Autophagic Genes Research Articles

Exogenous Melatonin Improves Pear Resistance to Botryosphaeria dothidea by Increasing Autophagic Activity and Sugar/Organic Acid Levels.

Pear is an important fruit tree worldwide, but it is often infected by the pathogen Botryosphaeria dothidea, which causes pear ring rot disease. To explore the effect of exogenous melatonin on the disease resistance of pear, we treated inoculated pear fruits with different concentrations of melatonin. The results showed that 100 μΜ of melatonin had the most significant effect with resistance to B. dothidea. In addition, melatonin treatment significantly reduced the diameter of disease lesions and enhanced the endogenous melatonin content in pears inoculated with B. dothidea. Compared with the control treatment, melatonin treatment suppressed increases in reactive oxygen species (ROS) and activated ROS-scavenging enzymes. Treatment with exogenous melatonin maintained ascorbic acid-glutathione at more reductive status. The expression levels of core autophagic genes and autophagosome formation were elevated by melatonin treatment in pear fruits. Silencing of PbrATG5 in Pyrus pyrifolia conferred sensitivity to inoculation that was only slightly attenuated by melatonin treatment. After inoculation with B. dothidea, exogenous melatonin treatment led to higher levels of soluble sugars and organic acids in pear fruits than H2O treatment. Overall, our results demonstrate that melatonin enhances resistance to B. dothidea by increasing autophagic activity and soluble sugar/organic acid accumulation.

The Role of Autophagy in the Mother-to-Child Transmission of Pregnant Women With a High Level of HBV DNA.

BackgroundMother-to-child transmission (MTCT) is the most common propagation mode of hepatitis B virus (HBV) transmission. Exploring the mechanisms of HBV MTCT is the key to protect infant from infection. In this study, we aim to clarify the important role of autophagy complicated in HBV MTCT.MethodsA total of 169 placental samples were collected in this study, includes 144 HBV positive pregnant women and 25 normal pregnant women. In vitro, JEG-3 cells were treated with serum contained different HBV viral loads. Electron microscope was used to observed the number of autophagosome. RT-qPCR and western blotting were used to measure the expression level of autophagy relative genes and proteins respectively. Immunofluorescence was used to analyzed the expression of LC-3 of the frozen section of placental tissue.ResultsAccording to the number of autophagosomes and the expression level of autophagic genes mRNA and protein, autophagy was increased in HBV maternal placenta. Among the control, low viral load, medium viral load and high viral load groups, autophagy was significantly up-regulated with the increase of HBV viral loads. Also, autophagy was increased in the HBeAg positive pregnant women compared with their HBeAg negative counterparts. Also, autophagy in infant-infected group was up-regulated compared with infant-uninfected group. In vitro, choriocarcinoma JEG-3 cells were treated with the different HBV viral loads or different time incubation, the mRNA and protein of autophagy related genes was maximum expression in the medium viral load or treatment in a short period, but decreased in the high viral load treatment or with long-term HBV exposure.ConclusionOur study determines the high levels of viremia could be the cause of both increase autophagy activities and MTCT. Autophagy was significantly up-regulated in pregnant women with high viral load or HBeAg positive, which plays an important part in the HBV MTCT.

The Effect of Autophagic Gene Inhibition on Exosomal Tau

Philip Yu is an undergraduate student researcher passionate about translational research in various fields. The following is an independent experimental study on genetic control of the cellular processes that regulate protein expression and transport in neurodegeneration. The study was conducted under the supervision of Dr. Derrick Gibbings in the Department of Cellular and Molecular Medicine at the University of Ottawa. Throughout the study, Philip conducted all experiments involved, in addition to collecting and processing the generated data. The misfolding of the protein tau contributes to the development of Alzheimer’s disease (AD). Misfolded tau is thought to propagate through a homeostatic degradation process known as autophagy, resulting in the export of cellular materials to the extracellular space via extracellular vesicles, called exosomes. By inhibiting the ATG7 and p62 genes necessary for autophagy to occur, the effects on the amount of exosomal and intracellular tau can be observed. Following the analysis of western blot and protein assay data, it was determined that the inhibition of the ATG7 and p62 genes results in a 70% and 60% reduction in the concentration of tau found in exosomes, respectively. These results suggest potential therapeutic applications of autophagic gene inhibition for the treatment of AD.

Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression

ABSTRACT Macroautophagy/autophagy is a conserved cellular process associated with tumorigenesis and aggressiveness, while mechanisms regulating expression of autophagic machinery genes in cancers still remain elusive. Herein, we identified E2F4 (E2F transcription factor 4) as a novel transcriptional activator of cytoprotective autophagy crucial for zinc homeostasis in cancer cells. Gain- and loss-of-function studies showed that E2F4 promoted autophagy in a cell cycle-dependent manner, resulting in facilitated degradation of MT (metallothionein) proteins, elevated distribution of Zn2+ within autophagosomes, decreased labile intracellular zinc ions, and increased growth, invasion, and metastasis of gastric cancer cells. Mechanistically, E2F4 directly regulated the transcription of ATG2A (autophagy related 2A) and ULK2 (unc-51 like autophagy activating kinase 2), leading to autophagic degradation of MT1E, MT1M, and MT1X, while USP2 (ubiquitin specific peptidase 2) stabilized E2F4 protein to induce its transactivation via physical interaction and deubiquitination in cancer cells. Rescue experiments revealed that USP2 harbored oncogenic properties via E2F4-facilitated autophagy and zinc homeostasis. Emetine, a small chemical inhibitor of autophagy, was able to block interaction between UPS2 and E2F4, increase labile intracellular zinc ions, and suppress tumorigenesis and aggressiveness. In clinical gastric cancer specimens, both USP2 and E2F4 were upregulated and associated with poor outcome of patients. These findings indicate that therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression. Abbreviations: 3-MA: 3-methyladenine; ANOVA: analysis of variance; ATG2A: autophagy related 2A; ATG5: autophagy related 5; ATP: adenosine triphosphate; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CCND1: cyclin D1; CDK: cyclin dependent kinase; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; E2F4: E2F transcription factor 4; eATP: extracellular adenosine triphosphate; EBSS: Earle’s balanced salt solution; FP: first progression; FRET: fluorescence resonance energy transfer; FUCCI: fluorescent ubiquitination-based cell cycle indicator; GFP: green fluorescent protein; GST: glutathione S-transferase; HA: hemagglutinin; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDM2: MDM2 proto-oncogene; MKI67/Ki-67: marker of proliferation Ki-67; MT: metallothionein; MT1E: metallothionein 1E; MT1M: metallothionein 1M; MT1X: metallothionein 1X; MTT: 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide; OS: overall survival; PECAM1/CD31: platelet and endothelial cell adhesion molecule 1; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; qPCR: quantitative PCR; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; UBXN1: UBX domain protein 1; Ub: ubiquitin; ULK2: unc-51 like autophagy activating kinase 2; USP14: ubiquitin specific peptidase 14; USP2: ubiquitin specific peptidase 2; USP5: ubiquitin specific peptidase 5; USP7: ubiquitin specific peptidase 7; ZnCl2: zinc chloride.

Combined training increases thermogenic fat activity in patients with overweight and type 2 diabetes.

