90 results on '"Autophagy Protein 5"'
Search Results
2. Serum autophagy protein 5 is positively related to T helper 2/T helper 1 ratio, inflammation, and exacerbation in adult asthma patients.
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Ke, Changjiang and Xie, Sheng
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ASTHMATICS , *BLOOD proteins , *TH2 cells , *FORCED expiratory volume , *ENZYME-linked immunosorbent assay - Abstract
Background: Autophagy protein 5 (ATG5) regulates airway epithelial cell autophagy, immune response, and inflammation, which is involved in asthma progression. This study aimed to evaluate ATG5 levels and its clinical roles in adult asthma patients. Methods: Totally, 200 adult asthma patients and 100 healthy controls (HCs) were enrolled in this case-control study. Subsequently, serum ATG5 was measured by enzyme-linked immunosorbent assay. Results: ATG5 was increased in asthma patients compared with HCs [median (interquartile range): 44.2 (31.7–77.8) vs. 23.2 (16.7–39.2) ng/mL] (P < 0.001). In asthma patients, ATG5 was positively related to male gender (P = 0.022), a family history of asthma (P = 0.035), eosinophil count (P < 0.001), and immune globulin E (P < 0.001), while it was negatively correlated with forced expiratory volume in 1 s (FEV1)/forced vital capacity (P < 0.001) and FEV1 (Predicted) (P < 0.001). Meanwhile, ATG5 was inversely associated with T helper (Th) 1 cells (P = 0.008), while it was positively linked with Th2 cells (P < 0.001), Th2/Th1 ratio (P < 0.001), interleukin (IL)-4 (P = 0.002), and IL-4/interferon-γ ratio (P = 0.015). Additionally, ATG5 was positively correlated with tumor necrosis factor-α (P < 0.001), IL-1β (P = 0.001), IL-6 (P = 0.003), and IL-17 (P = 0.029). Notably, ATG5 was elevated in asthma patients at exacerbation compared to those at remission [median (interquartile range): 53.6 (37.6–90.0) vs. 35.6 (28.2–51.5) ng/mL] (P < 0.001). It was also noteworthy that ATG5 was positively linked with exacerbation severity in asthma patients (P = 0.005). Conclusion: Serum ATG5 is related to increased Th2/Th1 ratio, inflammation, exacerbation risk and severity in adult asthma patients, which serves as a candidate marker for the management of asthma. However, further validation is still needed. [ABSTRACT FROM AUTHOR]
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- 2023
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3. Anti-inflammatory effects of N-cyclooctyl-5-methylthiazol-2-amine hydrobromide on lipopolysaccharide-induced inflammatory response through attenuation of NLRP3 activation in microglial cells
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Kyouk Hwang, Jee-Yin Ahn, Sung-Woo Cho, Soo Young Choi, Ji-Eun Kim, Eun-A Kim, and Seung-Ju Yang
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Bromides ,Lipopolysaccharides ,Lipopolysaccharide ,Inflammasomes ,ATG5 ,Pro-inflammatory cytokines ,Anti-Inflammatory Agents ,Nitric Oxide ,Biochemistry ,Pyrin domain ,Article ,Nitric oxide ,Inflammasome ,chemistry.chemical_compound ,Mice ,NLRP3 ,NLR Family, Pyrin Domain-Containing 3 Protein ,Animals ,Molecular Biology ,chemistry.chemical_classification ,Inflammation ,Reactive oxygen species ,Tumor Necrosis Factor-alpha ,NF-kappa B ,General Medicine ,Macrophage Activation ,Cell biology ,chemistry ,Autophagy Protein 5 ,Cytokines ,Tumor necrosis factor alpha ,Microglia ,Lipid Peroxidation ,Signal transduction ,Reactive Oxygen Species ,N-cyclooctyl-5-methylthiazol-2-amine hydrobromide ,Signal Transduction - Abstract
Microglial activation is closely associated with neuroinflammatory pathologies. The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasomes are highly organized intracellular sensors of neuronal alarm signaling. NLRP3 inflammasomes activate nuclear factor kappa-B (NF-κB) and reactive oxygen species (ROS), which induce inflammatory responses. Moreover, NLRP3 dysfunction is a common feature of chronic inflammatory diseases. The present study investigated the effect of a novel thiazol derivative, N-cyclooctyl-5-methylthiazol-2-amine hydrobromide (KHG26700), on inflammatory responses in lipopolysaccharide (LPS)-treated BV-2 microglial cells. KHG26700 significantly attenuated the expression of several pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, in these cells, as well as the LPS-induced increases in NLRP3, NF-κB, and phospho-IkBα levels. KHG26700 also suppressed the LPS-induced increases in protein levels of autophagy protein 5 (ATG5), microtubule- associated protein 1 light chain 3 (LC3), and beclin-1, as well as downregulating the LPS-enhanced levels of ROS, lipid peroxidation, and nitric oxide. These results suggest that the anti-inflammatory effects of KHG26700 may be due, at least in part, to the regulation of the NLRP3-mediated signaling pathway during microglial activation. [BMB Reports 2021; 54(11): 557-562].
- Published
- 2021
4. Inhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cells.
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ChEN, Jianhua, Zhang, LemENg, Zhou, Hui, Wang, Wei, Luo, Yongzhong, Yang, Hua, and Yi, Huihuang
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AUTOPHAGY , *CISPLATIN , *CELL death , *LUNG cancer , *SMALL interfering RNA - Abstract
Recent studies have indicated that autophagy contributes to tumorigenesis and participates in acquired chemotherapeutic resistance. The present study aimed to determine the function and underlying mechanism of cisplatin‑induced autophagy in A549 human lung cancer cells. Autophagy was measured by LC3B‑I/II conversion, LC3B puncta and autophagosomes formation. Apoptotic cell death was measured by caspase‑3 activity, caspase‑3 cleavage and LDH release. The transcriptional and expressional level of autophagy related proteins were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Beclin 1 and Atg 5 siRNA transfection was used to explore the function of cisplatin‑induced autophagy. The results demonstrated that cisplatin induces apoptotic cell death in A549 cells and triggers an autophagic response, as indicated by increased microtubule‑associated protein 1 light chain 3β (LC3B)‑I/II conversion, increased LC3B puncta and autophagosome formation. Mechanisms underlying cisplatin‑induced autophagic responses were also investigated. Cisplatin induced autophagy by upregulating the mRNA and protein expression levels of autophagy protein (Atg)5 and Beclin 1, whereas the mRNA and protein expression levels of serine/threonine‑protein kinase ULK1, Atg3, Atg7, Atg12, and sequestosome‑1 were not markedly upregulated. In addition, knockdown of Atg5 and Beclin 1 by small interfering RNA transfection impaired cisplatin‑induced activation of autophagic responses, increased caspase‑3 cleavage and inhibited cell viability. These findings suggested that disruption of autophagy via the inhibition of Atg5 and Beclin 1 may promote cisplatin‑induced apoptotic cell death in A549 human lung cancer cells. In conclusion, the present study demonstrated that targeting autophagy may be used in the future for the treatment of lung cancer. [ABSTRACT FROM AUTHOR]
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- 2018
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5. PEDF regulates lipid metabolism and reduces apoptosis in hypoxic H9c2 cells by inducing autophagy related 5-mediated autophagy via PEDF-R.
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Zhang, Yiqian, Li, YufENg, Liu, Zhiwei, Zhao, Qixiang, Zhang, Hao, Wang, Xiaoyu, Lu, PENg, Yu, Hongli, Wang, MENg, Dong, Hongyan, and Zhang, Zhongming
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APOPTOSIS , *GLYCOPROTEINS , *HEART cells , *LIPID metabolism , *METABOLIC disorders , *AUTOPHAGY - Abstract
Pigment epithelial‑derived factor (PEDF) is a multifunctional secreted glycoprotein, which exerts a variety of physiological activities. PEDF may protect against hypoxia‑induced cell death associated with its antioxidative effects and p53 mitochondrial translocation in cultured cardiomyocytes and H9c2 cells. Additionally, previous studies have suggested that autophagy is an important cell survival mechanism. However, the effect of PEDF on autophagy and the associated pathway in hypoxic H9c2 cells has not been fully established. Autophagy has been reported to regulate lipid metabolism; however, little is known about whether PEDF is able to regulate lipid metabolism by promoting autophagy. In the present study, western blotting results revealed that PEDF increased the level of microtubule‑associated protein 1A/1B‑light chain 3 (LC3)‑II. Transmission electron microscopy (TEM) and LC3 fluorescence demonstrated that PEDF increased the number of autophagosomes. PEDF also increased the viability of hypoxic H9c2 cells and decreased the level of cleaved caspase‑3 protein, as evidenced by CCK‑8 assays and western blotting, respectively. TEM and a triglyceride assay kit demonstrated that PEDF‑induced autophagy may stimulate lipid degradation. Western blotting results revealed a novel mechanism underlying PEDF‑induced H9c2 cell autophagy via the PEDF‑R‑mediated Atg5 pathway under hypoxic conditions. Furthermore, the results also suggest that PEDF‑induced autophagy may stimulate lipid degradation. The survival function of autophagy suggests that modulation of PEDF‑induced autophagy may be used as a therapeutic strategy to protect cells against lipid‑associated metabolic diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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6. Dopamine D5 receptor-mediated decreases in mitochondrial reactive oxygen species production are cAMP and autophagy dependent
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Ying Wang, Peiying Yu, Imran Perwaiz, Maik Hüttemann, Brian M. Polster, David R. Sibley, Zhiwei Yang, Xiaoliang Jiang, Robin A. Felder, Pedro A. Jose, Peng Qu, Hewang Lee, Jin Wang, Selim Rozyyev, and Ines Armando
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chemistry.chemical_classification ,Reactive oxygen species ,Fenoldopam ,Physiology ,Autophagy ,ATG5 ,HEK 293 cells ,030204 cardiovascular system & hematology ,Mitochondrion ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,chemistry ,Internal Medicine ,medicine ,Autophagy Protein 5 ,030212 general & internal medicine ,Cardiology and Cardiovascular Medicine ,Receptor ,medicine.drug - Abstract
Overproduction of reactive oxygen species (ROS) plays an important role in the pathogenesis of hypertension. The dopamine D5 receptor (D5R) is known to decrease ROS production, but the mechanism is not completely understood. In HEK293 cells overexpressing D5R, fenoldopam, an agonist of the two D1-like receptors, D1R and D5R, decreased the production of mitochondria-derived ROS (mito-ROS). The fenoldopam-mediated decrease in mito-ROS production was mimicked by Sp-cAMPS but blocked by Rp-cAMPS. In human renal proximal tubule cells with DRD1 gene silencing to eliminate the confounding effect of D1R, fenoldopam still decreased mito-ROS production. By contrast, Sch23390, a D1R and D5R antagonist, increased mito-ROS production in the absence of D1R, D5R is constitutively active. The fenoldopam-mediated inhibition of mito-ROS production may have been related to autophagy because fenoldopam increased the expression of the autophagy hallmark proteins, autophagy protein 5 (ATG5), and the microtubule-associated protein 1 light chain (LC)3-II. In the presence of chloroquine or spautin-1, inhibitors of autophagy, fenoldopam further increased ATG5 and LC3-II expression, indicating an important role of D5R in the positive regulation of autophagy. However, when autophagy was inhibited, fenoldopam was unable to inhibit ROS production. Indeed, the levels of these autophagy hallmark proteins were decreased in the kidney cortices of Drd5-/- mice. Moreover, ROS production was increased in mitochondria isolated from the kidney cortices of Drd5-/- mice, relative to Drd5+/+ littermates. In conclusion, D5R-mediated activation of autophagy plays a role in the D5R-mediated inhibition of mito-ROS production in the kidneys.
- Published
- 2021
7. Vascular Smooth Muscle Cell Plasticity and Autophagy in Dissecting Aortic Aneurysms
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Juliette Raffort, Annabel L. Taylor, Patrick Bruneval, Helle F. Jørgensen, Alison Finigan, Martin R. Bennett, James Harrison, Ziad Mallat, Marc Clement, Fabien Lareyre, Joel Chappell, Soraya Taleb, and Marie Vandestienne
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0303 health sciences ,Vascular smooth muscle ,Endoplasmic reticulum ,Autophagy ,Phenotypic switching ,ATG5 ,030204 cardiovascular system & hematology ,Biology ,musculoskeletal system ,Angiotensin II ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,cardiovascular system ,Autophagy Protein 5 ,Cardiology and Cardiovascular Medicine ,tissues ,030304 developmental biology - Abstract
Objective— Recent studies suggested the occurrence of phenotypic switching of vascular smooth muscle cells (VSMCs) during the development of aortic aneurysm (AA). However, lineage-tracing studies are still lacking, and the behavior of VSMCs during the formation of dissecting AA is poorly understood. Approach and Results— We used multicolor lineage tracing of VSMCs to track their fate after injury in murine models of Ang II (angiotensin II)–induced dissecting AA. We also addressed the direct impact of autophagy on the response of VSMCs to AA dissection. Finally, we studied the relevance of these processes to human AAs. Here, we show that a subset of medial VSMCs undergoes clonal expansion and that VSMC outgrowths are observed in the adventitia and borders of the false channel during Ang II–induced development of dissecting AA. The clonally expanded VSMCs undergo phenotypic switching with downregulation of VSMC differentiation markers and upregulation of phagocytic markers, indicative of functional changes. In particular, autophagy and endoplasmic reticulum stress responses are activated in the injured VSMCs. Loss of autophagy in VSMCs through deletion of autophagy protein 5 gene ( Atg5 ) increases the susceptibility of VSMCs to death, enhances endoplasmic reticulum stress activation, and promotes IRE (inositol-requiring enzyme) 1α-dependent VSMC inflammation. These alterations culminate in increased severity of aortic disease and higher incidence of fatal AA dissection in mice with VSMC-restricted deletion of Atg5 . We also report increased expression of autophagy and endoplasmic reticulum stress markers in VSMCs of human dissecting AAs. Conclusions— VSMCs undergo clonal expansion and phenotypic switching in Ang II–induced dissecting AAs in mice. We also identify a critical role for autophagy in regulating VSMC death and endoplasmic reticulum stress–dependent inflammation with important consequences for aortic wall homeostasis and repair.
