Knockdown Of ATG5 Research Articles

ATG7 upregulation contributes to malignant transformation of human bronchial epithelial cells by B[a]PDE via DNMT3B protein degradation and miR-494 promoter methylation

Lung cancer primarily arises from exposure to various environmental factors, particularly airborne pollutants. Among the various lung carcinogens, benzo(a)pyrene and its metabolite B[a]PDE are the strongest ones that actively contribute to lung cancer development. ATG7 is an E1-like activating enzyme and contributes to activating autophagic responses in mammal cells. However, the potential alterations of ATG7 and its role in B[a]PDE-caused lung carcinogenesis remain unknown. Here, we found that B[a]PDE exposure promoted ATG7 expression in mouse lung tissues, while B[a]PDE exposure resulted in ATG7 induction in human normal bronchial epithelial cells. Our studies also demonstrated a significant correlation between high ATG7 expression levels and poor overall survival in lung cancer patients. ATG7 knockdown significantly repressed Beas-2B cell transformation upon B[a]PDE exposure, and such promotive effect of ATG7 on cell transformation mediated the p27 translation inhibition. Further studies revealed that miR-373 inhibition was required to stabilize ATG7 mRNA, therefore increasing ATG7 expression following B[a]PDE exposure, while ATG7 induction led to the autophagic degradation of the DNA methyltransferase 3 Beta (DNMT3B) protein, in turn promoted miR-494 transcription via its promoter region methylation status suppression. We also found that the miR-494 upregulation inhibited p27 protein translation and promoted bronchial epithelial cell transformation via its directly targeting p27 mRNA 3′-UTR region. Current studies, to the best of our knowledge, are for the first time to identify that ATG7 induction and its mediated autophagy is critical for B[a]PDE-induced transformation of human normal epithelial cells.

Escin enhanced the efficacy of sorafenib by autophagy-mediated apoptosis in lung cancer cells.

Combining anti-cancer drugs has been exploited as promising treatment strategy to target lung cancer. Synergistic chemotherapies increase anti-cancer effect and reduce effective drug doses and side effects. In this study, therapeutic potential of escin in combination with sorafenib has been explored. 3-(4,5-Dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide assay was used to calculate IC50 values. The synergy was evaluated using Chou-Talaly algorithm. Cellular reactive oxygen species, mitochondrial membrane potential, annexin V, and cell-cycle studies were done by flow-cytometer, and autophagy biomarkers expression were determined using western blotting. Moreover, autophagy was knocked down using ATG5 siRNA to confirm its role, diethylnitrosamine-induced lung cancer model was used to check the synergy of sorafenib/escin. Escin significantly reduced the IC50 of sorafenib in A549 and NCIH460 cells. The combination of sorafenib/escin produced a 2.95 and 5.45 dose reduction index for sorafenib in A549 and NCI-H460 cells. The combination of over-expressed p62 and LC3-II reflects autophagy block-mediated late apoptosis. This phenomenon was reconfirmed by ATG5 knockdown. This combination also selectively targeted G0/G1 phase of cancer cells. In in vivo study, the combination reduced tumour load and lower elevated serum biochemical parameters. The combination of sorafenib/escin synergistically inhibits autophagy to induce late apoptosis in lung cancer cells' G0/G1 phase.

Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Intriguingly, KRAS-mutant cancers are particularly vulnerable to ferroptosis. Osthole is a natural coumarin extracted from Cnidium spp. and other Apiaceous plants. In the present study, we explored the antitumor potential of osthole in KRAS-mutant colorectal cancer (CRC) cells. Cell viability assay, EdU incorporation assay, flow cytometry, tumor xenograft model, western blot, immunochemistry staining, immunofluorescence, transcriptome RNA sequencing and quantitative reverse transcription-PCR were performed to evaluate the influence of osthole treatment on KRAS-mutant CRC cells. We found that osthole treatment suppressed proliferation and tumor growth of KRAS-mutant CRC cell lines HCT116 and SW480. Moreover, osthole treatment increased ROS production and induced ferroptosis. Osthole treatment also promoted autophagy, but inhibition of autophagy by ATG7 knockdown or 3-MA showed no influence on osthole-induced ferroptosis. In comparison, osthole increased lysosomal activation, and co-treatment with lysosome inhibitor Baf-A1 attenuated osthole-induced ferroptosis. Besides, osthole treatment reduced the phosphorylation of AMPK, Akt and mTOR in HCT116 and SW480 cells, while restored AMPK signaling by AMPK agonist AICAR partially abrogated ferroptosis induced by osthole treatment. Finally, co-treatment with osthole increased the cytotoxicity of cetuximab in KRAS-mutant CRC cells in vitro and in vivo. Our results suggested that the natural product osthole exerted its anticancer effects in KRAS-mutant CRC cells via inducing ferroptosis, and this was partially through inhibiting AMPK/Akt/mTOR signaling. Our results may expand our current knowledge for the use of osthole as an anticancer agent.