Exercise is an important strategy in the management of diabetes. Experimental studies have shown that exercise acts, at least in part, by inducing the production of myokines that improve metabolic control and activate brown/beige adipose tissue depots. Combined training (CT) is recommended by the major diabetes guidelines due to its metabolic and cardiovascular benefits, however, its impact on brown/beige adipose tissue activities has never been tested in humans with overweight and type 2 diabetes (T2D). Here, we evaluated the effects of 16-week combined training (CT) program on brown adipose tissue activity; browning and autophagy markers, and serum pro-thermogenic/inflammatory inducers in patients with overweight and T2D. Thirty-four patients with overweight and T2D were assigned to either a control group (CG) or a combined training group (CTG) in a randomized and controlled study. Functional/fitness parameters, anthropometry/body composition parameters, blood hormone/biochemical parameters, thermogenic/autophagic gene expression in subcutaneous adipose tissue were evaluated before and at the end of the intervention. In addition, cold-induced 18-Fluoroxyglucose Positron Emission Computed Tomography (18F-FDG PET/CT) was performed in the training group before and after the end of the intervention. CT increased cervical/supraclavicular brown adipose tissue (BAT) thermogenic activity (p = 0.03) as well as in perirenal adipose tissue (p = 0.02). In addition, CT increased the expression of genes related to thermogenic profile (TMEM26: + 95%, p = 0.04; and EPSTI1: + 26%, p = 0.03) and decreased autophagic genes (ULK1: -15%, p = 0.04; LC3: -5%, p = 0.02; and ATG4: -22%, p < 0.001) in subcutaneous adipose tissue. There were positive correlations between Δ% BAT activity with Δ% of post training energy expenditure cold exposure, HDL-c, IL4, adiponectin, irisin, meteorin-like, and TMEM26 and ZIC1 genes, besides negative correlations with LDL-c, total cholesterol and C-reactive protein. This is the first evidence of the beneficial actions of CT on adipose tissue thermogenic activity in humans, and it adds important support for the recommendation of CT as a strategy in the management of diabetes.

Resveratrol as a modulatory of apoptosis and autophagy in cancer therapy.

Cancer is one of the leading causes of death, with a heavy socio-economical burden for countries. Despite the great advances that have been made in the treatment of cancer, chemotherapy is still the most common method of treatment. However, many side effects, including hepatotoxicity, renal toxicity, and cardiotoxicity, limit the efficacy of conventional chemotherapy. Over recent years, natural products have attracted attention as therapeutic agents against various diseases, such as cancer. Resveratrol (RES), a natural polyphenol occurring in grapes, nuts, wine, and berries, exhibited potential for preventing and treating various cancer types. RES also ameliorates chemotherapy-induced detrimental effects. Furthermore, RES could modulate apoptosis and autophagy as the main forms of cancer cell deaths by targeting various signaling pathways and up/downregulation of apoptotic and autophagic genes. This review will summarize the anti-cancer effects of RES and focus on the fundamental mechanisms and targets for modulating apoptosis and autophagy by RES.

Orthodontic loading activates cell-specific autophagy in a force-dependent manner

Orthodontic tooth movement (OTM) relies on bone remodeling and controlled aseptic inflammation. Autophagy, a conserved homeostatic pathway, has been shown to play a role in bone turnover. We hypothesize that autophagy participates in regulating bone remodeling during OTM in a force-dependent and cell type-specific manner. A split-mouth design was used to load molars with 1 of 3 force levels (15, 30, or 45 g of force) in mice carrying a green fluorescent protein-LC3 transgene to detect cellular autophagy. Fluorescent microscopy and quantitative polymerase chain reaction analyses were used to evaluate autophagy activation and its correlation with force level. Cell type-specific antibodies were used to identify cells with green fluorescent protein-positive puncta (autophagosomes) in periodontal tissues. Autophagic activity increased shortly after loading with moderate force and was associated with the expression of bone turnover, inflammatory, and autophagy markers. Different load levels resulted in altered degrees of autophagic activation, gene expression, and osteoclast recruitment. Autophagy was specifically induced by loading in macrophages and osteoclasts found in the periodontal ligament and alveolar bone. Data suggest autophagy participates in regulating bone turnover during OTM. Autophagy is induced in macrophage lineage cells by orthodontic loading in a force-dependent manner and plays a role during OTM, possibly through modulation of osteoclast bone resorption. Exploring the roles of autophagy in OTM is medically relevant, given that autophagy is associated with oral and systemic inflammatory conditions.