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- 2019
8. Danthron suppresses autophagy and sensitizes pancreatic cancer cells to doxorubicin
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Xingjun Guo, Chunle Zhao, Renyi Qin, Yuhui Liu, Feng Zhu, Hua Chen, Min Zhou, Xu Li, Ruizhi He, and Min Wang
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0301 basic medicine ,medicine.medical_treatment ,ATG5 ,Anthraquinones ,Toxicology ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Pancreatic cancer ,Autophagy ,medicine ,Humans ,Doxorubicin ,Chemotherapy ,Antibiotics, Antineoplastic ,business.industry ,Cancer ,Drug Synergism ,General Medicine ,medicine.disease ,Pancreatic Neoplasms ,030104 developmental biology ,Apoptosis ,030220 oncology & carcinogenesis ,Cancer research ,Autophagy Protein 5 ,business ,medicine.drug - Abstract
In contrast to the steady increase in survival observed for most cancer types, advances have been slow for pancreatic cancers. Current chemotherapy has limited benefits for patients with pancreatic cancer. Therefore, there is an urgent need for effective pancreatic cancer treatment strategies. At present, targeting the autophagic pathway is regarded as a promising new strategy for cancer treatment. Danthron (1,8-dihydroxyanthrquinone), a component from Rheum palmatum L. (polygonaceae), has several biological activities. However, the inhibition of autophagy by danthron has never been recognized, previously.Here we find that danthron may prevent autophagy, inhibit proliferation and induce apoptosis in pancreatic cancer cells in vitro. Autophagy induced by doxorubicin plays a protective role in pancreatic cancer cells and inhibition of autophagy by chloroquine or silencing autophagy protein 5 (Atg5) may chemosensitize pancreatic cancer cell lines to doxorubicin. Similarly, inhibition of autophagy by danthron also enhances toxicity of doxorubicin to pancreatic cancer cells. These results indicate that danthron has an anticancer effect and can sensitize the chemotherapeutic effect of doxorubicin on pancreatic cancer cells. These findings also suggest that inhibition of autophagy may be an effective way to promote the chemotherapy of pancreatic cancer.
- Published
- 2019
9. Ginsenoside Rg2 protects cardiomyocytes against trastuzumab-induced toxicity by inducing autophagy
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Xingtao Li, Xiaoyong Qi, Guang Liu, and Fangyi Sun
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0301 basic medicine ,Cancer Research ,Cardiotoxicity ,autophagy ,ATG5 ,Autophagy ,apoptosis ,General Medicine ,Articles ,Cell cycle ,03 medical and health sciences ,chemistry.chemical_compound ,trastuzumab ,030104 developmental biology ,0302 clinical medicine ,ginsenoside Rg2 ,Immunology and Microbiology (miscellaneous) ,chemistry ,Annexin ,Apoptosis ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,Cancer research ,Propidium iodide - Abstract
Trastuzumab (TZM) significantly improves the outcomes of patients with breast cancer; however, it is associated with severe cardiotoxicity. Ginsenoside Rg2 was reported to exert protective effects against myocardial injury and apoptosis in human cardiomyocytes (HCMs). However, whether ginsenoside Rg2 protects HCMs against TZM-induced toxicity remains unclear. The present study investigated the proliferation of HCMs using a Cell Counting Kit-8 assay and Ki67 immunofluorescence staining. Apoptotic cells were detected by Annexin V/propidium iodide staining and flow cytometry. Furthermore, monodansylcadaverine staining was performed to detect cell autophagy. In addition, western blotting was used to detect the expression levels of phosphorylated (p)-Akt, p-mTOR, beclin 1, microtubule associated protein 1 light chain 3α (LC3) and autophagy protein 5 (ATG5) in HCMs. Pretreatment with ginsenoside Rg2 significantly protected HCMs against TZM-induced cytotoxicity by inhibiting apoptosis. Furthermore, pretreatment with ginsenoside Rg2 induced autophagy in HCMs by upregulating the expression levels of p-Akt, p-mTOR, beclin 1, LC3 and ATG5. The results obtained in the present study suggested that ginsenoside Rg2 could protect HCMs against TZM-induced cardiotoxicity by activating autophagy. Therefore, ginsenoside Rg2 may serve as a potential therapeutic agent to prevent TZM-related cardiotoxicity in patients with breast cancer.
- Published
- 2021
10. Oxymatrine exerts a protective effect in myocardial ischemia/reperfusion-induced acute lung injury by inhibiting autophagy in diabetic rats
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Xin Liu, Jiali Xu, and Zhen Xiong
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Male ,Cancer Research ,ATG5 ,Acute Lung Injury ,Myocardial Reperfusion Injury ,Pharmacology ,Lung injury ,Biochemistry ,Diabetes Mellitus, Experimental ,Diabetes Complications ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Alkaloids ,Lactate dehydrogenase ,Genetics ,medicine ,Autophagy ,Animals ,oxymatrine ,Molecular Biology ,medicine.diagnostic_test ,diabetes ,business.industry ,Articles ,inflammatory response ,ischemia/reperfusion ,Rats ,Bronchoalveolar lavage ,Oxymatrine ,Oncology ,chemistry ,Autophagy Protein 5 ,Molecular Medicine ,Tumor necrosis factor alpha ,business ,Quinolizines - Abstract
Oxymatrine (OMT) is the primary active component of Sophora flavescens Ait., and is widely used for the treatment of diabetic complications. The present study aimed to investigate the effects of OMT on acute lung injury (ALI) in diabetic rats subjected to myocardial ischemia/reperfusion (I/R). ALI in a myocardial I/R model was established in streptozocin‑induced diabetic rats. Enzyme‑linked immunosorbent assays were used to evaluate the levels of creatine kinase isoenzyme MB and lactate dehydrogenase, and the inflammatory response was assessed via leukocyte counts and the levels of tumor necrosis factor (TNF)‑α, interleukin (IL)‑6 and IL‑8 in the bronchoalveolar lavage (BAL) fluid. Hematoxylin and eosin staining was used to determine pathological changes to the lung tissue, and the autophagy‑related proteins LC‑3II/LC‑3I, Beclin‑1, autophagy protein 5 (Atg5) and p62 were detected by western blotting. Diabetic rats subjected to myocardial I/R showed increased levels of ALI with a higher lung injury score and WET/DRY ratio, and lower partial pressure of oxygen. This was accompanied by aberrant autophagy, indicated by an increased LC‑3II/LC‑3I ratio, decreased p62 expression levels, increased Atg5 and beclin‑1 expression levels, decreased superoxide dismutase activity and increased 15‑F2t‑isoprostane formation in lung tissues, as well as increased levels of leukocytes, TNF‑α, IL‑6 and IL‑8 in the BAL fluid. Administration of the autophagy inducer rapamycin significantly accelerated these alterations, while the autophagy inhibitor 3‑Methyladenine exerted the opposite effects. These results indicated that diabetic lungs are more vulnerable to myocardial I/R, which was associated with aberrant autophagy. Furthermore, oxymatrine was observed to reverse and alleviate ALI in diabetic rats with myocardial I/R in a concentration‑dependent manner, the mechanism of which may be associated with the inhibition of autophagy.
- Published
- 2021
11. Intestinal antiviral signaling is controlled by autophagy gene Epg5 independent of the microbiota
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Rachel Rodgers, Harshad Ingle, Gowri Kalugotla, Xia Yang, Qun Lu, Chandni Desai, Sanghyun Lee, Nicolette R. Borella, Lindsay Droit, Ryan Hill, Megan T. Baldridge, Stefan T. Peterson, Chunyan Wu, Yuhao Li, Hongju Deng, Dylan Lawrence, Ya-Ting Wang, and Jin Zhang
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education.field_of_study ,biology ,Interferon-stimulated gene ,Cell Biology ,Molecular biology ,Sequestosome 1 ,TANK-binding kinase 1 ,Interferon ,biology.protein ,Autophagy Protein 5 ,STAT protein ,medicine ,Interferon gamma ,STAT1 ,education ,Molecular Biology ,medicine.drug - Abstract
Mutations in the macroautophagy/autophagy gene EPG5 are responsible for Vici syndrome, a human genetic disease characterized by combined immunodeficiency. Previously, we found that epg5−/- mice exhibit hyperinflammation in the lungs mediated by IL1B/IL-1β and TNF/TNFα, resulting in resistance to influenza. Here, we find that disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. Gene expression analysis reveals IFNL/IFN-λ responsive genes as a key alteration. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free mouse studies indicate Epg5-associated inflammation of both the intestine and lung is microbiota-independent. Genetic studies support IFNL signaling as the primary mediator of resistance to enteric viruses, but not of microbial dysbiosis, in epg5−/- mice. This study unveils an important role, unexpectedly independent of the microbiota, for autophagy gene Epg5 in host organism protection by modulating intestinal IFNL responses. Abbreviations: CTNNB1: catenin (cadherin associated protein), beta 1; DAPI: 4′,6-diamidino-2-phenylindole; EPG5: ectopic P-granules autophagy protein 5 homolog (C. elegans); FT: fecal transplant; IFI44: interferon-induced protein 44; IFIT1: interferon-induced protein with tetratricopeptide repeats 1; IFNG/IFN-γ: interferon gamma; IFNL/IFN-λ: interferon lambda; IFNLR1: interferon lambda receptor 1; IL1B/IL-1β: interleukin 1 beta; ISG: interferon stimulated gene; GF: germ-free; LEfSe: linear discriminant analysis effect size; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MNoV: murine norovirus; MX2: MX dynamin-like GTPase 2; OAS1A: 2’-5’ oligoadenylate synthetase 1A; RV: rotavirus; SPF: specific-pathogen free; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TNF/TNFα: tumor necrosis factor
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- 2021
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12. Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II.
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Zhao, Wei, Li, Yulin, Jia, Lixin, Pan, Lili, Li, Huihua, and Du, Jie
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AUTOPHAGY , *PROTEIN deficiency , *HEART injuries , *INFLAMMATION , *ANGIOTENSIN II , *HYPERTENSION , *HOMEOSTASIS - Abstract
Abstract: Objective: Hypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II). Methods and Results: Autophagy protein 5 (Atg5) haplodeficiency (Atg5+/−) and age-matched wild-type (WT) C57BL/6J mice were infused with Ang II (1500ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5+/− mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis. Conclusion: Atg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage. [Copyright &y& Elsevier]
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- 2014
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13. MicroRNA‑22 regulates autophagy and apoptosis in cisplatin resistance of osteosarcoma
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Shi‑Bing Guo, Chao Sun, Yu‑Xing Wang, Zhen‑Qun Zhao, Chen‑Yang Meng, Wei Feng, Rui Bai, Wei Zhao, and Liang Sun
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Male ,0301 basic medicine ,autophagy ,Cancer Research ,Cell ,Mice, Nude ,cisplatin ,Antineoplastic Agents ,Apoptosis ,Bone Neoplasms ,Transfection ,Biochemistry ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Cell Proliferation ,Cisplatin ,Osteosarcoma ,Chemistry ,Cell growth ,Autophagy ,Membrane Proteins ,RNA-Binding Proteins ,MTDH ,Articles ,osteosarcoma cells ,Cell cycle ,Xenograft Model Antitumor Assays ,Tumor Burden ,MicroRNAs ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,Cancer research ,Molecular Medicine ,microRNA-22 ,medicine.drug - Abstract
Osteosarcoma (OS) is a primary malignant tumor of bone tissue. Effective chemotherapy may improve the survival of patients with OS. MicroRNAs (miRs) serve significant roles in the regulatory function of tumorigenesis and chemosensitivity of different types of cancer. miR-22 has been revealed to inhibit the proliferation and migration of OS cells, as well as increasing their sensitivity to cisplatin (CDDP). The mechanisms of action behind the functions of miR-22 in OS drug resistance require investigation. Therefore, in the present study, the human OS cell lines (MG-63, U2OS, Saos2 and OS9901) and a drug-resistant cell line (MG-63/CDDP) were cultured. Cell proliferation, apoptosis and autophagy assays were performed to investigate the proliferation, apoptosis and autophagy of cell lines transfected with miR-22 mimic. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to investigate the expression levels of associated genes. The results revealed that miR-22 inhibited the proliferation of MG-63 cells and MG-63/CDDP cells, and enhanced the anti-proliferative ability of CDDP. miR-22 induced apoptosis and inhibited autophagy of MG-63 cells and MG-63/CDDP cells. Apoptosis-related genes, including caspase-3 and Bcl-2-associated X protein were upregulated, while B-cell lymphoma-2 was downregulated in both cell lines transfected with the miR-22 mimic. Autophagy protein 5, beclin1 and microtubules-associated protein 1 light chain 3 were downregulated in both cell lines transfected with miR-22 mimic. Furthermore, the in vitro and in vivo expression levels of metadherin (MTDH) in the OS/OS-CDDP-resistant models were downregulated following transfection with the miR-22 mimic. Therefore, the results of the present study suggested that miR-22 promoted CDDP sensitivity by inhibiting autophagy and inducing apoptosis in OS cells, while MTDH may serve a positive role in inducing CDDP resistance of OS cells.
