the pro-inflammatory cytokines TNF-α, IL-6, HMGB1, and anti-inflammatory cytokine IL-10 in renal I/R. I/R can activated the autophagy, and autophagy increase mitigated the renal injury by decreasing kidney injury score, levels of Cr and BUN after renal I/R, and inflammation response via regulating the balance of pro-inflammation and anti-inflammation cytokines.

Topics: Acute Kidney Injury; Adenine; Animals; Anti-Inflammatory Agents; Autophagy; Beclin-1; Enzyme Activation; HMGB1 Protein; Inflammation; Interleukin-10; Interleukin-6; Kidney; Lysosomal-Associated Membrane Protein 2; Male; Microtubule-Associated Proteins; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Tumor Necrosis Factor-alpha

Hypoxic injury of cardiac microvascular endothelial cells (CMECs) is an important pathophysiological event in myocardial infarction, whereas, the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for normal cardiac function and also has been implicated in various cardiovascular diseases. Here we investigated the potential role of autophagy in CMEC dysfunction under hypoxia. CMECs were isolated from SD rats. Hypoxia (6-24h, 1% O2) induced autophagy in CMECs as evidenced by formation of punctate LC3, increased conversion of LC3-I to LC3-II and increased p62 degradation. Importantly, hypoxia-induced apoptosis in CMECs was attenuated by 3-Methyladenine (5mM), an autophagy inhibitor, and aggravated by rapamycin (1.0 μg/L), an autophagy inducer. Meanwhile, hypoxia increased the nuclear localization of FoxO3α, accompanying with the decreased phosphorylation of FoxO3α and Akt. FoxO3α silencing decreased hypoxia-induced autophagy and the resultant apoptosis. Furthermore, treatment with 3-Methyladenine (10mg/kg/day) improved the endothelial-dependent diastolic function of coronary artery in rats with myocardial infarction. These results indicated that hypoxia-induced autophagy formation in CMECs is mediated by FoxO3α and contributes to hypoxic injury of hearts.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Hypoxia; Cells, Cultured; Endothelial Cells; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Silencing; Male; Microvessels; Myocardial Infarction; Myocardium; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sirolimus

To study the correlation between the phosphorylation of keratin 18 (K18) and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin (OXA) and investigate its possible mechanism.. HCT116 cells were transfected with empty plasmid, wild-type K18 expression plasmid and 33, 52 phosphorylation site mutated K18 (Ser33/52A) expression plasmid separately, and all cells were then treated with 60 μmol/L OXA, followed by supplementation of autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide (PI) double staining combined with flow cytometry, calcein-AM/PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation, autophagy related proteins microtubule associated protein 1 light chain 3 (LC3) and beclin-1.. Transfection of Ser33/52A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA, the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group, while the apoptosis rate of Ser33/52A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group, the autophagy of K18 plasmid transfected group was significantly promoted, while the autophagy in Ser33/52A plasmid transfected group was significantly inhibited.. K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Autophagy; Blotting, Western; Colorectal Neoplasms; Flow Cytometry; HCT116 Cells; Humans; Keratin-18; Microscopy, Fluorescence; Mutation; Organoplatinum Compounds; Oxaliplatin; Phosphorylation; Serine; Sirolimus; Transfection

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) frequently occurs in many human cancers and hampers their therapeutic use. A large body of evidence has demonstrated the pro-survival role of autophagy in many human cancers. However, whether autophagy is involved in the induction of erlotinib resistance in tongue squamous cell carcinoma (TSCC) remains unknown. In this report, we found that autophagy prior to or induced by erlotinib treatment plays an important role in erlotinib resistance in tongue cancer cells. Using LC3 transfection, we observed that autophagy is upregulated and further induced when treated with erlotinib. Moreover, we found that autophagy plays a cytoprotective role by MTT analysis of the cell viability in TSCCs when treated with rapamycin or hydroxychloroquine (HCQ) in combination with erlotinib. However, 3-methyladenine (3-MA) did not influence the autophagy. Then, through siRNA technology and WB, we found that erlotinib-induced autophagy is mediated by ATG5 but not Beclin1. Also, knockdown of ATG5 significantly decreased the erlotinib resistance and knockdown of Beclin1 did not affect the sensitivity to erlotinib in TSCCs. Taken together, this indicates the critical role of non-canonical autophagy in erlotinib resistance in TSCCs.

Topics: Adenine; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Autophagy-Related Protein 5; Beclin-1; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Erlotinib Hydrochloride; Humans; Hydroxychloroquine; RNA, Small Interfering; Sirolimus; Tongue Neoplasms; Up-Regulation

The endoplasmic reticulum (ER) plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH).. ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs). Myocardial mechanical and intracellular Ca(2+) properties, ER stress, autophagy and associated cell signaling molecules were evaluated.. ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca(2+) homeostasis), oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62), along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene.. Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy.

Topics: Adenine; Alcohol Dehydrogenase; Animals; Autophagy; Calcium; Endoplasmic Reticulum Stress; MAP Kinase Signaling System; Mice; Mice, Inbred Strains; Mice, Transgenic; Myocardial Contraction; Myocardium; Oxidative Stress; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Recombinant Fusion Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transgenes; Tunicamycin; Ultrasonography; Ventricular Dysfunction, Left

Graphene oxide (GO), owing to its large surface area and abundance of oxygen-containing functional groups, is emerging as a potential adsorbent for polychlorinated biphenyls (PCBs), which accumulate over time and are harmful to both natural ecosystems and human health. However, the effect of GO against PCB-induced toxicity remains largely unexplored. The present study aimed to investigate the protective effect of GO against PCB 52 induced cytotoxic and genotoxic response in mammalian cells at various exposure conditions and clarify the protective role of autophagy. Pretreatment with GO dramatically decreased PCB 52 induced cytotoxicity and CD59 gene mutation in human-hamster hybrid (AL) cells. The toxic response in cells either pretreated with PCB 52 and then treated with GO or concurrently treated with GO and PCB 52 did not differ significantly from the toxic response in the cells treated with PCB 52 alone. Using autophagy inhibitors (3-methyladenine and wortmannin) and inducers (trehalose and rapamycin), we found that genuine autophagy induced by GO was involved in decreasing PCB 52 induced toxicity. These findings suggested that GO has an antagonistic effect against the toxicity of PCB 52 mainly by triggering a genuine autophagic process, which might provide new insights into the potential application of GO in PCB disposal and environmental and health risk assessment.

Topics: Adenine; Androstadienes; Animals; Autophagy; CD59 Antigens; Cell Line; Cricetinae; Graphite; Humans; Hybrid Cells; Mutagens; Oxides; Polychlorinated Biphenyls; Sirolimus; Trehalose; Wortmannin

Toxic DNA-protein crosslinks (DPCs) can result from exposure to radiation or chemotherapeutic agents. DPCs can also accumulate during aging or stress. However, the cellular mechanisms underlying clearance of DPCs remain largely unknown. Here, we have identified an important role of autophagy in the processing of DPCs induced by three representative agents: formaldehyde, a chemical used widely in industry; UV light; and camptothecin, a cytotoxic anticancer drug. Autophagy inhibitors, 3-methyladenine (3-MA) or chloroquine (CQ), promoted the accumulation of DPCs in damaged cells and injured organs. siRNA-mediated silencing of Atg5 or Atg7, two essential components for the formation of the autophagosome, gave similar results. In contrast, the autophagy inducer rapamycin (RAP) attenuated DPCs in vitro and in vivo. Our findings reveal the importance of autophagy in controlling the level of DPCs, and may open up a new avenue for understanding the formation and clearance of this detrimental DNA adduct.

Topics: Adenine; Animals; Antineoplastic Agents; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Camptothecin; Cell Line, Tumor; Cell Survival; Chloroquine; DNA Damage; Formaldehyde; Gene Silencing; Humans; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; RNA, Small Interfering; Sirolimus; Ubiquitin-Activating Enzymes; Ultraviolet Rays

Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Aggregated beta-amyloid (Aβ) disturbs Ca(2+) homeostasis and causes mitochondrial dysfunction and finally underlies AD. Recent evidence suggests that autophagy initiation by Beclin-1 protein might be involved in the pathogenesis of AD. However, the effects of Beclin-1 dependent autophagy on intracellular calcium ion concentration ([Ca(2+)]i) and mitochondrial membrane potential (MMP) is unclear. The effects of Beclin-1 dependent autophagy that were activated by a gradient concentration of autophagy activator rapamycin or inhibited by autophagy inhibitor 3-methyladenine (3-MA) on cell viability and cell morphology were examined. Pretreatment with rapamycin significantly up-regulated the expression of Beclin-1 in response to Aβ1-42 application, but after pretreatment with 3-MA it was significantly down-regulated. Moderate activation of Beclin-1 dependent autophagy had an up regulation effect on cell viability and could maintain the original morphology of cells. Furthermore, rapamycin or 3-MA on [Ca(2+)]i and MMP in Aβ1-42 treatment of PC12 cells were evaluated. We also report that PC12 cells treated with Aβ1-42 showed an increase in [Ca(2+)]i but a decrease in MMP when compared to the normal control. However the application of rapamycin prior to this prevented the increase in [Ca(2+)]i and the decrease in MMP in response to Aβ1-42. When 3-MA was applied this exacerbated the effect of Aβ1-42 on the [Ca(2+)]i and the MMP. This shows that moderate activation of Beclin-1 dependent autophagy by rapamycin can modulate Ca(2+) homeostasis and maintain MMP in response to Aβ1-42 induced cytotoxicity and so may have a preventive function in AD.

Topics: Adenine; Amyloid beta-Peptides; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Calcium; Cell Survival; Intracellular Space; Membrane Potential, Mitochondrial; Mitochondria; PC12 Cells; Peptide Fragments; Phagosomes; Rats; Sirolimus

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.

Topics: Adenine; Autophagy; Cell Line; Down-Regulation; Fibroblasts; Foreskin; Humans; Male; Organisms, Genetically Modified; Protein Kinase Inhibitors; Sirolimus; Toxoplasma

Bone marrow-derived mesenchymal stem cells (BMSCs) are being broadly investigated for treating numerous inflammatory diseases. However, the low survival rate of BMSCs during the transplantation process has limited their application. Autophagy can maintain cellular homeostasis and protect cells against environmental stresses. Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine that can induce both autophagy and apoptosis of BMSCs. However, the actual role of autophagy in TNF-α-induced apoptosis of BMSCs remains poorly understood. In the current study, BMSCs were treated with TNF-α/cycloheximide (CHX), and cell death was examined by the Cell Counting Kit-8, Hoechst 33342 staining, and flow cytometric analysis as well as by the level of caspase-3 and caspase-8. Meanwhile, autophagic flux was examined by analyzing the level of microtubule-associated protein light chain 3 B (LC3B)-II and SQSTEM1/p62 and by examining the amount of green fluorescent protein-LC3B by fluorescence microscopy. Then, the cell death and autophagic flux of BMSCs were examined after pretreatment and cotreatment with 3-methyladenine (3-MA, autophagy inhibitor) or rapamycin (Rap, autophagy activator) together with TNF-α/CHX. Moreover, BMSCs pretreated with lentiviruses encoding short hairpin RNA of beclin-1 (BECN1) were treated with TNF-α/CHX, and then cell death and autophagic flux were detected. We showed that BMSCs treated with TNF-α/CHX presented dramatically elevated autophagic flux and cell death. Furthermore, we showed that 3-MA and shBECN1 treatment accelerated TNF-α/CHX-induced apoptosis, but that Rap treatment ameliorated cell death. Our results demonstrate that autophagy protects BMSCs against TNF-α-induced apoptosis. Enhancing the autophagy of BMSCs may elevate cellular survival in an inflammatory microenvironment.

Topics: Adenine; Adult; Apoptosis; Autophagy; Beclin-1; Bone Marrow Cells; Cell Differentiation; Cycloheximide; Female; Humans; Male; Mesenchymal Stem Cells; Multipotent Stem Cells; Phenotype; RNA, Small Interfering; Sirolimus; Tumor Necrosis Factor-alpha

Peripheral neuropathies can result in cytoskeletal changes in axons, ultimately leading to Wallerian degeneration and cell death. Recently, autophagy has been studied as a potential target for improving axonal survival and growth during peripheral nerve damage. This study investigates the influence of autophagy on adult dorsal root ganglia (DRG) neuron survival and axonal growth under control and nutrient deprivation conditions. Constitutive autophagy was modulated with pharmacological activators (rapamycin; Rapa) and inhibitors (3-methyladenine, bafilomycin A1) in conjunction with either a nutrient-stable environment (standard culture medium) or a nutrient-deprived environment (Hank's balanced salt solution + Ca(2+) /Mg(2+) ). The results demonstrated that autophagy inhibition decreased cell viability and reduced neurite growth and branching complexity. Although autophagy was upregulated with nutrient deprivation compared with the control, it was not further activated by rapamycin, suggesting a threshold level of autophagy. Overall, both cellular and biochemical approaches combined to show the influence of autophagy on adult DRG neuron survival and growth. © 2016 Wiley Periodicals, Inc.

Topics: Adenine; Animals; Autophagy; Cell Survival; Culture Media; Ganglia, Spinal; Macrolides; Male; Nerve Regeneration; Neurites; Neurons; Phagosomes; Rats; Rats, Sprague-Dawley; Sirolimus

Autophagy plays an important role in spinal cord ischemia reperfusion (I/R) injury, but its neuroprotective or neurodegenerative role remains controversial. The extent and persistence of autophagy activation may be the critical factor to explain the opposing effects. In this study, the different roles and action mechanisms of autophagy in the early and later stages after I/R injury were investigated in rats. Thespinal cord I/R injury was induced by 14-min occlusion of the aortic arch, after which rats were treated with autophagic inhibitor (3-methyladenine, 3-MA) or agonist (rapamycin) immediately or 48h following the injury. Autophagy markers, microtubule-associated protein light chain 3-II (LC3-II) and Beclin 1 increased and peaked at the early stage (8h) and the later stage (72h) after spinal cord I/R injury. Beclin 1 was mostly expressed in neurons, but was also expressed to an extent in astrocytes, microglia and vascular endothelial cells. 8h after injury, rats treated with 3-MA showed a decrease in the hind-limb Basso-Beattie-Bresnahan (BBB) motor function scores, surviving motor neurons, and B-cell lymphoma-2 (Bcl-2) expression, and increase in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, Bcl-2-associated X protein (Bax), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) expression, and activation of microglia, while those treated with rapamycin showed opposing effects. However, 72h after injury, rats treated with 3-MA improved the BBB scores, and the surviving motor neurons, and reduced the autophagic cell death, while those treated with rapamycin had adverse effects. These findings provide the first evidence that early activated autophagy alleviates spinal cord I/R injury via inhibiting apoptosis and inflammation; however later excessively elevated autophagy aggravates I/R injury through inducing autophagic cell death.

Topics: Adenine; Animals; Apoptosis; Autophagy; Central Nervous System Agents; Disease Models, Animal; Male; Motor Activity; Neuroimmunomodulation; Random Allocation; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Spinal Cord; Spinal Cord Ischemia

People have known that autophagy plays a very important role in many physiological and pathological events. But the role of autophagy on embryonic angiogenesis still remains obscure. In this study, we demonstrated that Atg7, Atg8 and Beclin1 were expressed in the plexus vessels of angiogenesis at chick yolk sac membrane and chorioallantoic membrane. Interfering in autophagy with autophagy inducer or inhibitor could restrict the angiogenesis in vivo, which might be driven by the disorder of angiogenesis-related gene expressions, and also lead to embryonic hemorrhage, which was due to imperfection cell junctions in endothelial cells including abnormal expressions of tight junction, adheren junction and desmosome genes. Using HUVECs, we revealed that cell viability and migration ability changed with the alteration of cell autophagy exposed to RAPA or 3-MA. Interestingly, tube formation assay showed that HUVECs ability of tube formation altered with the change of Atg5, Atg7 and Atg8 manipulated by the transfection of their corresponding siRNA or plasmids. Moreover, the lost cell polarity labeled by F-actin and the absenced β-catenin in RAPA-treated and 3-MA-treated cell membrane implied intracellular cytoskeleton alteration was induced by the activation and depression of autophagy. Taken together, our current experimental data reveal that autophagy is really involved in regulating angiogenesis during embryo development.