Chloroquine enhances the efficacy of chemotherapy drugs against acute myeloid leukemia by inactivating the autophagy pathway.

Current therapy for acute myeloid leukemia (AML) is largely hindered by the development of drug resistance of commonly used chemotherapy drugs, including cytarabine, daunorubicin, and idarubicin. In this study, we investigated the molecular mechanisms underlying the chemotherapy drug resistance and potential strategy to improve the efficacy of these drugs against AML. By analyzing data from ex vivo drug-response and multi-omics profiling public data for AML, we identified autophagy activation as a potential target in chemotherapy-resistant patients. In THP-1 and MV-4-11 cell lines, knockdown of autophagy-regulated genes ATG5 or MAP1LC3B significantly enhanced AML cell sensitivity to the chemotherapy drugs cytarabine, daunorubicin, and idarubicin. In silico screening, we found that chloroquine phosphate mimicked autophagy inactivation. We showed that chloroquine phosphate dose-dependently down-regulated the autophagy pathway in MV-4-11 cells. Furthermore, chloroquine phosphate exerted a synergistic antitumor effect with the chemotherapy drugs in vitro and in vivo. These results highlight autophagy activation as a drug resistance mechanism and the combination therapy of chloroquine phosphate and chemotherapy drugs can enhance anti-AML efficacy.

Induction of autophagy by oleifolioside A in HCT-116 human colorectal cancer cells

In current study, we addressed the anti-cancer effect of oleifolioside A and its mechanism on the regulation of cell death in HCT-116 human colorectal cancer cells. Oleifolioside A inhibited HCT-116 cell proliferation and caused apoptosis associated with sequential activation of caspases 8 and 3, followed by PARP cleavage. Moreover, anti-LC3-positive granules and the increased LC3-II level were observed in HCT-116 cells treated with oleifolioside A, which is the specific characteristics of autophagy. Treatment of autophagy inhibiors, 3-MA and Wort, markedly accelerated the cell death by oleifolioside A and, furthermore, knockdown of Beclin-1 and Atg7 using shRNA increased oleifolioside A-induced apoptosis, suggesting a cytoprotective function of autophagy against oleifolioside A-triggered apoptosis. Treatment of HCT-116 cells with oleifolioside A time-dependently activated extracellular signal-regulated kinase (ERK). Oleifolioside A-induced autophagy was dramatically inhibited by pretreatment with an ERK inhibitor, U0126, which resulted in a marked reduction in cell viability. These findings indicate that oleifolioside A induce autophagy through ERK activation in HCT-116 cells and that autophagy suppression enhances apoptosis induced by oleifolioside A.

Peroxisome proliferator-activated receptor γ activation ameliorates liver fibrosis-differential action of transcription factor EB and autophagy on hepatocytes and stellate cells.

Peroxisome proliferator-activated receptor γ (PPARγ) activation suppresses HSC activation and liver fibrosis. Moreover, autophagy is implicated in hepatic lipid metabolism. Here, we determined whether PPARγ activation ameliorates HSC activation by downregulating transcription factor EB (TFEB)-mediated autophagy. Atg7 or Tfeb knockdown in human HSC line LX-2 cells downregulated the expression of fibrogenic markers including α smooth muscle actin, glial fibrillary acidic protein, and collagen type 1. Conversely, Atg7 or Tfeb overexpression upregulated fibrogenic marker expression. Rosiglitazone (RGZ)-mediated PPARγ activation and/or overexpression in LX-2 cells and primary HSCs decreased autophagy, as indicated by LC3B conversion, total and nuclear-TFEB contents, mRFP-LC3 and BODIPY 493/503 colocalization, and GFP-LC3 and LysoTracker colocalization. RGZ treatment decreased liver fat content, liver enzyme levels, and fibrogenic marker expression in high-fat high-cholesterol diet-fed mice. Electron microscopy showed that RGZ treatment restored the high-fat high-cholesterol diet-mediated lipid droplet decrease and autophagic vesicle induction in primary HSCs and liver tissues. However, TFEB overexpression in LX-2 cells offset the aforementioned effects of RGZ on autophagic flux, lipid droplets, and fibrogenic marker expression. Activation of PPARγ with RGZ ameliorated liver fibrosis and downregulation of TFEB and autophagy in HSCs may be important for the antifibrotic effects of PPARγ activation.

