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Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Elevated expression of family with sequence similarity 83 member D (Fam83D) has been found in various cancers; however, its role in pancreatic adenocarcinoma (PDAC) remains unclear. The current study was designed to elucidate the roles of Fam83D in pancreatic cancer., Method: The level of Fam83D was detected in PDAC tissues and adjacent no-tumorous tissues. Effects of Fam83D on proliferation, glycolysis and gemcitabine (GEM) sensitivity of pancreatic cancer cells were examined., Results: Fam83D was overexpressed in PDAC and associated with clinical stage, metastatic status and survival rates of PDAC patients. Function study showed that Fam83D knockdown (KD) caused inhibited proliferation, suppressed mitochondrial respiration capacity, reduced aerobic glycolysis, and down-regulation of nuclear β-catenin, proto-oncogene C-Myc, and lactate dehydrogenase A (LDHA). Fam83D KD enhanced the sensitivity of PDAC cells to GEM in vitro and in vivo. On the contrary, Fam83D overexpression displayed reverse effects on PDAC cells. Moreover, the Wnt/β-catenin inhibitor abolished the effects of Fam83D overexpression in PDAC cells., Conclusions: the current data suggest that enhanced Fam83D expression contributes to PDAC progression and the development of chemoresistance through the Wnt/β-catenin signaling., (Copyright © 2021. Published by Elsevier Inc.)

Objective: This work aims to determine the role of circular RNA (circRNA) AGFG1 and related molecular mechanism in esophageal squamous cell carcinoma (ESCC) cells., Methods: CircAGFG1 expression in ESCC cell lines was probed with qRT-PCR. ESCC cells were transfected/cotransfected with si-circAGFG1, pcDNA3.1-circAGFG1, si-Microtubule Associated Protein RP/EB Family Member 2 (MAPRE2), pcDNA3.1-circAGFG1 + miR-4306 mimic or pcDNA3.1-circAGFG1 + si-MAPRE2. The interactions between circAGFG1 and miR-4306 as well as miR-4306 and MAPRE2 were confirmed by dual-luciferase reporter assay. Cell proliferation, migration and invasion were detected by CCK-8, cell scratch and Transwell assays, respectively. Relative RNA expression levels of circAGFG1, miR-4306 and MAPRE2 in ESCC cells were measured by qRT-PCR. The protein level of MAPRE2 in ESCC cells was monitored by Western blot., Results: CircAGFG1 was observably upregulated in ESCC cell lines. Besides, circAGFG1 silencing hindered ESCC cell development in vitro, and these effects were enhanced by miR-4306 overexpression or MAPRE2 silencing. Mechanistic analysis evidenced that circAGFG1 might act as a competitive endogenous RNA of miR-4306 to relieve the repressive effect of miR-4306 on its target MAPRE2., Conclusion: CircAGFG1 facilitates ESCC progression via the miR-4306/MAPRE2 axis, and it may act as a possible biomarker for therapy and diagnosis in ESCC treatment., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Objective: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum., Methods: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells., Results: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally., Interpretation: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284., (© 2021 American Neurological Association.)

Emerging evidence suggests that microRNAs (miRNAs) participate in hepatocellular carcinoma (HCC) progression. Nevertheless, the mechanism of miR-7-5p in HCC cells has not been researched. In the research, the underlying biological function of miR-7-5p and SPC24 in HCC was explored. qRT-PCR was performed to measure the miR-7-5p and SPC24 level in HCC tissues and cells. The effect of miR-7-5p on HCC progression was detected by performing CCK-8, BrdU, and transwell assay. The relationship between miR-7-5p and SPC24 was determined using luciferase and RNA pull-down assays. Our findings showed that miR-7-5p was downregulated in HCC whereas SPC24 was upregulated in HCC. It was also showed that miR-7-5p upregulation restricted malignant behaviors of HCC cells, but this inhibitory effect of miR-7-5p could be relieved by its target gene SPC24. In conclusion, this research suggested that by inhibiting SPC24, miR-7-5p could act as a tumor inhibitory factor in HCC., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Collapsin response mediator protein 5 (CRMP5), a member of the CRMP family, is expressed in the brain, particularly in the hippocampus, an area of the brain that can modulate stress responses. Social stress has a well-known detrimental effect on health and can lead to depression, but not all individuals are equally sensitive to stress. To date, researchers have not conclusively determined how social stress increases the susceptibility of the brain to depression. Here, we used the chronic social defeat stress (CSDS) model and observed higher hippocampal CRMP5 expression in stress-susceptible (SS) mice than in control and stress-resilient (RES) mice. A negative correlation was observed between the expression levels of CRMP5 and the social interaction (SI) ratio. Reduced hippocampal CRMP5 expression increased the SI ratio in SS mice, whereas CRMP5 overexpression was sufficient to induce social avoidance behaviors in control mice following exposure to subthreshold social stress induced by lentivirus-based overexpression and inducible tetracycline-on strategies to upregulate CRMP5. Interestingly, increased CRMP5 expression in SS and lenti-CRMP5-treated mice also caused serum corticosterone concentrations to increase. These findings improve our understanding of the potential mechanism by which CRMP5 triggers susceptibility to social stress, and they support the further development of therapeutic agents for the treatment of stress disorders in humans.

Background: Shenmai Injection (SMI) has been widely used in the treatment of cardiovascular diseases and can reduce side effects when combined with chemotherapy drugs. However, the potential protective mechanism of SMI on the cardiotoxicity caused by anthracyclines has not been clear., Methods: We used network pharmacology methods to collect the compound components in SMI and myocardial injury targets, constructed a 'drug-disease' target interaction network relationship diagram, and screened the core targets to predict the potential mechanism of SMI in treating cardiotoxicity of anthracyclines. In addition, the rat model of doxorubicin cardiotoxicity was induced by injecting doxorubicin through the tail vein. The rats were randomized in the model group, miR-30a agomir group, SMI low-dose group, SMI high-dose group，and the control group. The cardiac ultrasound was used to evaluate the structure and function of the rat heart. HE staining was used to observe the pathological changes of the rat myocardium. Transmission electron microscopy was used to observe myocardial autophagosomes. The expression of miR-30a and Beclin 1 mRNA in the rat myocardium was detected by RT-qPCR. Western Blot detected the expression of LC3-II/LC3-I and p62 protein., Results: The network pharmacological analysis found that SMI could act synergistically through multiple targets and multiple pathways, which might exert a myocardial protective effect through PI3K-Akt signaling pathways and cancer microRNAs. In vivo, compared with the control group, the treatment group could improve the cardiac structure and function, and reduce myocardial pathological damage and the number of autophagosomes. The expression of miR-30a in the myocardium of rats in miR-30a agomir group and SMI group increased (P < 0.01)，Beclin 1 mRNA was decreased (P < 0.01)，LC3-Ⅱ/LC3-I protein was decreased (P < 0.01 or P < 0.05)，and p62 protein was increased (P < 0.01 or P < 0.05)., Conclusions: SMI has the characteristics of multi-component, multi-target, and multi-pathway. It can inhibit myocardial excessive autophagy by regulating the expression of miR-30a/Beclin 1 and alleviate the myocardial injury induced by doxorubicin., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Medulloblastoma (MB), the most common malignant childhood brain tumor, is a serious threat to life. Circular RNA (circRNA) is involved in the development of various cancers, including MB. We aimed to explore the role of circRNA spindle and kinetochore associated complex subunit 3 (circ-SKA3) in MB progression. Circ-SKA3 expression was elevated in MB tissues and cells. Depleted expression of circ-SKA3 inhibited MB cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest, and circ-SKA3 knockdown inhibited MB growth in vivo. Mechanism analyses revealed that circ-SKA3 directly targeted miR-326 that could bind to ID3, and circ-SKA3 decoyed miR-326 to increasing ID3 expression. Rescue experiments showed that miR-326 inhibition reversed the effects of circ-SKA3 knockdown, and ID3 overexpression recovered MB cell proliferation, migration and invasion blocked by miR-326 restoration. In conclusion, circ-SKA3 functioned as an oncogene to promote the development of MB by increasing ID3 expression via decoying miR-326, hinting that circ-SKA3 might be a therapeutic target of MB., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lipid overload of the mitochondria is linked to the development of insulin resistance in skeletal muscle which may be a contributing factor to the progression of type 2 diabetes during obesity. The targeted degradation of mitochondria through autophagy, termed mitophagy, contributes to the mitochondrial adaptive response to changes in dietary fat. Our previous work demonstrates long-term (2-4 months) consumption of a high-fat diet increases mitochondrial lipid oxidation capacity but does not alter markers of mitophagy in mice. The purpose of this study was to investigate initial stages of mitochondrial respiratory adaptations to high-fat diet and the activation of mitophagy. C57BL/6J mice consumed either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 3 or 7 days. We measured skeletal muscle mitochondrial respiration and protein markers of mitophagy in a mitochondrial-enriched fraction of skeletal muscle. After 3 days of HFD, mice had lower lipid-supported oxidative phosphorylation alongside greater electron leak compared with the LFD group. After 7 days, there were no differences in mitochondrial respiration between diet groups. HFD mice had greater autophagosome formation potential (Beclin-1) and greater activation of mitochondrial autophagy receptors (Bnip3, p62) in isolated mitochondria, but no difference in downstream autophagosome (LC3II) or lysosome (Lamp1) abundance after both 3 and 7 days compared with the LFD groups. In cultured myotubes, palmitate treatment decreased mitochondrial membrane potential and hydrogen peroxide treatment increased accumulation of upstream mitophagy markers. We conclude that several days of high-fat feeding stimulated upstream activation of skeletal muscle mitophagy, potentially through lipid-induced oxidative stress, without downstream changes in respiration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ehrlicher, Stierwalt, Newsom and Robinson.)