- Published
- 2020
14. Lycopene prevents oxygen-glucose deprivation-induced autophagic death in SH-SY5Y cells via inhibition of the oxidative stress-activated AMPK/mTOR pathway
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He Liu, Tan Li, Yang Zhang, and Yi Qi
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Cancer Research ,Programmed cell death ,Autophagic Cell Death ,ATG5 ,Apoptosis ,AMP-Activated Protein Kinases ,Biochemistry ,Neuroblastoma ,Lycopene ,Downregulation and upregulation ,Cell Line, Tumor ,Genetics ,Autophagy ,Humans ,Molecular Biology ,PI3K/AKT/mTOR pathway ,Neurons ,Cell Death ,Chemistry ,TOR Serine-Threonine Kinases ,AMPK ,Cell biology ,Up-Regulation ,carbohydrates (lipids) ,Oxygen ,Oxidative Stress ,Glucose ,Neuroprotective Agents ,nervous system ,Oncology ,Autophagy Protein 5 ,Molecular Medicine ,Signal Transduction - Abstract
Lycopene has been reported to exert a protective effect on the brain against transient ischemia‑induced damage; however, whether it could regulate autophagic neuronal death remains elusive. The present study aimed to investigate the role of autophagy in the protective effects of lycopene against neuronal damage and its underlying mechanism. Oxygen‑glucose deprivation (OGD) was used to simulate neuronal ischemic injury in human SH‑SY5Y cells. Lactate dehydrogenase (LDH) release assay revealed that OGD induced SH‑SY5Y cell death. Western blotting demonstrated that OGD upregulated the expression levels of the autophagy marker proteins autophagy protein 5 (ATG5) and LC3II, but downregulated the autophagy substrate p62 in a time‑dependent manner. By contrast, OGD‑induced cell death was significantly inhibited by the autophagy inhibitors 3‑methyladenine or bafilomycin A1 or by knockdown of ATG5, indicating that OGD may induce autophagic death in SH‑SY5Y cells. Notably, lycopene was shown not only to prevent OGD‑induced SH‑SY5Y cell death, but was also able to effectively inhibit OGD‑induced upregulation of ATG5 and LC3II, and downregulation of p62 in a dose‑dependent manner. Mechanistically, it was suggested that lycopene inhibited OGD‑induced activation of the AMPK/mTOR pathway via attenuation of oxidative stress by maintaining the intracellular antioxidant glutathione (GSH). Furthermore, the inhibitory role of lycopene in GSH depletion was found to be associated with the prevention of OGD‑induced depletion of intracellular cysteine and downregulation of xCT. Collectively, the present study demonstrated that lycopene protected SH‑SY5Y cells against OGD‑induced autophagic death by inhibiting oxidative stress‑dependent activation of the AMPK/mTOR pathway.
- Published
- 2020
15. Graphene oxide induces dose-dependent lung injury in rats by regulating autophagy
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Ouyang Shuge, Mingke Qiu, Hongbo Zhang, Yuxin Dai, Yang Wang, Lei Zhang, Jingmin Ou, and Shuqing Wang
- Subjects
0301 basic medicine ,Cancer Research ,ATG5 ,Inflammation ,Lung injury ,Pharmacology ,medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Immunology and Microbiology (miscellaneous) ,medicine ,medicine.diagnostic_test ,Autophagy ,General Medicine ,Articles ,respiratory system ,Malondialdehyde ,respiratory tract diseases ,030104 developmental biology ,Bronchoalveolar lavage ,chemistry ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,medicine.symptom ,Oxidative stress - Abstract
Graphene is a two-dimensional structured material with a hexagonal honeycomb lattice composed of carbon atoms. The biological effects of graphene oxide (GO) have been extensively investigated, as it has been widely used in biological research due to its increased hydrophilicity/biocompatibility. However, the exact mechanisms underlying GO-associated lung toxicity have not yet been fully elucidated. The aim of the present study was to determine the role of GO in lung injury induction, as well as its involvement in oxidative stress, inflammation and autophagy. The results revealed that lower concentrations of GO (5 and 10 mg/kg) did not cause significant lung injury, but the administration of GO at higher concentrations (50 and 100 mg/kg) induced lung edema, and increased lung permeability and histopathological lung changes. High GO concentrations also induced oxidative injury and inflammatory reactions in the lung, demonstrated by increased levels of oxidative products [malondialdehyde(MDA) and 8-hydroxydeoxyguanosine (8-OHdG)] and inflammatory factors (TNF-α, IL-6, IL-1β and IL-8). The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CLQ) inhibited autophagy in the lung and attenuated GO-induced lung injury, as demonstrated by a reduced lung wet-to-dry weight ratio, lower levels of protein in the bronchoalveolar lavage fluid, and a reduced lung injury score. Furthermore, 3-MA and CLQ significantly reduced the levels of MDA, 8-OHdG and inflammatory factors in lung tissue, suggesting that autophagy also mediates the development of oxidative injury and inflammation in the lung. Finally, autophagy was directly inhibited in BEAS-2B cells by short hairpin RNA-mediated autophagy protein 5 (ATG5) knockdown, which were then treated with GO. Cell viability, as well as the extent of injury (indicated by lactate dehydrogenase level) and oxidative stress were determined. The results revealed that ATG5 knockdown-induced autophagic inhibition significantly decreased cellular injury and oxidative stress, suggesting that autophagy induction is a key event that leads to lung injury during exposure to GO. In conclusion, the findings of the present study indicated that GO causes lung injury in a dose-dependent manner by inducing autophagy.
- Published
- 2020
16. MiR-208a-3p aggravates autophagy through the PDCD4-ATG5 pathway in Ang II-induced H9c2 cardiomyoblasts
- Author
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Fei Peng, Hexi Zhang, Nan Ye, Hui Gong, Xiaoyu Lian, and Li Wang
- Subjects
0301 basic medicine ,Programmed cell death ,ATG5 ,Down-Regulation ,Cardiomegaly ,Autophagy-Related Protein 5 ,Cell Line ,Muscle hypertrophy ,Myoblasts ,03 medical and health sciences ,Downregulation and upregulation ,Autophagy ,Animals ,Humans ,Myocytes, Cardiac ,Pharmacology ,Gene knockdown ,Chemistry ,Angiotensin II ,RNA-Binding Proteins ,General Medicine ,Rats ,Up-Regulation ,Cell biology ,MicroRNAs ,030104 developmental biology ,cardiovascular system ,Autophagy Protein 5 ,Apoptosis Regulatory Proteins - Abstract
Pathological cardiac hypertrophy is the main determinant of the development of heart failure, for which there is often no effective therapy. The dysregulation of autophagy is implicated in hypertrophy, but the mechanism linking these processes is unclear. In this study, we characterized the regulatory role of miR-208a-3p in autophagy in H9c2 cardiomyoblasts induced by Angiotensin II (Ang II). We found that miR-208a-3p was up-regulated in Ang II-induced H9c2 cardiomyoblasts and in starvation-induced autophagy. The overexpression of miR-208a-3p increased Ang II-induced autophagy, and this was accompanied by the inhibition of programmed cell death protein (PDCD4) and upregulation of autophagy protein 5 (ATG5). A dual-luciferase report assay confirmed the direct binding between miR-208a-3p and PDCD4. PDCD4 knockdown up-regulated autophagy, and its overexpression down-regulated this process. Moreover, the PDCD4-mediated regulation of autophagy was modulated by ATG5. Taken together, these findings indicate that miR-208a-3p promotes autophagy during Ang II-induced hypertrophy and provide a basis for the development of therapies for hypertrophic-induced cardiac dysfunction.
- Published
- 2018
17. NUPR1- CHOP experssion, autophagosome formation and apoptosis in the postmortem striatum of chronic methamphetamine user
- Author
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Fatemeh Sadat Tabatabaei Mirakabad, Mostafa Rezaei-Tavirani, Atefeh Shirazi Tehrani, Foozhan Tahmasebinia, Hojjat-Allah Abbaszadeh, Maryam Sadat Khoramgah, Shahrokh Khoshsirat, Somayeh Niknazar, Gholam-Reza Mahmoudiasl, and Mohammad-Amin Abdollahifar
- Subjects
Adult ,Male ,0301 basic medicine ,Programmed cell death ,Amphetamine-Related Disorders ,ATG5 ,Apoptosis ,Biology ,CHOP ,Methamphetamine ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,0302 clinical medicine ,Basic Helix-Loop-Helix Transcription Factors ,Humans ,Autophagy ,Autophagosomes ,Meth ,Corpus Striatum ,Neoplasm Proteins ,Cell biology ,030104 developmental biology ,chemistry ,TRIB3 ,Autophagy Protein 5 ,Central Nervous System Stimulants ,Autopsy ,Transcription Factor CHOP ,030217 neurology & neurosurgery - Abstract
Methamphetamine (Meth) is a neuro-stimulator substrate which might lead to neural cell death and the activation of several interconnected cellular pathways as well. However, the precise molecular mechanisms underlying Meth-induced neural cell death remained unclear yet. The current study aimed to assess the specific relationship between long-term Meth exposure and several endoplasmic reticulum stress, autophagy, and apoptosis associated markers including C/EBP homologous protein (CHOP), Tribbles homolog 3(Trib3), Nuclear protein 1(NUPR1), and Beclin-1 expression in postmortem human striatum. Therefore, the effects of long-term Meth exposure on autophagy and apoptosis in the striatum of postmortem users were evaluated and molecular, immunehistochemical, and histological examinations were performed on 10 control and 10 Meth-addicted brains. The level of CHOP, Trib3, NUPR1, and Beclin-1, Microtubule-associated proteins 1A/1B light chain 3B(LC3), Caspase 3, and Autophagy protein 5 (ATG5) were measured by using qPCR and immunohistochemistry. Stereological neural cell counting, Hematoxylin and Eosin, Nissl and Tunel staining were also performed. Based on our findings, the expression level of CHOP, Trib3, NUPR1, and Beclin-1 in the striatum of Meth group were significantly higher than the control group. Besides, the neuronal cell death was substantially increased in the striatum based on data obtained from the Tunel assay and the stereological analysis. Long-term presence of Meth in the brain can induce ER stress and overexpression of NUPR1 which is associated with the upregulation of CHOP, a pro-apoptotic transcription factor. Moreover, an increase in Trib3 expression is implicated in CHOP-dependent autophagic cell death during Meth-induced ER stress accompanied by an increase in neuronal cell death in the striatum of the postmortem human brains. Beclin 1 expression was also upregulated which may due to the activation of autophagic mechanisms upon prolonged Meth exposure.
- Published
- 2021
18. Autophagy mediates free fatty acid effects on MDA-MB-231 cell proliferation, migration and invasion
- Author
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Sheng‑Rong Sun, Juan‑Juan Li, Juan Wu, Si Sun, Qi Wu, Chuang Chen, and Chang‑Hua Wang
- Subjects
0301 basic medicine ,autophagy ,Cancer Research ,Programmed cell death ,Cell ,Biology ,Metastasis ,03 medical and health sciences ,0302 clinical medicine ,palmitate acid ,LC3 ,medicine ,chemistry.chemical_classification ,Cell growth ,Autophagy ,Fatty acid ,Cancer ,Articles ,medicine.disease ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,oleic acid ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,free fatty acid - Abstract
Epidemiological and animal studies indicate an association between high levels of dietary fat intake and an increased risk of breast cancer. The multifaceted role of autophagy in cancer has been revealed in previous years. However, the mechanism of this role remains unknown. In the present study, the two most common free fatty acids, palmitate acid (PA) and oleic acid (OA), were used to determine the effect on human breast cancer MDA-MB-231 cells, and the possible role of autophagy was investigated by detecting light chain 3 (LC3)-II/I. Bafliomycin A1 was used to detect autophagy flux. High palmitate acid condition-induced MDA-MB-231 cell death and invasion were mitigated by 3-methyladenine pretreatment or transfection with shRNA against autophagy protein 5. By contrast, high oleic acid condition induced MDA-MB-231 cell proliferation, migration and invasion were mitigated using rapamycin. The present results suggest that autophagy has an important role in the effects of PA and OA on breast cancer growth and metastasis in vitro.
- Published
- 2017
19. Overexpression of let-7a increases neurotoxicity in a PC12 cell model of Alzheimer's disease via regulating autophagy
- Author
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Guijun Song, Lan Li, Huizi Gu, Chen Cui, and Zihui Zhao
- Subjects
0301 basic medicine ,Cancer Research ,Autophagy ,Neurotoxicity ,Articles ,General Medicine ,Cell cycle ,Biology ,medicine.disease ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Immunology and Microbiology (miscellaneous) ,Downregulation and upregulation ,Autophagy Protein 5 ,medicine ,Signal transduction ,Protein kinase B ,PI3K/AKT/mTOR pathway - Abstract
Increased deposition of β-amyloid (Aβ) protein is one of the typical characteristics of Alzheimer's disease (AD). Recent evidence has demonstrated that the microRNA let-7 family, which is highly expressed in the central nervous system, participates in the regulation of pathologic processes of AD. In the present study, the effect of let-7a overexpression on Aβ1-40-induced neurotoxicity was evaluated in PC12 and SK-N-SH cells. The results indicated that overexpression of let-7a enhanced the neurotoxicity induced by Aβ1-40 in PC12 and SK-N-SH cells. In addition, the apoptosis induced by Aβ1-40 in PC12 and SK-N-SH cells was increased by let-7a overexpression. Furthermore, Aβ1-40 treatment increased the protein levels of microtubule-associated protein 1A/1B-light chain 3 (LC3) and beclin-1 and increased the LC3 II/I ratio. The mRNA expression levels of beclin-1, autophagy protein 5 (Atg-5) and Atg-7 were also increased by Aβ1-40 treatment in PC12 cells. Let-7a overexpression further upregulated the above autophagy-related markers. Furthermore, the protein level of p62 was increased by Aβ1-40 treatment, and this was further enhanced by let-7a overexpression. Finally, the present results demonstrated that the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was involved in the autophagy regulation by let-7a. In conclusion, the present study demonstrates that the neurotoxicity induced by Aβ1-40 is augmented by let-7a overexpression via regulation of autophagy, and the PI3K/Akt/mTOR signaling pathway also serves a function in this process.