Melatonin protects against developmental PBDE-47 neurotoxicity by targeting the AMPK/mitophagy axis.

The neurotoxicity of 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) is closely linked to mitochondrial abnormalities while mitophagy is vital for mitochondrial homeostasis. However, whether PBDE-47 disrupts mitophagy contributing to impaired neurodevelopment remain elusive. Here, this study showed that neonatal PBDE-47 exposure caused learning and memory deficits in adult rats, accompanied with striatal mitochondrial abnormalities, neuronal apoptosis and the resultant neuronal loss. Mechanistically, PBDE-47 suppressed PINK1/Parkin-mediated mitophagy induction and degradation, inducing mitophagosome accumulation and mitochondrial dysfunction in vivo and in vitro. Additionally, stimulation of mitophagy by adenovirus-mediated Parkin or Autophagy-related protein 7 (Atg7) overexpression aggravated PBDE-47-induced mitophagosome accumulation, mitochondrial dysfunction, neuronal apoptosis and death. Conversely, suppression of mitophagy by the siRNA knockdown of Atg7 rescued PBDE-47-induced detrimental consequences. Importantly, melatonin, a hormone secreted rhythmically by the pineal, improved PBDE-47-caused neurotoxicity via preventing neuronal apoptosis and loss by restoring mitophagic activity and mitochondrial function. These neuroprotective effects of melatonin depended on activation of the AMP-activated protein kinase (AMPK)/Unc-51-like kinase 1 (ULK1) signaling. Collectively, these data indicate that PBDE-47 impairs mitophagy to perturb mitochondrial homeostasis, thus triggering apoptosis, leading to neuronal loss and consequent neurobehavioral deficits. Manipulation of the AMPK-mitophagy axis via melatonin could be a novel therapeutic strategy against developmental PBDE-47 neurotoxicity.

Pretreatment of 3-MA prevents doxorubicin-induced cardiotoxicity through inhibition of autophagy initiation

Anthracycline antineoplastics are effective in the treatment of hematological malignancies and solid tumors. However, the anthracycline-induced cardiotoxicity (AIC) limits their use as chemotherapeutic agents. Autophagy-based therapies have been explored to prevent AIC. Yet, whether inhibition of autophagy during its early stage could alleviate AIC remains unclear. In this study, we firstly observed the activation of autophagy during AIC in both cardiomyocyte cell lines AC16 and H9c2. Moreover, knockdown of Atg7, a key regulatory factor in early autophagy, could ameliorate the effects of DOX-induced AIC. Importantly, the use of early autophagy inhibitor 3-MA protected cardiomyocyte cells from DOX-induced cardiotoxicity in vitro and in a chronic AIC mouse model. Our findings demonstrate that inhibiting early stage of autophagy may be an effective preventative therapeutic strategy to protect cardiac function from AIC.

Autophagy restricts Mycobacterium tuberculosis during acute infection in mice.

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

5613327 KNOCK-DOWN OF AUTOPHAGY-RELATED GENE 5 (ATG5) EXPRESSION IN THALASSEMIC CD34+ CELLS LEADS TO AMELIORATION OF ERYTHROPOIESIS

