miR-34a-5p was involved in chronic intermittent hypoxia-induced autophagy of human coronary artery endothelial cells via Bcl-2/beclin 1 signal transduction pathway

Autophagy refers to the genetically regulated process to regulate the survival and death of cells, which is conserved in evolution. Typically, autophagy exerts a vital part under physiopathological conditions. Whether autophagy can be resulted from chronic intermittent hypoxia (CIH), a prominent characteristic of obstructive sleep apnea-hypopnea syndrome (OSAHS), remains to be investigated. Furthermore, microRNAs (miRNAs) can serve as the regulating factors in a variety of benign and malignant diseases; nonetheless, it remains to be fully illustrated about the way by which miRNAs modulate autophagy. According to our results, for human coronary artery endothelial cells (HCAECs), CIH increased the expression of autophagy-associated proteins, which depended on the concentration and time; besides, it could promote autophagic vacuole (AV) formation. In addition, CIH could activate beclin 1, which was dependent on dose and time. In HCAECs, microRNA-34a-5p (miR-34a-5p) was overexpressed after exposed to CIH, and its target protein B-cell lymphoma 2 (Bcl-2) was downregulated. Moreover, inhibiting miR-34a-5p increased Bcl-2 and p62 expression, while downregulating beclin 1, Vps34, Atg5, and LC3 levels, implying the role of miR-34a-5p in CIH-induced autophagy. Moreover, exogenous upregulation of Bcl-2 could block miR-34a-5p influence on CIH-induced autophagy through suppressing beclin 1 expression. Additionally, beclin 1 could enhance the autophagy induced by CIH. In conclusion, overexpression of miR-34a-5p activated beclin 1 through Bcl-2 inhibition in CIH and participated in CIH-induced autophagy.     

MCM7 silencing promotes cutaneous melanoma cell autophagy and apoptosis by inactivating the AKT1/mTOR signaling pathway

Cutaneous melanoma (CM) has become a major public health concern. Studies illustrate that minichromosome maintenance protein 7 (MCM7) participate in various diseases including skin disease. Our study aimed to study the effects of MCM7 silencing on CM cell autophagy and apoptosis by modulating the AKT threonine kinase 1 (AKT1)/mechanistic target of rapamycin kinase (mTOR) signaling pathway. Initially, microarray analysis was used to screen the CM-related gene expression data as well as differentially expressed genes. Subsequently, MCM7 expression vector and lentivirus RNA used for MCM7 silencing (LV-shRNA-MCM7) were constructed, and these vectors, dimethyl sulfoxide (DMSO) and AKT activator SC79 were then introduced into CM cell line SK-MEL-2 to validate the role of MCM7 in cell autophagy, viability, apoptosis, cell cycle, migration, and invasion. To further investigate the regulatory mechanisms of MCM7 in CM progress, the expression of MCM7, AKT1, mTOR, cyclin D1, as well as autophagy and apoptosis relative factors, such as LC3B, SOD2, DJ-1, p62, Bcl-2, Bax, and caspase-3 in melanoma cells was determined. MCM7 might mediate the AKT1/mTOR signaling pathway to influence the progress of melanoma. MCM7 silencing contributed to the increased expression of Bax, capase-3, and autophagy-related genes (LC3B, SOD2, and DJ-1), but decreased the expression of Bcl-2, which suggested that MCM7 silencing promoted autophagy and cell apoptosis. At the same time, MCM7 silencing also attenuated cell viability, invasion, and migration, and reduced the cyclin D1 expression and protein levels of p-AKT1 and p-mTOR. Taken together, MCM7 silencing inhibited CM via inactivation of the AKT1/mTOR signaling pathway.     

LRP6 regulates Rab7-mediated autophagy through the Wnt/β-catenin pathway to modulate trophoblast cell migration and invasion