Nanoencapsulation of Cordycepin Induces Switching from Necroptosis to Apoptosis in Human Oral Cancer Cells (HSC-4) Through Inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3) and Autophagy Modulation

Human oral squamous carcinoma is considered the most common oral cancer; it imposes multiple oral and dental consequences as a result of oral cancer treatment. We previously found that the nanoencapsulation of cordycepin (CS) could inhibit oral cancer cells. However, the mechanism of action was not understood. The aim of this study was to investigate the signaling pathway by which CS and encapsulated nanoparticles (NPs) activate the inhibition of cancer cell growth. We demonstrated that human oral cancer (HSC-4) cells underwent necroptosis when incubated with high concentrations of CS, but not when incubated with either low concentrations of CS or encapsulated CS NPs. High concentrations of CS-induced necroptosis of HSC-4 cells, demonstrated by a reduction in apoptotic ( BAX, Caspase-3, Caspase-8, and Caspase-9) and autophagic genes ( LC3, Atg5, and Atg12). However, low concentrations of CS significantly induced the expression of autophagic gene LC3. Interestingly, encapsulated CS NPs induced a significant increase in apoptotic genes ( P53, BAX, Caspase-3, Caspase-8, and Caspase-9), but a significant decrease in autophagic ( P62, Atg5, and Atg12) and necroptotic genes ( receptor-interacting serine/threonine-protein kinase 3 [RIPK3]) . We also found that encapsulated CS NPs enhanced the accumulation of cellular protein and decreased secreted supernatant protein levels. Moreover, encapsulated CS NPs had higher efficacy in terms of reactive oxygen species (ROS) generation-mediated inhibition of autophagy compared to nonencapsulated CS, suggesting that nanoencapsulation of CS can switch the program of HSC-4 cell death from necroptosis to apoptosis. In conclusion, HSC-4 cells have a defense strategy against CS-induced cell apoptosis, but this problem can be solved through the use of encapsulation combined with nanotechnology.

Ovarian Toxicity Induced by Aluminum Chloride: Alteration of Cyp19a1, Pcna, Puma, and Map1lc3b genes Expression

Aluminum (Al) is an abundant metal with wide application in our daily lives including medicine, industry, cosmetics, and packaging. After entrance to the body, aluminum binds to transferrin and reaches different tissues. Al is a metalloestrogen that can lead to oxidative stress (OxS) and endocrine disruption. No detailed study can be found addressing the effect of Al on the ovary and granulosa cells (GCs). In this study, the focus is on the treated ovaries and GCs of NMRI mice exposed to low, middle, and high doses of aluminum chloride (AlCl3) via in vitro and in vivo assays. The steroidogenic, proliferative, apoptotic, and autophagic-related genes were examined. Up-regulated expression of steroidogenic and proliferative genes was detected. The observed apoptotic and autophagic genes had variable expression. Interrupted ovarian structure, disrupted folliculogenesis, presence of Call-Exner bodies, overexpression of steroidogenic gene, and unbalanced apoptosis/autophagy and proliferation resembled features of granulosa cell tumor (GCT).

Combined molecular, structural and memory data unravel the destructive effect of tramadol on hippocampus

Tramadol is a synthetic analogue of codeine and stimulates neurodegeneration in several parts of the brain that leads to various behavioral impairments. Despite the leading role of hippocampus in learning and memory as well as decreased function of them under influence of tramadol, there are few studies analyzing the effect of tramadol administration on gene expression profiling and structural consequences in hippocampus region. Thus, we sought to determine the effect of tramadol on both PC12 cell line and hippocampal tissue, from gene expression changes to structural alterations. In this respect, we investigated genome‐wide mRNA expression using high throughput RNA-seq technology and confirmatory quantitative real‐time PCR, accompanied by stereological analysis of hippocampus and behavioral assessment following tramadol exposure. At the cellular level, PC12 cells were exposed to 600 μM tramadol for 48 hrs, followed by the assessments of ROS amount and gene expression levels of neurotoxicity associated with neurodegenerative pathways such as apoptosis and autophagy. Moreover, the structural and functional alteration of the hippocampus under chronic exposure to tramadol was also evaluated. In this regard, rats were treated with tramadol at doses of 50 mg/kg for three consecutive weeks. In vitro data revealed that tramadol provoked ROS production and caused the increase in the expression of autophagic and apoptotic genes in PC12 cells. Furthermore, in-vivo results demonstrated that tramadol not only did induce hippocampal atrophy, but it also triggered microgliosis and microglial activation, causing upregulation of apoptotic and inflammatory markers as well as over-activation of neurodegeneration. Tramadol also interrupted spatial learning and memory function along with long-term potentiation (LTP). Taken all together, our data disclosed the neurotoxic effects of tramadol on both in vitro and in-vivo. Moreover, we proposed a potential correlation between disrupted biochemical cascades and memory deficit under tramadol administration.