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose physiological function is poorly understood. The AhR is highly expressed in barrier organs such as the skin, intestine, and lung. The lungs are continuously exposed to environmental pollutants such as cigarette smoke (CS) that can induce cell death mechanisms such as apoptosis, autophagy, and endoplasmic reticulum (ER) stress. CS also contains toxicants that are AhR ligands. We have previously shown that the AhR protects against apoptosis, but whether the AhR also protects against autophagy or ER stress is not known. Using cigarette smoke extract (CSE) as our in vitro surrogate of environmental tobacco exposure, we first assessed the conversion of LC3I to LC3II, a classic feature of both autophagic and ER stress-mediated cell death pathways. LC3II was elevated in CSE-exposed lung structural cells [mouse lung fibroblasts (MLFs), MLE12 and A549 cells] when AhR was absent. However, this heightened LC3II expression could not be explained by increased expression of key autophagy genes ( Gabarapl1 , Becn1 , Map1lc3b ), upregulation of upstream autophagic machinery (Atg5-12, Atg3), or impaired autophagic flux, suggesting that LC3II may be autophagy independent. This was further supported by the absence of autophagosomes in Ahr -/- lung cells. However, Ahr -/- lung cells had widespread ER dilation, elevated expression of the ER stress markers CHOP and GADD34, and an accumulation of ubiquitinated proteins. These findings collectively illustrate a novel role for the AhR in attenuating ER stress by a mechanism that may be autophagy independent.

This study aimed to clarify the regulation role of miR-708 and miR-335-3p in retinal ganglion cell (RGC) autophagy and apoptosis in glaucoma. Chronic glaucoma mice were established by laser photocoagulation. RGCs were isolated and transfected with a series of plasmids and the cultured in 60 mmHg pressure. miR-335-3p, miR-708, and ATG3 mRNA expressions were detected by qRT-PCR. Protein levels of ATG3, autophagy-related protein LC3, and p62 were detected by Western blot. The apoptosis of RGCs was detected by flow cytometry. The regulation role of miR-335-3p/miR-708 in ATG3 was confirmed by the dual-luciferase reporter gene. The expressions of several miRNAs were measured in retinal tissues from chronic glaucoma mice and RGCs under pressure conditions, and results showed that both miR-335-3p and miR-708 were down-regulated. Besides, the inhibition of miR-708 and miR-335-3p induced the apoptosis of RGCs through promoting autophagy. Also, miR-708 and miR-335-3p could bind to ATG3 and targeted regulated ATG3. Furthermore, the interference with miR-708/miR-335-3p induced RGC apoptosis by up-regulating ATG3 to promote autophagy. In general, the down-regulation of miR-708 and miR-335-3p contributed to the apoptosis of RGCs through promoting autophagy in glaucoma.

Endothelial monocyte‑activating polypeptide II (EMAP II) is a sensitive marker of neurotoxic injury, the expression of which increases significantly under conditions of stress, such as hypoxia or apoptosis. Studies have confirmed the extensive apoptosis of nerve cells in the brain following status epilepticus (SE), and the occurrence of SE can confer a hypoxic state on cells. The purpose of the present study was to observe the changes in the expression of EMAP II, and in the numbers and tight junction protein levels of microvascular endothelial cells in the hippocampus of rats with pilocarpine‑induced SE. The protein expression levels of EMAP II, CD31, zonula occludens 1 (ZO‑1) and occludin in the hippocampus were determined by immunofluorescence and western blot analyses. It was found that almost 75.6% of the rats in the SE group developed Racine stage IV‑V seizures at approximately 44.7±18.8 min after the pilocarpine administration, and the 24‑h mortality rate was almost 10.4%. The weight of the rats in the SE group was significantly decreased within 24 h following SE. Immunofluorescence staining revealed a low EMAP II expression in the hippocampus of the rats in the control group; however, the numbers of EMAP II‑positive cells were significantly increased in the SE group from 2 h to 21 days. The trend of EMAP II protein expression was consistent with that obtained with immunofluorescence staining. The numbers of CD31‑positive microvascular endothelial cells were significantly increased from 24 h to 21 days compared with the levels in the control group. The protein expression of ZO‑1 and occludin was most significantly decreased in the SE group. On the whole, the present study demonstrated that the expression of EMAP II in the rat hippocampus was upregulated in the SE model, which may promote angiogenesis and alter the TJ integrity of brain microvascular endothelial cells, with an increased number of CD31‑positive microvascular endothelial cells and a decreased expression of ZO‑1 and occludin.

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Plaza-Zabala, Sierra-Torre and Sierra.)