Pre-eclampsia is a common complication during pregnancy; however, the underlying mechanisms of the crosstalk between low-density lipoprotein receptor-related protein 6 (LRP6) and autophagy in trophoblast cells are still not fully explored. Messenger RNA (mRNA) and protein levels of LRP6, beclin 1, Unc-51-like autophagy activating kinase 1 (ULK1), p62, vimentin, matrix metallopeptidase-9 (MMP-9), β-catenin, c-Myc, and Rab7, as well as the ratio of LC3-II/LC3-I, were analysed by quantitative real-time polymerase chain reaction or Western blot analysis, respectively. An MTT assay was used to measure cell growth, and transwell and wound healing assays were carried out to evaluate the invasion and migration abilities of the trophoblasts used. An immunofluorescence assay was used to measure LC3. The mRFP-GFP-LC3 tandem fluorescence assay was applied to detect autophagic flow. LRP6 overexpression was achieved by constructing pcDNA3.1-LRP6 vectors. LRP6 was expressed at low levels in HTR-8/SVneo cells under hypoxia/reoxygenation (H/R) conditions. H/R inhibited the activation of autophagy. LRP6 overexpression promoted cell proliferation and activated autophagy, which led to the upregulation of beclin 1 and ULK1, as well as the ratio of LC3-II/LC3-I and the downregulation of p62. Furthermore, LRP6 overexpression elevated the migration and invasion abilities of the indicated cells and increased vimentin and MMP-9 expression levels. Furthermore, LRP6 upregulated Rab7 and activated autophagy through the Wnt/β-catenin pathway. The late autophagy inhibitor bafilomycin A1 (Baf-A1) and the Wnt/β-catenin pathway inhibitor PKF115-584 reversed the effects of LRP6 on trophoblast autophagy, migration and invasion. LRP6 promotes Rab7-mediated autophagy by activating the Wnt/β-catenin pathway, which leads to increasing migration and invasion of trophoblast cells. Our study paves a new avenue for clinical treatment, and LRP6 may serve as an essential target in pre-eclampsia.     

Autophagy activated by tuberin/mTOR/p70S6K suppression is a protective mechanism against local anaesthetics neurotoxicity

The local anaesthetics (LAs) are widely used for peripheral nerve blocks, epidural anaesthesia, spinal anaesthesia and pain management. However, exposure to LAs for long duration or at high dosage can provoke potential neuronal damages. Autophagy is an intracellular bulk degradation process for proteins and organelles. However, both the effects of LAs on autophagy in neuronal cells and the effects of autophagy on LAs neurotoxicity are not clear. To answer these questions, both lipid LAs (procaine and tetracaine) and amide LAs (bupivacaine, lidocaine and ropivacaine) were administrated to human neuroblastoma SH‐SY5Y cells. Neurotoxicity was evaluated by MTT assay, morphological alterations and median death dosage. Autophagic flux was estimated by autolysosome formation (dual fluorescence LC3 assay), LC3‐II generation and p62 protein degradation (immunoblotting). Signalling alterations were examined by immunoblotting analysis. Inhibition of autophagy was achieved by transfection with beclin‐1 siRNA. We observed that LAs decreased cell viability in a dose‐dependent manner. The neurotoxicity of LAs was tetracaine > bupivacaine > ropivacaine > procaine > lidocaine. LAs increased autophagic flux, as reflected by increases in autolysosome formation and LC3‐II generation, and decrease in p62 levels. Moreover, LAs inhibited tuberin/mTOR/p70S6K signalling, a negative regulator of autophagy activation. Most importantly, autophagy inhibition by beclin‐1 knockdown exacerbated the LAs‐provoked cell damage. Our data suggest that autophagic flux was up‐regulated by LAs through inhibition of tuberin/mTOR/p70S6K signalling, and autophagy activation served as a protective mechanism against LAs neurotoxicity. Therefore, autophagy manipulation could be an alternative therapeutic intervention to prevent LAs‐induced neuronal damage.     

Eldecalcitol induces apoptosis and autophagy in human osteosarcoma MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway

Eldecalcitol (ED-71) is a new type of vitamin D analog, and vitamin D has been reported to have therapeutic effects in infectious disease, autoimmune disease, and cancer. However, the anti-cancer effect of ED-71 remains unclear. The objective of this study was to explore the anti-cancer effect of ED-71 in human osteosarcoma cells and to identify the related mechanism. The CCK8 assay results showed that ED-71 inhibited MG-63 cell viability in dose and time dependent manners. Cloning and Transwell invasion assays showed that ED-71 inhibited clonal and invasion ability of MG-63 cells. Flow cytometry results showed ED-71 the G2/M cycle arrest rate, apoptosis, and intracellular ROS. Western blot was used to detect cleaved-caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and P62 levels and the mTOR pathway. The increase of LC3-II and P62 indicated that ED-71 induced the formation of autophagosomes and inhibited autophagy flux. Furthermore, ED-71-induced apoptosis was weakened after adding 3-methyladenine and ED-71-induced early autophagy was weakened by caspase-3 inhibitor (Z-VAD-FMK), which indicated the two processes active each other in the presence of ED-71. Furthermore, N-acetylcysteine (NAC) pretreatment reversed the ED-71-treatment outcomes, including increased apoptosis and autophagy and inhibition of the PI3K/Akt/mTOR pathway. In conclusion, our results reveal that ED-71 induced G2/M arrest, apoptosis and autophagy in MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway     