- Published
- 2017
20. Intrinsic Autophagy Is Required for the Maintenance of Intestinal Stem Cells and for Irradiation-Induced Intestinal Regeneration
- Author
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Toshiaki Ohteki, Taku Sato, Jumpei Asano, Shigeomi Shimizu, Mihoko Kajita, Shizuko Ichinose, and Nobuyuki Onai
- Subjects
inorganic chemicals ,0301 basic medicine ,ATG5 ,Mice, Transgenic ,digestive system ,General Biochemistry, Genetics and Molecular Biology ,Autophagy-Related Protein 5 ,Mice ,03 medical and health sciences ,Autophagy ,Animals ,Regeneration ,Intestinal Mucosa ,lcsh:QH301-705.5 ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Stem Cells ,Regeneration (biology) ,fungi ,Cell biology ,Radiation Injuries, Experimental ,030104 developmental biology ,lcsh:Biology (General) ,chemistry ,Gamma Rays ,biology.protein ,Autophagy Protein 5 ,Stem cell ,Reactive Oxygen Species ,Villin ,Homeostasis - Abstract
Summary: Autophagy is a lysosomal degradation pathway with important roles in physiological homeostasis and disease. However, the role of autophagy in intestinal stem cells (ISCs) is unclear. Here, we show that intrinsic autophagy in ISCs is important for ISC homeostasis. Mice lacking autophagy protein 5 (ATG5) in intestinal epithelial cells (iECs) (Villin-Cre: Atg5fl/fl, hereafter Atg5ΔIEC mice) or in all iECs except Paneth cells (Ah-Cre: Atg5fl/fl mice) had significantly fewer ISCs than did control mice and showed impaired ISC-dependent intestinal recovery after irradiation. Crypt ISCs from Atg5ΔIEC mice had significantly higher reactive oxygen species (ROS) levels than did those from control mice. A ROS-inducing reagent decreased the ISC number and impaired ISC regenerative capacity ex vivo, and treating Atg5ΔIEC mice with an antioxidant rescued their defects. Our results show that intrinsic autophagy supports ISC maintenance by reducing excessive ROS. Optimizing autophagy may lead to autophagy-based therapies for intestinal injuries. : Autophagy is a lysosomal degradation pathway with important roles in physiological homeostasis and disease. Asano et al. find that intrinsic autophagy is important for the maintenance of intestinal stem cells by reducing excessive reactive oxygen species. This stem cell maintenance is necessary to provide damage-induced intestinal regeneration. Keywords: intestinal epithelial cell (iEC), intestinal stem cell (ISC), autophagy, reactive oxygen species (ROS), regeneration, irradiation, 5-fluorouracil (5-FU)
- Published
- 2017
21. Regulated in Development and DNA Damage Response 1 Deficiency Impairs Autophagy and Mitochondrial Biogenesis in Articular Cartilage and Increases the Severity of Experimental Osteoarthritis
- Author
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Martin Lotz, Tokio Matsuzaki, Lars Plate, Oscar Alvarez-Garcia, Merissa Olmer, and Jeffery W. Kelly
- Subjects
030203 arthritis & rheumatology ,0301 basic medicine ,Pathology ,medicine.medical_specialty ,biology ,Cartilage homeostasis ,Chemistry ,Cartilage ,Immunology ,Autophagy ,TFAM ,Chondrocyte ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Rheumatology ,Mitochondrial biogenesis ,Autophagy Protein 5 ,medicine ,biology.protein ,Immunology and Allergy ,Mechanistic target of rapamycin - Abstract
Objective Regulated in development and DNA damage response 1 (REDD1) is an endogenous inhibitor of mechanistic target of rapamycin (mTOR) that regulates cellular stress responses. REDD1 expression is decreased in aged and osteoarthritic (OA) cartilage, and it regulates mTOR signaling and autophagy in articular chondrocytes in vitro. This study was undertaken to investigate the effects of REDD1 deletion in vivo using a mouse model of experimental OA. Methods OA severity was histologically assessed in 4-month-old wild-type and REDD1−/− mice subjected to surgical destabilization of the medial meniscus (DMM). Chondrocyte autophagy, apoptosis, mitochondrial content, and expression of mitochondrial biogenesis markers were determined in cartilage and cultured chondrocytes from wild-type and REDD1−/− mice. Results REDD1 deficiency increased the severity of changes in cartilage, menisci, subchondral bone, and synovium in the DMM model of OA. Chondrocyte death was increased in the cartilage of REDD1−/− mice and in cultured REDD1−/− mouse chondrocytes under oxidative stress conditions. Expression of key autophagy markers (microtubule-associated protein 1A/1B light chain 3 and autophagy protein 5) was markedly reduced in cartilage from REDD1−/− mice and in cultured human and mouse chondrocytes with REDD1 depletion. Mitochondrial content, ATP levels, and expression of the mitochondrial biogenesis markers peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were also decreased in REDD1-deficient chondrocytes. REDD1 was required for AMP-activated protein kinase–induced PGC-1α in chondrocytes. Conclusion Our findings suggest that REDD1 is a key mediator of cartilage homeostasis through regulation of autophagy and mitochondrial biogenesis and that REDD1 deficiency exacerbates the severity of injury-induced OA.
- Published
- 2017
22. Oestrogen Inhibits Arterial Calcification by Promoting Autophagy
- Author
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Xiao Lin, Yi-Qun Peng, Dan Xiong, Feng Xu, Ting Zhu, Min-Zhi Mao, Xiao-Bo Liao, Rong-Rong Cui, Feng Wu, Ling-Qing Yuan, and Jia-Yu Zhong
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Vascular smooth muscle ,Cellular differentiation ,Science ,Myocytes, Smooth Muscle ,ATG5 ,Biology ,Article ,Muscle, Smooth, Vascular ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,Internal medicine ,Autophagy ,medicine ,Animals ,Humans ,skin and connective tissue diseases ,Osteoblasts ,Multidisciplinary ,Calcinosis ,Cell Differentiation ,Estrogens ,Arteries ,Mice, Inbred C57BL ,Arterial calcification ,030104 developmental biology ,Endocrinology ,Mechanism of action ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,Medicine ,Female ,medicine.symptom - Abstract
Arterial calcification is a major complication of cardiovascular disease. Oestrogen replacement therapy in postmenopausal women is associated with lower levels of coronary artery calcification, but its mechanism of action remains unclear. Here, we show that oestrogen inhibits the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) in vitro and arterial calcification in vivo by promoting autophagy. Through electron microscopy, GFP–LC3 redistribution, and immunofluorescence analyses as well as measurement of the expression of the autophagosome marker light-chain I/II (LC3I/II) and autophagy protein 5 (Atg5), we show that autophagy is increased in VSMCs by oestrogen in vitro and in vivo. The inhibitory effect of oestrogen on arterial calcification was counteracted by 3-methyladenine (3MA) or knockdown of Atg5 and was increased by rapamycin. Furthermore, the inhibitory effect of oestrogen on arterial calcification and the degree of autophagy induced by oestrogen were blocked by a nonselective oestrogen receptor (ER) antagonist (ICI 182780), a selective oestrogen receptor alpha (ERα) antagonist (MPP), and ERα-specific siRNA. Our data indicate that oestrogen inhibits the osteoblastic differentiation of VSMCs by promoting autophagy through the ERα signalling pathway in vitro and arterial calcification in vivo by increasing autophagy. Our findings provide new insights into the mechanism by which oestrogen contributes to vascular calcification in vitro and in vivo.
- Published
- 2017
23. Combined inhibition of autophagy protein 5 and galectin-1 by thiodigalactoside reduces diet-induced obesity through induction of white fat browning
- Author
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Jong Won Yun and Hilal Ahmad Parray
- Subjects
0301 basic medicine ,medicine.medical_specialty ,biology ,Clinical Biochemistry ,ATG5 ,Lipid metabolism ,Cell Biology ,White adipose tissue ,Biochemistry ,03 medical and health sciences ,Fatty acid synthase ,030104 developmental biology ,0302 clinical medicine ,Endocrinology ,medicine.anatomical_structure ,Adipogenesis ,030220 oncology & carcinogenesis ,Internal medicine ,Brown adipose tissue ,Lipogenesis ,Genetics ,biology.protein ,medicine ,Autophagy Protein 5 ,Molecular Biology - Abstract
Our previous study demonstrated that thiodigalactoside (TDG) ameliorates obesity by targeted inhibition of galectin-1 (GAL1). Here, for the first time, we report the unexpected role of GAL1 and ATG5 inhibition by TDG in lipid metabolism. Core thermogenic marker proteins and genes were highly induced in white adipose tissue (WAT) of rats fed a high fat diet (HFD) and TDG, resulting in the significant development of brown fat-like adipocytes in inguinal WAT. TDG treatment reduced weight gain and fat mass as well as activated brown adipose tissue (BAT) in HFD-fed rats. TDG also reduced protein levels of LC3-II and increased protein levels of P62, suggesting its possible role in suppression of autophagy. Combined inhibition of GAL1 and ATG5 by TDG treatment protected rats against both HFD-induced adipogenesis as well as lipogenesis, as evidenced by suppression of CCAAT/enhancer-binding protein alpha, peroxisome proliferator-activated receptor gamma and fatty acid synthase. In conclusion, the present findings suggest that TDG plays a role in browning and lipid catabolism by combined inhibition of GAL1 and ATG5 and thus may have potential therapeutic implications in the regulation of energy homeostasis via its action in WAT. © 2017 IUBMB Life, 69(7):510-521, 2017.
- Published
- 2017
24. p62 Promotes Amino Acid Sensitivity of mTOR Pathway and Hepatic Differentiation in Adult Liver Stem/Progenitor Cells
- Author
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Shohei Yoshiya, Ken Shirabe, Shinji Itoh, Yoshihiko Maehara, Shinji Okano, Yoshihiro Matsumoto, Yuji Soejima, Tomoharu Yoshizumi, M. Sugiyama, Norifumi Harimoto, Toru Ikegami, Yuki Bekki, Yoshihiro Yoshida, and Takeo Toshima
- Subjects
0301 basic medicine ,Physiology ,Cellular differentiation ,Clinical Biochemistry ,ATG5 ,RPTOR ,Autophagy ,Cell Biology ,Biology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,Stem cell ,Progenitor cell ,PI3K/AKT/mTOR pathway - Abstract
Autophagy is a homeostatic process regulating turnover of impaired proteins and organelles, and p62 (sequestosome-1, SQSTM1) functions as the autophagic receptor in this process. p62 also functions as a hub for intracellular signaling such as that in the mammalian target of rapamycin (mTOR) pathway. Liver stem/progenitor cells have the potential to differentiate to form hepatocytes or cholangiocytes. In this study, we examined effects of autophagy, p62, and associated signaling on hepatic differentiation. Adult stem/progenitor cells were isolated from the liver of mice with chemically induced liver injury. Effects of autophagy, p62, and related signaling pathways on hepatic differentiation were investigated by silencing the genes for autophagy protein 5 (ATG5) and/or SQSTM1/p62 using small interfering RNAs. Hepatic differentiation was assessed based on increased albumin and hepatocyte nuclear factor 4α, as hepatocyte markers, and decreased cytokeratin 19 and SOX9, as stem/progenitor cell markers. These markers were measured using quantitative RT-PCR, immunofluorescence, and Western blotting. ATG5 silencing decreased active LC3 and increased p62, indicating inhibition of autophagy. Inhibition of autophagy promoted hepatic differentiation in the stem/progenitor cells. Conversely, SQSTM1/p62 silencing impaired hepatic differentiation. A suggested mechanism for p62-dependent hepatic differentiation in our study was activation of the mTOR pathway by amino acids. Amino acid activation of mTOR signaling was enhanced by ATG5 silencing and suppressed by SQSTM1/p62 silencing. Our findings indicated that promoting amino acid sensitivity of the mTOR pathway is dependent on p62 accumulated by inhibition of autophagy and that this process plays an important role in the hepatic differentiation of stem/progenitor cells. J. Cell. Physiol. 232: 2112-2124, 2017. © 2016 Wiley Periodicals, Inc.
- Published
- 2017
25. ATF4/ATG5 Signaling in Hypothalamic Proopiomelanocortin Neurons Regulates Fat Mass via Affecting Energy Expenditure
- Author
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Shanghai Chen, Hao Ying, Yalan Deng, Yi Liu, Zhigang Li, Yuzhong Xiao, Feixiang Yuan, Tingting Xia, Hao Liu, Zhixue Liu, Qiwei Zhai, and Feifan Guo
- Subjects
Blood Glucose ,Leptin ,0301 basic medicine ,medicine.medical_specialty ,Pro-Opiomelanocortin ,Adipose Tissue, White ,Endocrinology, Diabetes and Metabolism ,Blotting, Western ,ATG5 ,Hypothalamus ,Fluorescent Antibody Technique ,Activating Transcription Factor 4 ,Real-Time Polymerase Chain Reaction ,Energy homeostasis ,Autophagy-Related Protein 5 ,Mice ,Norepinephrine ,03 medical and health sciences ,Adipose Tissue, Brown ,Proopiomelanocortin ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Insulin ,Mice, Knockout ,Neurons ,biology ,ATF4 ,Organ Size ,Glucose Tolerance Test ,030104 developmental biology ,Endocrinology ,Adipose Tissue ,Growth Hormone ,Autophagy Protein 5 ,biology.protein ,Insulin Resistance ,Corticosterone ,Energy Metabolism ,Signal Transduction - Abstract
Although many biological functions of activating transcription factor 4 (ATF4) have been identified, a role of hypothalamic ATF4 in the regulation of energy homeostasis is poorly understood. In this study, we showed that hypothalamic proopiomelanocortin (POMC) neuron–specific ATF4 knockout (PAKO) mice are lean and have higher energy expenditure. Furthermore, PAKO mice were resistant to high-fat diet–induced obesity, glucose intolerance, and leptin resistance. Moreover, the expression of autophagy protein 5 (ATG5) was increased or decreased by ATF4 knockdown or overexpression, respectively, and ATF4 inhibited the transcription of ATG5 by binding to the basic zipper-containing protein sites on its promoter. Importantly, mice with double knockout of ATF4 and ATG5 in POMC neurons gained more fat mass and reduced energy expenditure compared with PAKO mice under a high-fat diet. Finally, the effect of ATF4 deletion in POMC neurons was possibly mediated via enhanced ATG5-dependent autophagy and α-melanocyte–stimulating hormone production in the hypothalamus. Taken together, these results identify the beneficial role of hypothalamic ATF4/ATG5 axis in the regulation of energy expenditure, obesity, and obesity-related metabolic disorders, which suggests that ATF4/ATG5 axis in the hypothalamus may be a new potential therapeutic target for treating obesity and obesity-related metabolic diseases.
