SARS-CoV-2 NSP13 Inhibits Type I IFN Production by Degradation of TBK1 via p62-Dependent Selective Autophagy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.

Vitamin D promotes autophagy in AML cells by inhibiting miR-17-5p-induced Beclin-1 overexpression.

MicroRNA (miR)-17-5p has been investigated in many diseases as a regulator of disease progression and is highly expressed in acute myeloid leukemia (AML). However, potential mechanisms underlying the function of miR-17-5p in AML need more elucidation. MiR-17-5p expression was augmented, while 25(OH)D3 and Beclin-1 levels were decreased in AML patients with the highest risk for disease progression. MiR-17-5p, 25(OH)D3 and Beclin-1 were determined to be clinically important in AML based on ROC curve analysis. Higher miR-17-5p expression as well as lower 25(OH)D3 and Beclin-1 expression were relevant with poor prognosis in AML. In addition, miR-17-5p was negatively correlated with and bound to BECN1. Vitamin D was found to diminish cell proliferation and enhance autophagy. Finally, through rescue assays, miR-17-5p facilitated the ability of cell proliferation, inhibited autophagy and apoptosis by modulating Beclin-1 in HL-60 cells following the treatment of 4 µM vitamin D. Vitamin D promoted autophagy in AML cells by modulating miR-17-5p and Beclin-1.

Hypertrophic Preconditioning Attenuates Myocardial Ischaemia-Reperfusion Injury by Modulating SIRT3-SOD2-mROS-Dependent Autophagy.

BACKGROUND: Ischaemic preconditioning elicited by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischaemic insult. Here, we test the hypothesis that short-term non-ischaemic stimulation of hypertrophy renders the heart resistant to subsequent ischaemic injury. METHODS AND RESULTS: Transient transverse aortic constriction (TAC) was performed for 3 days in mice and then withdrawn for 4 days by aortic debanding, followed by subsequent exposure to myocardial ischaemia-reperfusion (I/R) injury. Following I/R injury, myocardial infarct size and apoptosis were significantly decreased, and cardiac dysfunction was markedly improved in the TAC preconditioning group compared with the control group. Mechanistically, TAC preconditioning markedly suppressed I/R-induced autophagy and preserved autophagic flux by deacetylating SOD2 via a SIRT3-dependent mechanism. Moreover, treatment with an adenovirus encoding SIRT3 partially mimicked the effects of hypertrophic preconditioning, whereas genetic ablation of SIRT3 in mice blocked the cardioprotective effects of hypertrophic preconditioning. Furthermore, in vivo lentiviral-mediated knockdown of Beclin 1 in the myocardium ameliorated the I/R-induced impairment of autophagic flux and was associated with a reduction in cell death, whereas treatment with a lentivirus encoding Beclin 1 abolished the cardioprotective effect of TAC preconditioning. CONCLUSIONS: The present study identifies TAC preconditioning as a novel strategy for induction of an endogenous self-defensive and cardioprotective mechanism against cardiac injury. Specifically, TAC preconditioning reduced myocardial autophagic cell death in a SIRT3/SOD2 pathway-dependent manner.

Acetyl-L-carnitine confers neuroprotection against lipopolysaccharide (LPS) -induced neuroinflammation by targeting TLR4/NFκB, autophagy, inflammation and oxidative stress.