The persistence of HIV in the central nervous system leads to cognitive deficits in up to 50% of people living with HIV even with systemic suppression by antiretroviral treatment. The interaction of chronic inflammation with age-associated degeneration places these individuals at increased risk of accelerated aging and other neurodegenerative diseases and no treatments are available that effectively halt these processes. The adverse effects of aging and inflammation may be mediated, in part, by an increase in the expression of the p75 neurotrophin receptor (p75 NTR ) which shifts the balance of neurotrophin signaling toward less protective pathways. To determine if modulation of p75 NTR could modify the disease process, we treated HIV gp120 transgenic mice with a small molecule ligand designed to engage p75 NTR and downregulate degenerative signaling. Daily treatment with 50 mg/kg LM11A-31 for 4 months suppressed age- and genotype-dependent activation of microglia, increased microtubule associated protein-2 (MAP-2), reduced dendritic varicosities and slowed the loss of parvalbumin immunoreactive neurons in the hippocampus. An age related accumulation of microtubule associated protein Tau was identified in the hippocampus in extracellular clusters that co-expressed p75 NTR suggesting a link between Tau and p75 NTR . Although the significance of the relationship between p75 NTR and Tau is unclear, a decrease in Tau-1 immunoreactivity as gp120 mice entered old age (>16 months) suggests that the Tau may transition to more pathological modifications; a process blocked by LM11A-31. Overall, the effects of LM11A-31 are consistent with strong neuroprotective and anti-inflammatory actions that have significant therapeutic potential., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a malignant peripheral T-cell neoplasm associated with human T-cell leukemia virus type-1 (HTLV-1). The acute and lymphoma subtypes are regarded as aggressive ATLLs, and the overall survival (OS) of patients remains poor. Transforming acidic coiled-coil-containing protein 3 (TACC3) regulates microtubules, which are associated with cancer-related proteins overexpressed in various cancers. Such a relationship has not been reported in hematopoietic tumors, including ATLL., Methods: We examined tissue microarrays of histological samples from 92 cases of aggressive ATLL and assessed clinical features, including TACC3 protein expression levels., Results: Compared with TACC3-low, TACC3-high ATLL patients were significantly older (P < .001), with a tendency toward pleomorphic variant over other morphological classifications (P = .019). TACC3-high patients (median survival time [MST] 10.6 months, confidence interval [CI] [6.27-15.6]) had poorer OS compared to TACC3-low patients (MST 20 months, CI [9.43-38.5]) (P = .0168). Moreover, multivariate analysis on TACC3 expression levels suggests that TACC3-high is an independent significant prognostic factor (HR, 1.700; 95% CI, 1.037-2.753; P = .0355)., Conclusion: Certain drugs that inhibit TACC3-overexpressing neoplastic cells are used clinically. Further studies might highlight a key role for TACC3 in the oncogenesis and progression of ATLL., (© 2020 John Wiley & Sons Ltd.)

Fragile X mental retardation protein (FMRP), strongly associated with fragile X syndrome, plays important roles by regulating gene expression via interacting with other RNA binding proteins in the brain. However, the role of FMRP in hypothalamus, a central part responsible for metabolic control, is poorly known. Our study shows that FMRP is primarily located in the hypothalamic arcuate nucleus (ARC). Using proteomic analysis, we identified 56 up-regulated and 22 down-regulated proteins in the hypothalamus of Map1b KO mice, with microtubule-associated protein 1 B (MAP1B) being the most outstanding increased protein (more than 10 folds). Immunofluorescent assays showed that MAP1B significantly increased in the Map1b-KO ARC, in which the number of agouti-related peptide (AgRP)-staining neurons significantly reduced, but not altered for pro-opiomelanocortin (POMC) neurons. We further showed an age-dependent reduces in food intake and body weight of the KO mice, along with the decreases of MAP1B and AgRP at the same time points. In hypothalamic GT1-7 cells, the AgRP expression decreased upon knockdown of FMRP or overexpression of MAP1B, and increased in response to overexpression of FMRP or knockdown of MAP1B. Co-knockdown or co-overexpression of FMRP and MAP1B led to a reverse expression of AgRP compared to overexpression of knockdown of FMRP alone, demonstrating that MAP1B is essential for the regulatory effect of FMRP on AgRP expression. Taken together, these data suggest that FMRP-deficiency-induced increase of hypothalamic MAP1B and decrease of AgRP might be associated with reduces in food intake and body weight., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial-derived extracellular vesicles (M-EVs) to the maintenance of microglia homeostasis and how M-EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M-EVs is limited. Here, we analysed the effects of M-EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non-activated microglia BV2 cells. We showed that M-EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M-EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M-EVs increased the protein expression of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B isoform II (LC3B-II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M-EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M-EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia-microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that they have no conflict of interest., (© 2020 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background & Aims: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association., Methods: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2., Results: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage., Conclusion: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets., Lay Summary: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. All rights reserved.)

Photoreceptor cells undergo changes with aging. It is unknown if their microtubules are stable or not with aging. This study examined photoreceptor cell ultrastructure from 18 human donor retinas (32 eyes; age: 45-94 years) and quantified the photoreceptors with altered microtubules over six to ninth decades in four defined retinal regions. In addition, immunoreactivity (IR) to microtubule-associated protein-2 (MAP-2), tau and hyperphophorylated tau was performed in retinal sections from companion eyes. In young donor retinas below 75 years of age, microtubules appeared straight in photoreceptor inner segments and axons. With age, they appeared bent or misaligned in macular and mid-peripheral photoreceptors. In addition, dense granular materials were present in photoreceptor axons and synaptic terminals in advanced ages. In all decades, rod microtubules were affected more than their cone counterparts (28% vs 15%, p < 0.005). Both rods and cones were significantly affected in mid-peripheral retina (5-8 mm outside the macular border) in eighth decade, compared to other decades or retinal regions (parafoveal, perifoveal and nasal) examined (p < 0.005). IR showed a steady expression of MAP-2 in inner segments, and tau in inner segments to axons below 75 years of age, but was absent for both markers in scattered macular and mid-peripheral photoreceptors in advanced ages (>75 years). IR to hyperphosphorylated tau was present mainly in inner retina and increased with aging. Markers of oxidative stress, e.g., lipid peroxidation (4-hydroxy 2-nonenal) and nitrosative stress (nitrotyrosine) were immunopositive in aged photoreceptors. The sporadic loss of MAP-2 and tau-IR in photoreceptors may be due to microtubule changes; all these changes may affect intracellular transport and be partly responsible for photoreceptor death in aged human retina., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dengue virus infection is associated with the upregulation of metabolic pathways within infected cells. This effect is common to infection by a broad array of viruses. These metabolic changes, including increased glucose metabolism, oxidative phosphorylation and autophagy, support the demands of viral genome replication and infectious particle formation. The mechanisms by which these changes occur are known to be, in part, directed by viral nonstructural proteins that contact and control cellular structures and metabolic enzymes. We investigated the roles of host proteins with overarching control of metabolic processes, the transcriptional regulators, cyclin-dependent kinase 8 (CDK8) and its paralog, CDK19, as mediators of virally induced metabolic changes. Here, we show that expression of CDK8, but not CDK19, is increased during dengue virus infection in Huh7 human hepatocellular carcinoma cells, although both are required for efficient viral replication. Chemical inhibition of CDK8 and CDK19 with Senexin A during infection blocks virus-induced expression of select metabolic and autophagic genes, hexokinase 2 (HK2) and microtubule-associated protein 1 light chain 3 (LC3), and reduces viral genome replication and infectious particle production. The results further define the dependence of virus replication on increased metabolic capacity in target cells and identify CDK8 and CDK19 as master regulators of key metabolic genes. The common inhibition of CDK8 and CDK19 offers a host-directed therapeutic intervention that is unlikely to be overcome by viral evolution.