Synergistic effects of botanical curcumin-induced programmed cell death on the management of Spodoptera litura Fabricius with avermectin

Chemical pesticides and adjuvants have caused many negative effects. Botanical compounds provide solutions for the development of environment friendly pesticides and the management of increasing pest resistance. Curcumin, a natural polyphenol, showed synergistic effects on avermectin upon the destructive agricultural pest, Spodoptera litura. However, the botanical synergist and its relevant mechanisms remain unclear. In the article, curcumin significantly enhanced the growth inhibition and midgut structural damage of avermectin on the larvae of S. litura, and the synergistic effects were confirmed with pot experiments. There were only a few influences on the gene expression of avermectin targets, while apoptotic and autophagic related genes and proteins were accumulated in the avermectin/curcumin mixed regent (0.013/0.0013 μg/mL) treated group. Moreover, the potential mechanism was explored with an in vitro model, insect Spodoptera frugiperda Sf9 cell line. Morphology observation featured the damage on cells and Hoechst33258 staining revealed the fragments of DNA after treating with the avermectin/curcumin mixed regent (10/1 μg/mL). Dansylcadaverine and LysoTracker staining, as well as the gene expressions, supposed that curcumin exhibited autophagy inducing effects and the mixed regent possessed a higher ability to induce apoptosis and autophagy. All these results suggested that the synergistic effects of curcumin on the pest management of avermectin potentially mainly derived from the enhancement of programed cell death. It provides new sights for the application of natural compounds in integrated pest management and enriches examples of synergistic mechanisms.

Zeb1-mediated autophagy enhances resistance of breast cancer cells to genotoxic drugs

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy.Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.

Transcriptomic Studies Suggest a Coincident Role for Apoptosis and Pyroptosis but Not for Autophagic Neuronal Death in TBEV-Infected Human Neuronal/Glial Cells

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, Flavivirus genus, is responsible for neurological symptoms that may cause permanent disability or death. With an incidence on the rise, it is the major arbovirus affecting humans in Central/Northern Europe and North-Eastern Asia. Neuronal death is a critical feature of TBEV infection, yet little is known about the type of death and the molecular mechanisms involved. In this study, we used a recently established pathological model of TBEV infection based on human neuronal/glial cells differentiated from fetal neural progenitors and transcriptomic approaches to tackle this question. We confirmed the occurrence of apoptotic death in these cultures and further showed that genes involved in pyroptotic death were up-regulated, suggesting that this type of death also occurs in TBEV-infected human brain cells. On the contrary, no up-regulation of major autophagic genes was found. Furthermore, we demonstrated an up-regulation of a cluster of genes belonging to the extrinsic apoptotic pathway and revealed the cellular types expressing them. Our results suggest that neuronal death occurs by multiple mechanisms in TBEV-infected human neuronal/glial cells, thus providing a first insight into the molecular pathways that may be involved in neuronal death when the human brain is infected by TBEV.

PERK signaling activation restores nucleus pulposus degeneration by activating autophagy under hypoxia environment