Background: β-thalassemia is characterized by ineffective erythropoiesis due to increased apoptosis of the thalassemic erythroid precursors. Recent studies documented enhanced initiation of autophagy in β-thalassemia erythroblasts, which may contribute to the elevated levels of apoptosis.1,2 Therefore, genetic manipulation of key genes of autophagy employing shRNA technology, could improve the pathophysiology of the disease. Aim: To this end, our study aimed to investigate the role of ATG5 in autophagy and/or apoptosis in β-thalassemia, by downregulating its expression via shRNA technology, and potentially exploit this effect for the amelioration of the disease pathophysiology. Materials and Methods: Lentiviral vectors (LVs), harboring three distinct shRNAs (sh11840, sh11841 and sh11842) targeting discrete regions of endogenous ATG5 mRNA, were used to transduce K562 cells at a multiplicity of infection (MOI) 25. A lentiviral vector carrying a shRNA against no human gene (shNT; No Target), was used as a control. Flow cytometry and confocal microscopy using an ATG5-specific antibody were employed to determine ATG5 expression at the protein level, while qPCR assessed ATG5 knock-down at the mRNA level. The sh11842 LV exhibited the highest ATG5 knock-down and was selected for in vitro assessment in thalassemic cells, following initial transduction of CD34+ cells at MOI 25, for a differentiation time period to include the early autophagy activation at basophilic stage. Autophagic flux in untransduced, shNT-transduced and shATG5-transduced CD34+ cells, was assessed using confocal microscopy to determine the level of the LC3/LAMP co-localization, markers of autophagosomes and lysosomes, while flow cytometry was employed to determine erythroid differentiation and apoptosis in the differentiated cells. Results: Transduction of K562 cells with the sh11842 LV led to the highest ATG5 knock-down, reaching 93% and 38% at mRNA and protein level, respectively (n=4). Transduction of thalassemic CD34+ cells with the sh11842 LV at day 6 of differentiation showed advanced erythropoiesis compared to shNT-transduced cells. Specifically, the percentage of basophilic erythroblasts was significantly reduced following sh11842-transduction as compared to shNT-transduction, (43.5 ± 4.5% vs 59.6 ± 8.1%, p=0.0127, n=4, respectively), leading to a transient increase of 28.9 ± 5.9% in polychromatic erythroblasts, compared to 16.3 ± 3.7% (p=0.0118, n=4) in shNT-transduced cells. As a result of this accelerated erythropoiesis, the percentage of orthochromatic erythroblasts increased to 5.5 ± 2.4%, compared to 2.8 ± 1.7% observed in shNT-transduced cells (p=0.199, n=4). This was followed by a significant reduction of LC3/LAMP co-localization in sh11842-transduced cells, reaching a Pearson’s correlation coefficient of 0.103 ± 0.29%, compared to 0.165 ± 0.4% seen in shNT-transduced and 0.372 in untransduced cells (p<0.0001). Furthermore, no differences in apoptosis were observed between sh11842- and shNT- transduced cells (59.0 ± 18.2% vs 50.3 ± 8.6%, p=0.6618 n=3). Pilot studies indicate that transduction with sh11842 LV may lead to increased mitochondrial membrane potential, as demonstrated by increased Mitotracker® staining, suggesting a potential reduction in mitophagy, as well. Summary - Conclusion: Knock-down of ATG5 expression leads to improvement of the thalassemic erythropoiesis in vitro, highlighting its potential synergistic role in the amelioration of thalassemic pathophysiology.

Berberine Induces Combined Cell Death in Gastrointestinal Cell Lines.

Berberine (BBR) is a plant alkaloid that has various biological activities. The effects of BBR on gastrointestinal cancer (GIC) have also been investigated and anti-tumor effects such as induction of cell death have been reported. However, the mechanism of BBR-induced cell death has not been fully elucidated. To this end, we investigated the effects of BBR using three GIC cell lines. Our analyses revealed that BBR inhibited cell proliferation, invasion, sphere formation, and anticancer drug resistance in all of the cell lines. BBR also induced an increase in mitochondrial superoxide, lipid peroxide and Fe2+ levels, decreased mitochondrial membrane potential and respiration, decreased glutathione peroxidase 4 expression and glutathione and induced Parkin/PINK1-associated mitophagy. BBR, as well as rotenone, inhibited mitochondrial complex I and enhanced complex II, which were associated with autophagy, reactive oxidative species production, and cell death. Inhibition of complex II by malonate abrogated these changes. BBR-induced cell death was partially rescued by ferrostatin-1, deferoxamine, Z-VAD-FMK, and ATG5 knockdown. Furthermore, oral administration of BBR significantly reduced tumor weight and ascites in a syngeneic mouse peritoneal metastasis model using CT26 GIC cells. These findings suggest that BBR induced a combined type of cell death via complex I inhibition and autophagy. The marked anti-tumor and anti-stemness effects are expected to be useful as a new cell death-inducing agent for the treatment of GIC.