Autism spectrum disorder (ASD) is a heterogeneously childhood neurodevelopmental disorder, believed to be under development of various genetic and environmental factors. Autophagy and related pathways have also been implicated in the etiology of ASD. We aimed to investigate autophagic markers by generating the transgenerational inheritance of ASD-like behaviors in the Cc2d1a animal model of ASD. Cc2d1a (+/-) mouse model of ASD was built in two different groups by following three generations. After behavior test, bilateral hippocampus was sliced. Western Blot assay and quantitative real-time polymerase chain reaction (QRT-PCR) were used for measurement of LC3 and Beclin-1 as key regulators of autophagy. All of the animal and laboratory studies were conducted in the Erciyes University Genome and Stem Cell Center (GENKOK). Significant LC3 and Beclin-1 mRNA expression levels were observed in mouse hippocampus between groups and generations. Western blot confirmed the changes of the proteins in the hippocampus. LC3 expressions were increased for females and decreased for males compared to the control group. Beclin-1 expression levels were found to be significantly decreased in males and females compared to controls. This study could help explain a new pathway of autophagy in ASD mouse models. Future animal studies need to investigate sex differences in mouse modeling autism-relevant genes like CC2D1A. We anticipate our results to be a starting point for more comprehensive autophagy studies in this mouse model of ASD.

Recently, minicircle (MC)-based cell therapy has been emerging as a novel technology for nonviral genetic modification. In this study, we investigated the characteristics of granulocyte chemotactic protein-2 (GCP-2)-overexpressing fibroblasts (GCP-2/MC) using MC microporation technology, as well as its therapeutic mechanism in wound healing. GCP-2 parent plasmid and MC containing GCP-2 were generated. Human dermal fibroblasts (HDF) were transfected with MC containing GCP-2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), scratch wound assay, and in vivo wound healing assay were performed. Gene and protein expression analysis revealed that GCP-2/MC highly expressed epithelialization growth factor, epidermal growth factor (EGF), chemokines, GCP-2, interleukin (IL)-8, as well as wound healing-associated genes such as insulin growth factor (IGF)-1 and hepatocyte growth factor (HGF). An in vitro scratch wound closure and matrigel tube formation assays demonstrated that the culture medium derived from GCP-2/MC substantially accelerated the wound closure and matrigel network formation. Wounds in nude mice were created by skin excisions followed by injections of GCP-2/MC. Results showed high cell survival potential and that GCP-2/MC transplantation highly accelerated skin wound closure by increasing reepithelialization, capillary density, and enhancing angiogenic factors, suggesting direct benefits for cutaneous closure. Taken together, these data suggest that MC-based GCP-2 overexpression could be a promising alternative strategy for promoting wound healing., (© 2020 John Wiley & Sons, Ltd.)

Background: Microtubule-associated protein 1 light chain 3 (LC3), an autophagic gene, has been reported as a vital marker for many diseases and cancers. However, the role of LC3 in hepatocellular carcinoma (HCC) was not still investigated. Therefore, we conducted a meta-analysis to examine the association of LC3 with its clinicopathological and prognostic in HCC., Methods: We consulted the PubMed, Cochrane Library, Web of Science, EMBASE, China National Knowledge Infrastructure and Wan Fang databases for published studies on LC3 in HCC. Newcastle-Ottawa scale was used to screen the quality of the literature. The statistical analysis was calculated by STATA 14.2., Results: Of the 1329 titles identified, 10 articles involving 949 patients in HCC were included in this meta-analysis. The results of our study show that increased LC3 expression is related to size of tumor, but not to gender, age, number of tumor, liver cirrhosis, HBsAg, TNM stage, alpha fetoprotein, vascular invasion and histological grade. Positive LC3 expression was associated with overall survival by pooled hazard ratio., Conclusions: This meta-analysis indicated that positive LC3 expression was related to size of tumor, and could predict prognosis in human hepatocellular carcinoma.

Background Gonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival. Materials and methods Collected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed. Results Gonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced. Conclusions Different gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.

To investigate the expression patterns of autophagy marker light chain protein 3 (LC3A) in keratoacanthoma (KA). KAs are generally regarded as benign but malignant behavior, including rare metastases, may occur. 85 KAs were assessed for the LC3A autophagic protein by immunohistochemistry. Diffuse cytoplasmic staining and a "stone-like structure" (SLS) characterized positive expression. Thirty-four out of 85 KAs (40%) had diffuse cytoplasmic LC3A immunostaining (percentage of positive cells ranging from 5 to 60%). In contrast, only 4 of the 85 KAs (4.7%) expressed SLSs. Only one SLS was detected per histologic section of each tumor. The p53 oncoprotein was encountered in all cases with expression ranging from 1 to 90% of cells (median 30%). The Ki-67 index was expressed in 63 cases (74% of cases; range 1-50% of cells; median value 5%). Neither of these two parameters nor diffuse cytoplasmic LC3A staining was significantly correlated with SLS expression or lack thereof. Expression of SLSs, a hallmark of malignancy, was found in 4.7% of KAs. Further study is necessary to determine whether this fraction represents the exceptional cases that harbor latent malignant potential.

Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+ cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+ cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+ thyroid cancer cells to CD8 + T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+ thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gastric cancer (GC) is one of the most common causes of cancer-related death worldwide, and outstanding biomarkers for therapeutic targets or predicting GC survival are still lacking. Increasing evidence indicated that nucleolar and spindle associated protein 1 (NUSAP1) involved in regulating the progression of various cancers; however, its specific role in GC remained unclear. In this study, we found that NUSAP1 was upregulated in the GC tissues and cell lines via analysing data from The Cancer Genome Atlas (TCGA), gene expression omnibus (GEO), qRT-PCR, and western blot assays. Patients with high NUSAP1 expression levels showed shorter free-progression survival (FPS), larger tumour size, and higher lymphatic metastasis rate compared with those with low NUSAP1 expression. Further functional experiments revealed knockdown of NUSAP1 could inhibit the growth, migration, and invasion of GC cells in vitro and vivo. Additionally, silencing NUSAP1 induced G0/G1 phase arrest, apoptosis, and suppressed the epithelial-mesenchymal transition (EMT) process. Finally, we performed gene set enrichment analysis (GSEA) and observed NUSAP1 was positive with mTORC1 signalling pathway, which was verified by the subsequent immunoblotting. In conclusion, our findings suggested that NUSAP1 contributed to GC progression and may act as a potential therapeutic target for GC. SIGNIFICANCE OF THE STUDY: Our results firstly illuminated that NUSAP1 expression was significantly upregulated in GC tissues and predicted poor FPS. Silencing it could attenuate GC progression via inhibiting mTORC1 signalling pathway. Hence, NUSAP1 may act as a promising therapy target for GC., (© 2019 John Wiley & Sons, Ltd.)

Increased amyloid-β (Aβ) accumulation associated with abnormal autophagy-lysosomal activity and nutrient kinase dysregulation are common features in Alzheimer's disease (AD) brain. Recent studies have identified PRKAG2 and TIPRL genes that control nutrient kinase regulated autophagy, and here we determined if their expression is altered in postmortem AD brains. Gene and protein expression of TIPRL were unchanged. However, gene expression of PRKAG2 was increased 3-fold and its protein levels positively correlated with Aβ accumulation in the AD brain. In summary, our findings suggest that increased PRKAG2 is an important contributing factor to Aβ accumulation in the AD brain.