Topics: Adenine; Angiodysplasia; Animals; Autophagy; Autophagy-Related Proteins; Cell Movement; Chick Embryo; Chorioallantoic Membrane; Embryonic Development; Endothelium, Vascular; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Microtubule-Associated Proteins; Neovascularization, Physiologic; Sirolimus

Honokiol, an active constituent extracted from the bark of Magnolia officinalis, possesses anticancer effects. Apoptosis is classified as type I programmed cell death, while autophagy is type II programmed cell death. We previously proved that honokiol induces cell cycle arrest and apoptosis of U87 MG glioma cells. Subsequently in this study, we evaluated the effect of honokiol on autophagy of glioma cells and examined the molecular mechanisms. Administration of honokiol to mice with an intracranial glioma increased expressions of cleaved caspase 3 and light chain 3 (LC3)-II. Exposure of U87 MG cells to honokiol also induced autophagy in concentration- and time-dependent manners. Results from the addition of 3-methyladenine, an autophagy inhibitor, and rapamycin, an autophagy inducer confirmed that honokiol-induced autophagy contributed to cell death. Honokiol decreased protein levels of PI3K, phosphorylated (p)-Akt, and p-mammalian target of rapamycin (mTOR) in vitro and in vivo. Pretreatment with a p53 inhibitor or transfection with p53 small interfering (si)RNA suppressed honokiol-induced autophagy by reversing downregulation of p-Akt and p-mTOR expressions. In addition, honokiol caused generation of reactive oxygen species (ROS), which was suppressed by the antioxidant, vitamin C. Vitamin C also inhibited honokiol-induced autophagic and apoptotic cell death. Concurrently, honokiol-induced alterations in levels of p-p53, p53, p-Akt, and p-mTOR were attenuated following vitamin C administration. Taken together, our data indicated that honokiol induced ROS-mediated autophagic cell death through regulating the p53/PI3K/Akt/mTOR signaling pathway.

Topics: Adenine; Animals; Apoptosis; Ascorbic Acid; Autophagy; Biphenyl Compounds; Caspase 3; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Glioma; Lignans; Mice; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Macroautophagy/autophagy plays an important role against pathogen infection in mammals and plants. However, little has been known about the role of autophagy in the interactions of insect vectors with the plant viruses, which they transmit. Begomoviruses are a group of single-stranded DNA viruses and are exclusively transmitted by the whitefly Bemisia tabaci in a circulative manner. In this study, we found that the infection of a begomovirus, tomato yellow leaf curl virus (TYLCV) could activate the autophagy pathway in the Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex as evidenced by the formation of autophagosomes and ATG8-II. Interestingly, the activation of autophagy led to the subsequent degradation of TYLCV coat protein (CP) and genomic DNA. While feeding the whitefly with 2 autophagy inhibitors (3-methyladenine and bafilomycin A1) and silencing the expression of Atg3 and Atg9 increased the viral load; autophagy activation via feeding of rapamycin notably decreased the amount of viral CP and DNA in the whitefly. Furthermore, we found that activation of whitefly autophagy could inhibit the efficiency of virus transmission; whereas inhibiting autophagy facilitated virus transmission. Taken together, these results indicate that TYLCV infection can activate the whitefly autophagy pathway, which leads to the subsequent degradation of virus. Furthermore, our report proves that an insect vector uses autophagy as an intrinsic antiviral program to repress the infection of a circulative-transmitted plant virus. Our data also demonstrate that TYLCV may replicate and trigger complex interactions with the insect vector.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Proteins; Begomovirus; DNA Primers; Female; Genomics; Gossypium; Hemiptera; Insect Vectors; Male; Plant Diseases; Sirolimus

Autophagy is a cellular pathway involved in degradation of damaged organelles and proteins in order to keep cellular homeostasis. It plays vital role in podocytes. Titanium dioxide nanoparticles (nano-TiO

Topics: Adenine; AMP-Activated Protein Kinases; Animals; Antioxidants; Apoptosis; Autophagy; Cell Line; Cell Proliferation; Cytoprotection; Enzyme Activation; Mice; Nanoparticles; Oxidative Stress; Podocytes; Signal Transduction; Sirolimus; Titanium; TOR Serine-Threonine Kinases

Insulin resistance is correlated with the progress of albuminuria in diabetic patients, and podocytes are crucial for maintaining the normal function of the glomerular filtration barrier. In the present study, we aimed to investigate the high glucose-induced insulin resistance and cell injury in human podocytes and the putative role of autophagy in this process.. Human podocytes were cultured in high glucose-supplemented medium and low glucose and high osmotic conditions were used for the controls. Autophagy in the podocytes was regulated using rapamycin or 3-methyladenine stimulation. Next, autophagy markers including LC3B, Beclin-1, and p62 were investigated using western blot and qPCR, and the insulin responsiveness was analyzed based on glucose uptake and by using the phosphorylation of the insulin receptor with Nephrin as a podocyte injury marker.. The basal autophagy level decreased under the high glucose conditions, which was accompanied by a decrease in the glucose uptake and phosphorylation of the insulin receptor in the human podocytes. More interestingly, the glucose uptake and the phosphorylation of the insulin receptor were decreased by 3-MA stimulation and increased by rapamycin, illustrating that the responsiveness of insulin was regulated by autophagy. The activation of autophagy by rapamycin also ameliorated cell injury in the human podocytes.. The presence or activation of autophagy was found to play a protective role in human podocytes against high glucose-induced insulin resistance and cell injury, which indicates a novel cellular mechanism and provides a potential therapeutic target for diabetic nephropathy (DN).

Topics: Adenine; Autophagy; Cells, Cultured; Culture Media; Glucose; Humans; Insulin Resistance; Membrane Proteins; Osmolar Concentration; Phosphorylation; Podocytes; Receptor, Insulin; Sirolimus

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Topics: Adenine; Animals; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Bacterial Proteins; Beclin-1; Cell Membrane; Cell Proliferation; Class III Phosphatidylinositol 3-Kinases; Dogs; Ehrlichia chaffeensis; Ehrlichiosis; Enzyme Activation; Glutamic Acid; Glutamine; Guanosine Triphosphate; HEK293 Cells; Host-Pathogen Interactions; Humans; Huntingtin Protein; Inclusion Bodies; Mutant Proteins; Protein Binding; rab5 GTP-Binding Proteins; Signal Transduction; Sirolimus; Ubiquitination

The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2h, 4h and 6h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2h, 4h and 6h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Hypoxia; Cell Line; Cell Survival; Doxorubicin; Endoplasmic Reticulum Stress; Histone Deacetylase 6; Histone Deacetylases; Leupeptins; Lung; Male; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Rats; Reperfusion Injury; Sirolimus; Ubiquitin

Autophagy is a conserved catabolic process of the cell, which has been described to be involved in the development of various viral diseases. However, the role of autophagy in Orf virus (ORFV) replication remains unknown. In this study, we provide the first evidence that ORFV infection triggered autophagy in primary ovine fetal turbinate cells (OFTu) based on the appearance of abundant double- and single-membrane vesicles, the accumulation of LC3 fluorescent puncta, the enhancement of LC3-I/-II conversion, and autophagic flux. Moreover, modulation of ORFV-induced autophagy by rapamycin (RAPA), Earle's balanced salts solution (EBSS), chloroquine (CQ) or 3-methyladenime (3-MA) does not affect virus production. In conclusion, these results suggest that autophagy can be induced in host cells by ORFV infection, but which maybe not essential for ORFV replication.

Topics: Adenine; Animals; Autophagy; Cells, Cultured; Chloroquine; Ecthyma, Contagious; Host-Pathogen Interactions; Microscopy, Electron, Transmission; Orf virus; Sheep; Sirolimus; Virus Replication

Doxorubicin (Doxo) is one of the most effective anti-neoplastic agents but its cardiotoxicity has been an important clinical limitation. The major mechanism of Doxo-induced cardiotoxicity is associated to its oxidative capacity. However, other processes are also involved with significant consequences for the cardiomyocyte. In recent years, a number of studies have investigated the role of autophagy on Doxo-induced cardiotoxicity but to date it is not clear how Doxo alters that process and its consequence on cardiomyocytes viability. Here we investigated the effect of Doxo 1uM for 24h of stimulation on cultured neonatal rat cardiomyocytes. We showed that Doxo inhibits basal autophagy. This inhibition is due to both Akt/mTOR signaling pathway activation and Beclin 1 level decrease. To assess the role of autophagy on Doxo-induced cardiomyocyte death, we evaluated the effects 3-methyladenine (3-MA), bafilomycin A1 (BafA), siRNA Beclin 1 (siBeclin 1) and rapamycin (Rapa) on cell viability. Inhibition of autophagy with 3-MA, BafA and siBeclin 1 increased lactate dehydrogenase (LDH) release but, when autophagy was induced by Rapa, Doxo-induced cardiomyocyte death was decreased. These results suggest that Doxo inhibits basal autophagy and contributes to cardiomyocyte death. Activation of autophagy could be used as a strategy to protect the heart against Doxo toxicity.

Topics: Adenine; Animals; Antibiotics, Antineoplastic; Apoptosis; Autophagy; Beclin-1; Cardiotoxins; Cell Survival; Doxorubicin; L-Lactate Dehydrogenase; Macrolides; Microtubule-Associated Proteins; Myocytes, Cardiac; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Dysregulated neutrophil extracellular traps (NETs) formation contributes to the pathogenesis of anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV). Increasing evidence indicates that autophagy is involved in the process of NETs formation. In this study, we aimed to investigate whether ANCA could induce autophagy in the process of NETs formation. Autophagy was detected using live cell imaging, microtubule-associated protein light chain 3B (LC3B) accumulation and Western blotting. The results showed that autophagy vacuolization was detected in neutrophils treated with ANCA-positive IgG by live cell imaging. This effect was enhanced by rapamycin, the autophagy inducer, and weakened by 3-methyladenine (3-MA), the autophagy inhibitor. In line with these results, the autophagy marker, LC3B, showed a punctate distribution pattern in the neutrophils stimulated with ANCA-positive IgG. In the presence of rapamycin, LC3B accumulation was further increased; however, this effect was attenuated by 3-MA. Moreover, incubated with ANCA-positive IgG, the NETosis rate significantly increased compared with the unstimulated group. And, the rate significantly increased or decreased in the neutrophils pretreated with rapamycin or 3-MA, respectively, as compared with the cells incubated with ANCA-positive IgG. Overall, this study demonstrates that autophagy is induced by ANCA and promotes ANCA-induced NETs formation.

Topics: Adenine; Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis; Antibodies, Antineutrophil Cytoplasmic; Autophagy; Cells, Cultured; Extracellular Traps; Healthy Volunteers; Humans; Microtubule-Associated Proteins; Neutrophils; Sirolimus

Autophagy is essential in physiological and pathological processes, however, the role of autophagy in cutaneous wound healing and the underlying molecular mechanism remain elusive. We hypothesized that autophagy plays an important role in regulating wound healing. Here, we show that enhanced autophagy negatively impacts on normal cutaneous healing process and is related to chronic wounds as demonstrated by the increased LC3 in diabetic mice skin or patients' chronic wounds. In addition, inhibition of autophagy by 3-MA restores delayed healing in C57BL/6 or db/db mice, demonstrating that autophagy is involved in regulating wound healing. Furthermore, we identify that macrophage is a major cell type underwent autophagy in wounds and increased autophagy induces macrophages polarization into M1 with elevated CD11c population and gene expressions of proinflammatory cytokines. To explore the mechanism underlying autophagy-impaired wound healing, we tested the role of IRF8, a regulator of autophagy, in autophagy-modulated macrophages polarization. IRF8 activation is up-regulating autophagy and M1 polarization of macrophages after AGEs (advanced glycation endproducts) treatment, blocking the IRF8 with shIRF8 inhibits autophagic activity and M1 polarization. In summary, this study elucidates that AGEs induces autophagy and modulates macrophage polarization to M1 via IRF8 activation in impairment of cutaneous wound healing.

Topics: Adenine; Animals; Autophagosomes; Autophagy; CD11c Antigen; Cytokines; Diabetes Mellitus, Experimental; Glycation End Products, Advanced; Interferon Regulatory Factors; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Microscopy, Electron; Microscopy, Fluorescence; RAW 264.7 Cells; RNA Interference; RNA, Small Interfering; Sirolimus; Skin; Wound Healing

Myocardial ischemia/reperfusion (I/R) injury detrimentally alters the prognosis of patients undergoing revascularization after acute myocardial infarction. Our previous study demonstrated that NF-κB-induced autophagy plays a detrimental role in cardiac I/R injury using a rabbit myocardial I/R model. In this study, we sought to explore the specific mechanism of this autophagy-mediated cell damage in an in vitro simulated ischemia/reperfusion (sI/R) model using human umbilical vein endothelial cells. Our current study demonstrates that simulated I/R induces autophagy in a p65-Beclin 1-dependent manner, which can be suppressed with the inhibition of NF-κB. Furthermore, rapamycin which promotes autophagy, exacerbates sI/R-induced cell death. While 3-methyladenine rescues cell damage. Our data thus suggest that I/R promotes NF-κB p65 activity mediated Beclin 1-mediated autophagic flux, thereby exacerbating myocardial injury.

Topics: Adenine; Autophagy; Beclin-1; Cell Death; Human Umbilical Vein Endothelial Cells; Humans; Myocardial Reperfusion Injury; Sirolimus; Transcription Factor RelA

To study the impact of autophagy on alveolar macrophage apoptosis and its mechanism in the early stages of hypoxia, we established a cell hypoxia-reoxygenation model and orthotopic left lung ischemia-reperfusion model. Rat alveolar macrophages stably expressing RFP-LC3 were treated with autophagy inhibitor (3-methyladenine, 3-MA) or autophagy promoter (rapamycin), followed by hypoxia-reoxygenation treatment 2 h, 4 h or 6 h later. Twenty Sprague-Dawley male rats were randomly divided into four different groups: no blocking of left lung hilum (model group), left lung hilum blocked for 1h with DMSO lavage (control group), left lung hilum blocked for 1 h with 100 ml/kg 3-MA (5 μmol/L) lavage (3-MA group), and left lung hilum blocked for 1 h with 100 ml/kg rapamycin (250 nmol/L) lavage (rapamycin group). Rapamycin decreased the unfolded protein response, which reduced endoplasmic reticulum stress-mediated apoptosis in the presence of oxygen deficiency. Rapamycin increased superoxide dismutase activities and decreased malondialdehyde levels, whereas 3-MA decreased superoxide dismutase activities and increased malondialdehyde levels. Thus, autophagy decreases alveolar macrophage apoptosis by attenuating endoplasmic reticulum stress and oxidative stress in the early stage of hypoxia in vitro and in vivo. This could represent a new approach to protecting against lung ischemia-reperfusion injury.

Topics: Adenine; Animals; Apoptosis; Autophagy; Caspase 3; Cell Hypoxia; Cells, Cultured; Endoplasmic Reticulum Stress; Macrophages, Alveolar; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Superoxide Dismutase

Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction.. The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry.. The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells.. Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.

Topics: Adenine; Animals; Autophagy; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Foscarnet; Humans; Hyperphosphatemia; Male; Microtubule-Associated Proteins; Phosphates; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats, Wistar; Renal Insufficiency, Chronic; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage.

Topics: Adenine; Animals; Anti-Bacterial Agents; Apoptosis; Autophagy; Cell Line; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Mice; Models, Animal; Phagosomes; Podocytes; Sirolimus; Thapsigargin; Tunicamycin

Ubenimex is a low-molecular-weight dipeptide with the ability to inhibit aminopeptidase N (APN) activity, enhance the function of immunocompetent cells and confer antitumor effects. We sought to characterize the effects of ubenimex on renal cell carcinoma (RCC). The 786-O and OS-RC-2 human RCC cell lines were positive for APN expression and ubenimex decreased APN activity without affecting the expression. Ubenimex suppressed the proliferation of both cell lines in a concentration‑dependent manner, as assessed by curve growth analysis and WST-8 proliferation assay. Wound healing and Matrigel invasion assays demonstrated that the migration and invasion of the RCC cells were also markedly suppressed by ubenimex. Furthermore, ubenimex increased the mortality of both RCC cell lines as determined by the LDH cytotoxicity assay. This affect was accompanied by increased levels of LC3B with no apparent effect on Caspase3; and we observed that autophagy increased significantly after ubenimex treatment in both RCC cell lines by electron microscopy. Moreover, rapamycin enhanced the cytotoxic effect of ubenimex, while 3-methyladenine reversed the effect, indicating that ubenimex cytotoxicity occured through an autophagy-related mechanism. To further assess the potential applicability of ubenimex in the treatment of RCC, we performed immunohistochemistry using tissue microarrays representing 76 RCC patients that underwent radical nephrectomy. The results showed that APN was expressed in most, but not all of the RCC tissues and that the expression was reduced in RCC as compared to the normal kidney tissues, suggesting a potential role for APN in RCC development. Collectively, these results indicated that ubenimex inhibits proliferation, migration and invasion of RCC cells. Ubenimex may induce autophagy, which may be associated with its effect on the growth arrest and the cell death of RCC cells.

Topics: Adenine; Adult; Aged; Aged, 80 and over; Aminopeptidases; Antibiotics, Antineoplastic; Autophagy; Carcinoma, Renal Cell; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Leucine; Middle Aged; Neoplasm Invasiveness; Sirolimus; Wound Healing; Young Adult

Nonviral gene transfection mediated by cationic polymer/DNA polyplexes often imposes stress and toxicity to cells. To better understand the relationship between cellular stress responses and polyplex-mediated transfection, polyplex-induced early autophagy in mouse fibroblasts was characterized and the impact of autophagy modulation on transgene expression evaluated. Transmission electron microscopy revealed the formation of double-membraned autophagosome in the cytoplasm of polyplex-transfected cells. Immunofluorescence staining and microscopy revealed intracellular LC3 punctation that was characteristic of early autophagy activation. Elevated expression of autophagosome-associated LC3 II protein was also detected by Western blot. When cells were treated with small-molecule modulators of autophagy, polyplex-mediated gene transfection efficiency was significantly affected. 3-Methyladenine (3-MA), an early autophagy inhibitor, reduced transfection efficiency, whereas rapamycin, an autophagy inducer, enhanced transgene expression. Importantly, the observed functional impact on gene transfection by autophagy modulation was decoupled from that of other modes of cellular stress response (apoptosis/necrosis). Treatment of cells by 3-MA or rapamycin did not affect the level of intracellular reactive oxygen species (ROS) but did decrease or increase, respectively, nuclear localization of polyplex-delivered plasmid DNA. These findings suggest new possibilities of enhancing polyplex-mediated gene delivery by codelivery of small-molecule regulators of autophagy.