- Published
- 2017
26. Upregulation of NRF2 through autophagy/ERK 1/2 ameliorates ionizing radiation induced cell death of human osteosarcoma U-2 OS
- Author
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Ni Chen, Jun Wang, Teruaki Konishi, and Rui Zhang
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,Programmed cell death ,MAP Kinase Signaling System ,NF-E2-Related Factor 2 ,Health, Toxicology and Mutagenesis ,ATG5 ,Apoptosis ,Biology ,digestive system ,environment and public health ,03 medical and health sciences ,Downregulation and upregulation ,Cell Line, Tumor ,Radiation, Ionizing ,Autophagy ,Genetics ,Humans ,Gene knockdown ,Cell Death ,Kinase ,respiratory system ,Up-Regulation ,Cell biology ,030104 developmental biology ,Gene Knockdown Techniques ,Autophagy Protein 5 - Abstract
The antioxidative response mediated by transcription factor NRF2 is thought to be a pivotal cellular defense system against various extrinsic stresses. It has been reported that activation of the NRF2 pathway confers cells with resistance to ionizing radiation-induced damage. However, the underlying mechanism remains largely unknown. In the current research, it was found that α-particle radiation has the ability to stimulate NRF2 expression in human osteosarcoma U-2 OS cells. Knockdown of cellular NRF2 level by shRNA-mediated gene silencing decreased the survival rate, increased the micronucleus formation rate and apoptosis rate in irradiated cells. Consistently, knockdown of NRF2 resulted in decreased expression of p65 and Bcl-2, and increased expression of p53 and Bax. Besides, it was observed that increased expression of NRF2 was partially dependent on radiation induced phosphorylation of ERK 1/2. Further results showed that radiation promoted autophagy flux which leads to the enhanced phosphorylation of ERK 1/2, as evidenced by the resultls that knockdown of ATG5 (Autophagy protein 5) gene by shRNA suppressed both radiation induced ERK 1/2 phosphorylation and NRF2 upregulation. Based on these results, it is proposed that attenuation of NRF2 antioxidative pathway can sensitize U-2 OS cells to radiation, where NRF2 antioxidative response is regulated by autophagy mediated activation of ERK 1/2 kinases.
- Published
- 2017
27. Mitigated NSAID-induced apoptotic and autophagic cell death with Smad7 overexpression
- Author
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Jong-Min Park, Ki Baik Hahm, and Ho-Jae Lee
- Subjects
0301 basic medicine ,Programmed cell death ,autophagy ,p38 mitogen-activated protein kinases ,Clinical Biochemistry ,Cell ,Medicine (miscellaneous) ,03 medical and health sciences ,gastrointestinal damages ,0302 clinical medicine ,medicine ,Cytotoxic T cell ,Cytotoxicity ,Nutrition and Dietetics ,integumentary system ,Smad7 ,Chemistry ,Autophagy ,apoptosis ,NSAID ,030104 developmental biology ,medicine.anatomical_structure ,Apoptosis ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,Cancer research ,Original Article - Abstract
Non-steroidal anti-inflammatory drugs damaged gastrointestinal mucosa in cyclooxygenase-dependent and -independent pathway, among which apopototic or autophagic cell death in gastrointestinal cells might be one of key cytotoxic mechanisms responsible for NSAID-induced damages. Therefore, alleviating this cell death after NSAIDs can be a rescuing strategy. In this study, we explored the role of Smad7 on NSAID-induced cytotoxicity in gastric epithelial cells. Using RGM1 cells, we have compared biological changes between mock-transfected and Smad7-overexpressed cells. As results, significantly decreased cytotoxicity accompanied with decreased levels of cleaved caspase-3 and poly (ADP-ribose) polymerase, Bax, and autophagic vesicles concurrent with decreased expressions of autophagy protein 5 and microtubule-associated protein light chain 3B-II were noted in Smad7-overexpressed cells with indomethacin administration compared to mock-transfected cells. Contrast to mitigated apoptotic execution, anti-apoptotic Bcl-2 and Beclin-1 were significantly increased in Smad7-overexpressed cells compared to mock-transfected cells. Smad7 siRNA significantly reversed these protective actions of Smad7 against indomethacin, in which p38 mitogen-activated protein kinase was significantly intervened. Furthermore, indomethacin-induced Smad7 degradation through ubiquitin-proteasome pathway was relevant to increased cytotoxicity, while chloroquine as autophagy inhibitor significantly attenuated indomethacin-induced cytotoxicity through Smad7 preservation via repressed ubiquitination. Conclusively, either genetic overexpression or pharmacological induction of Smad7 significantly attenuated indomethacin-induced gastric cell damages.
- Published
- 2016
28. Fish Autophagy Protein 5 Exerts Negative Regulation on Antiviral Immune Response Against Iridovirus and Nodavirus
- Author
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Chen Li, Jiaxin Liu, Xin Zhang, Shina Wei, Xiaohong Huang, Youhua Huang, Jingguang Wei, and Qiwei Qin
- Subjects
0301 basic medicine ,Fish Proteins ,lcsh:Immunologic diseases. Allergy ,Transcription, Genetic ,ATG5 ,Immunology ,Ranavirus ,RGNNV ,grouper ,Autophagy-Related Protein 5 ,Cell Line ,Iridovirus ,03 medical and health sciences ,Fish Diseases ,0302 clinical medicine ,RNA Virus Infections ,Interferon ,medicine ,Autophagy ,Immunology and Allergy ,Animals ,Grouper ,Nodaviridae ,Original Research ,Inflammation ,biology ,Cell Cycle ,Fishes ,interferon ,Cell cycle ,biology.organism_classification ,Immunity, Innate ,Cell biology ,030104 developmental biology ,SGIV ,Viral replication ,Gene Expression Regulation ,Autophagy Protein 5 ,Ectopic expression ,Atg5 ,lcsh:RC581-607 ,030215 immunology ,medicine.drug - Abstract
Autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. However, little is known about the functions of fish autophagy-related genes (Atgs). In this study, we cloned and characterized Atg5, a key gene in the autophagy gene superfamily, from orange-spotted grouper (Epinephelus coioides) (EcAtg5). EcAtg5 encoded a 275-amino acid protein that shared 94 and 81% identity to seabass (Lates calcarifer) and humans (Homo sapiens), respectively. The transcription level of EcAtg5 was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (RGNNV). In cells infected with Singapore grouper iridovirus (SGIV), EcAtg5 expression declined during the early stage of infection and increased in the late stage. Fluorescence microscopy revealed that EcAtg5 mainly localized with a dot-like pattern in the cytoplasm of grouper cells. Overexpression of EcAtg5 significantly increased the replication of RGNNV and SGIV at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. Knockdown of EcAtg5 decreased the replication of RGNNV and SGIV. Further studies showed that overexpression EcAtg5 activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κB, IFN-sensitive response element, and IFNs. In addition, ectopic expression of EcAtg5 affected cell cycle progression by hindering the G1/S transition. Taken together, our results demonstrated that fish Atg5 exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral IFN responses, and affecting the cell cycle.
- Published
- 2019
29. Carbazole alkaloids from Murraya koenigii trigger apoptosis and autophagic flux inhibition in human oral squamous cell carcinoma cells
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Anan Athipornchai, Monthon Lertcanawanichakul, Tanyarath Utaipan, Canussanun Jirachotikoon, Warangkana Chunglok, Xiaohong Yuan, and Apichart Suksamrarn
- Subjects
0301 basic medicine ,Autophagosome ,Murraya ,Herbal Medicine ,Antineoplastic Agents ,Apoptosis ,03 medical and health sciences ,chemistry.chemical_compound ,Alkaloids ,0302 clinical medicine ,Cell Line, Tumor ,Humans ,Cytotoxic T cell ,biology ,Carbazole ,Autophagy ,biology.organism_classification ,Plant Leaves ,030104 developmental biology ,Biochemistry ,chemistry ,Cell culture ,030220 oncology & carcinogenesis ,Carcinoma, Squamous Cell ,Autophagy Protein 5 ,Cancer research ,Molecular Medicine ,Mouth Neoplasms - Abstract
Carbazole alkaloids, a major constituent of Murraya koenigii (L.) Sprengel (Rutaceae), exhibit biological effects such as anticancer activity via the induction of apoptosis, and they represent candidate chemotherapeutic agents. Oral squamous cell carcinoma (OSCC) is the most prevalent cancer of the oral cavity and a growing and serious health problem worldwide. In this study, we investigated the anticancer properties and mechanisms of action of two carbazole alkaloids derived from M. koenigii leaves, mahanine and isomahanine, in the OSCC cell line CLS-354. At 15 μM, mahanine and isomahanine were cytotoxic to CLS-354 cells, triggering apoptosis via caspase-dependent and -independent mechanisms. Autophagosomes, visualised using monodansylcadaverine (MDC) labelling, were numerous in carbazole alkaloid-treated cells. Mahanine and isomahanine markedly induced the expression of the autophagosome marker microtubule-associated protein 1 light chain 3, type II (LC3B-II). Genetic and chemical inhibition of autophagy via silencing of the Autophagy protein 5 gene and exposure to bafilomycin A1 (BafA1), respectively, did not arrest carbazole alkaloid-induced apoptosis, indicating that it occurs independently of autophagic activation. Surprisingly, both carbazole alkaloids caused increased accumulation of p62/sequestosome1 (p62/SQSTM1), with coordinated expression of LC3B-II and cleaved caspase-3, suggesting inhibition of autophagic flux. Our results suggest that inhibition of autophagic flux is associated with carbazole alkaloid-induced apoptosis. Our findings provide evidence of a novel cytotoxic action of natural carbazole alkaloids and support their use as candidate chemotherapeutic agents for the treatment of OSCC.
- Published
- 2016
30. EHHM, a novel phenolic natural product from Livistona chinensis, induces autophagy-related apoptosis in hepatocellular carcinoma cells
- Author
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Jie Zhou, Xinsheng Cheng, Feng Zhong, Shibo Sun, Kun He, and Hongbo Chen
- Subjects
0301 basic medicine ,autophagy ,Cancer Research ,EHHM ,Cell growth ,Autophagy ,ATG5 ,apoptosis ,P70-S6 Kinase 1 ,Articles ,hepatocellular carcinoma ,Biology ,Cell cycle ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Oncology ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,Cancer research ,biology.protein ,Mechanistic target of rapamycin ,Protein kinase B - Abstract
Hepatocellular carcinoma (HCC) ranks the second cause of cancer-associated mortality worldwide. In the present study, the effects and mechanisms of a new phenolic natural product E-[6′-(5′-hydroxypentyl)tricosyl]-4-hydroxy-3-methoxycinnamate (EHHM) isolated from Livistona chinensis on the growth of HCC cells were investigated. It was observed that EHHM treatment significantly suppressed cell proliferation and colony formation, and induced cell apoptosis via a mitochondria-dependent caspase pathway in HepG2 cells in a time- and dose-dependent manner. Meanwhile, EHHM treatment also led to upregulated expression of autophagy protein 5 (Atg5), Beclin 1 and light chain 3 (LC3)-II proteins, and accumulation of green fluorescent protein-LC3 punctate florescent foci in HCC cells, suggesting that EHHM-induced apoptosis is accompanied by autophagy induction. Western blotting revealed that EHHM-induced autophagy is related to the inhibition of the Akt/mechanistic target of rapamycin/p70 ribosomal protein S6 kinase signaling pathway. Furthermore, treatment with Atg5 small interfering RNA or autophagy inhibitors significantly enhanced EHHM-mediated growth inhibition and apoptotic cell death, indicating that autophagy serves as a self-protective mechanism in EHHM-treated HCC cells, and that combined treatment with EHHM and autophagy inhibitors may be an effective therapeutic strategy for HCC.
- Published
- 2016
31. RLIP76 Depletion Enhances Autophagic Flux in U251 Cells
- Author
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Yicheng Lu, Liuhua Hu, Chenran Zhang, Qi Wang, Guohan Hu, Zheng Cai, and Qiang Liang
- Subjects
0301 basic medicine ,Autophagosome ,Autolysosome ,Apoptosis ,Membrane Fusion ,Phosphatidylinositol 3-Kinases ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Cell Line, Tumor ,Glioma ,Autophagy ,Temozolomide ,medicine ,Humans ,RNA, Small Interfering ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Gene knockdown ,Brain Neoplasms ,Chemistry ,GTPase-Activating Proteins ,Autophagosomes ,Cell Biology ,General Medicine ,medicine.disease ,Cell biology ,Dacarbazine ,Enzyme Activation ,030104 developmental biology ,Gene Knockdown Techniques ,Autophagy Protein 5 ,ATP-Binding Cassette Transporters ,Lysosomes ,Proto-Oncogene Proteins c-akt ,Signal Transduction - Abstract
Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.