Despite the recent breakthrough in cataract drug development, further improvements have been limited by the lack of human in vitro cataract disease models. This study, therefore, aims to generate a qualified cataract disease model. Mature lentoid bodies (LBs) on Day 25 (D25), which were differentiated from human induced pluripotent stem cells (iPSCs) using the "fried egg" method, were continually culturing (control) or extra treated with either ultraviolet (UV) radiation or hydrogen peroxide (H 2 O 2 ). The LBs' shape alteration and opacity were examined using light microscopy and mean gray value evaluation. Their structure and crystallin expression were examined using immunofluorescence and transmission electron microscopy (TEM). Real-time polymerase chain reaction and western blot were used to investigate the potential role of autophagy in cloudy LBs. Mature LBs became cloudy with time which was accelerated by H 2 O 2 . Immunofluorescence examinations and TEM showed that the H 2 O 2 -treated and control LBs had similar shapes, lens capsule, and monolayer lens epithelial cell (LEC) structures. However, we were unable to do further assessment of the UV-treated LBs as the structures of LBs were easily damaged when treated with UV radiation. Cells containing aggregated protein (αA-crystallin and αB-crystallin) puncta were more abundant in the H 2 O 2 -treated LBs as compared with control LBs. Moreover, LC3B expression decreased with age in anterior lens capsules obtained from age-related cataracts (ARCs) patients as compared with LC3B levels in primary LECs, which is consistent with that LC3B expression in LBs was lower on D45 than on D25. Our study found that human iPSCs-derived LBs became cloudy with time which was accompanied by protein aggregation, and this phenomenon was accelerated by H 2 O 2 , suggesting that LBs with extending culture may serve as a human model for in vitro ARCs., (© 2019 Wiley Periodicals, Inc.)

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by several behavioral disturbances, especially cognitive decline and deficits in social competence. Previous studies revealed that decreased social activity would accelerate AD progression, whereas enhanced social interaction could rescue AD-induced memory impairment. Collapsin response mediator protein 5 (CRMP5), which belongs to a family of cytosolic proteins, is abundantly expressed in the brain and is involved in the regulation of neurodevelopment and the pathology of several neuropsychiatric diseases. However, the functions of CRMP5 in AD are still unclear. Here, we demonstrated that 9-month-old 3xTg-AD mice exhibited social behavioral deficits and increased hippocampal CRMP5 levels compared to control (B6129S) mice. Knockdown of CRMP5 reversed the social deficits in 9-month-old 3xTg-AD mice, whereas CRMP5 overexpression decreased social interaction in both 3xTg-AD and control mice at 6 months of age. Interestingly, decreased expression of CRMP5 rescued AD-induced memory impairment, but overexpression of CRMP5 accelerated memory loss only in 3xTg-AD mice. In addition, we found that CRMP5 could regulate surface GluA2 and GluA2 S880 phosphorylation. These results suggest that CRMP5 regulates social behavior via modulation of surface GluA2 trafficking and affects memory performance in 3xTg-AD mice., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Cytoplasmic linker-associated protein 2 (CLASP2) belongs to a family of microtubule plus-end tracking proteins that localize to the distal ends of microtubules and is involved in various microtubule-dependent processes. We previously showed that CLASP2 is involved in the epithelial-to-mesenchymal transition of bladder urothelial cancer. This research aimed to explore the significance of CLASP2 expression as a prognostic marker for muscle-invasive bladder urothelial cancer (MIBC) patients after radical cystectomy-pelvic lymph node dissection (RC-PLND)., Methods: CLASP2 expression was analyzed in 76 benign bladder tissues and 160 MIBC tissues by tissue immunohistochemistry. Survival analysis and multiple regression analysis following propensity score matching were performed to investigate the correlation between high CLASP2 expression and MIBC patients' survival., Results: CLASP2 expression was increased in MIBC patients, especially those with high-stage tumors or lymph node metastasis. In the follow-up of MIBC patients after propensity score matching, whether MIBC patients received adjuvant chemotherapy after RC-PLND, high CLASP2 expression was significantly associated with a poor prognosis. MIBC patients with low CLASP2 expression who received adjuvant chemotherapy tended to have an improved survival prognosis., Conclusion: CLASP2 expression is correlated with malignant progression of MIBC. High CLASP2 expression predicted a poor prognosis for MIBC patients after RC-PLND., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Spinal cord injury (SCI) is a specific type of damage to the central nervous system causing temporary or permanent changes in its function. The present aimed to identify the genetic changes in neuroplasticity following SCI in rats. The GSE52763 microarray dataset, which included 15 samples [3 sham (1 week), 4 injury only (1 week), 4 injury only (3 weeks), 4 injury + treadmill (3 weeks)] was downloaded from the Gene Expression Omnibus database. An empirical Bayes linear regression model in limma package was used to identify the differentially expressed genes (DEGs) in injury vs. sham and treadmill vs. non‑treadmill comparison groups. Subsequently, time series and enrichment analyses were performed using pheatmap and clusterProfile packages, respectively. Additionally, protein‑protein interaction (PPI) and transcription factor (TF)‑microRNA (miRNA)‑target regulatory networks were constructed using Cytoscape software. In total, 159 and 105 DEGs were identified in injury vs. sham groups and treadmill vs. non‑treadmill groups, respectively. There were 40 genes in cluster 1 that presented increased expression levels in the injury (1 week/3 weeks) groups compared with the sham group, and decreased expression levels in the injury + treadmill group compared with the injury only groups; conversely, 52 genes in cluster 2 exhibited decreased expression levels in the injury (1 week/3 weeks) groups compared with the sham group, and increased expression levels in the injury + treadmill group compared with the injury only groups. Enrichment analysis indicated that clusters 1 and 2 were associated with immune response and signal transduction, respectively. Furthermore, microtubule associated protein 1B, phosphofurin acidic cluster sorting protein 2 and adenosylhomocysteinase‑like 1 exhibited the highest degrees in the regulatory network, and were regulated by miRNAs including miR‑34A, miR‑34B, miR‑34C and miR‑449. These miRNAs and their target genes may serve important roles in neuroplasticity following traumatic SCI in rats. Nevertheless, additional in‑depth studies are required to confirm these data.