Deficiency of lipopolysaccharide binding protein facilitates adipose browning, glucose uptake and oxygen consumption in mouse embryonic fibroblasts via activating PI3K/Akt/mTOR pathway and inhibiting autophagy

ABSTRACT This study aimed to explore the role of lipopolysaccharide-binding protein (LBP) in adipose browning. Mouse embryonic fibroblasts (MEFs) were treated with differentiation induction reagents and Perifosine (Akt inhibitor), with the transfection of Atg5, short hairpin RNA targeting LBP (shLBP), and Atg5 (shAtg5). The expression levels of LBP, inflammatory markers , brown fat markers, lipid metabolism marker, autophagy markers, insulin signaling-related molecules , p-mTOR, mTOR, p-Akt, Akt, p-PI3K, and PI3K were quantified or determined by Western blot, qRT-PCR, and immunofluorescence assay. The formation of lipid was examined through Oil red O staining assay. The consumption of oxygen was assessed using a Seahorse XF96 analyzer, and the uptake of glucose was evaluated by [3H]-2-deoxy-D-glucose uptake assay. Deficiency of LBP promoted adipose browning, oxygen consumption, glucose uptake, and insulin sensitivity in differentiated MEFs, where it inhibited inflammation and autophagy. All of the effects above were reversed by Atg5 overexpression. Meanwhile, the knockdown of Atg5 strengthened the activation of PI3K/Akt/mTOR pathway induced by the depletion of LBP, while Perifosine partly reversed the activation of differentiated MEFs. The knockdown of LBP facilitated adipose browning, glucose uptake, and oxygen consumption in MEFs via the activation of PI3K/Akt/mTOR pathway and the inhibition of autophagy.

PS-B01-4: ANGIOTENSIN II TYPE1 RECEPTOR INHIBITION IMPROVES VASCULAR SENESCENCE THROUGH IMPROVEMENT OF MITOCHONDRIAL QUALITY CONTROL VIA CRAF/MEK/ERK PATHWAY

Introduction: Prevention of atherosclerotic cardiovascular disease has a problem of residual risk that is insufficient even if lipids are properly lowered by drugs such as statin. It has been reported that lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) associated with Angiotensin II Type 1 Receptor (AT1R). Mitochondria play crucial role in vascular senescence and atherosclerosis. We hypothesized receptor association with LOX-1 and AT1R might be a breakthrough to solve residual risk in terms of mitochondrial quality control. Methods: We performed in vitro and in vivo experiments using vascular smooth muscle cell (VSMC), C57BL6 (WT) and apolipoprotein E deficient (ApoE KO) mice. Results: Administration of ox-LDL to VSMC induced mitochondrial dysfunction and cellular senescence accompanied by excessive mitochondrial fission through Drp1 activation. This Drp1 activation was derived from the activation of CRAF/MEK/ERK pathway, which is a downstream of AT1R. AT1R inhibition inactivated ox-LDL-mediated Drp1 activation and retarded mitochondrial dysfunction, excessive mitochondrial fission and cellular senescence, indicating that LDL receptor LOX-1 associates with AT1R and activates CRAF/MEK/ERK/Drp1, leading to induction of mitochondrial fission and resulting in facilitating cellular senescence. AT1R inhibition also induced mitophagy assessed by electron microscopy and immunohistochemistry of LAMP2 and TOMM20. AT1R inhibition-induced mitophagy was not affected by Atg7 knockdown, whereas it was diminished by Rab9 knockdown. Immunohistochemistry showed TOMM20 dots were co-localized to LAMP2 and Rab9 but not LC3. These results suggest that AT1R signal induces mitophagy derived from Rab9-dependent alternative autophagy. Moreover, inhibition of either CRAF or MEK also decreased excessive mitochondrial fission and induced mitophagy, suggesting that AT1R signal followed by CRAF/MEK pathway modulates both mitochondrial dynamics and mitophagy. Taken together, inhibition of AT1R induces dual modification of mitochondrial dynamics and mitophagy through inactivation of CRAF/MEK/ERK axis. Finally, we confirmed that the degree of vascular senescence and the number of fragmented mitochondria were higher in ApoE KO than those of WT mice, and treatment with Candesartan to ApoE KO inhibited vascular senescence through induction of mitophagy and imbalance of mitochondrial dynamics. Conclusion: Receptor association of LOX-1 with AT1R plays a crucial role in cellular/vascular senescence induced by ox-LDL, and AT1R inhibition improves mitochondrial quality control through regulation of mitochondrial dynamics and mitophagy derived from Rab9-dependent alternative autophagy.