Acetyl-L-carnitine has been shown to exert neuroprotection against neurodegenerative diseases. The present study was performed to evaluate neuroprotection effects of acetyl-L-carnitine against lipopolysaccharide (LPS) -induced neuroinflammation and clarify possible mechanisms. A single dose (500 µg/kg) of LPS was intraperitoneally injected to rats to induce model. The animals were intraperitoneally treated with different doses of acetyl-L-carnitine (30, 60, and 100) for 6 days. Y-maze task, single-trial passive avoidance and novel object recognition tests were used to evaluate memory impairments. ELISA assay was used to evaluate the expression of TLR4/NFκB, autophagic and oxidative stress markers. Our result showed that intraperitoneal injection of LPS resulted in initiation of neuroinflammation by activation of TLR4/NFκB, suppression of autophagic markers such as LC3 II/ LC3 I ratio and becline-1, and excessive production of ROS and MDA. Intraperitoneal administration of acetyl-L-carnitine contributed to neuroprotection against LPS -induced neuroinflammation by suppression of TLR4/NFκB pathway, restoring activity of autophagy and inhibition of oxidative stress. Collectively, our findings show that acetyl-L-carnitine attenuated LPS-induced neuroinflammation by targeting TLR4/NFκB pathway, autophagy and oxidative stress.

Puerarin plays a protective role in chondrocytes by activating Beclin1-dependent autophagy.

Puerarin can protect chondrocytes, whereby ameliorating osteoarthritis. Puerarin also promotes autophagy. Autophagy maintains chondrocyte homeostasis. The role of autophagy in puerarin-protected chondrocytes is unknown. Puerarin promoted chondrocyte autophagy. Puerarin-protected chondrocytes were reversed by autophagy inhibitors and Beclin1 inhibitor. 3-MA or Beclin1 inhibitor in vivo reversed puerarin-ameliorated cartilage damage of osteoarthritis mice. Thus, puerarin can protect chondrocytes through Beclin1-dependent autophagy activation.

Lipophagy confers a key metabolic advantage that ensures protective CD8A T-cell responses against HIV-1.

Although macroautophagy/autophagy has been proposed as a critical defense mechanism against HIV-1 by targeting viral components for degradation, its contribution as a catabolic process in providing optimal anti-HIV-1 immunity has never been addressed. The failure to restore proper antiviral CD8A/CD8 T-cell immunity, especially against HIV-1, is still the major limitation of current antiretroviral therapies. Consequently, it is of clinical imperative to provide new strategies to enhance the function of HIV-1-specific CD8A T-cells in patients under antiretroviral treatments (ART). Here, we investigated whether targeting autophagy activity could be an optional solution to make this possible. Our data show that, after both polyclonal and HIV-1-specific activation, CD8A T-cells from ART displayed reduced autophagy-dependent degradation of lysosomal contents when compared to naturally HIV-1 protected elite controllers (EC). We further confirmed in EC, by using specific BECN1 gene silencing and lysosomal inhibitors, the critical role of active autophagy in superior CD8A T-cell protection against HIV-1. More importantly, we found that an IL21 treatment was effective in rescuing the antiviral CD8A T-cell immunity from ART in an autophagy-dependent manner. Finally, we established that IL21-dependent rescue occurred due to the enhanced degradation of endogenous lipids via autophagy, referred to as lipophagy, which fueled the cellular rates of mitochondrial beta-oxidation. In summary, our data show that autophagy/lipophagy can be considered as a therapeutic tool to elicit functional antiviral CD8 T-cell responses. Our results also provide additional insights toward the development of improved T-cell-based prevention and cure strategies against HIV-1.Abbreviations: ART: patients under antiretroviral therapy; BaF: bafilomycin A1; BECN1: beclin 1; CEF: cytomegalo-, Epstein-Barr- and flu-virus peptide pool; Chloro.: chloroquine; EC: elite controllers; FAO: fatty acid beta-oxidation; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBMC: peripheral blood mononuclear cells; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

Thymoquinone Inhibits Proliferation and Migration of MDA-MB-231 Triple Negative Breast Cancer Cells by Suppressing Autophagy, Beclin-1 and LC3.