Autophagy is a cellular bulk degradation process used as an alternative source of energy and metabolites and implicated in various diseases. Inefficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy making its modulation valuable as a therapeutic strategy for cancer treatment, especially in combination with chemotherapy. Dipyridamole (DIP) is a vasodilator and antithrombotic drug. Its major effects involve the block of nucleoside uptake and phosphodiestesase inhibition, leading to increased levels of intracellular cAMP. Here we report that DIP increases autophagic markers due to autophagic flux blockage, resembling autophagosome maturation and/or closure impairment. Treatment with DIP results in an increased number of autophagosomes and autolysosomes and impairs degradation of SQSTM1/p62. As blockage of autophagic flux decreases the recycling of cellular components, DIP reduced the intracellular ATP levels in cancer cells. Autophagic flux blockage was neither through inhibition of lysosome function nor blockage of nucleoside uptake, but could be prevented by treatment with a PKA inhibitor, suggesting that autophagic flux failure mediated by DIP results from increased intracellular levels of cAMP. Treatment with DIP presented antiproliferative effects in vitro alone and in combination with chemotherapy drugs. Collectively, these data demonstrate that DIP can impair autophagic degradation, by preventing the normal autophagosome maturation, and might be useful in combination anticancer therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Autophagy is an evolutionary conserved, self-eating process that targets cellular constituents for lysosomal degradation. Transcription factor EB (TFEB) is a master regulator of autophagy by inducing the expression of genes involved in autophagic and lysosomal degradation. In breast cancer, ligand-activated progesterone receptor has been reported to influence cancer development by manipulating the autophagy pathway. However, understanding of the mechanism that underlies this autophagic response remains limited. Herein, we report that prolonged treatment with progestin R5020 upregulates autophagy in MCF-7 human breast cancer cells via a novel interplay between progesterone receptor B (PRB) and TFEB. R5020 upregulates TFEB gene expression and protein levels in a PRB-dependent manner. Additionally, R5020 enhances the co-recruitment of PRB and TFEB to each other to facilitate TFEB nuclear localization. Once in the nucleus, TFEB induces the expression of autophagy and lysosomal genes to potentiate autophagy. Together, our findings highlight a novel functional connection between ligand-activated PRB and TFEB to modulate autophagy in MCF-7 breast cancer cells. As breast cancer development is controlled by autophagy, the progestin-PRB-TFEB transduction pathway warrants future attention as a potential therapeutic target in cancer therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chlorpyrifos (CPF) is an organophosphorus pesticide that can damage the central nervous system in children upon exposure. Taxifolin (Tax) exerts protective effects against neurotoxins; however, the mechanism has not yet been illustrated. The current study used BV2 cells to investigate the protective mechanism underlying the organophosphorus pesticide taxifolin on CPF-induced neurotoxicity, which might present a therapeutic potential for the prevention and treatment of the nervous system diseases in children. BV2 cells were randomly divided into 4 groups: DMSO, CPF, Tax, and Tax + CPF. The viability, morphocytology, oxidative stress, inflammatory reaction, and autophagocytosis have been studied in the cells using Western blot analysis, CCK-8 assay, enzyme-linked immunosorbent assay, and immunofluorescence to estimate the level of LC3 II. As a result, CPF was found to exert a significant toxic effect on BV2 cells that was characterized by rounded cell body, atrophic synapse, poor adhesion, cell aggregation, inflammation, oxidative reaction, and autophagy. Tax treatment has a protective effect on CPF-induced neurotoxicity via downregulation of ROS, TNF-α, IFN-γ, and p62 levels and increased LC3 II level, which in turn, improved the viability and activity of BV2 cells. This phenomenon suggested that Tax can reduce the inflammation and oxidative stress and promote autophagy. Furthermore, the current study suggested that the protective mechanism of Tax on CPF-induced BV2 cell toxicity was via up-regulation of pAMPK level and activation of Nrf2/HO-1 signaling pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Autism spectrum disorders (ASDs) comprise a number of heterogeneous neurodevelopmental diseases. Recent studies suggest that the abnormal transmission of neural signaling pathways is associated with the pathogenesis of autism. The aim of this study was to identify a link between the Notch signaling pathway and the pathogenesis of autism. In this study, we demonstrated that prenatal exposure to valproic acid (VPA) resulted in autistic-like behaviors in offspring rats and that the expression of the Notch signaling pathway-related molecules Notch1, Jagged1, Notch intracellular domain (NICD) and Hes1 increased in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA rats compared to those of controls. However, inhibiting the Notch pathway with (3,5-Difluorophenacetyl)-L-alanyl-S-phenylglycine-2-butyl Ester (Dapt) reduced the overexpression of Notch pathway-related molecules in offspring rats. Notably, Dapt improved autistic-like behaviors in a VPA-exposed rat model of autism. Furthermore, we investigated whether Dapt improved autistic-like behavior in a VPA rat model by regulating autophagy and affecting the morphology of dendritic spines. We found that the expression of the autophagy-related proteins Beclin 1, LC3B and phospho-p62 in the PFC, HC and CB of VPA model rats increased after Notch signal activation and was inhibited by Dapt compared to those of controls. Moreover, postsynaptic density-95 (PSD-95) protein expression also increased significantly compared to that of VPA model rats. The density of dendritic spines decreased in the PFC of VPA rats treated with Dapt compared to that of VPA model rats. Our present results suggest that VPA induces an abnormal activation of the Notch signaling pathway. The inhibition of excessive Notch signaling activation by Dapt can alleviate autistic-like behaviors in VPA rats. Our working model suggests that the Notch signaling pathway participates in the pathogenesis of autism by regulating autophagy and affecting dendritic spine growth. The results of this study may help to elucidate the mechanism underlying autism and provide a potential strategy for treating autism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, expression of the SPC25 gene was characterized in breast cancer (BC), and its effects on BC development and progression, functions in BC cells, and potential underlying mechanisms were examined. Data from TCGAportal and FIREBROWSE indicated that SPC25 was upregulated in BC tissues compared to normal tissues, and CANCERTOOL indicated that higher SPC25 mRNA levels were associated with increased probability of recurrence and poorer survival in BC patients. BC patients with higher SPC25 expression displayed shorter distant metastasis-free survival, relapse-free survival, and overall survival. Colony formation and CCK-8 experiments confirmed that SPC25 promoted proliferation of BC cells. Single-cell analysis indicated that SPC25 is associated with cell cycle regulation, DNA damage and repair, and BC cell proliferation. SPC25 knockdown suppressed proliferation of BC cells. MiRNAs, circRNAs, RNA-binding proteins, transcription factors, and immune factors that might interact with SPC25 mRNA to promote BC were also identified. These findings suggest that SPC25 levels are higher in more malignant BC subtypes and are associated with poor prognosis in BC patients. In addition, DNA methyltransferase inhibitor and transcription factors inhibitor treatments targeting SPC25 might improve survival in BC patients.

Objective: To explore the role of autophagy in PM2.5-induced inflammation in human nasal epithelial cells and related mechanism. Methods: Human nasal epithelial cells were exposed to different concentration of PM2.5 for different times, and the expression levels of microtubule-associated protein-1 light chain-3 Ⅱ (LC3 Ⅱ) and Beclin1 proteins were measured by Western blot. The typical autophagosome and autolysosome were observed by using transmission electron microscopy (TEM). To observe autophagic flux, mRFP-GFP-LC3 plasmid was transfected to nasal epithelial cells and the punctate staining of mRFP-GFP-LC3 were determined by confocal laser scanning microscope. The expression of inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). To assess the role of autophagy in PM2.5-mediated inflammation, autophagy related gene Atg5 and Beclin-1 were silenced by siRNA knockdown, and inflammatory cytokines were analyzed.GraphPad Prism 6.0 was used for statistical analysis. Results: PM2.5 exposure increased the expression of LC3 Ⅱ and Beclin-1 proteins in a dose- (in PM2.5 group with concentration of 0, 15, 30, 60, 120 μg/ml, the expression of LC3 Ⅱ was 0.021±0.001( x ± s ), 0.037±0.002, 0.058±0.005, 0.075±0.006, 0.085±0.004, respectively, F= 126.8, P< 0.05; the expression of Beclin-1 was 0.002±0.000, 0.003±0.000, 0.005±0.000, 0.007±0.001, 0.008±0.001, respectively, F= 137.3, P< 0.05) and time-dependent manner (in PM2.5 group with exposure time of 0, 3, 6, 12, 24 h, the expression of LC3Ⅱ was 0.160±0.007, 0.222±0.003, 0.251±0.015, 0.483±0.029, 0.585±0.035, respectively, F= 215.3, P< 0.05; the expression of Beclin-1 was 0.059±0.002, 0.080±0.002, 0.087±0.002, 0.183±0.007, 0.228±0.005, respectively, F= 137.3, P< 0.05) in human nasal epithelial cells. TEM analysis showed typical autophagosome and autolysosome in cells after PM2.5 exposure for 24 h. PM2.5 significantly increased the number of yellow and red dots representing autophagosomes and autolysosomes respectively, indicating autophagic flux was elevated. Moreover, PM2.5 enhanced the secretion of inflammatory cytokines such as IL-6 and TNF-α, which was dramatically prevented by Atg5-siRNA and Beclin-1-siRNA. Conclusion: Autophagy plays an important role in PM2.5-caused inflammation response in nasal epithelial cells, which can induce release of inflammatory factors such as IL-6 and TNF-α and advance the inflammatory reaction.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Background: Leukemia inhibitory factor, a novel myokine, is known to be associated with neural function, but the underlying molecular mechanism remains unclear., Methods: HT-22 mouse hippocampal cells, primary hippocampal cells, and Drosophila Alzheimer's disease model were used to determine the effect of leukemia inhibitory factor on neurons. Immunoblot analysis and immunofluorescence method were used to analyze biological mechanism., Results: Leukemia inhibitory factor increased Akt phosphorylation in a phosphoinositide-3-kinase-dependent manner in hippocampal cells. Leukemia inhibitory factor also increased the phosphorylation of the mammalian target of rapamycin and the downstream S6K. Leukemia inhibitory factor stimulated the phosphorylation of signal transducer and activator of transcription via extracellular signal-regulated kinases. Leukemia inhibitory factor increased c-fos expression through both Akt and extracellular signal-regulated kinases. Leukemia inhibitory factor blocked amyloid β-induced neural viability suppression and inhibited amyloid β-induced glucose uptake impairment through the block of amyloid β-mediated insulin receptor downregulation. Leukemia inhibitory factor blocked amyloid β-mediated induction of the autophagy marker, microtubule-associated protein 1A/1B-light chain 3. Additionally, in primary prepared hippocampal cells, leukemia inhibitory factor stimulated Akt and extracellular signal-regulated kinase, demonstrating that leukemia inhibitory factor has physiological relevance in vivo. Suppression of the autophagy marker, light chain 3II, by leukemia inhibitory factor was observed in a Drosophila model of Alzheimer's disease., Conclusions: These results demonstrate that leukemia inhibitory factor protects against amyloid β-induced neurotoxicity via Akt/extracellular signal-regulated kinase-mediated c-fos induction, and thus suggest that leukemia inhibitory factor is a potential drug for Alzheimer's disease., (© The Author(s) 2019. Published by Oxford University Press on behalf of CINP.)