Intervertebral disc (IVD) degeneration is an important disease with no efficient biological therapy identified. Autophagy, a wildly known therapeutic target for human disease, has been demonstrated to be activated under hypoxia, with underlying mechanism remains elusive. Thus, this study aims to specify the role of autophagy in IVD degeneration, the regulating mechanism of hypoxia-inducing autophagy, and the therapeutic value of autophagy for IVD degeneration. RNA-seq was used to screen the primary pathway affected in NP cells under hypoxia, the specific link between hypoxia and autophagy were investigated using ChIP-seq and dual luciferase reporter assay. Conditional ATG7 knockout mice (ATG7-/-) were constructed for assessing the effect of autophagy on IVD degeneration, and puncture induced mice model of IVD degeneration were used for intradiscal injection to evaluate the therapeutic value of autophagy. We demonstrated that hypoxia induces autophagy by transcriptional activation of autophagic gene LC3B and ATG7, which is controlled by PERK signaling. Then, we observed that inhibiting autophagy or PERK signaling leads to impaired NP cell viability and function, furthermore, using ATG7 knockout (ATG7-/-) mice, we identified the protective role of autophagy in IVD. Furthermore, we found that intradiscal injection of PERK signaling agonist, CCT020312, significantly restores the degeneration level of needle punctured mice IVD. We showed that the activation of PERK signaling upon hypoxia serves as a vital mechanism to induce autophagy and identified the therapeutic value of PERK signaling agonist for IVD degeneration treatment.

Systematic Construction of an Autophagic Risk Model in Bone Marrow for Prognostic Prediction in Multiple Myeloma

Autophagy is an intracellular self-degradative process that balances cell energy source and regulates tissue homeostasis, which plays critical role in the pathogenesis of multiple myeloma (MM). However, the prognostic role of autophagy-related genes (ARGs) in MM remains undefined.In the present study, the ARGs were obtained from Gene Expression Omnibus datasets (accession GSE24080, GSE136337, GSE57317), which contains 1038 samples of patients with MM. Univariate Cox regression analysis identified 38 ARGs that were significantly associated with overall survival of MM. Furthermore, a risk score model with 11 prognosis-associated ARGs was developed using multivariate Cox regression analysis, including ARNT, ATG4D, BIRC5, BNIP3L, CDKN1A, EIF2S1, IRGM, ITGA3, NCKAP1, NRG1 and TM9SF1. The 3-year area under the curve (AUC) values for the receiver operating characteristic curves were 0.717(0.662, 0.758), 0.646(0.587, 0.703) and 0.906(0.694, 1.000) for GSE24080, GSE136337, GSE57317 prognosis predictions, respectively (Figure A-C). Using this prognostic signature, patients with MM could be separated into high- and low-risk groups with distinct clinical outcomes (Figure D-F). Moreover, autophagy risk score was an independent prognostic factor by multivariate analysis. KEGG revealed that most pathways were related to autophagy and metabolism. Furthermore, we validated the expression of 11 genes and ARNT in bone marrow of MM patients (Figure G-I) and showed the critical role of ARNT-mediated autophagy in the proliferation and drug resistance of bortezomib in myeloma cells (Figure J-M).In conclusion, we constructed ARGs-based prognostic model to predict the prognosis of MM, targeting specific autophagic gene such as ARNT might provide therapeutic clues for MM treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and chemotherapy with gemcitabine has limited effects and is associated with development of drug resistance. Treatment of Panc1 and MiaPaca2 pancreatic cancer cells with gemcitabine induced expression of the orphan nuclear receptor 4A2 (NURR1) and analysis of the cancer genome atlas indicated the NURR1 is overexpressed in pancreatic tumors and is a negative prognostic factor for patient survival. Results of NURR1 knockdown or treatment with the NURR1 antagonist 1,1-bis(3΄-indolyl)-1-(p-chlorophenyl)methane (C-DIM 12) demonstrated that NURR1 was pro-oncogenic in pancreatic cancer cells and regulated cancer cell and tumor growth and survival. NURR1 is induced by gemcitabine and serves as a key drug-resistance factor and is also required for gemcitabine-induced cytoprotective autophagy. NURR1 regulated genes were determined by RNA sequencing of mRNAs expressed in MiaPaCa2 cells expressing NURR1 and in CRISPR/Cas9 gene edited cells for NURR1 knockdown and KEGG enrichment analysis of the differentially expressed genes showed that autophagy was the major pathway regulated by NURR1. Moreover, NURR1 regulated expression of two major autophagic genes ATG7 and ATG12 which are also overexpressed in pancreatic tumors and like NURR1 are negative prognostic factors for patient survival. Thus, gemcitabine-induced cytoprotective autophagy is due to the NURR1 - ATG7/ATG12 axis and this can be targeted and disrupted by NURR1 antagonist C-DIM12 demonstrating the potential clinical applications for combination therapies with gemcitabine and NURR1 antagonists.