BACKGROUND: Triple Negative Breast Cancer (TNBC) is an aggressive and highly heterogeneous subtype of breast cancer associated with poor prognosis. A better understanding of the biology of this complex cancer is needed to develop novel therapeutic strategies for the improvement of patient survival. We have previously demonstrated that Thymoquinone (TQ), the major phenolic compound found in Nigella sativa, induces anti-proliferative and anti-metastatic effects and inhibits in vivo tumor growth in orthotopic TNBC models in mice. Also, we have previously shown that Beclin-1 and LC3 autophagy genes contributes to TNBC cell proliferation, migration and invasion, suggesting that Beclin-1 and LC3 genes provide proto-oncogenic effects in TNBC. However, the role of Beclin-1 and LC3 in mediating TQ-induced anti-tumor effects in TNBC is not known. OBJECTIVE: To investigate the effects of TQ on the major autophagy mediators, Beclin-1 and LC3 expression, as well as autophagic activity in TNBC cells. METHODS: Cell proliferation, colony formation, migration and autophagy activity were evaluated using MTS cell viability, colony formation assay, wound healing and acridine orange staining assays, respectively. Western blotting and RT-PCR assays were used to investigate LC3 and Beclin-1 protein and gene expressions, respectively, in MDA-MB-231 TNBC cells in response to TQ treatments. RESULTS: TQ treatment significantly inhibited cell proliferation, colony formation, migration and autophagic activity of MDA-MB-231 cells and suppressed LC3 and Beclin-1 expressions. Furthermore, TQ treatment led to the inhibition of Integrin-ß1, VEGF, MMP-2 and MMP-9 in TNBC cells. CONCLUSION: TQ inhibits autophagic activity and expression of Beclin-1 and LC3 in TNBC cells and suppresses pathways related to cell migration/invasion and angiogenesis, including Integrin-ß1, VEGF, MMP-2 and MMP- 9, suggesting that TQ may be used to control autophagic activity and oncogenic signaling in TNBC.

Beclin 1-ATG14L Protein-Protein Interaction Inhibitor Selectively Inhibits Autophagy through Disruption of VPS34 Complex I.

Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.

MicroRNA-384-5p/Beclin-1 As Potential Indicators For Epigallocatechin Gallate Against Cardiomyocytes Ischemia Reperfusion Injury By Inhibiting Autophagy Via PI3K/Akt Pathway.

BACKGROUND/AIMS: Epigallocatechin gallate (EGCG) has established protective actions against myocardial ischemia/reperfusion (I/R) injury by regulating autophagy. However, little is known about the mechanisms of EGCG in posttranscriptional regulation in the process of cardioprotection. Here we studied whether microRNAs play a role in EGCG-induced cardioprotection. METHODS: The myocardial I/R injury in vitro and in vivo model were made, with or without EGCG pretreatment. The upregulation and silencing of microRNA-384-5p (miR-384) and Beclin-1 in H9c2 cell lines were established. Rats were transfected with miR-384 specific shRNA. Dual-luciferase reporter gene assay was conducted to verify the relationship between miR-384 and Beclin-1. TTC staining was performed to analyze the area of myocardial infarct size. Cell viability was monitored by cell counting kit-8 (CCK-8). The release of cardiac troponin-I (cTnI) was examined by ELISA. The levels of autophagy-related genes or proteins expression were evaluated by qRT-PCR or Western blotting. Autophagosomes of myocardial cells were detected by transmission electron microscopy and laser scanning confocal microscope. RESULTS: I/R increased both autophagosomes and autolysosomes, thereby increasing autophagic flux both in vitro and in vivo. Pretreatment with EGCG attenuated I/R-induced autophagic flux expression, accompanied by an increase in cell viability and a decrease in the size of myocardial infarction. MiR-384 expression was down-regulated in H9c2 cell lines when subjected to I/R, while this suppression could be reversed by EGCG pretreatment. The dual-luciferase assay verified that Beclin-1 was a target of miR-384. Both overexpression of miR-384 and knocking down of Beclin-1 significantly inhibited I/R-induced autophagy, accompanied by the activation of PI3K/Akt pathway, thus enhanced the protective effect of EGCG. However, these functions were abrogated by the PI3K inhibitor, LY294002. CONCLUSION: We confirmed that EGCG has a protective role in microRNA-384-mediated autophagy by targeting Beclin-1 via activating the PI3K/Akt signaling pathway. Our results unveiled a novel role of EGCG in myocardial protection, involving posttranscriptional regulation with miRNA-384.