Topics: Active Transport, Cell Nucleus; Adenine; Animals; Autophagy; Biopharmaceutics; DNA; Gene Expression; Gene Transfer Techniques; Mice; Microscopy, Electron, Transmission; NIH 3T3 Cells; Phagosomes; Polymers; Reactive Oxygen Species; Sirolimus; Transfection

Much evidence demonstrated that autophagy played an important role in neural inflammation response after ischemia stroke. However, the specific effect of microglia autophagy in cerebral ischemia is still unknown. In the current study, we constructed focal cerebral ischemia model by permanent middle cerebral artery occlusion (pMCAO) in mice. We detected microglia autophagy and inflammation response in vivo, and observed infarct brain areas, edema formation, and neurological deficits of mice. We found that pMCAO induced microglia autophagy and inflammatory response. The suppression of autophagy using either pharmacologic inhibitor (3-MA) not only decreased the microglia autophagy and inflammatory response, but also significantly decreased infarct size, edema formation and neurological deficits in vivo. Taken together, these results suggested that cerebral ischemia induced microglia autophagy contributed to ischemic neural inflammation and injury. In addition, our findings also provided novel therapeutic strategy for ischemic stroke.

Topics: Adenine; Animals; Autophagy; Brain; Cell Hypoxia; Cerebral Arteries; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Interleukin-1beta; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Microglia; Sirolimus; Stroke; Tumor Necrosis Factor-alpha

Blindness and visual impairments are heavy loads for modern society. Visual prosthesis is a promising therapy to treat these diseases. However, electric stimulation (ES)-induced damage of the optic nerve and adjacent cells are problems that must not be overlooked. In the current study, we aimed to investigate the effects of ES on cultured microglia cells and the potential protective mechanisms from a natural compound Lycium barbarum polysaccharide (LBP). Cellular injuries were induced by 9 mA bipolar pulse current in BV-2 cells for 15 min. Treatment with LBP alone or in association with either autophagic inhibitor 3-MA or autophagic agonist rapamycin was preadded for 2 h before the ES challenge. After that, morphological and molecular changes of the cells were measured at 2 h or 6 h postchallenges. We found that ES induced evident morphological and pathological changes of BV-2 cells, including oxidative stress, inflammation, and apoptosis. Pretreatment with LBP significantly attenuated these injuries with enhanced endogenous autophagy. When cellular autophagy was inhibited or enhanced by corresponding drug, the protective properties of LBP were partly inhibited or maintained, respectively. In addition, we demonstrated that ERK and p38 MAPK exerted diversified roles in the protection of LBP against ES-induced cellular damages. In conclusion, LBP improves bipolar pulse current-induced microglia cell injury through modulating autophagy and MAPK pathway.

Topics: Adenine; Animals; Apoptosis; Autophagy; Caspase 3; Caspase 7; Cell Line; Drugs, Chinese Herbal; Electric Stimulation; Extracellular Signal-Regulated MAP Kinases; Inflammation; Lycium; Mice; Microglia; Microscopy, Fluorescence; Microtubule-Associated Proteins; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Sirolimus; Time-Lapse Imaging

Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future.

Topics: Acute Kidney Injury; Adenine; Animals; Apoptosis; Autophagy; Blood Urea Nitrogen; Caspase 3; Creatinine; Disease Models, Animal; In Situ Nick-End Labeling; Male; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus

Traumatic brain injury (TBI) is caused by both primary and secondary injury mechanisms, all of which cause neuronal cell death and functional deficits. Both apoptosis and autophagy participated in neuronal cell death and functional loss induced following TBI. Preclinical findings implicate that 17-allylamino-demethoxygeldanamycin (17-AAG), an anticancer drug in clinical, present neuroprotection actions in multiple neurological disorders, but whether 17-AAG is capable of modulating neuronal autophagy has never been addressed. The present study was designed to determine the hypothesis that17-AAG treatment could confer neuroprotection in a rat model of TBI. We also used an autophagy inhibitor 3-methyladenine (3-MA) as well as an autophagy inducer rapamycin (RAPA) to test its underlining mechanisms. Our results showed that post-TBI administration of 17-AAG could attenuate brain edema, decrease neuronal death, as well as improve the recovery of motor function. Afterwards, in our model, 17-AAG treatment protected against TBI-induced apoptosis activation as well as enhanced neuronal autophagy. The present study provides novel clues in understanding the mechanisms of which 17-AAG exerts its neuroprotective activity on neurological disorders.

Topics: Adenine; Animals; Apoptosis; Autophagy; Benzoquinones; Brain Edema; Brain Injuries; Cell Survival; Cerebral Cortex; Female; Lactams, Macrocyclic; Motor Skills; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Sirolimus

Autophagy is a dynamic catabolic process that maintains cellular homeostasis. Whether it plays a role in promoting cell survival or cell death in the process of renal ischemia/reperfusion (I/R) remains controversial, partly because renal autophagy is usually examined at a certain time point. Therefore, monitoring of the whole time course of autophagy and apoptosis may help better understand the role of autophagy in renal I/R.. Autophagy and apoptosis were detected after mice were subjected to bilateral renal ischemia followed by 0-h to 7-day reperfusion, exposure of TCMK-1 cells to 24-h hypoxia, and 2 to 24-h reoxygenation. The effect of autophagy on apoptosis was assessed in the presence of autophagy inhibitor 3-methyladenine (3-MA) and autophagy activator rapamycin.. Earlier than apoptosis, autophagy increased from 2-h reperfusion, reached the maximum at day 2, and then began declining from day 3 when renal damage had nearly recovered to normal. Exposure to 24-h hypoxia induced autophagy markedly, but it decreased drastically after 4 and 8-h reoxygenation, which was accompanied with increased cell apoptosis. Inhibition of autophagy with 3-MA increased the apoptosis of renal tubular cells during I/R in vivo and hypoxia/reoxygenation (H/R) in vitro. In contrast, activation of autophagy by rapamycin significantly alleviated renal tissue damage and tubular cell apoptosis in the two models.. Autophagy was induced in a time-dependent manner and occurred earlier than the onset of cell apoptosis as an early response that played a renoprotective role during renal I/R and cell H/R. Up-regulation of autophagy may prove to be a potential strategy for the treatment of acute kidney injury.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Survival; Cells, Cultured; Gene Expression Regulation; Kidney Tubules; Male; Mice; Reperfusion Injury; Sirolimus; Time Factors

Intra-articular injection of glucocorticoids (GCs) has been widely used in the management of osteoarthritis and rheumatoid arthritis. Nevertheless, several studies showed that GCs had toxic effects on chondrocytes as well as synovial cells. Previously we reported the protective role of autophagy in the degeneration of meniscal tissues. However, the effects of GCs on autophagy in the meniscal cells have not been fully elucidated. To investigate whether GCs can regulate autophagy in human meniscal cells, the meniscal cells were cultured in vitro and exposed in the presence of dexamethasone. The levels of apoptosis and autophagy were investigated via flow cytometry as well as western blotting analysis. The changes of the aggrecanases were measured using real-time PCR. The role of autophagy in dexamethasone-induced apoptosis was investigated using pharmacological agents and RNA interference technique. An agonist of inositol 1,4,5-trisphosphate receptor (IP3R) was used to investigate the mechanism of dexamethasone-induced autophagy. The results showed that dexamethasone induced autophagy as well as apoptosis in normal human meniscal cells. Using RNA interference technique and pharmacological agents, our results showed that autophagy protected the meniscal cells from dexamethasone-induced apoptosis. Our results also indicated that dexamethasone increased the mRNA levels of aggrecanases. This catabolic effect of dexamethasone was enhanced by 3-MA, the autophagy inhibitor. Furthermore, our results showed that dexamethasone induced autophagy via suppressing the phosphorylation of IP3R. In summary, our results indicated that autophagy protected meniscal cells from GCs-induced apoptosis via inositol trisphosphate receptor signaling.

Topics: Adenine; Apoptosis; Autophagy; Cells, Cultured; Dexamethasone; Endopeptidases; Extracellular Matrix; Female; Humans; Inositol 1,4,5-Trisphosphate Receptors; Menisci, Tibial; Osteosarcoma; Phosphorylation; RNA, Messenger; Signal Transduction; Sirolimus

Cardiomyocyte death is one major factor in the development of heart dysfunction, thus, understanding its mechanism may help with the prevention and treatment of this disease. Previously, we reported that anti-β1-adrenergic receptor autoantibodies (β1-AABs) decreased myocardial autophagy, but the role of these in cardiac function and cardiomyocyte death is unclear. We report that rapamycin, an mTOR inhibitor, restored cardiac function in a passively β1-AAB-immunized rat model with decreased cardiac function and myocardial autophagic flux. Next, after upregulating or inhibiting autophagy with Beclin-1 overexpression/rapamycin or RNA interference (RNAi)-mediated expression of Beclin-1/3-methyladenine, β1-AAB-induced autophagy was an initial protective stress response before apoptosis. Then, decreased autophagy contributed to cardiomyocyte death followed by decreases in cardiac function. In conclusion, proper regulation of autophagy may be important for treating patients with β1-AAB-positive heart dysfunction.

Topics: Adenine; Adrenergic beta-1 Receptor Antagonists; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autoantibodies; Autophagy; Beclin-1; Cardiomyopathies; Cell Line; Gene Expression Regulation; Humans; Immunization, Passive; Male; Myocytes, Cardiac; Rats; Rats, Wistar; Receptors, Adrenergic, beta-1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Dysfunction, Left; Ventricular Pressure

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cadaverine; Cell Line; Cytoprotection; Dose-Response Relationship, Drug; Haloperidol; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Microtubule-Associated Proteins; Myocardial Reperfusion Injury; Myocytes, Cardiac; Protective Agents; Rats; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann-Sträussler-Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent.

Topics: Adenine; Animals; Autophagy; Cell Line; Gerstmann-Straussler-Scheinker Disease; Humans; MAP Kinase Signaling System; Mice; Protein Folding; Proteolysis; PrPSc Proteins; Sirolimus

Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT), CYP2E1 knockout (KO) or CYP2E1 humanized transgenic knockin (KI), mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA), an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These results suggest that autophagy is protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. We speculate that autophagy-dependent processes such as mitophagy and lipophagy help to minimize ethanol-induced CYP2E1-dependent oxidative stress and therefore the subsequent liver injury and steatosis. Attempts to stimulate autophagy may be helpful in lowering ethanol and CYP2E1-dependent liver toxicity.

Topics: Adenine; Animals; Autophagy; Cytochrome P-450 CYP2E1; Ethanol; Liver; Liver Diseases, Alcoholic; Male; Mice; Sirolimus

In human neonates, immature GABAergic interneurons are markedly affected by an excitotoxic insult. While in adults the interest of cell transplantation has been demonstrated in several neurological disorders, few data are available regarding the immature brain. The low survival rate constitutes a strong limitation in the capacity of transplanted neurons to integrate the host tissue. Because i) autophagy is an adaptive process to energetic/nutrient deprivation essential for cell survival and ii) literature describes cross-talks between autophagy and apoptosis, we hypothesized that regulation of autophagy would represent an original strategy to favor long-term survival of GABAergic precursors grafted in the immature neocortex. Morphological, neurochemical, and functional data showed that in control conditions, few grafted Gad67-GFP precursors survived. The first hours following transplantation were a critical period with intense apoptosis. Experiments performed on E15.5 ganglionic eminences revealed that Gad67-GFP precursors were highly sensitive to autophagy. Rapamycin and 3-MA impacted on LC3 cleavage, LC3II translocation, and autophagosome formation. Quantification of Bax, mitochondrial integrity, caspase-3 cleavage, and caspase-3 immunolocalization and activity showed that 3-MA induced a significant decrease of Gad67-GFP precursor apoptosis. In vivo, 3-MA induced, within the first 24 h, a diffuse LC3 pattern of grafted Gad67-GFP precursors, an increase of precursors with neurites, a reduction of the density of caspase-3 immunoreactive cells. A twofold increase in the survival rate occurred 15 days after the graft. Surviving neurons were localized in the cortical layers II-IV, which were still immature when the transplantation was done. Altogether, these data indicate that inhibition of autophagy represents an original strategy to allow GABAergic interneurons to overpass the first critical hours following transplantation and to increase their long-term survival in mice neonates.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Differentiation; Cell Survival; Cell Transplantation; Embryonic Stem Cells; Female; GABAergic Neurons; Glutamate Decarboxylase; Green Fluorescent Proteins; Interneurons; Male; Mice; Mice, Transgenic; Neocortex; Neural Stem Cells; Pregnancy; Recombinant Fusion Proteins; Sirolimus

Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long-lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3-methyladenine administration or as occurs in activating molecule in Beclin-1-regulated autophagy transgenic mice (Ambra1(+/gt)), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox-sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; CD11b Antigen; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Immunosuppressive Agents; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Pain Measurement; Schwann Cells; Sciatic Nerve; Sciatica; Sirolimus; Time Factors

Dengue virus (DENV) is one of the major infectious pathogens worldwide. DENV infection is a highly dynamic process. Currently, no antiviral drug is available for treating DENV-induced diseases since little is known regarding how the virus interacts with host cells during infection. Advanced molecular imaging technologies are powerful tools to understand the dynamics of intracellular interactions and molecular trafficking. This study exploited a single-virus particle tracking technology to address whether DENV interacts with autophagy machinery during the early stage of infection. Using confocal microscopy and three-dimensional image analysis, we showed that DENV triggered the formation of green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta, and DENV-induced autophagosomes engulfed DENV particles within 15-min postinfection. Moreover, single-virus particle tracking revealed that both DENV particles and autophagosomes traveled together during the viral infection. Finally, in the presence of autophagy suppressor 3-methyladenine, the replication of DENV was inhibited and the location of DENV particles spread in cytoplasma. In contrast, the numbers of newly synthesized DENV were elevated and the co-localization of DENV particles and autophagosomes was detected while the cells were treated with autophagy inducer rapamycin. Taken together, we propose that DENV particles interact with autophagosomes at the early stage of viral infection, which promotes the replication of DENV.

Topics: Adenine; Autophagy; Cell Line, Tumor; Dengue; Dengue Virus; Host-Pathogen Interactions; Humans; Molecular Imaging; Phagosomes; Sirolimus; Virion

Autophagy plays an important role in cellular responses to pathogens. However, the impact of the autophagy machinery on classical swine fever virus (CSFV) infection is not yet confirmed. In this study, we showed that CSFV infection significantly increases the number of autophagy-like vesicles in the cytoplasm of host cells at the ultrastructural level. We also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including LC3-I/LC3-II conversion and ATG12-ATG5 conjugation, which are considered important indicators of autophagy. Meanwhile, high expression of ATG5 and BECN1 was detected in CSFV-infected cells; conversely, degradation of SQSTM1 was observed by immunoblotting, suggesting that CSFV infection triggered a complete autophagic response, most likely by the NS5A protein. Furthermore, by confocal immunofluorescence analysis, we discovered that both envelope protein E2 and nonstructural protein NS5A colocalized with LC3 and CD63 during CSFV infection. Examination by immunoelectron microscopy further confirmed the colocalization of both E2 and NS5A proteins with autophagosome-like vesicles, indicating that CSFV utilizes the membranes of these vesicles for replication. Finally, we demonstrated that alteration of cellular autophagy by autophagy regulators and shRNAs affects progeny virus production. Collectively, these findings provide strong evidence that CSFV infection needs an autophagy pathway to enhance viral replication and maturity in host cells.

Topics: Adenine; Animals; Autophagy; Biomarkers; Cell Line; Cell Survival; Classical Swine Fever; Classical Swine Fever Virus; Intracellular Membranes; Membrane Proteins; Microtubule-Associated Proteins; Phagosomes; RNA, Small Interfering; RNA, Viral; Sirolimus; Swine; Viral Proteins; Virus Replication

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.