- Published
- 2016
32. The Nuclear Receptor, Nor-1, Induces the Physiological Responses Associated With Exercise
- Author
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Shu-Ching M. Wang, Zewen K. Tuong, Emily Shao, Joel M. Goode, Tae Gyu Oh, George E.O. Muscat, and Michael A. Pearen
- Subjects
0301 basic medicine ,Muscle tissue ,Receptors, Steroid ,medicine.medical_specialty ,Transgene ,Neovascularization, Physiologic ,Mice, Transgenic ,Nerve Tissue Proteins ,mTORC1 ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,Models, Biological ,Cell Line ,Muscle hypertrophy ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Physical Conditioning, Animal ,Internal medicine ,Autophagy ,medicine ,Animals ,RNA, Messenger ,Muscle, Skeletal ,Molecular Biology ,Original Research ,Sirolimus ,Receptors, Thyroid Hormone ,Calcineurin ,Autophagosomes ,Skeletal muscle ,Hypertrophy ,General Medicine ,DNA-Binding Proteins ,Mice, Inbred C57BL ,Phenotype ,030104 developmental biology ,medicine.anatomical_structure ,Autophagy Protein 5 ,Calcium ,Signal transduction ,Lysosomes ,Microtubule-Associated Proteins ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Skeletal muscle remodels metabolic capacity, contractile and exercise phenotype in response to physiological demands. This adaptive remodeling response to physical activity can ameliorate/prevent diseases associated with poor diet and lifestyle. Our previous work demonstrated that skeletal muscle-specific transgenic expression of the neuron-derived orphan nuclear receptor, Nor-1 drives muscle reprogramming, improves exercise endurance, and oxidative metabolism. The current manuscript investigates the association between exercise, Nor-1 expression and the role of Nor-1 in adaptive remodeling. We demonstrate that Nor-1 expression is induced by exercise and is dependent on calcium/calcineurin signaling (in vitro and in vivo). Analysis of fatigue-resistant transgenic mice that express Nor-1 in skeletal muscle revealed increased hypertrophy and vascularization of muscle tissue. Moreover, we demonstrate that transgenic Nor-1 expression is associated with increased intracellular recycling, ie, autophagy, involving 1) increased expression of light chain 3A or LC3A-II, autophagy protein 5, and autophagy protein 12 in quadriceps femoris muscle extracts from Tg-Nor-1 (relative to Wild-type (WT) littermates); 2) decreased p62 expression indicative of increased autophagolysosome assembly; and 3) decreased mammalian target of rapamycin complex 1 activity. Transfection of LC3A-GFP-RFP chimeric plasmid demonstrated that autophagolysosome formation was significantly increased by Nor-1 expression. Furthermore, we demonstrated a single bout of exercise induced LC3A-II expression in skeletal muscle from C57BL/6 WT mice. This study, when combined with our previous studies, demonstrates that Nor-1 expression drives multiple physiological changes/pathways that are critical to the beneficial responses of muscle to exercise and provides insights into potential pharmacological manipulation of muscle reprogramming for the treatment of lifestyle induced chronic diseases.
- Published
- 2016
33. Trichosanthin-induced autophagy in gastric cancer cell MKN-45 is dependent on reactive oxygen species (ROS) and NF-κB/p53 pathway
- Author
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Qiaoying Huang, Bin Wei, Xiaogang Zhong, Wei Mai, and Shunrong Huang
- Subjects
0301 basic medicine ,Trichosanthin ,ATG5 ,Mice, Nude ,Apoptosis ,Biology ,03 medical and health sciences ,Stomach Neoplasms ,Cell Line, Tumor ,medicine ,Autophagy ,Animals ,Humans ,Cell Proliferation ,Pharmacology ,chemistry.chemical_classification ,Mice, Inbred BALB C ,Reactive oxygen species ,Caspase 3 ,Cell growth ,fungi ,lcsh:RM1-950 ,NF-kappa B ,Cancer ,medicine.disease ,Antineoplastic Agents, Phytogenic ,030104 developmental biology ,lcsh:Therapeutics. Pharmacology ,Biochemistry ,chemistry ,Cancer cell ,Cancer research ,Autophagy Protein 5 ,Molecular Medicine ,Female ,Tumor Suppressor Protein p53 ,Gastric cancer - Abstract
Trichosanthin (TCS), isolated from the root tuber of Trichosanthes kirilowii tubers in the Cucurbitaceae family, owns a great deal of biological and pharmacological activities including anti-virus and anti-tumor. TCS has been reported to induce cell apoptosis of a diversity of cancers, including cervical cancer, choriocarcinoma, and gastric cancer, etc. However, whether TCS would induce autophagy in gastric cancer cells was seldom investigated. In current study, human gastric cancer MKN-45 cell growth was significantly inhibited by TCS. The anti-proliferation effect of TCS was due to an increased autophagy, which was confirmed by monodansylcadervarine (MDC) staining, up-regulation of Autophagy protein 5 (Atg5), and conversion of LC3 I to LC3 II (autophagosome marker). In addition, TCS induced reactive oxygen species (ROS) in MKN-45 cells and ROS scavenger N-acetylcysteine (NAC) significantly reversed TCS-induced autophagy. Furthermore, NF-κB/p53 pathway was activated during the process of autophagy induced by TCS and the ROS generation was mediated by it in MKN-45 cells. In vivo results showed that TCS exerted significantly anti-tumor effect on MKN-45 bearing mice. Considering the clinical usage of TCS on other human diseases, these research progresses provided a new insight into cancer research and new therapeutic avenues for patients with gastric cancer.
- Published
- 2016
34. MOLECULAR CHARACTERIZATION OF AUTOPHAGY-RELATED GENE 5 FROMSpodoptera exiguaAND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS
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Kaiyu Liu, Yu-Qian Xia, Dandan Lu, Jing Zhou, Ning-Zhao Zhang, Li-Lin Zhou, Xu-Sheng Liu, Hui Ai, and Zuwen Chen
- Subjects
0301 basic medicine ,Regulation of gene expression ,biology ,Physiology ,fungi ,ATG5 ,Spodoptera litura ,General Medicine ,Spodoptera ,biology.organism_classification ,Biochemistry ,Molecular biology ,ATG12 ,03 medical and health sciences ,030104 developmental biology ,Rapid amplification of cDNA ends ,Insect Science ,Exigua ,Autophagy Protein 5 - Abstract
Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry-SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl-HP cells and partially co-localized with BmATG6-GFP, but it almost has no significant co-localization with GFP-HaATG8. Then, the Western blot analysis demonstrated that GFP-SeATG5 conjugated with ATG12. Moreover, real-time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.
- Published
- 2016
35. A comparative proteomics study of a synovial cell line stimulated with TNF‐α
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Tsuyoshi Iwasaki, Miki Karasaki, Mitsuyoshi Ueda, Seiji Shibasaki, Yumiko Takeda, Shunsuke Aburaya, Masayasu Kitano, Hajime Sano, Hironobu Morisaka, Wataru Aoki, and Sachie Kitano
- Subjects
rheumatoid arthritis ,030203 arthritis & rheumatology ,0301 basic medicine ,apoptosis ,gene ontology analysis ,Stimulation ,Biology ,Proteomics ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,comparative proteomics ,Pathogenesis ,03 medical and health sciences ,TNF‐α ,030104 developmental biology ,0302 clinical medicine ,Synovial Cell ,Downregulation and upregulation ,Apoptosis ,Autophagy Protein 5 ,Tumor necrosis factor alpha ,Research Articles ,Research Article ,synovial cell line - Abstract
To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor‐α (TNF‐α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. On the basis of the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF‐α. Next, we selected 269 differentially produced proteins that were detected only under TNF‐α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF‐α‐stimulated MH7A cells, we observed substantial production of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins such as BH3‐interacting domain death agonist, autophagy protein 5, apolipoprotein E, and caspase‐3. These results indicate that the upregulation of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins in synovial cells in response to TNF‐α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.
- Published
- 2016
36. Macrophage migration inhibitory factor induces autophagy to resist hypoxia/serum deprivation-induced apoptosis via the AMP-activated protein kinase/mammalian target of rapamycin signaling pathway
- Author
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Meng Hou and Wenzheng Xia
- Subjects
Male ,0301 basic medicine ,Cancer Research ,Programmed cell death ,Apoptosis ,AMP-Activated Protein Kinases ,Biology ,Biochemistry ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Autophagy ,Genetics ,Animals ,Macrophage Migration-Inhibitory Factors ,Molecular Biology ,PI3K/AKT/mTOR pathway ,TOR Serine-Threonine Kinases ,RPTOR ,AMPK ,Mesenchymal Stem Cells ,Cell Hypoxia ,Rats ,Cell biology ,Intramolecular Oxidoreductases ,030104 developmental biology ,Oncology ,Autophagy Protein 5 ,Cancer research ,Molecular Medicine ,Macrophage migration inhibitory factor ,Signal transduction ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Macrophage migration inhibitory factor (MIF) is an anti‑apoptotic agent in various cell types and protects the heart from stress‑induced injury by modulating autophagy. Autophagy, a conserved pathway for bulk degradation of intracellular proteins and organelles, helps to preserve and recycle energy and nutrients for cells to survive during starvation. The present study hypothesized that MIF protects bone marrow‑derived mesenchymal stem cells (MSCs) from apoptosis by modulating autophagy via the AMP‑activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. MSCs were obtained from rat bone marrow and cultured. Apoptosis was induced by hypoxia/serum deprivation for 24 h and was assessed using flow cytometry. MIF protected MSCs from apoptosis by modulating autophagy via the AMPK/mTOR signaling pathway resulting in increased expression of autophagy‑associated proteins (including LC3BI/LC3BII, Beclin‑1 and autophagy protein 5), and by increased phosphorylation of AMPK and decreased phosphorylation of mTOR. The MIF anti‑apoptotic effects were blocked by autophagy inhibitor, 3‑methyladenine or AMPK inhibitor, Compound C. These results indicate that MIF exerts a permissive role in protecting MSCs from apoptosis by regulation of autophagy via the AMPK/mTOR signaling pathway.
- Published
- 2016
37. C-myb Regulates Autophagy for Pulp Vitality in Glucose Oxidative Stress
- Author
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Junhyub Jeon, Young Hee Lee, H. Kim, Sung-Dae Cho, Mi-Kyung Yu, Jahae Kim, and Ho-Keun Yi
- Subjects
Male ,0301 basic medicine ,medicine.medical_specialty ,Blotting, Western ,AMP-Activated Protein Kinases ,Transfection ,medicine.disease_cause ,Diabetes Mellitus, Experimental ,Rats, Sprague-Dawley ,Glucose Oxidase ,Proto-Oncogene Proteins c-myb ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,Dentin sialophosphoprotein ,Internal medicine ,Autophagy ,medicine ,Animals ,Humans ,RNA, Small Interfering ,General Dentistry ,Protein kinase B ,Cells, Cultured ,Dental Pulp ,PI3K/AKT/mTOR pathway ,Chemistry ,030206 dentistry ,Dentinogenesis ,Immunohistochemistry ,Rats ,Oxidative Stress ,stomatognathic diseases ,Glucose ,030104 developmental biology ,Endocrinology ,Autophagy Protein 5 ,Pulp (tooth) ,Oxidative stress ,Signal Transduction - Abstract
Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B–light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases.
- Published
- 2015
38. Inhibition of glycogen synthase kinase 3β protects liver against ischemia/reperfusion injury by activating 5' adenosine monophosphate-activated protein kinase-mediated autophagy
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Qiang Xia, Jianjun Zhang, Xiangwei Hua, Defu Kong, Kang He, and Tian Qin
- Subjects
Liver injury ,Programmed cell death ,Hepatology ,Chemistry ,Autophagy ,AMPK ,Pharmacology ,medicine.disease ,Cytoprotection ,03 medical and health sciences ,0302 clinical medicine ,Infectious Diseases ,GSK-3 ,030220 oncology & carcinogenesis ,Autophagy Protein 5 ,medicine ,030211 gastroenterology & hepatology ,Protein kinase A - Abstract
Aim Autophagy has been found to play an important role in hepatic ischemia/reperfusion (I/R) injury. Our previous study has also clarified that rictor deficiency aggravated hepatic I/R injury by suppressing autophagy. Here, we explore whether autophagy participates in glycogen synthase kinase 3β (GSK3β)-mediated cytoprotection in liver I/R. Methods Mice were treated with SB216763 to inhibit GSK3β before being subjected to hepatic I/R. Liver injury was evaluated by liver and blood samples. Autophagy was measured by detecting expression of microtubule-associated protein 1 light chain-3B (LC3B) II and autophagy protein 5 (ATG-5), as well as the number of autophagosomes by transmission electron microscope. Primary hepatocytes pretreated with SB216763 for 2 h were subjected to hypoxia/reoxygenation to induce autophagy. The lactate dehydrogenase level was used to evaluate cell death and survival. Autophagy inhibitors and 5' adenosine monophosphate-activated protein kinase (AMPK) inhibitor were given in vivo or in vitro. Results SB216763 significantly increased the number of autophagosomes and the protein levels of LC3B II and ATG-5 in liver I/R models, which was accompanied by a decline of hepatic necrosis and apoptosis. Consistent with the in vivo study, autophagy and cytoprotection were induced by the inhibition of GSK3β in the in vitro study. Moreover, pretreatment with autophagy inhibitors attenuated the cytoprotective role of autophagy in the GSK3β-treated liver I/R models. Further analysis showed that pretreatment with an AMPK inhibitor increased mammalian target of rapamycin (mTOR) activity, decreased autophagy, and abrogated GSK3β- mediated liver protection. Conclusion Autophagy was induced by GSK3β inhibition through the AMPK/mTOR pathway and could substantially ameliorate liver I/R injury. Therefore, our findings strongly renew the therapeutic value of the GSK3β/autophagy axis in hepatic I/R injury.