Autophagic Survival Precedes Programmed Cell Death in Wheat Seedlings Exposed to Drought Stress.

Although studies have shown the concomitant occurrence of autophagic and programmed cell death (PCD) in plants, the relationship between autophagy and PCD and the factors determining this relationship remain unclear. In this study, seedlings of the wheat cultivar Jimai 22 were used to examine the occurrence of autophagy and PCD during polyethylene glycol (PEG)-8000-induced drought stress. Autophagy and PCD occurred sequentially, with autophagy at a relatively early stage and PCD at a much later stage. These findings suggest that the duration of drought stress determines the occurrence of PCD following autophagy. Furthermore, the addition of 3-methyladenine (3-MA, an autophagy inhibitor) and the knockdown of autophagy-related gene 6 (ATG6) accelerated PEG-8000-induced PCD, respectively, suggesting that inhibition of autophagy also results in PCD under drought stress. Overall, these findings confirm that wheat seedlings undergo autophagic survival under mild drought stress, with subsequent PCD only under severe drought.

Autophagy drives osteogenic differentiation of human gingival mesenchymal stem cells.

BACKGROUND/AIM: Autophagy is a macromolecular degradation process playing a pivotal role in the maintenance of stem-like features and in the morpho-functional remodeling of the tissues undergoing differentiation. In this work we investigated the involvement of autophagy in the osteogenic differentiation of mesenchymal stem cells originated from human gingiva (HGMSC). METHODS: To promote the osteogenic differentiation of HGMSCs we employed resveratrol, a nutraceutical known to modulate autophagy and cell differentiation, together with osteoblastic inductive factors. Osteoblastic differentiation and autophagy were monitored through western blotting and immunofluorescence staining of specific markers. RESULTS: We show that HGMSCs can differentiate into osteoblasts when cultured in the presence of appropriate factors and that resveratrol accelerates this process by up-regulating autophagy. The prolonged incubation with dexamethasone, ß-glycerophosphate and ascorbic acid induced the osteogenic differentiation of HGMSCc with increased expression of autophagy markers. Resveratrol (1 µM) alone elicited a less marked osteogenic differentiation yet it greatly induced autophagy and, when added to the osteogenic differentiation factors, it provoked a synergistic effect. Resveratrol and osteogenic inductive factors synergistically induced the AMPK-BECLIN-1 pro-autophagic pathway in differentiating HGMSCs, that was thereafter downregulated in osteoblastic differentiated cells. Pharmacologic inhibition of BECLIN-1-dependent autophagy precluded the osteogenic differentiation of HGMSCs. CONCLUSIONS: Autophagy modulation is instrumental for osteoblastic differentiation of HGMSCs. The present findings can be translated into the regenerative cell therapy of maxillary / mandibular bone defects.

The role of Beclin 1 in IR-induced crosstalk between autophagy and G2/M cell cycle arrest.