Topics: Adenine; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Deoxycytidine; DNA Fragmentation; Drug Resistance, Neoplasm; Everolimus; Gemcitabine; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Microtubule-Associated Proteins; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Securin; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

Mammalian target of rapamycin (mTOR) is known to be a major negative regulator of autophagy. Recent studies have shown that mTOR activity is abnormally increased in endometriotic lesions. In endometriosis, abnormal mTOR activity may contribute to the alteration of endometrial cell autophagy, which may affect apoptosis because endometrial cell autophagy is directly involved in the regulation of apoptosis. To test this hypothesis, we investigated whether endometrial cell autophagy is altered by aberrant mTOR activity and is associated with apoptosis in ovarian endometriotic cysts. Our results show that endometrial cell autophagy induction was increased by mTOR inhibition as the menstrual cycle progresses in the normal endometrium, and that it is correlated with apoptosis. However, in endometriotic tissues from ovarian endometriotic cysts, autophagy, mTOR activity and apoptosis were constant throughout the menstrual cycle, suggesting that a constant level of autophagy is maintained by disinhibition of mTOR activity during the menstrual cycle in endometriotic tissues and is related to decreased apoptosis. Indeed, compared with normal endometrium, increased mTOR activity during the secretory phase in endometriotic tissues inhibited autophagy and apoptosis induction. In addition, to determine the direct effect of autophagy induction mediated by mTOR on endometriotic cell apoptosis, endometriotic cells were treated with rapamacin (mTOR inhibitor) with and without 3-methyladenine (3-MA, autophagy inhibitor). Although rapamycin treatment induced autophagy and led to apoptosis promotion, the pro-apoptotic effect of rapamycin was reversed by the addition of 3-MA, suggesting that mTOR inhibition promotes endometriotic cell apoptosis via autophagy induction. In conclusion, our results suggest that aberrant mTOR activity in ovarian endometriotic cysts leads to alteration of endometrial cell autophagy, which is associated with abnormal apoptosis.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cysts; Endometriosis; Endometrium; Female; Gene Expression Regulation; Humans; Menstrual Cycle; Primary Cell Culture; Sirolimus; Stromal Cells; TOR Serine-Threonine Kinases

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin-regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)-induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy-mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time- and viral dose-dependent manner in control L929 cells (L-EV), whereas virus-induced autophagy was delayed in axin-expressing L929 cells (L-axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3-methyladenine (3MA) and beclin-1 knockdown facilitated viral replication in L-EV cells. In addition, preventing autophagy with 3MA suppressed virus-induced cytotoxicity in L-EV cells. In contrast, HSV replication in L-axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV-infection induced autophagy, leading to enhanced viral replication.

Topics: Adenine; Apoptosis Regulatory Proteins; Autophagy; Axin Protein; Beclin-1; Cell Line; Gene Expression; Gene Knockdown Techniques; Humans; Membrane Proteins; Simplexvirus; Sirolimus; Virus Replication

In the current study, we showed that the combination of mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and Akt inhibitor MK-2206 exerted synergistic cytotoxic effects against low-phosphatase and tensin homolog (PTEN) gastric cancer cells (HGC-27 and SNU-601 lines). In HGC-27 cells, RAD001 and MK-2206 synergistically induced G1/S cell cycle arrest, growth inhibition, cell death but not apoptosis. RAD001 and MK-2206 synergistically induced light chain 3B (LC3B) and beclin-1 expression, two important autophagy indicators. Meanwhile, the autophagy inhibitor 3-methyladenine (3-MA) and chloroquine inhibited the cytotoxic effects by RAD001 and MK-2206, suggesting that autophagic, but not apoptotic cell death was important for the cytotoxic effects by the co-administration. We observed that the combination of RAD001 and MK-2206 exerted enhanced effects on Akt/mTOR inhibition, cyclin D1 down-regulation and ERK/MAPK(extracellular signal-regulated kinase/mitogen-activated protein kinases) activation. Intriguingly, MEK/ERK inhibitors PD98059 and U0126 suppressed RAD001 plus MK-2206-induced beclin-1 expression, autophagy induction and cytotoxicity in HGC-27 cells. In conclusion, these results suggested that the synergistic anti-gastric cancer cells ability by RAD001 and MK-2206 involves ERK-dependent autophagic cell death pathway.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Butadienes; Cell Line, Tumor; Chloroquine; Cyclin D1; Drug Synergism; Everolimus; Flavonoids; Gastric Mucosa; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Membrane Proteins; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Nitriles; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Autophagy plays a protective role in endotoxemic mice. Heat shock factor 1 (HSF-1) also plays a crucial protective role in endotoxemic mice by decreasing inflammatory cytokines. The purpose of this study was to determine whether HSF-1 is involved in attenuating the release of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated mice and peritoneal macrophages (PMs) through regulating autophagy activity. Autophagosome formation in HSF-1(+/+) and HSF-1(-/-) mice and PMs stimulated by LPS was examined by Western blotting and immunofluorescence. Lipopolysaccharide-induced autophagy and inflammatory cytokines were examined in HSF-1(+/+) and HSF-1(-/-) PMs treated with 3-methyladenine (3-MA) or rapamycin. Results showed that LPS-induced autophagy was elevated transiently at 12 h but declined at 24 h in the livers and lungs of mice. Higher levels of inflammatory cytokines and lower autophagy activity were detected in HSF-1(-/-) mice and PMs compared with HSF-1(+/+) mice and PMs. Interestingly, LPS-induced release of inflammatory cytokines did not further increase in HSF-1(-/-) PMs treated with 3-MA but aggravated in HSF-1(+/+) PMs. Lipopolysaccharide-induced autophagy did not decrease in HSF-1(-/-) PMs treated with 3-MA but decreased in HSF-1 PMs(+/+). Taken together, our results suggested that HSF-1 attenuated the release of inflammatory cytokines induced by LPS by regulating autophagy activity.

Topics: Adenine; Animals; Autophagy; Cytokines; DNA-Binding Proteins; Heat Shock Transcription Factors; Immunohistochemistry; Lipopolysaccharides; Mice; Mice, Knockout; Sirolimus; Transcription Factors

To explore the effects of modulating autophagy on neuroamyloidogenesis in an ischemic stroke model of cultured neuroblastoma 2a (N2a)/Amyloid precursor protein (APP)695 cells.. The ischemic stroke model of N2a/APP695 cells was made by 6h oxygen-glucose deprivation/12h reperfusion (OGDR). Drug administration of 3-methyladenine (3-MA), rapamycin or dl-3-n-butylphthalide (NBP) was started at the beginning of the OGDR and lasted until the end of reperfusion, in order to explore their effects on N2a/APP695 cells under OGDR conditions. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 12h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-κB) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-β (Aβ)1-42 with Beclin 1 was performed to investigate their cellular co-localization relationship; β-secretase and γ-secretase activity assay and Aβ1-42 enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis.. The results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and Aβ generation in N2a/APP695 cells; down-regulating autophagy by 3-MA and NBP increased cell viability, decreased lactate dehydrogenase (LDH) production, inhibited the activation of NF-κB pathway, suppressed β- and γ-secretase activities and Aβ generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated Aβ1-42(+) signal was co-localized with Beclin 1(+) signal.. Our data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-κB pathway. This study might help us to find a new therapeutic strategy to prevent brain ischemic damage and depress the risk of post-stroke dementia.

Topics: Adenine; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Autophagy; Benzofurans; Brain Ischemia; Cell Survival; Mice; Neuroblastoma; Sirolimus; Stroke; Tumor Cells, Cultured

The maximum lifespan of the naked mole rat is over 28.3 years, which exceeds that of any other rodent species, suggesting that age-related changes in its body composition and functionality are either attenuated or delayed in this extraordinarily long-lived species. However, the mechanisms underlying the aging process in this species are poorly understood. In this study, we investigated whether long-lived naked mole rats display more autophagic activity than short-lived mice.. Hepatic stellate cells isolated from naked mole rats were treated with 50 nM rapamycin or 20 mM 3-methyladenine (3-MA) for 12 or 24 h. Expression of the autophagy marker proteins LC3-II and beclin 1 was measured with western blotting and immunohistochemistry. The induction of apoptosis was analyzed by flow cytometry.. Our results demonstrate that one-day-old naked mole rats have higher levels of autophagy than one-day-old short-lived C57BL/6 mice, and that both adult naked mole rats (eight months old) and adult C57BL/6 mice (eight weeks old) have high basal levels of autophagy, which may be an important mechanism inhibiting aging and reducing the risk of age-related diseases.. Here, we report that autophagy facilitated the survival of hepatic stellate cells from the naked mole rat, and that treatment with 3-MA or rapamycin increased the ratio of apoptotic cells to normal hepatic stellate cells.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Hepatic Stellate Cells; Immunosuppressive Agents; Longevity; Mice; Microtubule-Associated Proteins; Mole Rats; Sirolimus

Recently, evidence indicated that the rapamycin-eluting stent which was used worldwide may contribute to an increased risk for thrombosis. On the contrary, other researchers found it was safe. Thus, it is necessary to clarify the effect of rapamycin on thrombosis and the corresponding mechanisms.. The effects of rapamycin in vivo were evaluated by modified deep vein thrombosis animal model. The platelets were from healthy volunteers and the platelet-endothelium (purchased from ATCC) adhesion in cultured endothelial cells was assessed. Membrane rufflings in endothelial cells were examined by confocal and electron microscope. Thrombus formation increased in rats that were injected with rapamycin. Electron microscope analysis exhibited microvilli on the rapamycin-treated endothelium in rats. Rapamycin enhanced membrane ruffling in human umbilical vein endothelial cells (HUVECs) and adhesion of platelets to HUVECs. The platelet-HUVECs adhesion was attenuated when cells were treated with cytochalacin B. Inhibition of autophagy by 3-methyladenine led to suppression of membrane ruffles in HUVECs and augmentation of platelet-endothelial adhesion.. In conclusion, we found that endothelial membrane remodeling induced by rapamycin is crucial for the adhesion of platelets to endothelial cells and thereby for thrombosis in vivo, and that the endothelial membrane remodeling is autophagy dependent.

Topics: Adenine; Animals; Autophagy; Blood Platelets; Cell Adhesion; Cell Membrane; Cytochalasin B; Disease Models, Animal; Human Umbilical Vein Endothelial Cells; Humans; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Thrombosis

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1 expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increased Bcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Survival; Cells, Cultured; Female; Male; Neurons; Rats; Rats, Wistar; Sirolimus; Spinal Cord; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Increasing studies have suggested that rapamycin has inhibitory effect for cancer cell proliferation.. The present study aimed to investigate the effect of rapamycin on the proliferation of A549 lung cancer cells and try to elucidate its probable mechanism.. A549 cells were randomly divided into the following 3 groups (n=6): the Dulbecco's modified Eagle's medium (DMEM) culture solution administered alone group (C group), the 10 nmol/l rapamycin administered alone group (R group) and the 5 mmol/l 3-methyladenine (3-MA) plus 10 nmol/l rapamycin administered group (MR group). Death percentage of A549 cells was observed and the levels of caspase-3, Beclin-1, and microtubule-associated protein 1 light chain 3-II (LC3-II) were determined.. Compared with C group, percentage of cell death, caspase-3, Beclin-1 and LC3-II both showed a significant increase in R group (p < 0.05). On the contrary, as compared with R group, percentage of cell death, caspase-3, Beclin-1 and LC3-II both showed a significant decrease in MR group (p < 0.05).. Rapamycin has an inhibitory effect for the proliferation of A549 cells, and its mechanism is likely related to the activation of autophagic pathway.

Topics: Adenine; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Caspase 3; Cell Death; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Membrane Proteins; Microtubule-Associated Proteins; Sirolimus

Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 μm (PM2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-α)-induced pathway as evidenced by TNF-α secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-α secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-α with an anti-TNF-α special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM2.5 exposure.

Topics: Adenine; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cell Line, Tumor; Cell Survival; Epithelial Cells; Humans; Lung; Oxidative Stress; Particulate Matter; Reactive Oxygen Species; Signal Transduction; Sirolimus

Autophagy is involved in cell differentiation. We present evidence that autophagy is activated during β-mercaptoethanol (β-ME)-induced neuronal differentiation of bone marrow mesenchymal stem cells (MSCs), in which mammalian target of rapamycin (mTOR) signaling is important. mTOR activity declined after being transported from the nucleus to the cytoplasm. Using 3-methyladenine (3-MA) and rapamycin to regulate the activity of mTOR, it was found that the efficiency of neuronal differentiation was affected.

Topics: Adenine; Animals; Autophagy; Bone Marrow Cells; Cell Differentiation; Down-Regulation; Mercaptoethanol; Mesenchymal Stem Cells; Neurogenesis; Rats; Rats, Wistar; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Parkinson's disease is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing Parkinson's disease. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10-100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 h, caused cell death at 24 h with an LD50 of 10 nM. Overall, autophagic flux was decreased by 10 nM rotenone at both 2 and 24 h, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 h, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Up-regulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine exacerbated cell death. Interestingly, while 3-methyladenine exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor.

Topics: Adenine; Animals; Autophagy; Cell Survival; Cells, Cultured; Cerebral Cortex; DNA Damage; DNA, Mitochondrial; Dose-Response Relationship, Drug; Embryo, Mammalian; Energy Metabolism; Enzyme Inhibitors; Insecticides; Lactosylceramides; Neurons; Oligomycins; Oxygen Consumption; Rats; Rotenone; Sirolimus

Remote ischemic perconditioning (RIPer) has been proved to provide potent cardioprotection. However, there are few studies on neuroprotection of RIPer. This study aims to clarify the neuroprotective effect of RIPer and the role of autophagy induced by RIPer against cerebral ischemia reperfusion injury in rats. Using a transient middle cerebral artery occlusion (MCAO) model in rats to imitate focal cerebral ischemia. RIPer was carried out 4 cycles of 10 min ischemia and 10 min reperfusion, with a thin elastic band tourniquet encircled on the bilateral femoral arteries at the start of 10 min after MCAO. Autophagy inhibitor 3-methyladenine (3-MA) and autophagy inducer rapamycin were administered respectively to determine the contribution of autophagy in RIPer. Neurologic deficit scores, infarct volume, brain edema, Nissl staining, TUNEL assay, immunohistochemistry and western blot was performed to analyze the neuroprotection of RIPer and the contribution of autophagy in RIPer. RIPer significantly exerted neuroprotective effects against cerebral ischemia reperfusion injury in rats, and the autophagy-lysosome pathway was activated by RIPer treatment. 3-MA reversed the neuroprotective effects induced by RIPer, whereas rapamycin ameliorated the brain ischemic injury. Autophagy activation contributes to the neuroprotection by RIPer against focal cerebral ischemia in rats.

Topics: Adenine; Animals; Autophagy; Brain Ischemia; Cerebral Infarction; Ischemic Postconditioning; Ischemic Preconditioning; Male; Models, Animal; Neuroprotective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Autophagy is important for cell renewing for its contribution to the degradation of bulk cytoplasm, long-lived proteins, and entire organelles and its role in embryonic development is largely unknown. In our study, we investigated the function of autophagy in gastrulation of the chick embryo using both in vivo and in vitro approaches, especially in the EMT process, and we found that autophagy gene Atg7 was expressed on the apical side of the ectoderm and endoderm. Over-expression of Atg7 could enhance the expression of Atg8 and the E-cadherin, the latter of which is a crucial marker of the EMT process. We also found that the disturbance of autophagy could retard the development of chick embryos in HH4 with shorter primitive steak than that in the control group, which is a newly formed structure during EMT process. So we assumed that autophagy could affect EMT process by adhesion molecule expression. Moreover, more molecules, such as slug, chordin, shh et., which were all involved in EMT process, were detected to address the mechanism of this phenomena. We established that the inhibition of autophagy could cause developmental delay by affecting EMT process in gastrulation of chick embryos.

Topics: Adenine; Animals; Autophagy; Cadherins; Cell Adhesion Molecules; Chick Embryo; Epithelial-Mesenchymal Transition; Gastrula; Gastrulation; Gene Expression Regulation, Developmental; Germ Layers; HCT116 Cells; Humans; Microtubule-Associated Proteins; Models, Biological; Sirolimus

Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Beta-amyloid (Aβ) plays an important role in the pathogenesis of AD. Autophagy is a self-degradative process and its related protein Beclin-1 is involved in the initiation of autophagy. However, the role of Beclin-1 in the pathogenesis of AD is rarely reported. In this study, we examined cell viability and medium levels of neuron-specific enolase (NSE) in PC12 cells incubated with gradient concentrations of Aβ(1-42) (0.625, 1.25, 2.5, 5, 10 μM) for 6, 12, 24, 48, and 72 h, drew the index changes curves, and investigated the correlation between them. The result showed that cell viability was negatively correlated with NSE levels. Based on this study, Beclin-1 expression was quantitatively detected in Aβ1-42-treated PC12 cells and the dynamic changes curve of Beclin-1 was drawn from 3 to 72 h. Beclin-1 expression was positively correlated with cell viability. Furthermore, both autophagy inhibitor 3-methyladenine (3-MA) and autophagy activator rapamycin were used to investigate the effect of autophagy on Aβ(1-42)-induced cell injury. Aβ(1-42)-induced Beclin-1 expression was further upregulated by rapamycin but was downregulated by 3-MA. Moreover, cell viability was increased by rapamycin but was decreased by 3-MA, and NSE was decreased by rapamycin but was increased by 3-MA, suggesting that activation of Beclin-1-dependent autophagy before the damage occurred can prevent neuronal cell death, while inhibition of Beclin-1-dependent autophagy can hastened cell death. These findings indicate that increasing Beclin-1-dependent autophagy may have a preventive effect before the AD occurred.