Cucurbitacin I Induces Protective Autophagy in Glioblastoma in Vitro and in Vivo

There is an urgent need for new therapeutic avenues to improve the outcome of patients with glioblastoma multiforme (GBM). Current studies have suggested that cucurbitacin I, a natural selective inhibitor of JAK2/STAT3, has a potent anticancer effect on a variety of cancer cell types. This study showed that autophagy and apoptosis were induced by cucurbitacin I. Exposure of GBM cells to cucurbitacin I resulted in pronounced apoptotic cell death through activating bcl-2 family proteins. Cells treatment with cucurbitacin I up-regulated Beclin 1 and triggered autophagosome formation and accumulation as well as conversion of LC3I to LC3II. Activation of the AMP-activated protein kinase/mammalian target of rapamycin/p70S6K pathway, but not the PI3K/AKT pathway, occurred in autophagy induced by cucurbitacin I, which was accompanied by decreased hypoxia-inducible factor 1α. Stable overexpression of hypoxia-inducible factor 1α induced by FG-4497 prevented cucurbitacin I-induced autophagy and down-regulation of bcl-2. Knockdown of beclin 1 or treatment with the autophagy inhibitor 3-methyladenine also inhibited autophagy induced by cucurbitacin I. A coimmunoprecipitation assay showed that the interaction of Bcl-2 and Beclin 1/hVps34 decreased markedly in cells treated with cucurbitacin I. Furthermore, knockdown of beclin 1 or treatment with the lysosome inhibitor chloroquine sensitized cancer cells to cucurbitacin I-induced apoptosis. Finally, a xenograft model provided additional evidence for the occurrence of cucurbitacin I-induced apoptosis and autophagy in vitro. Our findings provide new insights into the molecular mechanisms underlying cucurbitacin I-mediated GBM cell death and may provide an efficacious therapy for patients harboring GBM.     

Ganoderic acid A holds promising cytotoxicity on human glioblastoma mediated by incurring apoptosis and autophagy and inactivating PI3K/AKT signaling pathway

Ganoderic acid A (GA‐A), recognized as a lanostanetriterpene isolated from Ganoderma lucidum, demonstrates an efficient antitumor activity in multiple cancers. To date, it is unclear whether and how GA‐A functions on human glioblastoma (GBM). To unravel the functional significance of GA‐A on human glioblastoma (GBM), the cell‐counting kit‐8 and transwell assays were used to detect proliferation, migration, and invasion of human GBM cell after GA‐A treatment. Then, we utilized the flow cytometry and western blot to further evaluate the effect of GA‐A on GBM cell. Further, activities of autophagy and PI3K/AKT signaling were assessed by Western blot assay. We found that GA‐A significantly inhibited proliferation, migration, and invasion of GBM cell. Additionally, GA‐A markedly triggered cell apoptosis, which incarnated an elevation trend in apoptotic percentage, simultaneously, an increased level of proapoptosis protein (Bax and active caspase‐3) and a decreased level of antiapoptosis protein (Bcl‐2), induced by GA‐A treatment. Meanwhile, levels of two well‐known autophagy markers (beclin 1 and LC3 II) increased while another autophagic substrate (P‐62) was reduced. Moreover, the expressions levels of phosphorylated AKT, mTOR, p‐P70S6K, and cyclin D1 in the PI3K/AKT pathway were significantly reduced, which revealed GA‐A repressed the activation of PI3K/AKT signaling pathway. Collectively, these results indicate that GA‐A may encourage U251 cell growth and invasion/migration inhibition, apoptosis, and autophagy through the inactivation of PI3K/AKT signaling pathway in human GBM. Hence, GA‐A may be a potent antitumorigenic agent for human GBM in future clinical practice.     