OBJECTIVES: Beclin 1 is a well-established core mammalian autophagy protein. Autophagy has been demonstrated to play roles in cellular responses to DNA damage, such as cell cycle regulation and apoptosis. In the present study, we investigated the exact mechanism by which Beclin 1 acts as a bridge between autophagy and cell cycle, when cells are exposed to ionizing radiation (IR). MATERIALS AND METHODS: Western blotting and coimmunoprecipitation were performed to investigate protein expression levels and interactions. Immunofluorescence was used to monitor the localization and distribution of the indicated proteins. The levels of apoptosis and cell cycle changes were evaluated by flow cytometry. Double thymidine deoxyribonucleoside (TdR) blocking was conducted to differentiate G2 from mitotic delay. In vitro kinase assays using ATM kinase were performed to elucidate the specific phosphorylation site in Beclin 1. RESULTS: In this study, we show that Beclin 1 knockdown reduces IR-induced autophagy. IR enhanced Beclin 1/PIK3CIII complex activity as demonstrated by the results of coimmunoprecipitation and immunofluorescence assays. An investigation to assess the possible relationship between autophagy and G2/M arrest showed that, similar to the autophagy inhibitor 3MA, Beclin 1 knockdown delayed IR-induced G2/M arrest. Furthermore, the interactions between Beclin 1 and several G2/M checkpoint-related proteins, namely, PLK1 and CDC25C, were observed to increase. In addition, we observed that both 3MA and Beclin 1 inhibition decreased IR-induced apoptosis. Regarding the potential mechanism associated with this phenomenon, we showed that IR induced the interaction between Beclin 1 and Tip60 as well as their redistribution. Furthermore, we demonstrated that Beclin 1 T57 may be a targeted phosphorylation site for ATM. CONCLUSIONS: In the present study, we demonstrate the crucial and intricate roles of Beclin 1 in IR-induced autophagy, G2/M cell cycle arrest, and apoptosis. Additionally, Tip60 and ATM were identified as important molecular regulators of Beclin 1. Our findings show the precise mechanism of crosstalk between IR-induced autophagy and G2/M cell cycle arrest.

Beclin1 inhibition enhances paclitaxel­mediated cytotoxicity in breast cancer in vitro and in vivo.

Beclin1, a key regulator of autophagy, has been demonstrated to be associated with cancer cell resistance to chemotherapy. Paclitaxel is a conventional chemotherapeutic drug used in the clinical treatment of breast cancer. However, the function and mechanism of Beclin1 in paclitaxel­mediated cytotoxicity in breast cancer are not well defined. The present study demonstrated that paclitaxel suppressed cell viability and Beclin1 expression levels in BT­474 breast cancer cells in a dose­ and time­dependent fashion. Compared with the control, the knockdown of Beclin1 significantly enhanced breast cancer cell death via the induction of caspase­dependent apoptosis following paclitaxel treatment in vitro (P<0.05). In a BT­474 xenograft model, paclitaxel achieved substantial inhibition of tumor growth in the Beclin1 knockdown group compared with the control group. Furthermore, analysis of the publicly available Gene Expression Omnibus datasets revealed a clinical correlation between Beclin1 levels and the response to paclitaxel therapy in patients with breast cancer. Collectively, the present results suggest that Beclin1 protects breast cancer cells from apoptotic death. Thus, the inhibition of Beclin1 may be a novel way to improve the effect of paclitaxel. Additionally, Beclin1 may function as a favorable prognostic biomarker for paclitaxel treatment in patients with breast cancer.

VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy.

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l-cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS-ER stress-autophagy axis in the vascular endothelial cells.

Oncogene SRSF3 suppresses autophagy via inhibiting BECN1 expression.

Autophagy is an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been demonstrated to be associated with many human diseases, including cancer. Alternative splicing of pre-mRNA is also an evolutionarily conserved regulatory mechanism of gene expression. Dysregulation of alternative splicing is increasingly linked to cancer. However, the association between these two cellular conserved processes is unclear. Splicing factors are critical players in the regulation of alternative splicing of pre-mRNA. We analyzed the expression of 28 splicing factors during hypoxia-induced autophagy in three oral squamous cell carcinoma (OSCC) cell lines. We discovered that oncogenes SRSF3 and SRSF1 are significantly downregulated in all three cell lines. Moreover, knockdown of SRSF3 increased autophagic activity, whereas overexpression of SRSF3 inhibited hypoxia-induced autophagy. Loss-of-function and gain-of-function assays also showed that SRSF3 inhibits the expression of p65 and FoxO1 and their downstream target gene BECN1, a key regulator of autophagy. Our results demonstrated that splicing factor SRSF3 is an autophagy suppressor.

Autophagy Activation in Asthma Airways Remodeling.