- Published
- 2018
39. PEDF regulates lipid metabolism and reduces apoptosis in hypoxic H9c2 cells by inducing autophagy related 5-mediated autophagy via PEDF-R
- Author
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Yufeng Li, Qixiang Zhao, Hongli Yu, Meng Wang, Yiqian Zhang, Hao Zhang, Peng Lu, Zhong-Ming Zhang, Xiaoyu Wang, Zhiwei Liu, and Hongyan Dong
- Subjects
Receptors, Neuropeptide ,0301 basic medicine ,autophagy ,Cancer Research ,Programmed cell death ,ATG5 ,Cell ,Apoptosis ,Biochemistry ,Autophagy-Related Protein 5 ,Cell Line ,03 medical and health sciences ,PEDF ,pigment epithelial-derived factor receptor ,autophagy protein 5 ,Genetics ,medicine ,Animals ,Nerve Growth Factors ,Eye Proteins ,Molecular Biology ,Serpins ,pigment epithelial-derived factor ,Chemistry ,Autophagy ,Lipid metabolism ,Articles ,Lipid Metabolism ,Cell Hypoxia ,Rats ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Autophagy Protein 5 ,Molecular Medicine ,lipid degradation ,Signal Transduction - Abstract
Pigment epithelial-derived factor (PEDF) is a multifunctional secreted glycoprotein, which exerts a variety of physiological activities. PEDF may protect against hypoxia-induced cell death associated with its antioxidative effects and p53 mitochondrial translocation in cultured cardiomyocytes and H9c2 cells. Additionally, previous studies have suggested that autophagy is an important cell survival mechanism. However, the effect of PEDF on autophagy and the associated pathway in hypoxic H9c2 cells has not been fully established. Autophagy has been reported to regulate lipid metabolism; however, little is known about whether PEDF is able to regulate lipid metabolism by promoting autophagy. In the present study, western blotting results revealed that PEDF increased the level of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II. Transmission electron microscopy (TEM) and LC3 fluorescence demonstrated that PEDF increased the number of autophagosomes. PEDF also increased the viability of hypoxic H9c2 cells and decreased the level of cleaved caspase-3 protein, as evidenced by CCK-8 assays and western blotting, respectively. TEM and a triglyceride assay kit demonstrated that PEDF-induced autophagy may stimulate lipid degradation. Western blotting results revealed a novel mechanism underlying PEDF-induced H9c2 cell autophagy via the PEDF-R-mediated Atg5 pathway under hypoxic conditions. Furthermore, the results also suggest that PEDF-induced autophagy may stimulate lipid degradation. The survival function of autophagy suggests that modulation of PEDF-induced autophagy may be used as a therapeutic strategy to protect cells against lipid-associated metabolic diseases.
- Published
- 2018
40. Inhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cells
- Author
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Lemeng Zhang, Hui Zhou, Jianhua Chen, Yongzhong Luo, Wei Wang, Hua Yang, and Huihuang Yi
- Subjects
0301 basic medicine ,Cancer Research ,Small interfering RNA ,Lung Neoplasms ,ATG5 ,Antineoplastic Agents ,Apoptosis ,Biochemistry ,Autophagy-Related Protein 5 ,ATG12 ,03 medical and health sciences ,Autophagy ,Genetics ,Humans ,RNA, Small Interfering ,Protein kinase A ,Molecular Biology ,A549 cell ,Chemistry ,Cell cycle ,Up-Regulation ,Cell biology ,030104 developmental biology ,Oncology ,A549 Cells ,Gene Knockdown Techniques ,Autophagy Protein 5 ,Molecular Medicine ,Beclin-1 ,RNA Interference ,Cisplatin - Abstract
Recent studies have indicated that autophagy contributes to tumorigenesis and participates in acquired chemotherapeutic resistance. The present study aimed to determine the function and underlying mechanism of cisplatin‑induced autophagy in A549 human lung cancer cells. Autophagy was measured by LC3B‑I/II conversion, LC3B puncta and autophagosomes formation. Apoptotic cell death was measured by caspase‑3 activity, caspase‑3 cleavage and LDH release. The transcriptional and expressional level of autophagy related proteins were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Beclin 1 and Atg 5 siRNA transfection was used to explore the function of cisplatin‑induced autophagy. The results demonstrated that cisplatin induces apoptotic cell death in A549 cells and triggers an autophagic response, as indicated by increased microtubule‑associated protein 1 light chain 3β (LC3B)‑I/II conversion, increased LC3B puncta and autophagosome formation. Mechanisms underlying cisplatin‑induced autophagic responses were also investigated. Cisplatin induced autophagy by upregulating the mRNA and protein expression levels of autophagy protein (Atg)5 and Beclin 1, whereas the mRNA and protein expression levels of serine/threonine‑protein kinase ULK1, Atg3, Atg7, Atg12, and sequestosome‑1 were not markedly upregulated. In addition, knockdown of Atg5 and Beclin 1 by small interfering RNA transfection impaired cisplatin‑induced activation of autophagic responses, increased caspase‑3 cleavage and inhibited cell viability. These findings suggested that disruption of autophagy via the inhibition of Atg5 and Beclin 1 may promote cisplatin‑induced apoptotic cell death in A549 human lung cancer cells. In conclusion, the present study demonstrated that targeting autophagy may be used in the future for the treatment of lung cancer.
- Published
- 2018
41. Countervailing, time-dependent effects on host autophagy promote intracellular survival of Leishmania
- Author
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Sneha A. Thomas, Neil E. Reiner, Jennifer Kass, and Devki Nandan
- Subjects
0301 basic medicine ,Autophagosome ,ATG5 ,Leishmania donovani ,Biology ,Biochemistry ,Autophagy-Related Protein 5 ,Host-Parasite Interactions ,03 medical and health sciences ,parasitic diseases ,Autophagy ,Humans ,Molecular Biology ,Mechanistic target of rapamycin ,PI3K/AKT/mTOR pathway ,Macrophages ,TOR Serine-Threonine Kinases ,Molecular Bases of Disease ,Cell Biology ,Leishmania ,biology.organism_classification ,Phosphoric Monoester Hydrolases ,3. Good health ,Cell biology ,030104 developmental biology ,Autophagy Protein 5 ,biology.protein ,Leishmaniasis, Visceral ,Microtubule-Associated Proteins - Abstract
Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection—and via activation of the Akt pathway—Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.
- Published
- 2017
42. Effect of β‑ecdysterone on glucocorticoid‑induced apoptosis and autophagy in osteoblasts
- Author
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Lin‑Ru Zeng, Can‑Da Xu, Da‑Wei Xin, Guo‑Song Li, Zhen‑Shuang Yue, Yang‑Hua Tang, and Zhong‑Qing Hu
- Subjects
Male ,0301 basic medicine ,autophagy ,Cancer Research ,Cellular differentiation ,Biology ,Biochemistry ,Mice ,03 medical and health sciences ,β-ecdysterone ,Genetics ,medicine ,Animals ,Glucocorticoids ,Molecular Biology ,PI3K/AKT/mTOR pathway ,mammalian target of rapamycin ,Osteoblasts ,Cell growth ,Autophagy ,apoptosis ,Cell Differentiation ,Mesenchymal Stem Cells ,Osteoblast ,Articles ,Cell biology ,Ecdysterone ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Apoptosis ,steoporosis ,Cancer research ,Autophagy Protein 5 ,Osteocalcin ,biology.protein ,Molecular Medicine - Abstract
The aim of the present study was to investigate the effect of glucocorticoids in osteoblasts and to examine the role of β‑ecdysterone in the pathogenesis of glucocorticoid‑induced osteoporosis. Osteoblasts were induced from bone marrow mesenchymal stem cells, which were isolated from C57BL/6 mice. Cell viability and apoptosis of osteoblasts were measured by Cell Counting Kit‑8 and flow cytometry analysis, respectively. The expression of related genes and proteins was measured by reverse transcription quantitative polymerase chain reaction and western blot analysis respectively. Dose‑dependent decreases in the cell proliferation and differentiation were observed in dexamethasone (Dex)‑treated osteoblasts, evidenced by downregulation in the activity of alkaline phosphatasedecreased expression levels of Runt‑related transcription factor 2 and osteocalcin, and upregulated expression of RANK ligand. Dex also induced apoptosis and inhibited autophagy of osteoblasts, evidenced by upregulated B‑cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio and the activation of mammalian target of rapamycin (mTOR), and decreased expression levels of Beclin‑1, autophagy protein 5 and microtubule‑associated protein 1 light chain 3 II. The effects on cell proliferation, apoptosis and autophagy induced by Dex were reversed by β‑ecdysterone in a dose‑dependent manner. Therefore, β‑ecdysterone may be a promising candidate drug for the treatment of osteoporosis through inducing osteoblast autophagic activity by inactivating mTOR.
- Published
- 2017
43. Proflavin suppresses the growth of human osteosarcoma MG63 cells through apoptosis and autophagy
- Author
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Mao‑Shu Zhang, Fu‑Wen Niu, and Kun Li
- Subjects
Cancer Research ,Messenger RNA ,Programmed cell death ,Oncogene ,Cell ,Autophagy ,Articles ,Biology ,Cell cycle ,Cell biology ,medicine.anatomical_structure ,Oncology ,Apoptosis ,Immunology ,Autophagy Protein 5 ,medicine - Abstract
Proflavin is one of the novel acridine derivatives that possess various pharmacological effects. Although numerous studies have been performed to investigate proflavin, its effects have not been investigated on the human osteosarcoma MG63 cell line. The core aim of the present study was to test the effects of proflavin on the viability of MG63 cells and the induction of apoptosis and autophagy in MG63 cells. The induction of apoptosis was examined by measuring the changes in the expression of the B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein mRNA and proteins. Apoptotic cell death was identified by the proteolytic cleavage of poly (adenosine diphosphate-ribose) polymerase and caspase-3 and caspase-9. In addition, the autophagic effects of proflavin were examined by the quantitation of the mRNA expression of autophagy protein 5 and Beclin 1, in addition to the identification of the accumulation of microtubule-associated protein 1 light chain 3-II. The present results revealed that proflavin inhibited the proliferation of MG63 cells in a dose-dependent manner. Proflavin-induced cell death was attributed to apoptosis and autophagy. Overall, the present results indicated that the antiseptic agent proflavin exerts anticancer potential through the synergistic activity of apoptosis and autophagy.
- Published
- 2015
44. MiRNA-30a-mediated autophagy inhibition sensitizes renal cell carcinoma cells to sorafenib
- Author
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Dong-hua Gu, Boxin Xue, Dongrong Yang, Lin Qian, Xiao-dong Pan, Hua Zhu, Bing Zheng, Yuxi Shan, and Jundong Zhou
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Male ,Niacinamide ,Sorafenib ,Biophysics ,Down-Regulation ,Antineoplastic Agents ,Apoptosis ,Biology ,urologic and male genital diseases ,Biochemistry ,Autophagy-Related Protein 5 ,Downregulation and upregulation ,Cell Line, Tumor ,Autophagy ,medicine ,Humans ,RNA, Neoplasm ,RNA, Small Interfering ,Cytotoxicity ,Carcinoma, Renal Cell ,neoplasms ,Molecular Biology ,Aged ,Gene knockdown ,Phenylurea Compounds ,Membrane Proteins ,Cell Biology ,Middle Aged ,Kidney Neoplasms ,female genital diseases and pregnancy complications ,Up-Regulation ,Cell biology ,MicroRNAs ,Drug Resistance, Neoplasm ,Cell culture ,Gene Knockdown Techniques ,Autophagy Protein 5 ,Beclin-1 ,Female ,Apoptosis Regulatory Proteins ,Microtubule-Associated Proteins ,Signal Transduction ,medicine.drug - Abstract
Chemotherapy-induced autophagy activation often contributes to cancer resistance. MiRNA-30a (miR-30a) is a potent inhibitor of autophagy by downregulating Beclin-1. In this study, we characterized the role of miR-30a in sorafenib-induced activity in renal cell carcinoma (RCC) cells. We found that expression of miR-30a was significantly downregulated in several human RCC tissues and in RCC cell lines. Accordingly, its targeted gene Beclin-1 was upregulated. Sorafenib activated autophagy in RCC cells (786-0 and A489 lines), evidenced by p62 degradation, Beclin-1/autophagy protein 5 (ATG-5) upregulation and light chain (LC)3B-I/-II conversion. Exogenously expressing miR-30a in 786-0 or A489 cells inhibited Beclin-1 expression and enhanced sorafenib-induced cytotoxicity. In contrast, knockdown of miR-30a by introducing antagomiR-30a increased Beclin-1 expression, and inhibited sorafenib-induced cytotoxicity against RCC cells. Autophagy inhibitors, including chloroquine, 3-methyaldenine or Bafliomycin A1, enhanced sorafenib activity, causing substantial cell apoptosis. Meanwhile, knockdown of Beclin-1 or ATG-5 by targeted siRNAs also increased sorafenib-induced cytotoxicity in above RCC cells. These findings indicate that dysregulation of miR-30a in RCC may interfere with the effectiveness of sorafenib-mediated apoptosis by an autophagy-dependent pathway, thus representing a novel potential therapeutic target for RCC.
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- 2015
45. PPARγ Maintains Homeostasis through Autophagy Regulation in Dental Pulp
- Author
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Young Hee Lee, Nan-Hee Lee, Ho-Keun Yi, Tae Gun Kim, Mi-Kyung Yu, and H.Y. Lee
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Adult ,Lipopolysaccharides ,Aging ,Cell Survival ,Metalloporphyrins ,Sialoglycoproteins ,Protoporphyrins ,Vascular Cell Adhesion Molecule-1 ,Biology ,Rats, Sprague-Dawley ,Young Adult ,Dentin sialophosphoprotein ,Autophagy ,Animals ,Homeostasis ,Humans ,Viability assay ,Enzyme Inhibitors ,General Dentistry ,Cells, Cultured ,Dental Pulp ,Aged ,Extracellular Matrix Proteins ,Cell adhesion molecule ,Middle Aged ,Intercellular Adhesion Molecule-1 ,Phosphoproteins ,Mitochondria ,Rats ,Cell biology ,PPAR gamma ,Dentinogenesis ,Autophagy Protein 5 ,Pulp (tooth) ,Apoptosis Regulatory Proteins ,Microtubule-Associated Proteins ,Cell aging ,Heme Oxygenase-1 - Abstract
This study investigated the relevance between pulp vitality and autophagy in aged human dental pulp cells (HDPCs) and whether peroxisome proliferator-activated receptor gamma (PPARγ) affects autophagy regulation for homeostasis in the aging progress. In vivo experiments were used in human and Sprague-Dawley rat teeth obtained from young and adult individuals. Aging- and autophagy-related molecules were determined by immunohistochemistry and hematoxylin and eosin staining. HDPCs were serially subcultured until spontaneously arrested for in vitro aging, and the replication deficiency adenovirus was introduced for PPARγ overexpression. Subsequently, the effect of PPARγ on regulation of autophagy molecules, mitochondria activity, and cell viability was assessed using Western blotting, confocal microscopy, and the MTT assay, respectively. In adult pulp tissue, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B light chain, and Beclin-1) were increased, but aging-related (PPARγ and heme oxygenase 1 [HO-1]) and dentinogenesis (dentin sialophosphoprotein and dentin matrix acidic phosphoprotein) molecules were decreased. In aged HDPCs, autophagy and intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were increased, while PPARγ and HO-1 were decreased. Under stimulation with lipopolysaccharide, autophagy- and aging-related molecules were differentially expressed between young and aged cells. PPARγ induced HO-1 and autophagy molecules but reduced inflammatory molecules in aged cells. In addition, PPARγ activated strong mitochondrial activity and cell viability in aging cells. Inhibition of HO-1 by tin protoporphyrin IX exacerbated autophagy and mitochondrial activity as well as cell viability in young cells. This study indicates that PPARγ maintains pulp homeostasis through the regulation of autophagy molecules during the life span of HDPCs.