Topics: Adenine; Amyloid beta-Peptides; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Death; Cell Survival; Dose-Response Relationship, Drug; Neurons; PC12 Cells; Peptide Fragments; Phosphopyruvate Hydratase; Rats; Sirolimus; Up-Regulation

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

Topics: Adenine; Animals; Autophagy; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Cells, Cultured; Hypoxia; Hypoxia-Ischemia, Brain; Ischemic Preconditioning; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Neuroprotective Agents; Prosencephalon; Proteins; Rats; Rats, Wistar; RNA Interference; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Autophagy is a catabolic process involving the degradation of cells' own unnecessary, injured, or aged proteins and recycling of degraded products to maintain hemostasis. Recently, studies indicated that autophagy plays a crucial role in cancer development. However, the role of autophagy in tongue squamous cell carcinoma (TSCC) has not been well documented. This study aims to assess the expression of autophagy-related protein and investigate its effect on TSCC.. Archival 50 TSCC samples were enrolled. Immunohistochemistry were performed to examine the expression of Beclin1 and LC3. Statistical analyses were carried out to assess the associations among clinicopathologic parameters. In vitro, cells were treated with rapamycin or 3-MA. Then, qPCR, western blot and immunofluorescence were performed to detect the expression of Beclin1 and LC3. Transmission electron microscopy was utilized to identify autophagsomes. For functional analysis, cell proliferation and cell cycle were evaluated with MTT assay and flow cytometer, respectively. At last, cell migration and invasion potentials were assessed by wound healing assay and transwell assay.. We confirmed that down-regulation of Beclin1 and LC3 is a frequent event in TSCC. Then, we demonstrated that decreased expression of Beclin1 was associated with T stage, clinical stage and differentiation. Furthermore, we showed that activation of autophagy by rapamycin suppressed proliferation, migration and invasion while inhibition of autophagy by 3-MA promoted proliferation, migration and invasion in TSCC cells.. Taken together, these data suggest that autophagy plays a pivotal role in the progression of TSCC.

Topics: Adenine; Antibiotics, Antineoplastic; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Female; Humans; Male; Membrane Proteins; Microtubule-Associated Proteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Phagosomes; Sirolimus; Tongue Neoplasms

We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides "cell-fate control" due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.

Topics: Adenine; Animals; Antigens, CD19; Apoptosis; Autophagy; CD4-Positive T-Lymphocytes; Cell Lineage; Cell Proliferation; Cell Survival; Genetic Therapy; Humans; Lentivirus; Mice; Nucleoside-Phosphate Kinase; Phenotype; Sirolimus; Transduction, Genetic; Transgenes; Zidovudine

To explore the effects of 3-methyladenine (3-MA) and rapamycin (Rapa) on autophagy and uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) induced by sulforaphane (SFN) in human colon cancer Caco-2 cells.. Western blot was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and UGT1A1 proteins. And immunocytochemistry was employed to observe the intracellular distribution of LC3 and nuclear localization of NF-E2-related factor 2 (Nrf2). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the mRNA expression of UGT1A1 and human pregnane X receptor (hPXR).. After the treatment of SFN, the LC3-II protein was induced in a dose and time-dependent manner. SFN-induced LC3-II protein could be attenuated and enhanced by 3-MA and Rapa respectively. In comparison with the control group, UGT1A1 mRNA levels increased significantly after the treatment of Rapa, SFN or their combination (2.4, 4.12 and 2.41 folds respectively, all P < 0.01). And the combination of SFN and Rapa possessed the highest level. UGT1A1 protein band intensity was also enhanced in three groups. There was no obvious nuclear staining of Nrf2 in control group while intense nuclear fluorescent labeling of Nrf2 could be observed in the SFN-treated groups, especially the combination group of SFN and Rapa. The hPXR mRNA levels increased significantly in the Rapa and combination groups (1.82 and 1.4 folds respectively, both P < 0.01).. The treatment of 3-MA or Rapa may attenuate or enhance SFN-induced autophagy respectively. And Rapa also potentiates SFN-induced UGT1A1 expression. The mechanism for the synergic effect of Rapa and SFN on UGT1A1 induction may be a simultaneous activation of Nrf2 and hPXR signaling pathway.

Topics: Adenine; Autophagy; Caco-2 Cells; Glucuronosyltransferase; Humans; Isothiocyanates; Sirolimus; Sulfoxides

In addition to their action of lowering blood cholesterol levels, statins modulate biological characteristics and functions of arterial myocytes such as viability, proliferation, apoptosis, survival and contraction. The present study tested whether simvastatin, as a prototype statin, enhances autophagy in coronary arterial myocytes (CAMs) to thereby exert their beneficial effects in atherosclerosis.. Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR) activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries.. Our results indicate that simvastatin inhibits the Rac1-mTOR pathway and thereby increases autophagy in CAMs which may stabilize CAMs in the contractile phenotype to prevent proliferation and growth of these cells.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Protein 7; Calcium-Binding Proteins; Calponins; Cell Proliferation; Cells, Cultured; Coronary Vessels; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lysosomes; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microtubule-Associated Proteins; Muscle Cells; Phenotype; rac1 GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Signal Transduction; Simvastatin; Sirolimus; TOR Serine-Threonine Kinases

Sublethal ischemic preconditioning (IPC) is a powerful inducer of ischemic brain tolerance. However, its underlying mechanisms are still not well understood. In this study, we chose four different IPC paradigms, namely 5 min (5 min duration), 5×5 min (5 min duration, 2 episodes, 15-min interval), 5×5×5 min (5 min duration, 3 episodes, 15-min intervals), and 15 min (15 min duration), and demonstrated that three episodes of 5 min IPC activated autophagy to the greatest extent 24 h after IPC, as evidenced by Beclin expression and LC3-I/II conversion. Autophagic activation was mediated by the tuberous sclerosis type 1 (TSC1)-mTor signal pathway as IPC increased TSC1 but decreased mTor phosphorylation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin staining confirmed that IPC protected against cerebral ischemic/reperfusion (I/R) injury. Critically, 3-methyladenine, an inhibitor of autophagy, abolished the neuroprotection of IPC and, by contrast, rapamycin, an autophagy inducer, potentiated it. Cleaved caspase-3 expression, neurological scores, and infarct volume in different groups further confirmed the protection of IPC against I/R injury. Taken together, our data indicate that autophagy activation might underlie the protection of IPC against ischemic injury by inhibiting apoptosis.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 3; Cerebrum; Immunosuppressive Agents; In Situ Nick-End Labeling; Ischemic Preconditioning; Male; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Amiodarone, bi-iodinated benzofuran derivative, is one of the most frequently prescribed and efficacious antiarrhythmic drugs. Despite its low incidence, amiodarone-induced pulmonary toxicity is of great concern and the leading cause of discontinuation. Autophagy is an essential homeostatic process that mediates continuous recycling of intracellular materials when nutrients are scarce. It either leads to a survival advantage or initiates death processes in cells under stress. In the present study, we investigated the role of autophagy in amiodarone-induced pulmonary toxicity. Amiodarone treatment-induced autophagy in H460 human lung epithelial cells and BEAS-2B normal human bronchial epithelial cells was demonstrated by increased LC3-II conversion, Atg7 upregulation, and autophagosome formation. Autophagic flux, as determined by the lysosomal inhibitor bafilomycin A1, was also increased following amiodarone treatment. To determine the role of autophagy in amiodarone toxicity, amiodarone-induced cell death was evaluated in the presence of 3-methyladenine or by knocking down the autophagy-related genes Atg7. Inhibition of autophagy decreased cellular viability and significantly increased apoptosis. Intratracheal instillation of amiodarone in rats increased the number of inflammatory cells recovered from bronchoalveolar lavage fluid, and periodic acid-Schiff-positive staining in bronchiolar epithelial cells. However, induction of autophagy by rapamycin treatment inhibited amiodarone-induced pulmonary toxicity. In conclusion, amiodarone treatment induced autophagy, which is involved in protection against cell death and pulmonary toxicity.

Topics: Adenine; Administration, Inhalation; Amiodarone; Animals; Anti-Arrhythmia Agents; Apoptosis; Autophagy; Autophagy-Related Protein 7; Bronchoalveolar Lavage Fluid; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Epithelial Cells; Gene Knockdown Techniques; Humans; Lung; Macrolides; Male; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Inbred F344; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Ubiquitin-Activating Enzymes

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects.

Topics: Adenine; Adult; Aged; Aged, 80 and over; Bone Marrow Cells; Cytogenetics; Cytokines; Female; Humans; Indazoles; Leukemia, Myeloid, Acute; Male; Middle Aged; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; RNA, Messenger; Signal Transduction; Sirolimus; Stromal Cells; Sulfonamides; TOR Serine-Threonine Kinases; Young Adult

We and others have reported that autophagy is induced by dengue viruses (DVs) in various cell lines, and that it plays a supportive role in DV replication. This study intended to clarify whether DV infection could induce autophagy in vivo. Furthermore, the effect of DV induced autophagy on viral replication and DV-related pathogenesis was investigated.. The physiopathological parameters were evaluated after DV2 was intracranially injected into 6-day-old ICR suckling mice. Autophagy-related markers were monitored by immunohistochemical/immunofluorescent staining and Western blotting. Double-membrane autophagic vesicles were investigated by transmission-electron-microscopy. DV non-structural-protein-1 (NS1) expression (indicating DV infection) was detected in the cerebrum, medulla and midbrain of the infected mice. In these infected tissues, increased LC3 puncta formation, LC3-II expression, double-membrane autophagosome-like vesicles (autophagosome), amphisome, and decreased p62 accumulation were observed, indicating that DV2 induces the autophagic progression in vivo. Amphisome formation was demonstrated by colocalization of DV2-NS1 protein or LC3 puncta and mannose-6-phosphate receptor (MPR, endosome marker) in DV2-infected brain tissues. We further manipulated DV-induced autophagy by the inducer rapamycin and the inhibitor 3-methyladenine (3MA), which accordingly promoted or suppressed the disease symptoms and virus load in the brain of the infected mice.We demonstrated that DV2 infection of the suckling mice induces autophagy, which plays a promoting role in DV replication and pathogenesis.

Topics: Adenine; Animals; Animals, Newborn; Antimetabolites; Autophagy; Blotting, Western; Dengue; Dengue Virus; Fluorescent Antibody Technique; Immunochemistry; Immunosuppressive Agents; Mice; Mice, Inbred ICR; Microscopy, Electron, Transmission; Sirolimus; Viral Load; Virus Replication

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by severe striatal atrophy with extensive neuronal loss and gliosis. Although the molecular mechanism is not well understood, experimental studies use the irreversible mitochondrial inhibitor 3-nitropropionic acid (3-NP) to mimic the neuropathological features of HD. In this study, the role of autophagy as a neuroprotective mechanism against 3-NP-induced astrocyte cytotoxicity was evaluated. Autophagy is a catabolic process that is essential for the turnover of cytosolic proteins and organelles and is involved in the modulation of cell death and survival. We showed that 3-NP-induced apoptosis, which was accompanied by Bax and Beclin-1 upregulation, was dependent on acidic vesicular organelle (AVO) formation after a continuous exposure to 3-NP for 12 h. The upregulation of Bax and Beclin-1 as well as AVO formation were normalized 24 h after 3-NP exposure.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Astrocytes; Autophagy; bcl-2-Associated X Protein; Beclin-1; Disease Models, Animal; Huntington Disease; Mice; Nitro Compounds; Propionates; Sirolimus

The present study aimed to analyze the responses to autophagy in osteoarthritis (OA) and aging chondrocytes in order to elucidate the role of autophagy in the pathogenesis of OA. We used multiple assays to confirm that autophagic activity was downregulated in chondrocytes of aged articular cartilage. Surprisingly, we found that the expression of autophagy-related proteins was not decreased in the tissues of patients with OA. We also observed that rapamycin-induced autophagy prevented the accumulation of subdiploid cells in young chondrocytes, while it induced cell death by autophagy in OA chondrocytes. Our results demonstrate that autophagic activity decreases with aging, and may be responsible for the cytoprotective effects in young cartilage. However, we found that autophagic activity in patients with OA was higher than in the aging group, and reported autophagic cell death in OA chondrocytes. These results suggest that autophagy plays both a cytoprotective and death-promoting role in the pathogenesis of OA.

Topics: Adenine; Aged; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cartilage, Articular; Cell Cycle; Cell Hypoxia; Cell Survival; Cells, Cultured; Chondrocytes; Female; Green Fluorescent Proteins; Humans; Male; Membrane Proteins; Microtubule-Associated Proteins; Middle Aged; Osteoarthritis; Sirolimus; Young Adult

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.

Topics: Adenine; Animals; Autophagy; Axons; Cell Line; Green Fluorescent Proteins; Heat-Shock Proteins; Intraocular Pressure; Male; Microtubule-Associated Proteins; Nerve Degeneration; Neuroprotective Agents; Nicotinamide-Nucleotide Adenylyltransferase; Optic Nerve; Protein Transport; Rats; Rats, Wistar; Retinal Ganglion Cells; Sequestosome-1 Protein; Sirolimus; Subcellular Fractions; Transfection; Tumor Necrosis Factor-alpha

Autophagy has been shown to be protective against drug and alcohol-induced liver injury. CYP2E1 plays a role in the toxicity of ethanol, carcinogens and certain drugs. Inhibition of autophagy increased ethanol-toxicity and accumulation of fat in wild type and CYP2E1 knockin mice but not in CYP2E1 knockout mice as well as in HepG2 cells expressing CYP2E1 (E47 cells) but not HepG2 cells lacking CYP2E1 (C34 cells). The goal of the current study was to evaluate whether modulation of autophagy can affect CYP2E1-dependent cytotoxicity in the E47 cells. The agents used to promote CYP2E1 -dependent toxicity were a polyunsaturated fatty acid, arachidonic acid (AA), buthionine sulfoximine (BSO), which depletes GSH, and CCl4, which is metabolized to the CCl3 radical. These three agents produced a decrease in E47 cell viability which was enhanced upon inhibition of autophagy by 3-methyladenine (3-MA) or Atg 7 siRNA. Toxicity was lowered by rapamycin which increased autophagy and was much lower to the C34 cells which do not express CYP2E1. Toxicity was mainly necrotic and was associated with an increase in reactive oxygen production and oxidative stress; 3-MA increased while rapamycin blunted the oxidative stress. The enhanced toxicity and ROS formation produced when autophagy was inhibited was prevented by the antioxidant N-Acetyl cysteine. AA, BSO and CCl4 produced mitochondrial dysfunction, lowered cellular ATP levels and elevated mitochondrial production of ROS. This mitochondrial dysfunction was enhanced by inhibition of autophagy with 3-MA but decreased when autophagy was increased by rapamycin. The mitogen activated protein kinases p38 MAPK and JNK were activated by AA especially when autophagy was inhibited and chemical inhibitors of p38 MAPK and JNK lowered the elevated toxicity of AA produced by 3-MA. These results show that autophagy was protective against the toxicity produced by several agents known to be activated by CYP2E1. Since CYP2E1 plays an important role in the toxicity of ethanol, drugs and carcinogens and is activated under various pathophysiological conditions such as diabetes, NASH and obesity, attempts to stimulate autophagy may be beneficial in preventing/lowering CYP2E1/ethanol liver injury.

Topics: Adenine; Arachidonic Acid; Autophagy; Autophagy-Related Protein 7; Buthionine Sulfoximine; Carbon Tetrachloride; Cytochrome P-450 CYP2E1; Hep G2 Cells; Humans; MAP Kinase Signaling System; Mitochondria; Oxidative Stress; Sirolimus; Ubiquitin-Activating Enzymes

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemia-reperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line, Tumor; Cell Survival; Mechanistic Target of Rapamycin Complex 1; Mice; Mitochondria; Multiprotein Complexes; Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Reperfusion Injury; Sirolimus; TOR Serine-Threonine Kinases

Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, can elicit anti-tumor effects in various malignancies. Here, we sought to clarify the role of autophagy in celecoxib-induced cytotoxicity in human urothelial carcinoma (UC) cells. The results shows celecoxib induced cellular stress response such as endoplasmic reticulum (ER) stress, phosopho-SAPK/JNK, and phosopho-c-Jun as well as autophagosome formation in UC cells. Inhibition of autophagy by 3-methyladenine (3-MA), bafilomycin A1 or ATG7 knockdown potentiated celecoxib-induced apoptosis. Up-regulation of autophagy by rapamycin or GFP-LC3B-transfection alleviated celecoxib-induced cytotoxicity in UC cells. Taken together, the inhibition of autophagy enhances therapeutic efficacy of celecoxib in UC cells, suggesting a novel therapeutic strategy against UC.