A vital constituent of the centrosome involved in regulating the activity of the organelle during the cell cycle is centrosomal protein (CEP)-72, whose function in the case of human cancer yet lacks clarity. The expression dynamics of CEP72 and its clinical impact were examined in a large cohort of bladder tissues. Several experiments at both the in vitro and in vivo levels on urothelial carcinoma of the bladder (UCB) cells were conducted to understand the role of this molecule along with the mechanisms. Overexpression of CEP72 in UCB was linked with the acquisition of an aggressive phenotype, which was associated with poor prognosis. In UCB cell lines, knockdown of CEP72 using shRNA was sufficient to inhibit cell invasiveness/metastasis, whereas ectopic overexpression of CEP72 promoted cell invasiveness and/or metastasis both in vitro and in vivo. CEP72 was demonstrated to induce UCB cell aggressiveness via up-regulation of an important target downstream, the serpin family member 1 gene (SERPINE1) (alias plasminogen activator inhibitor, PAI1), ultimately leading to increased cancer cell invasiveness. Particularly, overexpression of CEP72 was associated with a sizable increase in cAMP response element-binding protein binding at the SERPINE1 promoter, leading to increased SERPINE1 transcription. Such observations are suggestive of the potential use of CEP72 as a therapeutic tool for UCB., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

This study investigated the influence of autophagy on the expression of Collagen type II and light chain 3 (LC-3) in the articular cartilage of osteoarthritis (OA) models. The expression of OA associated biomarkers namely Matrix metalloproteinase (MMP-13), NOD-, LRR- and pyrin domain-containing 3 (NLRP3) induced by destabilizing the medial meniscus operation (DMM) were also investigated. A total of 60 C57BL/6 mice were divided into (1) control; (2) DMM2; (3) DMM8; (4) rapamycin 2 weeks; and (5) rapamycin 8 weeks groups. Saffranin O-Fast green staining, histomorphometry and immunohistochemical methods were used for analysis. In the DMM group, the expression of the OA biomarkers MMP-13, NLRP3 significantly increased, whilst Collagen II and LC-3B levels were significantly lower than other experimental groups. We hypothesized that NLRP3 inhibits autophagy activation and delays disease progression.

Dyneins are major components of microtubules. Dynein assembly is modulated by a heterogeneous group of dynein axonemal assembly factors (DNAAFs). The present study analyzes dynein axonemal assembly factor 1 (DNAAF1) and leucine-rich repeat-containing protein 50 (LRRC50), the corresponding encoded protein, in lower motor neurons in spinal cord of sALS postmortem samples and hSOD1-G93A transgenic mice compared with controls. DNAAF1 mRNA is significantly reduced in the anterior horn in sALS, and LRRC50 immunoreactivity is significantly reduced in C-boutons of the remaining motor neurons of the anterior horn, dorsal nucleus of the vagus nerve, and hypoglossal nuclei at terminal stages of ALS. LRRC50 immunoreactivity has a perinuclear distribution in motor neurons in sALS thus suggesting a disorder of transport. The number of LRRC50-/S1R-immunoreactive structures is also significantly decreased in hSOD1-G93A transgenic mice at the age of 90 days (preclinical stages), and the number of motor neurons with LRRC50-immunoreactive structures is significantly reduced in animals aged 150 days (clinical stages). These observations suggest cholinergic denervation of motor neurons as a pathogenic factor in motor neuron disease. LRRC50 protein levels were not detected in human CSF., (© 2019 American Association of Neuropathologists, Inc. All rights reserved.)

Background: With the rapid development of the high throughput detection techniques, tumor-related Omics data has become an important source for studying the mechanism of tumor progression including breast cancer, one of the major malignancies worldwide. A previous study has shown that the G2 and S phase-expressed-1 (GTSE1) can act as an oncogene in several human cancers. However, its functional roles in breast cancer remain elusive., Method: In this study, we analyzed breast cancer data downloaded from The Cancer Genome Atlas (TCGA) databases and other online database including the Oncomine, bc-GenExMiner and PROGgeneV2 database to identify the molecules contributing to the progression of breast cancer. The GTSE1 expression levels were investigated using qRT-PCR, immunoblotting and IHC. The biological function of GTSE1 in the growth, migration and invasion of breast cancer was examined in MDA-MB-231, MDA-MB-468 and MCF7 cell lines. The in vitro cell proliferative, migratory and invasive abilities were evaluated by MTS, colony formation and transwell assay, respectively. The role of GTSE1 in the growth and metastasis of breast cancer were revealed by in vivo investigation using BALB/c nude mice., Results: We showed that the expression level of GTSE1 was upregulated in breast cancer specimens and cell lines, especially in triple negative breast cancer (TNBC) and p53 mutated breast cancer cell lines. Importantly, high GTSE1 expression was positively correlated with histological grade and poor survival. We demonstrated that GTSE1 could promote breast cancer cell growth by activating the AKT pathway and enhance metastasis by regulating the Epithelial-Mesenchymal transition (EMT) pathway. Furthermore, it could cause multidrug resistance in breast cancer cells. Interestingly, we found that GTSE1 could regulate the p53 function to alter the cell cycle distribution dependent on the mutation state of p53., Conclusion: Our results reveal that GTSE1 played a key role in the progression of breast cancer, indicating that GTSE1 could serve as a novel biomarker to aid in the assessment of the prognosis of breast cancer.