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- 2015
46. A Nontoxic Concentration of Cisplatin Induces Autophagy in Cervical Cancer
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Benjamin Loos, Gina Leisching, Anna-Mart Engelbrecht, and M H Botha
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Programmed cell death ,Pathology ,medicine.medical_specialty ,ATG5 ,Uterine Cervical Neoplasms ,Antineoplastic Agents ,Autophagy-Related Protein 5 ,HeLa ,Autophagy ,medicine ,Humans ,Enzyme Inhibitors ,RNA, Small Interfering ,Cisplatin ,biology ,business.industry ,Carcinoma ,RNA-Binding Proteins ,Obstetrics and Gynecology ,biology.organism_classification ,Oncology ,Drug Resistance, Neoplasm ,Apoptosis ,Cancer cell ,Autophagy Protein 5 ,Cancer research ,Female ,Macrolides ,Squamous Intraepithelial Lesions of the Cervix ,business ,Microtubule-Associated Proteins ,HeLa Cells ,medicine.drug - Abstract
Background Increasing resistance to cisplatin as well as the severity of the adverse effects limit the use of this drug, particularly at high doses. Evidence has implicated the importance of autophagy in cancer resistance as well as the fact that various chemotherapy agents induce autophagy in cancer cells. We therefore aimed to first assess the role of autophagy in cisplatin treatment and second to assess whether a nontoxic concentration of cisplatin, together with autophagy inhibition, is able to maintain its cancer-specific cytotoxic action. Methods Three human cervical cell lines were used: a noncancerous ectocervical epithelial cell line (Ect1/E6E7) and 2 cancerous cervical cell lines (HeLa and CaSki). Autophagy was monitored through the presence of the classical protein markers LC-3 II and p62 under basal and treatment conditions, and inhibited using bafilomycin and autophagy protein 5 (ATG5) siRNA under treatment conditions. Cell death was analyzed through examination of the apoptotic markers PARP and caspase-3 through Western blotting, as well as the Caspase-Glo assay to confirm caspase-3/7 activity. Cervical biopsies were analyzed for the presence of LC-3 using Western blotting and immunofluorescence to determine if a correlation between autophagic levels and the progression of the disease exists. Results Cervical cancer cells exhibit increased basal autophagic levels in comparison to the noncancerous counterparts. Cisplatin treatment enhanced autophagic activity in all 3 cell lines. Inhibition of this autophagic response together with cisplatin treatment leads to significant increases in cancer cell death. Expression profiles of LC-3 in normal, premalignant (low-grade and high-grade squamous intraepithelial lesion), and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma cervical tissue, which emphasized the role of autophagy in the progression of the disease. Conclusions The inhibition of autophagy improves the cytotoxicity of a nontoxic concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer.
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- 2015
47. Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (α-Synuclein)-Environment (Paraquat) Interactions
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Oleh Khalimonchuk, Annadurai Anandhan, Mihalis I. Panayiotidis, Rodrigo Franco, Roman M. Levytskyy, Robert Powers, Aglaia Pappa, Ronald L. Cerny, and Shulei Lei
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0301 basic medicine ,Paraquat ,Programmed cell death ,Glucose uptake ,Neuroscience (miscellaneous) ,Glucose Transport Proteins, Facilitative ,Nitric Oxide Synthase Type II ,B100 ,B200 ,Article ,Cell Line ,Pentose Phosphate Pathway ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Dopaminergic Cell ,Autophagy ,Animals ,Mice, Knockout ,biology ,Cell Death ,Dopaminergic Neurons ,Adenylate Kinase ,C100 ,Glucose transporter ,AMPK ,Brain ,Biological Transport ,A100 ,C700 ,C900 ,Cell biology ,Rats ,Enzyme Activation ,Mice, Inbred C57BL ,B900 ,030104 developmental biology ,Glucose ,Neurology ,Autophagy Protein 5 ,biology.protein ,Metabolome ,alpha-Synuclein ,Gene-Environment Interaction ,030217 neurology & neurosurgery ,GLUT4 ,Signal Transduction - Abstract
While environmental exposures are not the single cause of Parkinson’s disease (PD), their interaction with genetic alterations is thought to contribute to neuronal dopaminergic degeneration. However, the mechanisms involved in dopaminergic cell death induced by gene-environment interactions remain unclear. In this work, we have revealed for the first time the role of central carbon metabolism and metabolic dysfunction in dopaminergic cell death induced by paraquat (PQ)-α-synuclein interaction. The toxicity of PQ in dopaminergic N27 cells was significantly reduced by glucose deprivation, inhibition of hexokinase with 2-deoxy-D-glucose (2-DG), or equimolar substitution of glucose with galactose, which evidenced the contribution of glucose metabolism to PQ-induced cell death. PQ also stimulated an increase in glucose uptake, and the translocation of glucose transporter type 4 (GLUT4) and Na+-glucose transporters isoform 1 (SGLT1) proteins to the plasma membrane, but only inhibition of GLUT-like transport with STF-31 or ascorbic acid reduced PQ-induced cell death. Importantly, while autophagy protein 5 (ATG5)/unc-51 like autophagy activating kinase 1 (ULK1)-dependent autophagy protected against PQ toxicity, the inhibitory effect of glucose deprivation on cell death progression was largely independent of autophagy or mammalian target of rapamycin (mTOR) signaling. PQ selectively induced metabolomic alterations and adenosine monophosphate-activated protein kinase (AMPK) activation in the midbrain and striatum of mice chronically treated with PQ. Inhibition of AMPK signaling led to metabolic dysfunction and an enhanced sensitivity of dopaminergic cells to PQ. In addition, activation of AMPK by PQ was prevented by inhibition of the inducible nitric oxide syntase (iNOS) with 1400W, but PQ had no effect on iNOS levels. Overexpression of wild type or A53T mutant α-synuclein stimulated glucose uptake and PQ toxicity, and this toxic synergism was reduced by inhibition of glucose metabolism/transport and the pentose phosphate pathway (6-aminonicotinamide). These results demonstrate that glucose metabolism and AMPK regulate dopaminergic cell death induced by gene (α-synuclein)-environment (PQ) interactions.
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- 2017
48. Investigation of the expression of apoptosis-inducing factor-mediated apoptosis in Hirschsprung's disease
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Chunyan Hu, Yu-Ying Zhao, Xin Ge, Peng Li, Weili Yang, Qingqing Guo, Wei Li, Yudu Wang, Qiang Huang, and Ya Gao
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0301 basic medicine ,Pathology ,medicine.medical_specialty ,ATG5 ,Blotting, Western ,Gene Expression ,Apoptosis ,Real-Time Polymerase Chain Reaction ,Autophagy-Related Protein 5 ,03 medical and health sciences ,Microscopy, Electron, Transmission ,medicine ,In Situ Nick-End Labeling ,Humans ,Hirschsprung Disease ,RNA, Messenger ,Intestinal Mucosa ,Hirschsprung's disease ,Caspase ,Neurons ,biology ,Calpain ,Caspase 3 ,General Neuroscience ,Autophagy ,Apoptosis Inducing Factor ,medicine.disease ,Molecular biology ,Immunohistochemistry ,Blot ,Intestines ,030104 developmental biology ,Proto-Oncogene Proteins c-bcl-2 ,Autophagy Protein 5 ,biology.protein ,Apoptosis-inducing factor - Abstract
One of the widely accepted hypotheses of Hirschsprung's disease (HD) is that the absence of ganglion cells in the distal part of the intestine is caused by the death of enteric neural crest-derived cells following migration. Although a caspase-dependent pathway has not yet been detected in the HD bowel, it is unclear whether a caspase-independent pathway contributes toward aganglionosis. In the current study, we observed highly condensed marginal heterochromatin in nuclei only in the transitional segment using electron microscopy and a high proportion of TUNEL-positive cells were observed in the transitional segment. Activation of caspase was not observed in any segments of the HD bowel upon characterization of the apoptotic pathway. Rather, real-time PCR results showed that apoptosis-inducing factor (AIF) and calpain-1 mRNAs were highly expressed in the transitional segment, whereas autophagy protein 5 (Atg5) was highly expressed in the narrow segment. Western blot results were consistent with mRNA levels, with increased AIF, calpain-1, and Atg5 expressions in the transitional segment compared with the dilated segment. Furthermore, correlation analysis indicated an inverse correlation between calpain-1 and Atg5 mRNA levels in both the narrow segment and the transitional segment. These results indicated that apoptosis occurs in the HD bowel. The detection of related genes indicates that the AIF-mediated apoptotic pathway may be responsible for the absence of ganglion cells in HD and calpain-1 may act as the regulatory switch between autophagy and apoptosis.
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- 2017
49. Acetylated cyclophilin A is a major mediator in hypoxia-induced autophagy and pulmonary vascular angiogenesis
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Xiufeng Yu, Yao Fu, Daling Zhu, Jiali Li, Qian Li, Min Mao, Xiaodong Zheng, Rui Gu, Tingting Shen, Xin Ge, Shasha Song, and Xuecang Li
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0301 basic medicine ,Male ,Physiology ,Angiogenesis ,Hypertension, Pulmonary ,Cellular homeostasis ,Cypa ,Pulmonary Artery ,Autophagy-Related Protein 7 ,Muscle, Smooth, Vascular ,Autophagy-Related Protein 5 ,03 medical and health sciences ,Cell Movement ,Internal Medicine ,Autophagy ,Medicine ,Animals ,Endothelium ,Hypoxia ,Cells, Cultured ,Tube formation ,biology ,Neovascularization, Pathologic ,business.industry ,Endothelial Cells ,Acetylation ,Hypoxia (medical) ,biology.organism_classification ,Cell biology ,Rats ,Endothelial stem cell ,030104 developmental biology ,Cytoprotection ,Autophagy Protein 5 ,Cattle ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business ,Cyclophilin A - Abstract
Background Autophagy is a major intracellular degradation and recycling process that maintains cellular homeostasis, which is involved in structural and functional abnormalities of pulmonary vasculature in hypoxic pulmonary arterial hypertension (HPAH). Cyclophilin A (CyPA) is a secreted, oxidative stress-induced factor. Its role in inducing autophagy and augmenting endothelial cell dysfunction has never been explored. Methods Lungs from rats exposed to chronic hypoxia were examined for autophagy with electron microscopy, western blotting, and fluorescence microscopy. Results Activated autophagy was seen in the endothelium of the pulmonary artery from experimental rat models of HPAH and cultured bovine pulmonary arterial endothelial cells under hypoxia. Inhibiting autophagy attenuated the pathological progression of HPAH and repressed endothelial cell migration and angiogenesis. We also showed that CyPA was upregulated and acetylated under hypoxia and led to the abnormal occurrence of autophagy through its interaction with autophagy protein 5 and autophagy protein 7. Moreover, acetylated CyPA was essential for the excessive proliferation, migration, and tube formation networks of pulmonary arterial endothelial cells. Conclusion Our results indicate the crucial role of acetylated CyPA in the abnormal occurrence of autophagy and subsequent pulmonary vascular angiogenesis.
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- 2017
50. Acriflavine suppresses the growth of human osteosarcoma cells through apoptosis and autophagy
- Author
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Xin Yang, Zhenggang Bi, and Jingzhang Fan
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Osteosarcoma ,Programmed cell death ,Reverse Transcriptase Polymerase Chain Reaction ,Cell growth ,Poly ADP ribose polymerase ,Immunoblotting ,Autophagy ,ATG5 ,Antineoplastic Agents ,Apoptosis ,General Medicine ,Biology ,Real-Time Polymerase Chain Reaction ,Molecular biology ,Cell biology ,chemistry.chemical_compound ,chemistry ,Cell Line, Tumor ,Autophagy Protein 5 ,Humans ,Acriflavine ,Cell Proliferation - Abstract
The aim of this study was to investigate the effects of acriflavine on viability and induction of apoptosis and autophagy in human osteosarcoma cell lines MG63. Inhibition of cell proliferation by acriflavine was determined using MTT assay. Induction of apoptosis was examined by measuring the changes in expression of Bcl-2 and Bax in messenger RNA (mRNA) and protein levels. Identification of the proteolytic cleavage of poly (ADP)-ribose polymerase (PARP) and caspase-3 and caspase-9 was carried out to study apoptotic cell death. Autophagic effects were examined by quantitation of mRNA expression of autophagy protein 5 (Atg5) and Beclin1 and identifying accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II. The results showed acriflavine inhibited cell proliferation of osteosarcoma cells in dose-dependent fashion. Acriflavine-induced cell death was attributed to both apoptosis and autophagy. Moreover, it was associated with changes in the levels of Bcl-2 and Bax in the osteosarcoma cells. The antiseptic agent, acriflavine, has anticancer potential through synergistic activity of apoptosis and autophagy.
- Published
- 2014
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