Topics: Adenine; Antineoplastic Agents; Autophagy; Autophagy-Related Protein 7; Carcinoma, Transitional Cell; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Macrolides; MAP Kinase Kinase 4; Phosphoproteins; Pyrazoles; RNA, Small Interfering; Signal Transduction; Sirolimus; Sulfonamides; Ubiquitin-Activating Enzymes; Urinary Bladder Neoplasms

Autophagy is an evolutionarily conserved lysosomal degradation pathway and plays a critical role in the homeostatic process of recycling proteins and organelles. Functional relationships have been described between apoptosis and autophagy. Perturbations in the apoptotic machinery have been reported to induce autophagic cell deaths. Inhibition of autophagy in cancer cells has resulted in cell deaths that manifested hallmarks of apoptosis. However, the molecular relationships and the circumstances of which molecular pathways dictate the choice between apoptosis and autophagy are currently unknown. This study aims to identify specific gene expression of rapamycin-induced autophagy and the effects of rapamycin when the autophagy process is inhibited. In this study, we have demonstrated that rapamycin is capable of inducing autophagy in T-47D breast carcinoma cells. However, when the autophagy process was inhibited by 3-MA, the effects of rapamycin became apoptotic. The Phlda1 gene was found to be up-regulated in both autophagy and apoptosis and silencing this gene was found to reduce both activities, strongly suggests that Phlda1 mediates and positively regulates both autophagy and apoptosis pathways.

Topics: Adenine; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Humans; Oligonucleotide Array Sequence Analysis; Sirolimus; Transcription Factors; Up-Regulation

Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Apoptosis; Autophagy; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Survival; Chlorpyrifos; Humans; Insecticides; Microtubule-Associated Proteins; Neurotoxicity Syndromes; Proto-Oncogene Proteins c-bcl-2; Sequestosome-1 Protein; Sirolimus; Up-Regulation

Autophagy is a cellular pathway involved in protein and organelle degradation. It is relevant to many types of cellular homeostasis and human diseases. High level of glucose is known to inflict podocyte injury, but little is reported about the relationship between high concentrations of glucose and autophagy in these cells. The present study demonstrates that high glucose promotes autophagy in podocytes. Rapamycin further enhances this effect, but 3-methyadenine inhibits it. The proautophagic effect of high glucose manifested in the form of enhanced podocyte expression of LC3-2 and beclin-1; interestingly, antioxidants such as NAC were found to inhibit high glucose-induced autophagy. High glucose induced the generation of ROS by podocytes in a time-dependent manner. High glucose also enhanced podocyte expression of MnSOD and catalase. These findings indicate that high glucose-induced autophagy is mediated through podocyte ROS generation.

Topics: Acetylcysteine; Adenine; Animals; Antioxidants; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blood Glucose; Catalase; Culture Media; Diabetes Mellitus, Experimental; Glucose; Male; Microscopy, Electron; Microtubule-Associated Proteins; Podocytes; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sirolimus; Streptozocin; Superoxide Dismutase; Tumor Cells, Cultured

Autophagy is a lysosomal degradation process of redundant or faulty cell components in normal cells. However, certain diseases are associated with dysfunctional autophagy. Rapamycin, a major immunosuppressant used in islet transplantation, is an inhibitor of mammalian target of rapamycin and is known to cause induction of autophagy. The objective of this study was to evaluate the in vitro and in vivo effects of rapamycin on pancreatic β cells. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β cells of green fluorescent protein-microtubule-associated protein 1 light chain 3 transgenic mice. Rapamycin reduced the viability of isolated β cells and down-regulated their insulin function, both in vitro and in vivo. In addition, rapamycin increased the percentages of apoptotic β cells and dead cells in both isolated and in vivo intact islets. Treatment with 3-methyladenine, an inhibitor of autophagy, abrogated the effects of rapamycin and restored β-cell function in both in vitro experiments and animal experiments. We conclude that rapamycin-induced islet dysfunction is mediated through upregulation of autophagy, with associated downregulation of insulin production and apoptosis of β cells. The results also showed that the use of an autophagy inhibitor abrogated these effects and promoted islet function and survival. The study findings suggest that targeting the autophagy pathway could be beneficial in promoting islet graft survival after transplantation.

Topics: Adenine; Animals; Autophagy; Cells, Cultured; Green Fluorescent Proteins; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Mice; Mice, Transgenic; Microscopy, Fluorescence; Sirolimus; Up-Regulation

Rapamycin, a lipophilic macrolide antibiotic, has been found to reduce injury in different models of neurodegenerative disorders. We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) the neuroprotective effect of rapamycin was associated with increased autophagy and decreased caspase-3 activation. We show here that the strong reduction of caspase-3 activation after rapamycin was due, at least in part, to its effect on the intrinsic apoptotic mitochondrial pathway because after rapamycin treatment there was a marked reduction of Bax and Bad translocation to mitochondria, cytochrome c release, and caspase-3 activation. Poly (ADP-ribose) polymerase 1 (PARP-1) cleavage and the number of terminal dUDP nick-end labeling (TUNEL)-positive cells were also reduced. To assess how the antiapoptotic effect of rapamycin was linked to the strong autophagy signal induced by the drug, we blocked the formation of autophagosomes with 3-methyladenine (3MA). 3MA administered 10 min after rapamycin, elicited again Bax and Bad translocation to the mitochondria but did not cause cytochrome c release and caspase-3 activation. After 3MA treatment, cells underwent necrotic cell death. These data indicate that rapamycin administered before HI prevents the apoptotic signaling taking place through the mitochondrial pathway. We hypothesize that rapamycin confers a preconditioning-like protection and suggest that caution is necessary before using pharmacological agents targeting autophagy in neuroprotection because they could interfere with endogenous protective mechanisms.

Topics: Adenine; Animals; Autophagy; bcl-2-Associated X Protein; Caspase 3; Cell Death; Mitochondria; Necrosis; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus

Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.

Topics: Adenine; Amino Acids; Animals; Autophagy; Cell Survival; Chlorocebus aethiops; Culture Media; Energy Metabolism; Host-Parasite Interactions; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Prokaryotic Initiation Factor-2; Protein Biosynthesis; Signal Transduction; Sirolimus; Stress, Physiological; Toxoplasma; Vero Cells

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 12; Caspase 3; Cells, Cultured; Cerebral Cortex; Cinnamates; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Mice; Neurons; Oxygen; Rats; Rats, Sprague-Dawley; Sirolimus; Thiourea; Transcription Factor CHOP

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Earlier ultrastructural studies indicated the formation of autophagic vacuoles in neurons after TMT intoxication. However, no evaluation has been attempted to determine the role of the autophagic pathway in TMT neurotoxicity. To assess the contribution of autophagy to TMT-induced neuronal cell death, we checked the vulnerability of neuronal cultures to TMT after activation or inhibition of autophagy. Our results show that autophagy inhibitors (3-methyladenine and L-asparagine) greatly enhanced TMT neurotoxicity. Conversely, known activators of autophagy, such as lithium and rapamycin, displayed neuroprotection against this toxic compound. Due to its diverse targets, the action of lithium was complex. When lithium was administered according to a chronic treatment protocol (6 days pretreatment) it was able to rescue both hippocampal and cortical neurons from TMT (or from glutamate toxicity used as reference). This effect was accompanied by an increased phosphorylation of glycogen synthase kinase 3 which is a known target for lithium neuroprotection. If the pre-incubation time was reduced to 2 h (acute treatment protocol), lithium was still able to counteract TMT toxicity in hippocampal but not in cortical neurons. The neuroprotective effect of lithium acutely administered against TMT in hippocampal neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. These data indicate that TMT neurotoxicity can be dramatically modified, at least in vitro, by lithium addition which seems to act through different mechanisms if acutely or chronically administered.

Topics: Adenine; Adjuvants, Immunologic; Aldehydes; Analysis of Variance; Animals; Asparagine; Autophagy; Brain; Cell Count; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; L-Lactate Dehydrogenase; Lithium Chloride; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Mitochondria; Neurons; Phosphorylation; Serine; Sirolimus; Tetrazolium Salts; Thiazoles; Trimethyltin Compounds; Vacuoles

Hyperglycemia is linked to increased heart failure among diabetic patients. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Autophagy is a cellular degradation pathway that plays important roles in cellular homeostasis. Autophagic activity is altered in the diabetic heart, but its functional role has been unclear. In this study, we determined if mimicking hyperglycemia in cultured cardiomyocytes from neonatal rats and adult mice could affect autophagic activity and myocyte viability. High glucose (17 or 30 mM) reduced autophagic flux compared with normal glucose (5.5 mM) as indicated by the difference in protein levels of LC3-II (microtubule-associated protein 1 light chain 3 form II) or the changes of punctate fluorescence patterns of GFP-LC3 and mRFP-LC3 in the absence and presence of the lysosomal inhibitor bafilomycin A(1). Unexpectedly, the inhibited autophagy turned out to be an adaptive response that functioned to limit high glucose cardiotoxicity. Indeed, suppression of autophagy by 3-methyladenine or short hairpin RNA-mediated silencing of the Becn1 or Atg7 gene attenuated high glucose-induced cardiomyocyte death. Conversely, upregulation of autophagy with rapamycin or overexpression of Becn1 or Atg7 predisposed cardiomyocytes to high glucose toxicity. Mechanistically, the high glucose-induced inhibition of autophagy was mediated at least partly by increased mTOR signaling that likely inactivated ULK1 through phosphorylation at serine 467. Together, these findings demonstrate that high glucose inhibits autophagy, which is a beneficial adaptive response that protects cardiomyocytes against high glucose toxicity. Future studies are warranted to determine if autophagy plays a similar role in diabetic heart in vivo.

Topics: Adenine; Animals; Animals, Newborn; Autophagy; Autophagy-Related Protein 7; Cardiotonic Agents; Cell Death; Cell Survival; Cytoprotection; Ether-A-Go-Go Potassium Channels; Gene Knockdown Techniques; Glucose; Humans; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Myocytes, Cardiac; Phagosomes; Phosphorylation; Proteins; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitin-Activating Enzymes

Autophagy, the bulk intracellular degradation of cytoplasmic constituents, can be a pro-survival or a pro-death mechanism depending on the context. A recent study showed that autophagy was activated in the phase of early brain injury following subarachnoid hemorrhage (SAH). However, whether autophagy activation after SAH is protective or harmful is still elusive. This study was undertaken to determine the potential role of autophagy pathway activation in early brain injury following SAH. The rats were pretreated with intracerebral ventricular infusion of either the autophagy inducer rapamycin (RAP) or inhibitor 3-methyladenine (3-MA) before SAH onset. The results from electron microscopic examinations showed that RAP administration caused the formation of autophagosomal vacuoles, and 3-MA induced neuronal apoptosis. RAP treatment significantly increased the expression of autophagic proteins Atg5 and Beclin 1, the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I and reduced caspase-3 activity, the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells, brain edema and neurological deficits after SAH. Conversely, 3-MA treatment exacerbated early brain injury. RAP treatment significantly increased the expression of the autophagic proteins Atg5 and Beclin 1, the ratio of LC3-II to LC3-I and reduced caspase-3 activity, the number of TUNEL-positive cells, brain edema and neurological deficits after SAH. Conversely, 3-MA treatment reversed these changes and exacerbated early brain injury. To further clarify the mechanism of autophagy protection, we investigated the expression levels of key apoptosis-related molecules. The results showed that RAP administration decreased Bax translocation to the mitochondria and downstream cytochrome c release from the mitochondria to the cytosol. Taken together, our study indicates that activation of autophagic pathways reduces early brain injury after SAH. This neuroprotective effect is likely exerted by anti-apoptotic mechanisms.

Topics: Adenine; Animals; Apoptosis; Autophagy; Blotting, Western; Disease Models, Animal; In Situ Nick-End Labeling; Male; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Sirolimus; Subarachnoid Hemorrhage

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.

Topics: Adenine; Animals; Autophagy; Cell Line; Cell Survival; Endocytosis; Gene Knockdown Techniques; Lysosomes; Membrane Fusion; Microtubule-Associated Proteins; Phagosomes; Porcine Reproductive and Respiratory Syndrome; Porcine respiratory and reproductive syndrome virus; RNA Interference; Sirolimus; Swine; Viral Nonstructural Proteins; Virus Replication

Coxsackievirus B3 (CVB3) has previously been shown to utilize autophagy in an advantageous manner during the course of infection of the host cell. However, few studies have determined whether stem cells induce autophagy in a similar fashion, and whether virus-induced autophagy occurs following infection of stem cells. Therefore, we compared the induction of autophagy following CVB3 infection of neural progenitor and stem cells (NPSCs), which we have recently shown to be highly susceptible to CVB3 infection, to HL-1 cells, a transformed cardiomyocyte cell line. As previously demonstrated for other susceptible host cells, HL-1 cells showed an increase in the activity of autophagic signaling following infection with a CVB3 expressing dsRed protein (dsRed-CVB3). Furthermore, viral titers in HL-1 cells increased in the presence of an inducer of autophagy (CCPA), while viral titers decreased in the presence of an inhibitor of autophagy (3-MA). In contrast, no change in autophagic signaling was seen in NPSCs following infection with dsRed-CVB3. Also, basal levels of autophagy in NPSCs were found to be highly elevated in comparison to HL-1 cells. Autophagy could be induced in NPSCs in the presence of rapamycin without altering levels of dsRed-CVB3 replication. In differentiated NPSC precursors, autophagy was activated during the differentiation process, and a decrease in autophagic signaling was observed within all three CNS lineages following dsRed-CVB3 infection. Hence, we conclude that the role of autophagy in modulating CVB3 replication appears cell type-specific, and stem cells may uniquely regulate autophagy in response to infection.

Topics: Adenine; Adenosine; Animals; Autophagy; Cell Differentiation; Coxsackievirus Infections; Enterovirus B, Human; Fibroblast Growth Factors; Green Fluorescent Proteins; HeLa Cells; Humans; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Neural Stem Cells; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; Transduction, Genetic; Viral Load; Viral Proteins; Virus Replication

Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Blastocyst; Cell Cycle Proteins; Ectogenesis; Female; Gene Expression Regulation, Developmental; In Vitro Oocyte Maturation Techniques; Meiosis; Microtubule-Associated Proteins; Oocytes; Parthenogenesis; RNA Stability; RNA, Messenger, Stored; Sirolimus; Sus scrofa; Transcription Factors

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp ( 0/0) ) hippocampal neuronal cells in comparison to cellular prion protein (PrP (C) )-expressing (Prnp (+/+) ) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP (C) is associated with autophagy using Prnp ( 0/0) hippocampal neuronal cells under hydrogen peroxide (H 2O 2)-induced oxidative stress. We found that Prnp ( 0/0) cells were more susceptible to oxidative stress than Prnp (+/+) cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H 2O 2-treated Prnp ( 0/0) cells compared with H 2O 2-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H 2O 2-treated Prnp ( 0/0) cells, while the H 2O 2-treated Prnp (+/+) cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H 2O 2. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp (+/+) cells contributes to the prosurvival effect of autophagy against H 2O 2 cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP (C) may impair autophagic flux via H 2O 2-induced oxidative stress.

Topics: Adenine; Animals; Apoptosis; Autophagy; Caspases; Enzyme Activation; Gene Knockdown Techniques; Hippocampus; Hydrogen Peroxide; Mice; Models, Biological; Neurons; Oxidative Stress; Prions; Protective Agents; Sirolimus; Time Factors; Trehalose

TNF-alpha levels in prostate cancer correlate with the extent of disease and are significantly elevated in the metastatic stage. TNF receptor superfamily controls two distinct signalling cascades, leading to opposite effects, i.e. apoptosis and survival; in prostate cancer TNF-alpha-mediated signalling induces cell survival and resistance to therapy. The apoptosis of prostate epithelial cancer cells LNCaP and PC3 was investigated upon treatment with the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin, in combination with TNF-alpha. Cells were exposed to these molecules for 18, 24 and 48 h. Autophagy was assessed via LC3 Western blot analysis; propidium iodide and TUNEL stainings followed by flow cytometry or caspase-8 and caspase-3 activation assays were performed to evaluate apoptosis. TNF-alpha-induced apoptosis was potentiated by 3-methyladenine in the androgen-responsive LNCaP cells, whereas no effect was observed in the androgen-insensitive PC3 cells. Interestingly such pro-apoptosis effect in LNCaP cells was associated with reduced c-Flip levels through proteasomal degradation via increased reactive oxygen species production and p38 activation; such c-Flip reduction was reversed in the presence of either the proteasome inhibitor MG132 or the reactive oxygen species scavenger N-acetyl-cysteine. Conversely in PC3 but not in LNCaP cells, rapamycin stimulated TNF-alpha-dependent apoptosis; such effect was associated with reduced c-Flip promoter activity and FoxO3a activation. We conclude that TNF-alpha-induced apoptosis may be potentiated, in prostate cancer epithelial cells, through autophagy modulators. Increased sensitivity to TNF-alpha-dependent apoptosis correlates with reduced c-Flip levels which are consequent to a post-transcriptional and a transcriptional mechanism in LNCaP and PC3 cells respectively.

Topics: Adenine; Apoptosis; Autophagy; Blotting, Western; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Drug Screening Assays, Antitumor; Enzyme Activation; Epithelial Cells; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Male; Microtubule-Associated Proteins; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Sirolimus; Transcription, Genetic; Tumor Necrosis Factor-alpha

Autophagy is the primary mechanism of degradation of cellular proteins and at least two functions can be attributed to this biological phenomenon: increased nutrient supply via recycling of the products of autophagy under nutrient starvation; and antimicrobial response involved in the innate immune system. Many microorganisms induce host cell autophagy and it has been proposed as a pathway by which parasites compete with the host cell for limited resources. In this report we provide evidence that the intracellular parasite Leishmania amazonensis induces autophagy in macrophages. Using western blotting, the LC3II protein, a marker of autophagosomes, was detected in cell cultures with a high infection index. Macrophages infected with L. amazonensis were examined by transmission electronic microscopy, which revealed enlarged myelin-like structures typical late autophagosome and autolysosome. Other evidence indicating autophagy was Lysotracker red dye uptake by the macrophages. Autophagy also occurs in the leishmaniasis skin lesions of BALB/c mice, detected by immunohistochemistry with anti-LC3II antibody. In this study, autophagy inhibitor 3-methyladenine (3MA) reduced the infection index, while autophagy inductors, such as rapamycin or starvation, did not alter the infection index in cultivated macrophages, suggesting that one aspect of the role of autophagy could be the provision of nutritive support to the parasite.