Methyltransferase-like 3 suppresses phenotypic switching of vascular smooth muscle cells by activating autophagosome formation.

Prevention of neointima formation is the key to improving long-term outcomes after stenting or coronary artery bypass grafting. RNA N6 -methyladenosine (m6 A) methylation has been reported to be involved in the development of various cardiovascular diseases, but whether it has a regulatory effect on neointima formation is unknown. Herein, we revealed that methyltransferase-like 3 (METTL3), the major methyltransferase of m6 A methylation, was downregulated during vascular smooth muscle cell (VSMC) proliferation and neointima formation. Knockdown of METTL3 facilitated, while overexpression of METTL3 suppressed the proliferation of human aortic smooth muscle cells (HASMCs) by arresting HASMCs at G2/M checkpoint and the phosphorylation of CDC2 (p-CDC2) was inactivated by METTL3. On the other hand, the migration and synthetic phenotype of HASMCs were enhanced by METTL3 knockdown, but inhibited by METTL3 overexpression. The protein levels of matrix metalloproteinase 2 (MMP2), MMP7 and MMP9 were reduced, while the expression level of tissue inhibitor of metalloproteinase 3 was increased in HASMCs with METTL3 overexpression. Moreover, METTL3 promoted the autophagosome formation by upregulating the expression of ATG5 (autophagy-related 5) and ATG7. Knockdown of either ATG5 or ATG7 largely reversed the regulatory effects of METTL3 overexpression on phenotypic switching of HASMCs, as evidenced by increased proliferation and migration, and predisposed to synthetic phenotype. These results indicate that METTL3 inhibits the phenotypic switching of VSMCs by positively regulating ATG5-mediated and ATG7-mediated autophagosome formation. Thus, enhancing the level of RNA m6 A or the formation of autophagosomes is the promising strategy to delay neointima formation.

Hernandezine induces autophagic cell death in human pancreatic cancer cells via activation of the ROS/AMPK signaling pathway

Hernandezine (Her) is a bisbenzylisoquinoline alkaloid extracted from the traditional Chinese herbal medicine Thalictrum glandulosissimum. Evidence shows that Her is a natural agonist of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and induces apoptosis and autophagy in tumor cells. In this study, we investigated the role of autophagy in Her-induced cell death in human pancreatic cancer cell lines. We showed that Her dose-dependently suppressed cell proliferation, promoted autophagy and induced autophagic death in pancreatic ductal adenocarcinoma (PDAC) cell lines Capan-1 and SW1990. The IC50 values of Her in inhibition of Capan-1 and SW1990 cells were 47.7 μM and 40.1 μM, respectively. Immunoblotting showed that Her (1−40 μM) promoted the conversion of LC3-I to LC3-II, and Her exerted concentration-dependent and time-dependent effects on autophagy activation in PDAC cells. In transmission electron microscopy and fluorescence image analysis, we found that autophagic vacuoles were significantly increased in Her-treated cells. Knockdown of ATG5, a key gene in the autophagy pathway, alleviated the activation of autophagy by Her. These results demonstrated that Her induced autophagy in PDAC cells. Intensely activated autophagy could promote cell death. The autophagy inhibitors, BafA1 and HCQ significantly inhibited Her-induced cell death, implying that Her induced autophagic cell death in PDAC cells. Moreover, we showed that Her activated autophagy by increasing the phosphorylation of AMPK and decreasing the phosphorylation of mTOR/p70S6K. Knockdown of AMPKα relieves the autophagic cell death induced by Her. Furthermore, Her concentration-dependently enhanced reactive oxygen species (ROS) generation in PDAC cells. Antioxidants could reduce the phosphorylation of AMPK and suppress autophagic cell death induced by Her. Our study provides evidence for the development of Her as a therapeutic agent for the treatment of pancreatic cancer.

Basal Gp78-dependent mitophagy promotes mitochondrial health and limits mitochondrial ROS

Mitochondria are major sources of cytotoxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, that when uncontrolled contribute to cancer progression. Maintaining a finely tuned, healthy mitochondrial population is essential for cellular homeostasis and survival. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating damaged ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. E3 ubiquitin ligase Gp78 is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume, elevated ROS production and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Xenograft tumors of HT-1080 knockout cells show increased labeling for mitochondria and the cell proliferation marker Ki67 and reduced labeling for the TUNEL cell death reporter. Basal Gp78-dependent mitophagic flux is, therefore, selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population, limiting ROS production and tumor cell proliferation.