Lack of blood or glucose supply is the most common pathological factor in the brain. To cope with such an energy stress, initiating programmed autophagic processes in neurons is required. However, the mechanisms controlling neuronal autophagy during starvation remain far from clear. Here, we report an essential role of 14-3-3γ in starvation-activated neuronal autophagic influx signaling and elucidate the underlying mechanism. Double-fluorescent immunostaining demonstrates that 14-3-3γ protein elevation is well co-localized with Beclin-1 and LC3 elevation in cortical neurons in ischemic brains. Starvation treatment activates autophagic influx and upregulates Beclin-1 and only the γ isoform of 14-3-3 in N2a cells and cultured cortical neurons. Suppressing overall 14-3-3 function by difopein overexpression or knocking-out the γ isoform of 14-3-3 is sufficient to abolish starvation-induced Beclin-1 induction and LC3 activation while overexpressing 14-3-3γ but no other 14-3-3 isoform significantly upregulate Beclin-1-LC3 signaling. Upon starvation, 14-3-3γ binds more p-β-catenin but less Beclin-1. Finally, overexpressing 14-3-3γ reactivates β-catenin-suppressed Beclin-1-LC3 signaling in neuronal cells. Taken together, our data reveal that starvation-induced 14-3-3γ is required for β-catenin-Beclin-1-LC3-autophagy in starved neurons in vitro and in vivo, which may provide insights in the treatment of neurologic diseases such as stoke.

Purpose: We have previously reported on the prognostic importance of the calpain family of proteins in ovarian cancer, especially calpain-2. Spleen tyrosine kinase (Syk) phosphorylates a variety of cytoskeletal proteins with studies suggesting potential interactions between Syk and conventional calpains. Microtubule-associated protein 4 (MAP4) has been reported to be regulated by Syk., Methods: The current study assessed Syk and MAP4 protein expression, by immunohistochemistry on a tissue microarray comprised of cores from primary ovarian carcinomas (n = 575), to evaluate associations with patient clinical outcomes and other clinicopathological factors and sought to determine whether there were any correlations between the expression of Syk, MAP4 and the calpain system., Results: MAP4 expression was significantly associated with ovarian cancer histological subtype (P < 0.001), stage (P = 0.001), grade (P < 0.001) and residual tumour (P = 0.005). Despite this finding, we found no significant association existing between MAP4 expression and overall survival. Syk expression was also found significantly associated with histological subtype (P < 0.001). Syk seems to play a contradictory role with respect to tumour progression: low cytoplasmic Syk expression was significantly associated with low stage (P = 0.013), and low nuclear Syk expression with chemo-resistance in patients treated with taxane-containing therapy (P = 0.006). Interestingly, despite the lack of association in the whole cohort, high nuclear Syk expression was significantly associated with better overall survival in certain subgroups (P = 0.001)., Conclusions: The current study indicates a lack of correlation between calpain-2 expression and Syk and MAP4. Syk, MAP4 and calpain-1 appeared to significantly correlate with each other in the whole cohort, with calpain-1 being more highly associated with MAP4 and Syk in mucinous carcinomas. Overall, the current results suggest that Syk, MAP4, and calpain-1 expression are correlated with each other and these proteins may be involved in early stages of tumour spread.

Purpose: We aimed to analyze the expression of ZWINT, NUSAP1, DLGAP5, and PRC1 in tumor tissues and adjacent tissues with public data., Methods: The expression patterns of four genes were detected in cancer tissues and adjacent tissues by qRT-PCR. The overall survival analysis was used to explore these genes in lung adenocarcinoma and squamous cell carcinoma patients. Knockdown assays were used to select the most suitable gene among these four genes. Cell function assays with the knockdown gene were conducted in A549 and NCL H226 cells. The role of the knockdown gene in lung cancer was dissected in a mice tumor model. Transcriptome sequencing analyses with the knockdown gene were analyzed., Results: Overexpression of these genes was significantly detected in cancer tissues (P < 0.01). Overall survival revealed that high expression of these genes is closely related with poor prognosis of lung adenocarcinoma patients (P < 0.05). Knockdown of ZWINT reduced proliferation in NCI H226 and A549 cells (P < 0.05). Knockdown also inhibited cell migration, invasion, apoptosis, and colony formation (P < 0.05). ZWINT knockdown reduced tumor volume (P < 0.05). Transcriptome sequencing of ZWINT knockdown-treated A549 and NCI H226 cells indicated that 100 and 426 differentially expressed genes were obtained, respectively. Gene ontology analysis suggested that binding, biological regulation, and multicellular organismal processes were the most enriched. KEGG analysis revealed that TNF, P53, and PI3K signal networks would be the most potential ZWINT-related pathways and were identified by Western blot analysis., Conclusions: ZWINT may be a novel target for lung cancer therapy.

Objective: Cell autophagy reduces the sensitivity of cancer cells to therapeutic reagents in various types of human cancer. Therefore, the aim of our study was to use human colorectal cancer HCT116 cells to explore whether inhibition of autophagy by 3-Methyladenine (3-MA, an autophagy inhibitor) is able to enhance hypoxia-induced apoptosis in vitro., Materials and Methods: HCT116 cells were treated with 3-MA, hypoxia, or 3-MA plus hypoxia, and the autophagy, apoptosis and proliferation of the HCT116 cells were investigated. Western blot analysis was used to detect autophagy specificity protein microtubule-associated protein light chain 3 (LC3) expression. Effects on apoptosis were evaluated by using flow cytometry (JC-1 staining to measure mitochondrial membrane potential) and annexin V-propidium iodide (PI) staining., Results: The results showed that the treatment of HCT116 cells in vitro with hypoxia alone increased autophagy as well as apoptosis, whereas combination treatment with 3-MA and hypoxia markedly inhibited hypoxia-induced autophagy, but increased hypoxia-induced cell apoptosis., Conclusions: Autophagy might play a role as a self-defense mechanism in hypoxia-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.

Introduction and Objectives: Multiple androgen receptor (AR)-dependent and -independent resistance mechanisms limit the efficacy of current castration-resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR-targeting compounds, including EPI-001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD-AR treatment may upregulate autophagy and that a combination of NTD-AR and autophagy inhibition might therefore increase antitumor effects., Methods: AR-expressing prostate cancer cell lines (LNCaP, LNCaP-EnzR) were treated with different concentrations of EPI (10, 25, 50 μM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 μM) or 3-methyladenine (3-MA, 5 mM). Cell proliferation was assessed by WST-1-assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3-I/II and p62., Results: Treatment with EPI resulted in a dose-dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP-EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3-II expression and a decreased p62 expression in all EPI-treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines., Conclusions: Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer., (© 2018 Wiley Periodicals, Inc.)

Diethylstilbestrol (DES) is an endocrine disruptor that was used to prevent adverse effects of pregnancy in women in late 1940s until early 1970s. Its use was banned following significant toxicity and negative effects not only in the mothers but also transgenerationally. Previous studies from our laboratory showed that DES induces thymic atrophy and immunosuppression in mice. In this study, we investigated the molecular mechanisms through which DES triggers thymic atrophy, specifically autophagy. To that end, we treated C57BL/6 mice with DES, and determined expression of two autophagy-related proteins, microtubule-associated protein-1 light chain 3 (LC3) and Beclin-1 (Becn1). We observed that DES-induced thymic atrophy was associated with increased autophagy in thymocytes and significant upregulation in the expression of both Becn1 and LC3. DES also caused downregulation in the expression of miR-30a in thymocytes, and transfection studies revealed that miR-30a targeted Becn1. Upon examination of methylation status of Becn1, we noted hypomethylation of Becn1 in thymocytes of mice exposed to DES. Together, these data demonstrate for the first time that DES induces autophagy in thymocytes potentially through epigenetic changes involving hypomethylation of Becn1 and down-regulation of miR-30a expression., (Copyright © 2018. Published by Elsevier B.V.)