MiR‐19a Affects Hepatocyte Autophagy via Regulating lncRNA NBR2 and AMPK/PPARα in D‐GalN/Lipopolysaccharide‐Stimulated Hepatocytes

This study aims to evaluate the potential involvement and regulatory mechanism of miR‐19a in hepatocytes autophagy of acute liver failure (ALF). The in vitro hepatocytes injury model of primary hepatocyte and hepatocytes line HL‐7702 was established by D‐galactosamine (D‐GalN) and lipopolysaccharide (LPS) co‐treatment. Relative expression level of miR‐19a and NBR2 was determined by qRT‐PCR. Protein expression of AMPK/PPARα and autophagy‐related gene was determined by Western blot. In hepatic tissue of 20 ALF patients and D‐GalN/LPS‐stimulated hepatocytes, miR‐19a was upregulated and NBR2 was downregulated. D‐GalN/LPS stimulation caused the inactivation of AMPK/PPARα signaling and the decrease of autophagy‐related LC3‐II/LC3‐I ratio and beclin‐1 expression in hepatocytes. The expression of both AMPK/PPARα and NBR2 were negatively controlled by miR‐19a overexpression or knockdown. Moreover, both NBR2 and PPARα were targeted regulated by miR‐19a according to luciferase reporter assay. In D‐GalN/LPS‐stimulated hepatocytes, AMPK activation promoted PPARα expression. AMPK inactivation inhibited the pro‐autophagy effect of miR‐19a and caused the decrease of LC3‐II/LC3‐I ratio and beclin‐1 expression. PPARα activation abrogated the anti‐autophagy effect of miR‐19a mimic and caused the increase of LC3‐II/LC3‐I ratio and beclin‐1 expression. NBR2 knockdown reversed the anti‐autophagy impact of miR‐19a inhibitor and caused the decrease of LC3‐II/LC3‐I ratio and beclin‐1 expression. In summary, our data suggested that miR‐19a negatively controlled the autophagy of hepatocytes attenuated in D‐GalN/LPS‐stimulated hepatocytes via regulating NBR2 and AMPK/PPARα signaling. J. Cell. Biochem. 119: 358–365, 2018. © 2017 Wiley Periodicals, Inc.     

Necrostatin-1 Suppresses Autophagy and Apoptosis in Mice Traumatic Brain Injury Model

Traumatic brain injury (TBI) results in neuronal apoptosis, autophagic cell death and necroptosis. Necroptosis is a newly discovered caspases-independent programmed necrosis pathway which can be triggered by activation of death receptor. Previous works identified that necrostatin-1 (NEC-1), a specific necroptosis inhibitor, could reduce tissue damage and functional impairment through inhibiting of necroptosis process following TBI. However, the role of NEC-1 on apoptosis and autophagy after TBI is still not very clear. In this study, the amount of TBI-induced neural cell deaths were counted by PI labeling method as previously described. The expression of autophagic pathway associated proteins (Beclin-1, LC3-II, and P62) and apoptotic pathway associated proteins (Bcl-2 and caspase-3) were also respectively assessed by immunoblotting. The data showed that mice pretreated with NEC-1 reduced the amount of PI-positive cells from 12 to 48?h after TBI. Immunoblotting results showed that NEC-1 suppressed TBI-induced Beclin-1 and LC3-II activation which maintained p62 at high level. NEC-1 pretreatment also reversed TBI-induced Bcl-2 expression and caspase-3 activation, as well as the ratio of Beclin-1/Bcl-2. Both 3-MA and NEC-1 suppressed TBI-induced caspase-3 activation and LC3-II formation, Z-VAD only inhibited caspase-3 activation but increased LC3-II expression at 24?h post-TBI. All these results revealed that multiple cell death pathways participated in the development of TBI, and NEC-1 inhibited apoptosis and autophagy simultaneously. These coactions may further explain how can NEC-1 reduce TBI-induced tissue damage and functional deficits and reflect the interrelationship among necrosis, apoptosis and autophagy.     

ZFYVE26/SPASTIZIN and SPG11/SPATACSIN mutations in hereditary spastic paraplegia types AR-SPG15 and AR-SPG11 have different effects on autophagy and endocytosis

ZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis.

Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A₁; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.     

p62/Sequestosome-1 Up-regulation Promotes ABT-263-induced Caspase-8 Aggregation/Activation on the Autophagosome

Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, their functional relationship remains unclear. p62/sequestosome 1 is a multifunctional protein and a signaling hub that shuttles ubiquitinated proteins to the lysosome during autophagy. Autophagy inhibition up-regulates p62, and prior data suggest that p62 may mediate apoptosis. Here, we demonstrate that p62 can regulate a caspase-8-dependent apoptosis in response to the BH3 mimetic agent, ABT-263. Up-regulation of p62 was shown to enhance ABT-263-induced caspase-8 activation that was Bax-dependent and resulted from mitochondrial amplification. Dependence upon caspase-8 was confirmed using caspase-8-deficient cells and by caspase-8 siRNA. Ectopic wild-type p62, but not p62 mutants with loss of ability to promote apoptosis, was shown to co-localize with caspase-8 and to promote its self-aggregation in ABT-263-treated cells, shown using a bimolecular fluorescence complementation assay. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 plus an autophagy inhibitor. Caspase-8 was shown to interact and co-localize with the autophagosome marker, LC3II. Knockdown of p62 attenuated binding between caspase-8 and LC3II, whereas p62 overexpression enhanced the co-localization of caspase-8 aggregates with LC3. LC3 knockdown did not affect interaction between caspase-8 and p62, suggesting that p62 may facilitate caspase-8 translocation to the autophagosomal membrane. A direct activator of caspase-8, i.e., TRAIL, alone or combined with ABT-263, induced caspase-8 aggregation and co-localization with p62 that was associated with a synergistic drug interaction. Together, these results demonstrate that up-regulation of p62 can mediate apoptosis via caspase-8 in the setting of autophagy inhibition.     

1-Methyl-4-Phenylpyridinium-Induced Cell Death via Autophagy Through a Bcl-2/Beclin 1 Complex-Dependent Pathway

Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson’s disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP⁺) toxicity. Therefore, we hypothesized that the MPP⁺ toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP⁺ increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP⁺ can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

Molecular mechanisms of oridonin-induced apoptosis and autophagy in murine fibrosarcoma L929 cells

Apoptosis and autophagy are genetically regulated, evolutionarily-conserved processes that can jointly seal the fate of cancer cells; however, substantial gaps remain in our understanding of the molecular mechanisms that mediate the two cellular processes. In the present study, the exposure of murine fibrosarcoma L929 cells to oridonin led to the generation of intracellular reactive oxygen species (ROS) and, subsequently, the ROS triggered apoptosis by Bax translocation, cytochrome c release and ERK activations. In addition, oridonin induced autophagy in L929 cells, and the inhibition of autophagy by 3-MA or siRNA against LC3 and beclin 1 promoted oridonin-induced apoptosis. Furthermore, p38 and NF-κB were confirmed to have roles in inhibiting apoptosis but promoting autophagy. Moreover, the inhibition of autophagy could reduce oridonin-induced activation of p38. Finally, NF-κB activation was inhibited by blocking the p38 pathway. In conclusion, these findings indicate that oridonin-induced apoptosis can be regulated by ROS-mediated signaling pathways, and oridonin-induced autophagy may block apoptosis by up-regulating p38 and NFκB activation.     

Autophagy protects intestinal epithelial Cells against Deoxynivalenol toxicity by alleviating oxidative stress via IKK signaling pathway

Autophagy is an intracellular process of homeostatic degradation that promotes cell survival under various stressors. Deoxynivalenol (DON), a fungal toxin, often causes diarrhea and disturbs the homeostasis of the intestinal system. To investigate the function of intestinal autophagy in response to DON and associated mechanisms, we firstly knocked out ATG5 (autophagy-related gene 5) in porcine intestinal epithelial cells (IPEC-J2) using CRISPR-Cas9 technology. When treated with DON, autophagy was induced in IPEC-J2 cells but not in IPEC-J2.Atg5ko cells. The deficiency in autophagy increased DON-induced apoptosis in IPEC-J2.atg5ko cells, in part, through the generation of reactive oxygen species (ROS). The cellular stress response can be restored in IPEC-J2.atg5ko cells by overexpressing proteins involved in protein folding. Interestingly, we found that autophagy deficiency downregulated the expression of endoplasmic reticulum folding proteins BiP and PDI when IPEC-J2.atg5ko cells were treated with DON. In addition, we investigated the molecular mechanism of autophagy involved in the IKK, AMPK, and mTOR signaling pathway and found that Bay-117082 and Compound C, specific inhibitors for IKK and AMPK, respectively, inhibited the induction of autophagy. Taken together, our results suggest that autophagy is pivotal for protection against DON in pig intestinal cells.     