Topics: Adenine; Amines; Animals; Autophagy; Blotting, Western; Bone Marrow Cells; Cells, Cultured; Female; Leishmania mexicana; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microtubule-Associated Proteins; Sirolimus; Staining and Labeling

Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.. A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.. The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain; Brain Ischemia; Immunosuppressive Agents; Ischemic Postconditioning; Male; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Sirolimus; Up-Regulation

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cnidaria; In Situ Nick-End Labeling; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Models, Biological; Necrosis; Sirolimus

Mammalian target of rapamycin (mTOR) is involved in a caspase independent form of programmed cell death called autophagy. The aim of this research was to investigate the effects of rapamycin and 3-methyladenine (3-MA) on autophagy, proliferation, apoptosis, and cell-cycle parameters of rat bone marrow-derived endothelial progenitor cells (EPCs).. Mononuclear cells isolated from rat bone marrow were treated with rapamycin (0.01, 0.1, 1, or 10 µg/L) or 3-MA (1.25, 2.5, 5, or 10 mmol/L) for 24 hours. Expression of the autophagy marker protein LC3-II was analyzed by Western blotting. Apoptosis and cell-cycle progression were analyzed by flow cytometry. Cell proliferation was measured using the MTT assay.. Rapamycin treatment of EPCs induced apoptosis and autophagy and inhibited proliferation and cell-cycle progression in a dose-dependent manner. Treatment with 5 mmol/L 3-MA promoted cell proliferation; in contrast, treatment with 10 mmol/L 3-MA promoted apoptosis and induced S-phase arrest.. Rapamycin treatment of EPCs induced apoptosis and autophagy. Low concentrations of 3-MA had no significant effect on the proliferation and apoptosis of EPCs; The 5 mmol/L group promoted cell proliferation, but had no effect on the apoptosis; the 10 mmol/L group inhibited the proliferation and promoted apoptosis through the cell cycle.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Cycle; Cell Proliferation; Cells, Cultured; Rats; Sirolimus

To investigate the influence of autophagy on the survival and proliferation of multiple myeloma (MM) cells.. Multiple myeloma (MM) cell line U266 cell autophagy was induced by serum-free culture condition, and adding rapamycin or 3-MA respectively. The cells proliferation was observed. U266 cells, lymphoma cell Jurket under normal culture condition, and serum-free cultured Jurket cell were used as control group. The proliferation and apoptosis of cells were determined by CCK8 and flow cytometry, respectively. MDC staining were employed to detect the autophagy. The mRNA expression of Mtor and Beclin1 gene of U266 cells were assayed by RT-PCR. Protein LC3I/LCII and LAMP1 was analyzed by western blot.. There was low level of autophagy in U266 cells, sera starvation increased the level of autophagy. Rapamycin upregulated autophagy of the U266 cells and stimulated their proliferation. But the autophagy level of sera starvation and rapamycin group declined when culture for 96h.3-MA had the same effects on U266 cells, although it was on 24 h. But rapamycin and 3-MA could inhibit cell proliferation under normal culture condition. Compared with normal culture condition, apoptosis of U266 cells increased significantly after 24h incubation in medium without sera \\[(1.33 ± 0.09)% and (17.90 ± 1.46)%, respectively\\] (P < 0.01). Rapamycin and 3-MA could inhibit the serum-free induced apoptosis \\[(6.23 ± 0.12)% and (6.97 ± 0.03)%, respectively\\](P < 0.01), but cell apoptosis was at the same level after 72 hour incubation \\[(30.37 ± 0.27)%, (30.13 ± 1.93)% and (28.57 ± 2.83)%, respectively\\] (P > 0.05). However, apoptosis of U266 cells decreased to 18.7% and 12.6% after removal of rapamycin and 3-MA.. There is basically level of autophagy in MM cells which is higher than those in the Jurkat cells. Both Rapamycin and 3-MA can inhibit the cells proliferation under normal culture condition. Up-regulated autophagy promotes survival and proliferation of MM cells under sera deletion. Rapamycin strengthens this effect with limited duration. 3-MA has dual effects on cell autophagy.

Topics: Adenine; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Humans; Multiple Myeloma; Sirolimus

Autophagy is a survival pathway required for cellular viability during starvation through catabolic self-digestion of damaged proteins and organelles; however, autophagy may result in cell death if it proceeds to completion. Although the exact mechanism of this process is not clear, it seems that proper regulation of autophagy can potentially contribute to the therapeutics of cancers. This study was designed to examine the role of autophagy in the death of human acute promyelocytic leukemia NB4 cells initiated by arsenic trioxide. Furthermore, the effects of autophagy inhibition and augmentation on cell viability were also compared. Our data suggested that both augmentation and suppression of autophagy could enhance the treatment effects while the latter was preferable. This study indicated that autophagy regulation augmented the treatment effects initiated by arsenic trioxide in NB4 cells, and that the selection of regulator should be precisely considered.

Topics: Adenine; Antibiotics, Antineoplastic; Apoptosis; Arsenic Trioxide; Arsenicals; Autophagy; Cell Line, Tumor; Cell Survival; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Oxides; Sirolimus

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.

Topics: Adenine; Animals; Apoptosis; Autophagy; Blotting, Western; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; MAP Kinase Kinase 1; NF-kappa B; Nitroprusside; Oncogene Protein p21(ras); PC12 Cells; Phosphatidylinositol 3-Kinases; raf Kinases; Rats; Reactive Oxygen Species; Silybin; Silymarin; Sirolimus

Autophagyis, the bulk degradation of proteins and organelles, is essential for cellular maintenance, cell viability, and development, and is often involved in type II programmed cell death in mammals. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on the in vitro development and apoptosis of mouse embryos. LC3, which is essential for the formation of autophagosomes, was widely expressed in mouse embryos, and high levels of transcript were present from 1 to 4 cells but gradually decreased through the morula and blastocyst stages. 3-MA-treated embryos exhibited significantly reduced developmental rates and total cell numbers, but increased rates of apoptosis. Furthermore, both the expression of Lc3, Gabarap, Atg4A, and Atg4B, and the synthesis of LC3 were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect developmental rates, cell numbers decreased, and the apoptosis rate increased. Expression of Lc3, Gabarap, Atg4A, and Atg4B, and synthesis of LC3 increased as well. Modulation of Lc3 mRNA and LC3 protein levels using 3-MA or rapamycin significantly increased apoptotic cell death through the disruption of mitochondrial morphology and reduction of mtDNA copy number at the blastocyst stage. Interestingly, the inner cell mass, detected by immunostaining with POU5F1 (OCT3/4) after 3-MA or rapamycin treatment of embryos, was significantly increased compared to controls. These results suggest that autophagy influences developmental patterning and apoptosis, and may play a role in early mouse embryogenesis.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Blastocyst; Cell Nucleus; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Histocytochemistry; Male; Mice; Mice, Inbred ICR; Microscopy, Confocal; Mitochondria; Morula; Octamer Transcription Factor-3; Sirolimus

Chikungunya Virus (ChikV) surprised by a massive re-emerging outbreak in Indian Ocean in 2006, reaching Europe in 2007 and exhibited exceptional severe physiopathology in infants and elderly patients. In this context, it is important to analyze the innate immune host responses triggered against ChikV. Autophagy has been shown to be an important component of the innate immune response and is involved in host defense elimination of different pathogens. However, the autophagic process was recently observed to be hijacked by virus for their own replication. Here we provide the first evidence that hallmarks of autophagy are specifically found in HEK.293 infected cells and are involved in ChikV replication.. To test the capacity of ChikV to mobilize the autophagic machinery, we performed fluorescence microscopy experiments on HEK.GFP.LC3 stable cells, and followed the LC3 distribution during the time course of ChikV infection. To confirm this, we performed electron microscopy on HEK.293 infected cells. To test the effect of ChikV-induced-autophagy on viral replication, we blocked the autophagic process, either by pharmacological (3-MA) or genetic inhibition (siRNA against the transcript of Beclin 1, an autophagic protein), and analyzed the percentage of infected cells and the viral RNA load released in the supernatant. Moreover, the effect of induction of autophagy by Rapamycin on viral replication was tested.. The increasing number of GFP-LC3 positive cells with a punctate staining together with the enhanced number of GFP-LC3 dots per cell showed that ChikV triggered an autophagic process in HEK.293 infected cells. Those results were confirmed by electron microscopy analysis since numerous membrane-bound vacuoles characteristic of autophagosomes were observed in infected cells. Moreover, we found that inhibition of autophagy, either by biochemical reagent and RNA interference, dramatically decreases ChikV replication.. Taken together, our results suggest that autophagy may play a promoting role in ChikV replication. Investigating in details the relationship between autophagy and viral replication will greatly improve our knowledge of the pathogenesis of ChikV and provide insight for the design of candidate antiviral therapeutics.

Topics: Adenine; Alphavirus Infections; Antimetabolites; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Chikungunya Fever; Chikungunya virus; Disease Outbreaks; Europe; Gene Silencing; HEK293 Cells; Host-Pathogen Interactions; Humans; Immunosuppressive Agents; Indian Ocean; Membrane Proteins; Microscopy, Electron; Microscopy, Fluorescence; Phagosomes; Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Sirolimus; Virus Replication

Glioma stem/progenitor cells (GSPCs) are considered to be responsible for the initiation, propagation, and recurrence of gliomas. The factors determining their differentiation remain poorly defined. Accumulating evidences indicate that alterations in autophagy may influence cell fate during mammalian development and differentiation. Here, we investigated the role of autophagy in GSPC differentiation. SU-2 cells were treated with rapamycin, 3-methyladenine (3-MA) plus rapamycin, E64d plus rapamycin, or untreated as control. SU-2 cell xenografts in nude mice were treated with rapamycin or 3-MA plus rapamycin, or untreated as control. Western blotting and immunocytochemistry showed up-regulation of microtubule-associated protein light chain-3 (LC3)-II in rapamycin-treated cells. The neurosphere formation rate and the number of cells in each neurosphere were significantly lower in the rapamycin treatment group than in other groups. Real-time PCR and immunocytochemistry showed down-regulation of stem/progenitor cell markers and up-regulation of differentiation markers in rapamycin-treated cells. Transmission electron microscopy revealed autophagy activation in rapamycin-treated tumor cells in mice. Immunohistochemistry revealed decreased Nestin-positive cells and increased GFAP-positive cells in rapamycin-treated tumor sections. These results indicate that rapamycin induces differentiation of GSPCs by activating autophagy.

Topics: Adenine; Animals; Antibiotics, Antineoplastic; Autophagy; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Female; Glial Fibrillary Acidic Protein; Glioma; Humans; Leucine; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microtubule-Associated Proteins; Neoplastic Stem Cells; RNA, Messenger; Sirolimus; Xenograft Model Antitumor Assays

Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure. The transcription factor GATA4 antagonizes DOX-induced cardiotoxicity. However, the protective mechanism remains obscure. Autophagy is the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles, and its activation could be either protective or detrimental depending on specific pathophysiological conditions. Here we investigated the ability of GATA4 to inhibit autophagy as a potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat cardiomyocytes. DOX markedly increased autophagic flux in cardiomyocytes as indicated by the difference in protein levels of LC3-II (microtubule-associated protein light chain 3 form 2) or numbers of autophagic vacuoles in the absence and presence of the lysosomal inhibitor bafilomycin A1. DOX-induced cardiomyocyte death determined by multiple assays was aggravated by a drug or genetic approach that activates autophagy, but it was attenuated by manipulations that inhibit autophagy, suggesting that autophagy contributes to DOX cardiotoxicity. DOX treatment depleted GATA4 protein levels, which predisposed cardiomyocytes to DOX toxicity. Indeed, GATA4 gene silencing triggered autophagy that rendered DOX more toxic, whereas GATA4 overexpression inhibited DOX-induced autophagy, reducing cardiomyocyte death. Mechanistically, GATA4 up-regulated gene expression of the survival factor Bcl2 and suppressed DOX-induced activation of autophagy-related genes, which may likely be responsible for the anti-apoptotic and anti-autophagic effects of GATA4. Together, these findings suggest that activation of autophagy mediates DOX cardiotoxicity, and preservation of GATA4 attenuates DOX cardiotoxicity by inhibiting autophagy through modulation of the expression of Bcl2 and autophagy-related genes.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Doxorubicin; GATA4 Transcription Factor; Gene Expression Regulation; Gene Knockdown Techniques; Microtubule-Associated Proteins; Myocytes, Cardiac; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sirolimus

Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.

Topics: Adenine; Androstadienes; Autophagy; Blastocystis; Microscopy, Confocal; Microscopy, Electron, Transmission; Sirolimus; Vacuoles; Wortmannin

Despite of similarities between glioma stem/progenitor cells (GSPCs) and neural stem/progenitor cells (NSPCs), inhibition of differentiation is a distinct characteristic of GSPCs. In this study, we investigated the effects of autophagy impairment on inhibition of differentiation of GSPC, and its molecular mechanism. GSPCs were kept by our laboratory; NSPCs were isolated from human fetal brain tissue. We found that the autophagic activity in GSPCs was significantly lower than that in NSPCs. However, the autophagic activity markedly increased after GSPCs were induced to differentiate by fetal calf serum (FCS). The autophagy inhibitors 3-methyladenine and Bafilomycin A1 (BFA) inhibited the FSC-induced differentiation of GSPCs. And autophagy activator Rapamycin could promote differentiation of GSPCs. In order to disclose whether the loss of PTEN in GSPC is related to the deficiency of autophagic activity in GSPCs (for PTEN being lost in the GSPCs studied by us), we introduced the wild type gene of PTEN into GSPCs, and found that the autophagic activity was restored significantly after the gene transduction. The low autophagic activity in GSPCs leads to the inhibition of differentiation of GSPCs, and the loss of PTEN in GSPCs probably is an underlying mechanism for the low autophagic activity in GSPCs. These results suggest that bust autophagic activity target at PTEN might be a potential therapy target for glioma therapy.

Topics: Adenine; Autophagy; Blotting, Western; Cadaverine; Cell Differentiation; Cells, Cultured; Central Nervous System Agents; Glioma; Humans; Immunohistochemistry; Macrolides; Microscopy, Electron, Transmission; Neurons; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Stem Cells; Transduction, Genetic

The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.

Topics: Adenine; Autophagy; Cadaverine; Chromones; Coloring Agents; Escherichia coli; Guanosine; Humans; Hydrogen-Ion Concentration; Inflammation; Microscopy, Confocal; Morpholines; Neutrophils; Phagosomes; Poly I-C; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Small Ubiquitin-Related Modifier Proteins; Tetradecanoylphorbol Acetate; Toll-Like Receptors; Transcription, Genetic; Vacuoles

Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPC's neuroprotective effects can be mimicked by autophagy inducers.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain Ischemia; Disease Models, Animal; Down-Regulation; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Microtubule-Associated Proteins; Neostriatum; Neurogenesis; Neurons; Neuroprotective Agents; Phagosomes; Rats; Rats, Sprague-Dawley; Sequestosome-1 Protein; Sirolimus; Up-Regulation

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Differentiation; Cell Polarity; Drug Resistance; Gene Knockdown Techniques; Graft vs Host Disease; Humans; Immunologic Memory; Interferon-alpha; Interleukin-12; Lipopolysaccharides; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mice, Transgenic; Phenotype; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sirolimus; STAT Transcription Factors; T-Lymphocytes, Cytotoxic; Th1 Cells; Tumor Necrosis Factor-alpha

We reported that dihydrocapsaicin (DHC) induces autophagy in a catalase-regulated manner. In this study, we further examined the role of DHC-induced autophagy in lung cell lines. DHC-induced cytotoxicity was higher in WI38 and H1299 cells than in H460 and A549 cells, and was related to the loss of cell membrane integrity. However, apoptotic cells markedly increased in H460 and A549 cells. In WI38 and H1299 cells, DHC-induced catalase was correlated with a decrease of intracellular reactive oxygen species (ROS) and an increase in the level of LC3II, an autophagy marker, and LC3 conversion was attenuated by the catalase inhibitor 3-amino-1,2,4-triazole (3AT) or by knockdown of the catalase gene. In A549 cells, DHC downregulated catalase, led to ROS accumulation, and blocked LC3 conversion. In H460 cells expressing limited amount of catalase, DHC caused ROS accumulation and blocked LC3 conversion. However, H460 cells overexpressing catalase were able to induce autophagy. In contrast to Earle's balanced salt solution and rotenone, H(2)O(2) treatment caused ROS accumulation and did not promote upregulation of catalase and LC3II in lung cell lines. Cytoplasmic vacuolization in WI38 and H1299 cells was blocked by treatment of 3AT and which enhanced caspase-3 activity and LDH release. Suppression of autophagy by 3-methyladenine also enhanced DHC-induced cell death through apoptotic and necrotic cell death. In A549 and H460 cells, treatment of rapamycin attenuated DHC-induced cell death. Collectively, these results suggest that catalase regulates autophagy, which helps protect cells against apoptotic and necrotic cell death.