Akt Inhibition Promotes Autophagy and Clearance of Group B Streptococcus from the Alveolar Epithelium.

Group B Streptococcus (GBS) is a gram-positive bacterium that is harmless for healthy individuals but may provoke invasive disease in young infants and immunocompromised hosts. GBS invades the epithelial barriers to enter the bloodstream, and thus strategies that enhance epithelial cell responses may hamper GBS invasion. In the present study, we sought to investigate whether the inhibition of Akt, a kinase that regulates host inflammatory responses and autophagy via suppression of mTOR, can enhance the response of non-phagocytic alveolar epithelial cells against GBS. Treatment of the alveolar epithelial cell line A549 with the Akt inhibitor MK-2206 resulted in the enhanced production of reactive oxygen species and inflammatory mediators in response to GBS. Additionally, Akt inhibition via MK-2206 resulted in elevated LC3II/I ratios and increased autophagic flux in alveolar epithelial cells. Importantly, the inhibition of Akt promoted GBS clearance both in alveolar epithelial cells in vitro and in lung tissue in vivo in a murine model of GBS pneumonia. The induction of autophagy was essential for GBS clearance in MK-2206 treated cells, as knockdown of ATG5, a critical component of autophagy, abrogated the effect of Akt inhibition on GBS clearance. Our findings highlight the role of Akt kinase inhibition in promoting autophagy and GBS clearance in the alveolar epithelium. The inhibition of Akt may serve as a promising measure to strengthen epithelial barriers and prevent GBS invasion in susceptible hosts.

Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication

ABSTRACT Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.

Transient Systemic Autophagy Inhibition Is Selectively and Irreversibly Deleterious to Lung Cancer.

Transient loss of systemic autophagy causes irreversible damage to tumors by suppressing cancer cell metabolism and promoting antitumor immunity, supporting autophagy inhibition as a rational strategy for treating lung cancer. See related commentary by Gan, p. 4322.

Autophagy inhibition restores CD200 expression under IL-1β microenvironment in placental mesenchymal stem cells of fetal origin and improves its pulmonary fibrosis therapeutic potential

BackgroundMesenchymal stem cells (MSCs) are promising remedies for various inflammatory disease including pulmonary fibrosis (PF). However, the properties of MSCs in PF pathological microenvironment remain unclear. In this study, the efficacy of autophagy in placental mesenchymal stem cells of fetal origin (fPMSCs) in either IL-1β treatment or BLM induced pulmonary fibrosis mice model was examined. MethodsThe characteristic of fPMSCs was identified by morphological observation, flow cytometry and differentiation potential. In vitro experiments, fPMSCs were stimulated with IL-1β, to mimic inflammatory microenvironment of pulmonary fibrosis. The immunosuppressive properties and autophagic function in fPMSCs treated with IL-1β were evaluated by both macrophage cells THP-1 activation and the expression of CD200 situation, autophagy marker and MAPK signaling pathway. The in vivo anti-fibrotic activity of fPMSCs interfering autophagy was evaluated by using BLM induced pulmonary fibrosis mice model. ResultsfPMSCs belonged to CD73+CD90+CD105+/CD14- CD34-CD45-HLA-DR- cells, and capable differentiation to adipogenic, osteogenic and chondrogenic cells. In addition, immunoinhibitory activity of fPMSCs for macrophage was restrained by IL-1β treatment in CD200 dependent manner. Suppression of autophagy by sh-Atg5 lentivirus increased the expression of CD200 and ratio of CD200 positive fPMSCs, and enhanced fPMSCs immunosuppression for THP-1 activation. Mechanistically, IL-1β induced autophagy regulated by p38 signaling cascade. In vivo, autophagy inhibition induced by Atg5 knockdown in fPMSCs resulted in strengthening antifibrotic effects on PF mice model. ConclusionsCollectively, autophagy derived from inflammatory microenvironment hampered the immunoinhibitory properties of MSCs. Based on this, adjustment of autophagy may be a valid approach to facilitate their immunomodulatory and anti-fibrotic efficacy.