TXNIP在高糖诱导的小鼠视网膜Müller细胞自噬中的作用及相关机制

目的:探讨硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)对高糖诱导的小鼠视网膜Müller细胞自噬的影响及其可能机制。方法:采用高糖诱导体外培养的小鼠视网膜Muller细胞,通过RNA干扰降低TXNIP的表达,免疫荧光、Western blot和Real-time PCR检测自噬相关蛋白及丝氨酸/苏氨酸激酶/雷帕霉素靶蛋白(serine/threonine kinase 1/mechanistic target of rapamycin kinase,AKT/m TOR)的表达。结果:高糖诱导的Muller细胞中TXNIP、微管相关蛋白1轻链3α(microtubule associated protein 1 light chain 3 alpha,LC3Ⅱ)、Sequestosome1(p62/SQSTMl)的表达均显著增加(P<0.05);而TXNIP敲降的Muller细胞中自噬相关特征性蛋白(LC3Ⅱ、P62)的表达则显著降低(P<0.05)。结论:TXNIP可能通过AKT/m TOR信号通路来抑制糖尿病性视网膜病变中Müller细胞自噬活性,并引起细胞发生凋亡。     

Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

过表达微小RNA-130a-3P对LPS诱导心肌细胞损伤的干预作用及其机制*

目的: 探讨miRNA-130a-3p对脂多糖(LPS)诱导的心肌细胞自噬与凋亡的影响及分子机制。方法: H9C2心肌细胞随机分为5组,即正常对照组,LPS模型组,miRNA阴性对照组(miRNA-negative control组),miRNA-130a-3p mimics组(过表达miRNA-130a-3p),miRNA-130a-3p mimics+LY294002组(过表达miRNA-130a-3p + PI3K抑制)。LPS模型组即终浓度为10 μg/ml的LPS诱导24 h,miRNA阴性对照组与miRNA-130a-3p mimics组是利用lipo3000将阴性对照miRNA及miRNA-130a-3p mimics转染至H9C2细胞,培养24 h后,再将LPS加入培养基中培养24 h。miRNA-130a-3p mimics + LY294002组是利用lipo3000将miRNA-130a-3p mimics转染至H9C2细胞,同时在培养基中加入10 μmol/L(终浓度)的LY294002,培养24 h后,再将浓度为10 μg/ml的LPS加入培养基中培养24 h。所有实验均重复5次以上。利用RT-qPCR检测细胞中miRNA-130a-3p mRNA的表达水平,利用CCK-8实验检测细胞活性,利用ELISA实验检测细胞培养液中肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),白细胞介素-1β (IL-1β)的含量,利用比色法检测细胞培养液中超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)的含量;利用Western blot检测细胞中p-PI3K蛋白,p-AKT蛋白,Bax蛋白,Bcl-2蛋白,cleaved-caspase-3蛋白,LC3蛋白,p62蛋白的表达水平。结果: 结果显示,与正常组相比较,LPS模型细胞中miRNA-130a-3p mRNA水平,p-PI3K蛋白与p-AKT蛋白的水平显著低于正常对照组(P<0.01);与LPS组相比较,miRNA-130a-3p mimics组细胞中p-PI3K,p-AKT蛋白的表达显著升高(P<0.01,P<0.05);与正常对照组相比较,LPS组细胞活性显著降低,细胞培养液中TNF-α,IL-6,IL-1β及 LDH的含量显著升高(P<0.01), SOD的含量显著降低(P<0.01),细胞中Bax蛋白,cleaved caspase-3蛋白,p62蛋白的表达显著升高(P<0.01),Bcl-2蛋白的表达和LC3II/I的比率显著降低(P<0.01);与LPS组相比较,miRNA-130a-3p mimics可提高细胞活性,降低细胞培养液中TNF-α,IL-6,IL-1β及LDH的含量(P<0.01,P<0.05),提高SOD的含量(P<0.05),降低细胞中Bax蛋白,cleaved caspase-3蛋白,p62蛋白的表达(P<0.01),促进Bcl-2蛋白的表达(P<0.01),提高LC3II/I的比率(P<0.05);与miRNA-130a-3p mimics组相比较,miRNA-130a-3p mimics+LY294002组,可部分逆转miRNA-130a-3p mimics对细胞的作用。结论: 过表达miRNA-130a-3p可部分通过激活PI3K/AKT信号通路促进细胞的自噬与抑制细胞凋亡,减轻LPS诱导的心肌细胞损伤。     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.