Topics: Adenine; Amitrole; Apoptosis; Autophagy; Capsaicin; Catalase; Cell Line, Tumor; Enzyme Activation; Epithelial Cells; Humans; Lung; Microtubule-Associated Proteins; Reactive Oxygen Species; RNA, Small Interfering; Sirolimus

Helicobacter pylori has developed several mechanisms to evade the intracellular killing after phagocytosis. In this study, we reported that some Taiwanese clinical isolated H. pylori can multiply in human monocytic cells, such as THP-1 or U937 cells, but not in murine macrophage Raw264.7 cells. After internalization, there was a 5- to 10-fold increment of re-cultivable H. pylori from the infected THP-1 cells at 12 hrs post infection. The dividing H. pylori was found in a double-layer vesicle, which is characteristic of autophagosome. The formation of autophagosomes is associated with the multiplication of H. pylori in THP-1 cells. Its modulation with rapamycin or 3-MA affects the level of H. pylori replication. Furthermore, the VacA or CagA mutants of H. pylori have lower levels of multiplication in macrophages. We conclude that H. pylori infection induces autophagosome formation, and these autophagic vesicles were adapted for the multiplication of H. pylori in the host.

Topics: Adenine; Animals; Autophagy; Bacterial Proteins; Cell Line; Cell Proliferation; Helicobacter Infections; Helicobacter pylori; Humans; Macrophages; Mice; Microbial Viability; Microtubule-Associated Proteins; Monocytes; Mutation; Sirolimus; Vacuoles

Autophagy is a process to engulf aberrant organelles or protein aggregates into double-membrane vesicles for lysosomal breakdown. Autophagy is a protective process against some intracellular bacteria and viruses; however, it is also used for replication by some viruses, such as poliovirus. We recently found that coxsackievirus B4 (CVB4) also induces the autophagy pathway and activates the calpain system for replication in neurons. Notably, the inhibition of autophagy with 3-methyladenine (3MA) reduced calpain activation and virus replication. Calpain inhibitors also reduced autophagosome formation and virus replication. This finding indicates that calpain and the autophagy pathway are closely connected with each other during the infection. Interestingly, we also found that 3MA and calpain inhibitors enhanced the caspase-3 specific cleavage of spectrin during CVB4 infection, suggesting that autophagy inhibition by these drugs triggered apoptosis. Thus, autophagy and apoptosis may balance each other in CVB4-infected neurons. Here, we show that inhibition of caspase with zVAD increased autophagosome formation, further proposing the cross-talk between autophagy and apoptosis in CVB4-infected neurons.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Autophagy; Calpain; Caspases; Coxsackievirus Infections; Cysteine Proteinase Inhibitors; Models, Biological; Neurons; Rats; Sirolimus; Virus Replication

Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress-inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib.

Topics: Adenine; Antifungal Agents; Antimalarials; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Boronic Acids; Bortezomib; Cell Proliferation; Chloroquine; Drug Therapy, Combination; Enzyme Inhibitors; Humans; Immunoblotting; Membrane Proteins; Microscopy, Fluorescence; Multiple Myeloma; Pyrazines; RNA, Small Interfering; Sirolimus; Thapsigargin; Tumor Cells, Cultured

Penta-1,2,3,4,6-O-galloyl-beta-d-glucose (PGG) suppresses the in vivo growth of human DU145 and PC-3 prostate cancer xenografts in nude mice, suggesting potential utility as a prostate cancer chemotherapeutic or chemopreventive agent. Our earlier work implicates caspase-mediated apoptosis in DU145 and LNCaP prostate cancer cells as one mechanism for the anticancer activity. We show here that, in the more aggressive PC-3 prostate cancer cell line, PGG induced programmed cell deaths lacking the typical caspase-mediated apoptotic morphology and biochemical changes. In contrast, PGG induced patent features of autophagy, including formation of autophagosomes and lipid modification of light chain 3 after 48 hours of PGG exposure. The "autophagic" responses were also observed in the murine TRAMP-C2 cells. Caspase inhibition exacerbated PGG-induced overall death. As for molecular changes, we observed a rapid inhibition of the phosphorylation of mammalian target of rapamycin-downstream targets S6K and 4EBP1 by PGG in PC-3 and TRAMP-C2 cells but not that of mammalian target of rapamycin itself, along with increased AKT phosphorylation. Whereas the inhibition of phosphatidylinositol 3-kinase increased PGG-induced apoptosis and autophagy, experiments with pharmacologic inducer or inhibitor of autophagy or by knocking down autophagy mediator Beclin-1 showed that autophagy provided survival signaling that suppressed caspase-mediated apoptosis. Knocking down of death receptor-interacting protein 1 kinase increased overall death without changing light chain 3-II or caspase activation, thus not supporting death receptor-interacting protein 1-necroptosis for PGG-induction of autophagy or other programmed cell death. Furthermore, PGG-treated PC-3 cells lost clonogenic ability. The induction by PGG of caspase-independent programmed cell death in aggressive prostate cancer cell lines supports testing its merit as a potential drug candidate for therapy of caspase-resistant recurrent prostate cancer.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Enzyme Activation; Gene Knockdown Techniques; GTPase-Activating Proteins; Humans; Hydrolyzable Tannins; Male; Membrane Proteins; Mice; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Protein Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Sirolimus; TOR Serine-Threonine Kinases; Tumor Stem Cell Assay

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.

Topics: Adenine; Autophagy; Cell Line, Tumor; Cell Membrane; Chloroquine; Culture Media; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hypoxia; Lysosomes; Macrolides; Neuropilin-1; Neuropilin-2; Sirolimus

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.

Topics: Adenine; Adenosine Triphosphate; Animals; Autophagy; Cell Shape; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Glucose; Leupeptins; Male; Microtubule-Associated Proteins; Myocytes, Cardiac; Necrosis; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Sirolimus; Time Factors; Vacuoles

Autophagy, an intracellular bulk degradation process of cellular constituents, plays a key role in cell homeostasis and can be induced by stresses, such as nutrient depletion, closed head injury or focal cerebral ischemia. This study focuses on the role of autophagy in neonatal hypoxia-ischemia (HI). Enhanced beclin 1 expression, a Bcl-2-interacting protein required for autophagy, has been used as a marker of autophagy. Beclin 1 was significantly increased at short times after HI, both in the hippocampus and in the cerebral cortex. Beclin 1-positive cells were found in the injured but not in the contralateral side and co-localized with MAP2 but not with GFAP or ED1, indicating that the protein is over-expressed in neurons. Beclin 1-positive cells were also TUNEL-positive. 3-Methyladenine and wortmannin, that inhibit autophagy, significantly reduced beclin 1 expression and switched the mechanism of the cell death mode from apoptosis to necrosis. Conversely, rapamycin, that increases autophagy, augmented beclin 1 expression, reduced necrotic cell death, and decreased brain injury. A prophylactic treatment with simvastatin or hypoxic preconditioning also increased beclin 1 expression. Taken together, these data indicate that autophagy is increased in neuronal cells after neonatal hypoxia-ischemia and suggest that over-activation of autophagic pathways represents a potential protective mechanism in the early stage of the brain injury.

Topics: Adenine; Analysis of Variance; Androstadienes; Animals; Animals, Newborn; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blotting, Western; Cell Count; Cerebral Cortex; Hippocampus; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Nick-End Labeling; Necrosis; Neurons; Rats; Rats, Sprague-Dawley; Simvastatin; Sirolimus; Wortmannin

Autophagy was induced in human neuroblastoma SH-SY5Y cells by two different procedures: deprivation of fetal serum in culture medium, or treatment with dopamine. 3-methyladenine prevented autophagy in the two procedures. Although it is usually considered that the conversion of soluble LC3-I to lipid bound LC3-II is associated with the formation of autophagosomes, the inhibition of autophagy with 3-methyladenine prevented this transformation in serum-deprived but not in dopamine-treated cells. While the PI3K-mTOR pathway was inhibited by serum deprivation, dopamine increased the phosphorylation of Akt but inhibited mTOR activity in a similar way to rapamycin. Dopamine and rapamycin increased LC3-II levels by a mechanism not prevented by 3-methyladenine. The activation of LC3-I to LC3-II may then be necessary but not sufficient to trigger cell autophagy. Thus, the increase in LC3-II, as the main biochemical parameter for autophagy at present, should be considered with caution.

Topics: Adenine; Autophagy; Blotting, Western; Dopamine; Humans; Microscopy, Electron; Microscopy, Fluorescence; Microtubule-Associated Proteins; Neuroblastoma; Phosphatidylinositol 3-Kinases; Protein Kinases; Serum; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligatory intracellular pathogen. After entry into host cells, the bacterium is diverted from the endosomal pathway and replicates in a membrane-bound compartment devoid of endosomal or lysosomal markers. Here, we show that several hallmarks of early autophagosomes can be identified in A. phagocytophilum replicative inclusions, including a double-lipid bilayer membrane and colocalization with GFP-tagged LC3 and Beclin 1, the human homologues of Saccharomyces cerevisiae autophagy-related proteins Atg8 and Atg6 respectively. While the membrane-associated form of LC3, LC3-II, increased during A. phagocytophilum infection, A. phagocytophilum-containing inclusions enveloped with punctate GFP-LC3 did not colocalize with a lysosomal marker. Stimulation of autophagy by rapamycin favoured A. phagocytophilum infection. Inhibition of the autophagosomal pathway by 3-methyladenine did not inhibit A. phagocytophilum internalization, but reversibly arrested its growth. Although autophagy is considered part of the innate immune system that clears a variety of intracellular pathogens, our study implies that A. phagocytophilum subverts this system to establish itself in an early autophagosome-like compartment segregated from lysosomes to facilitate its proliferation.

Topics: Adenine; Anaplasma phagocytophilum; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line; Haplorhini; Humans; Inclusion Bodies; Intracellular Membranes; Lipid Bilayers; Membrane Proteins; Microscopy, Confocal; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Sirolimus

Cell differentiation is often associated with decreased cell growth, indicating an altered rate of macromolecule synthesis and degradation. In this study, we present evidence that autophagy, a process for bulk degradation of cytoplasm, is activated during retinoic acid-induced neuronal differentiation of neuroblastoma N2a cells. Chemical inhibitors of autophagy, including 3-MA and LY294002, abrogate cell differentiation. RNA interference of autophagy gene beclin 1 markedly delays the process of differentiation. We also find that cell differentiation is accompanied by decreased activity of mTOR, a major controller of cell growth and a negative regulator of autophagy. However, completely inhibiting mTOR by rapamycin decreases neurite outgrowth, cell size and the immunoreactivity for neuronal markers. Our study suggests that an appropriate level of mTOR activity is important in cell differentiation for a balance between macromolecule synthesis and degradation.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Size; Chromones; Dose-Response Relationship, Drug; Down-Regulation; Mice; Morpholines; Neurites; Neuroblastoma; Neurons; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tretinoin

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Protein 5; Caspase 12; Cell Death; eIF-2 Kinase; Endoplasmic Reticulum; Enzyme Activation; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Leucine; Lysosomes; Mice; Microtubule-Associated Proteins; Models, Biological; Pepstatins; Peptides; Phosphorylation; Protein Structure, Quaternary; RNA, Messenger; Sirolimus

Induction of autophagy has been shown to be beneficial for the replication of poliovirus, a phenomenon that might also apply for other picornaviruses. We demonstrate that de novo synthesis of human rhinovirus type 2 (HRV2), an HRV of the minor receptor group, is unaffected by tamoxifen, rapamycin, and 3-methyladenine (3-MA), drugs either stimulating (tamoxifen and rapamycin) or inhibiting (3-MA) autophagic processes. Furthermore, LC3-positive vesicles (i.e., autophagosomes) are not induced upon infection. Therefore, multiplication of this particular picornavirus is not dependent on autophagy.

Topics: Adenine; Autophagy; Cells, Cultured; Humans; Microtubule-Associated Proteins; Phagosomes; Rhinovirus; Sirolimus; Tamoxifen; Virus Replication

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Androstadienes; Animals; Apoptosis; Autophagy; Dinoflagellida; Dose-Response Relationship, Drug; Hot Temperature; RNA Interference; Sea Anemones; Sirolimus; Symbiosis; Wortmannin

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.

Topics: Adenine; Animals; Antibiotics, Antineoplastic; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; HeLa Cells; Humans; Membrane Proteins; Mice; Microtubule-Associated Proteins; Neoplasms; Neurodegenerative Diseases; NIH 3T3 Cells; Pancreatitis, Acute Necrotizing; Phagosomes; Protein Binding; Proteins; RNA, Small Interfering; Sirolimus

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Topics: Acetylcysteine; Adenine; Animals; Autophagy; Cell Line; Chromones; Epidermal Growth Factor; Glyceraldehyde-3-Phosphate Dehydrogenases; Kidney Tubules; Leupeptins; Lysosomes; Methylamines; Morpholines; PAX2 Transcription Factor; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In mammalian cells, amino acids affect the phosphorylation state and function of several proteins involved in mRNA translation that are regulated via the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. These include ribosomal protein S6 kinase, S6K1, and eukaryotic initiation factor 4E-binding protein, 4E-BP1. Amino acids, especially branched-chain amino acids, such as leucine, promote phosphorylation of 4E-BP1 and S6K1, and permit insulin to further increase their phosphorylation. However, it is not clear whether these effects are exerted by extracellular or intracellular amino acids. Inhibition of protein synthesis is expected to increase the intracellular level of amino acids, whereas inhibiting proteolysis has the opposite effect. We show in the present study that inhibition of protein synthesis by any of several protein synthesis inhibitors tested allows insulin to regulate 4E-BP1 or S6K1 in amino-acid-deprived cells, as does the addition of amino acids to the medium. In particular, insulin activates S6K1 and promotes initiation factor complex assembly in amino-acid-deprived cells treated with protein synthesis inhibitors, but cannot do so in the absence of these compounds. Their effects occur at concentrations commensurate with their inhibition of protein synthesis and are not due to activation of stress-activated kinase cascades. Inhibition of protein breakdown (autophagy) impairs the ability of insulin to regulate 4E-BP1 or S6K1 under such conditions. These and other data presented in the current study are consistent with the idea that it is intracellular amino acid levels that regulate mTOR signalling.

Topics: Adenine; Amino Acids; Animals; Anisomycin; Blotting, Western; Carrier Proteins; CHO Cells; Cricetinae; Cycloheximide; Enzyme Inhibitors; Gene Expression Regulation; Immunosuppressive Agents; Insulin; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Peptide Initiation Factors; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Synthesis Inhibitors; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In nutrient-deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane-bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell-cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole-arrested pseudo-prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)-kinase inhibitors wortmannin, LY294002 and 3-methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1-/- embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3-kinases or other wortmannin/LY294002-sensitive kinases in mitotic membrane traffic regulation.

Topics: Adenine; Anaphase; Androstadienes; Animals; Autophagy; Blotting, Western; Cell Line; Chromones; Endoplasmic Reticulum; Enzyme Inhibitors; Golgi Apparatus; HeLa Cells; Humans; Immunohistochemistry; Metaphase; Microscopy, Immunoelectron; Microscopy, Phase-Contrast; Mitosis; Morpholines; Nocodazole; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Telophase; Time Factors; TOR Serine-Threonine Kinases; Wortmannin

In rat hepatocytes, autophagy is known to be inhibited by amino acids. Insulin and cell swelling promote inhibition by amino acids. Each of the conditions leading to inhibition of autophagic proteolysis was found to be associated with phosphorylation of a 31-kDa protein that we identified as ribosomal protein S6. A combination of leucine, tyrosine, and phenylalanine, which efficiently inhibits autophagic proteolysis, was particularly effective in stimulating S6 phosphorylation. The relationship between the percentage inhibition of proteolysis and the degree of S6 phosphorylation was linear. Thus, inhibition of autophagy and phosphorylation of S6 are under the control of the same signal transduction pathway. Stimulation of S6 phosphorylation by the presence of amino acids was due to activation of S6 kinase and not to inhibition of S6 phosphatase. The inhibition by amino acids of both autophagic proteolysis and autophagic sequestration of electro-injected cytosolic [14C]sucrose was partially prevented by rapamycin, a compound known to inhibit activation of p70 S6 kinase. In addition, rapamycin partially inhibited the rate of protein synthesis. We conclude that the fluxes through the autophagic and protein synthetic pathways are regulated in an opposite manner by the degree to which S6 is phosphorylated. Possible mechanisms by which S6 phosphorylation can cause inhibition of autophagy are discussed.

Topics: Adenine; Animals; Autophagy; Male; Phosphorylation; Polyenes; Proteins; Rats; Rats, Wistar; Ribosomal Protein S6; Ribosomal Proteins; Ribosomes; Sirolimus