Current asthma therapies fail to target airway remodeling that correlates with asthma severity driving disease progression that ultimately leads to loss of lung function. Macroautophagy (hereinafter "autophagy") is a fundamental cell-recycling mechanism in all eukaryotic cells; emerging evidence suggests that it is dysregulated in asthma. We investigated the interrelationship between autophagy and airway remodeling and assessed preclinical efficacy of a known autophagy inhibitor in murine models of asthma. Human asthmatic and nonasthmatic lung tissues were histologically evaluated and were immunostained for key autophagy markers. The percentage area of positive staining was quantified in the epithelium and airway smooth muscle bundles using ImageJ software. Furthermore, the autophagy inhibitor chloroquine was tested intranasally in prophylactic (3 wk) and treatment (5 wk) models of allergic asthma in mice. Human asthmatic tissues showed greater tissue inflammation and demonstrated hallmark features of airway remodeling, displaying thickened epithelium (P < 0.001) and reticular basement membrane (P < 0.0001), greater lamina propria depth (P < 0.005), and increased airway smooth muscle bundles (P < 0.001) with higher expression of Beclin-1 (P < 0.01) and ATG5 (autophagy-related gene 5) (P < 0.05) together with reduced p62 (P < 0.05) compared with nonasthmatic control tissues. Beclin-1 expression was significantly higher in asthmatic epithelium and ciliated cells (P < 0.05), suggesting a potential role of ciliophagy in asthma. Murine asthma models demonstrated effective preclinical efficacy (reduced key features of allergic asthma: airway inflammation, airway hyperresponsiveness, and airway remodeling) of the autophagy inhibitor chloroquine. Our data demonstrate cell context-dependent and selective activation of autophagy in structural cells in asthma. Furthermore, this pathway can be effectively targeted to ameliorate airway remodeling in asthma.

Beclin1 overexpression suppresses tumor cell proliferation and survival via an autophagy­dependent pathway in human synovial sarcoma cells.

Beclin1 is an important autophagy­related prot-ein, which is involved in both autophagy and apoptosis. In recent years, the antitumor effect of Beclin1 has received increased attention. In the present study, we established a stable Beclin1­overexpressing cell line with SW982 human synovial sarcoma cells. We found that Beclin1 overexpression decreased the cell viability, inhibited proliferation and induced apoptosis in SW982 cells. The expression levels of Bcl­2 and PCNA were decreased, while the levels of cleaved­caspase­3 and cleaved­PARP were increased. Beclin1 is closely related with autophagy, thus the autophagy­related markers LC3 and p62 were detected by western blot analysis, and transmission electron microscopy was used to observe autophagosomes. The results showed that the expression level of LC3II was increased and that of p62 was decreased. Moreover, many double membrane­enclosed autophagosomes were found in cells with Beclin1 overexpression, which indicated that the autophagic activity was enhanced. To explore the effect of autophagy on the viability of SW982 cells, Atg5 was knocked down using siRNA to inhibit the autophagic activity. We found that autophagy contributed to the decrease in cell viability. Knockdown of Atg5 increased the viability and decreased the apoptotic rate of SW982 cells with Beclin1 overexpression. The expression level of Bcl­2 was increased, while the expression levels of cleaved­caspase­3 and cleaved­PARP were decreased. We also found that the Akt/Bcl­2/caspase­9 pathway was involved. The phosphorylation of AKT was positively correlated with cell viability. The cleavage of caspase­9 was increased by Beclin1 overexpression and decreased by inhibition of autophagy. Altogether, our results suggested that both autophagy and apoptosis contributed to the antitumor effect of Beclin1 in SW982 cells.

Nano-sized Al2O3 particle-induced autophagy reduces osteolysis in aseptic loosening of total hip arthroplasty by negative feedback regulation of RANKL expression in fibroblasts.

Aseptic loosening is mainly caused by wear debris generated by friction that can increase the expression of receptor activation of nuclear factor (NF)-κB (RANKL). RANKL has been shown to support the differentiation and maturation of osteoclasts. Although autophagy is a key metabolic pathway for maintaining the metabolic homeostasis of cells, no study has determined whether autophagy induced by Al2O3 particles is involved in the pathogenesis of aseptic loosening. The aim of this study was to evaluate RANKL levels in patients experiencing aseptic loosening after total hip arthroplasty (THA) and hip osteoarthritis (hOA) and to consequently clarify the relationship between RANKL and LC3II expression. We determined the levels of RANKL and autophagy in fibroblasts treated with Al2O3 particles in vitro while using shBECN-1 interference lentivirus vectors to block the autophagy pathway and BECN-1 overexpression lentivirus vectors to promote autophagy. We established a novel rat model of femoral head replacement and analyzed the effects of Al2O3 particles on autophagy levels and RANKL expression in synovial tissues in vivo. The RANKL levels in the revision total hip arthroplasty (rTHA) group were higher than those in the hOA group. In patients with rTHA with a ceramic interface, LC3II expression was high, whereas RANKL expression was low. The in vitro results showed that Al2O3 particles promoted fibroblast autophagy in a time- and dose-dependent manner and that RANKL expression was negatively correlated with autophagy. The in vivo results further confirmed these findings. Al2O3 particles induced fibroblast autophagy, which reduced RANKL expression. Decreasing the autophagy level promoted osteolysis and aseptic prosthetic loosening, whereas increasing the autophagy level reversed this trend.

Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence.

Cancer recurrence after initial diagnosis and treatment is a major cause of breast cancer (BC) mortality, which results from the metastatic outbreak of dormant tumour cells. Alterations in the tumour microenvironment can trigger signalling pathways in dormant cells leading to their proliferation. However, processes involved in the initial and the long-term survival of disseminated dormant BC cells remain largely unknown. Here we show that autophagy is a critical mechanism for the survival of disseminated dormant BC cells. Pharmacologic or genetic inhibition of autophagy in dormant BC cells results in significantly decreased cell survival and metastatic burden in mouse and human 3D in vitro and in vivo preclinical models of dormancy. In vivo experiments identify autophagy gene autophagy-related 7 (ATG7) to be essential for autophagy activation. Mechanistically, inhibition of the autophagic flux in dormant BC cells leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis.

The mTOR Kinase Inhibitor CZ415 Inhibits Human Papillary Thyroid Carcinoma Cell Growth.

BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) plays an important role in papillary thyroid carcinoma (PTC) cell progression. CZ415 is a novel, highly-efficient and specific mTOR kinase inhibitor. The current study tested the potential anti-tumor activity of CZ415 in human PTC cells. METHODS: The established (TPC-1 cell line) and primary human PTC cells were treated with CZ415. Cell survival and growth were tested by Cell Counting Kit-8 assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by caspase-3/-9 activity assay, Hoechst-33342 staining assay and single-stranded DNA ELISA assay. Cell cycle progression was tested by propidium iodide-FACS assay. The mTOR signaling was tested by Western blotting assay and co-immunoprecipitation assay. The mouse xenograft tumor model was applied to study the effect of CZ415 in vivo. RESULTS: In cultured human PTC cells, treatment with CZ415 at nM concentrations significantly inhibited cell survival and growth. CZ415 induced apoptosis activation and cell cycle arrest in human PTC cells. CZ415 disrupted assembling of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-GßL association) in TPC-1 cells, which led to de-phosphorylation of the mTORC1 substrates (S6K1 and 4E-BP1) and the mTORC2 substrate AKT (Ser-473). Further studies show that the autophagy inhibitor 3-methyladenine (3-MA) or Beclin-1 shRNA aggravated CZ415-induced cytotoxicity against PTC cells. In vivo, CZ415 oral administration inhibited TPC-1 xenograft tumor growth in mice. CONCLUSION: Our results show that mTOR blockage by CZ415 inhibits PTC cell growth in vitro and in vivo.