Autophagy-Related Chemoprotection against Sorafenib in Human Hepatocarcinoma: Role of FOXO3 Upregulation and Modulation by Regorafenib.

Early acquisition of sorafenib resistance is responsible for the dismal prognosis of advanced hepatocarcinoma (HCC). Autophagy, a catabolic process involved in liver homeostasis, has been associated with chemosensitivity modulation. Forkhead box O3 (FOXO3) is a transcription factor linked to HCC pathogenesis whose role on autophagy-related sorafenib resistance remains controversial. Here, we unraveled the linkage between autophagy and sorafenib resistance in HCC, focusing on the implication of FOXO3 and its potential modulation by regorafenib. We worked with two HepG2-derived sorafenib-resistant HCC in vitro models (HepG2S1 and HepG2S3) and checked HCC patient data from the UALCAN database. Resistant cells displayed an enhanced basal autophagic flux compared to HepG2, showing higher autophagolysosome content and autophagy markers levels. Pharmacological inhibition of autophagy boosted HepG2S1 and HepG2S3 apoptosis and subG1 cells, but reduced viability, indicating the cytoprotective role of autophagy. HCC samples displayed higher FOXO3 levels, being associated with shorter survival and autophagic genes expression. Consistently, chemoresistant in vitro models showed significant FOXO3 upregulation. FOXO3 knockdown suppressed autophagy and caused resistant cell death, demonstrating that overactivation of such pro-survival autophagy during sorafenib resistance is FOXO3-dependent; a cytoprotective mechanism that the second-line drug regorafenib successfully abolished. Therefore, targeting FOXO3-mediated autophagy could significantly improve the clinical efficacy of sorafenib.

Expression of BECN1 gene in Acute Myeloid Leukemia: Association with Hematological and Clinical Parameters

Abstract Background In acute myeloid leukemia (AML), there is copy number loss in autophagic genes such as BECN1. Accordingly, decreased autophagy and the development of AML are related. BECN1 is a critical mediator that influences the onset and progress of autophagy. Objective To investigate the expression status of BECN1 gene in newly diagnosed adult AML patients and its association with various hematological parameters and clinical outcomes. Methods Case control study to study BECN1 gene expression variability between 50 newly diagnosed adult AML patients and 20 healthy age and sex matched controls, with follow up of the patients to detect its effect on induction therapy. All AML patients underwent full history taking, through clinical examination, laboratory investigations such as complete blood count (CBC) with examination of peripheral blood and bone marrow Leishman stained films, immunophenotyping, cytogenetic analysis (karyotyping/FISH analysis) and BECN1 gene expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR). Results In our study, a highly significant difference was found as regards reduced expression of BECN1 gene in patients group compared to control group. We also found reduced BECN1 gene expression in both intermediate and adverse risk groups compared to favorable risk group. Reduced expression of BECN1 gene was associated with increasing age and total leukocytic count (TLC), peripheral blood (PB) and bone marrow (BM) blasts, the presence of FLT3-ITD mutation, CD34 and CD117 and in non-responders group. No statistically significant difference was found as regards haemoglobin (Hb) level, platelet (PLT) count and FAB subtypes. Conclusion Autophagy plays an important role in the pathogenesis of AML. Furthermore; the reductive regulation of the BECN1 gene may carry a poor prognosis and is associated with many well established bad prognostic factors especially FLT3-ITD mutation. Targeting autophagy pathways especially its major regulator (BECN1 gene) may become an effective and promising new line of therapy for AML patients.

Development and Validation of an Autophagy-Related LncRNA Prognostic Signature in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the greatest public challenges because of delayed diagnosis and poor prognosis. In this study, we established an autophagy-associated long non-coding (Lnc)RNA prognostic signature to assess the prognosis of HNSCC patients. The LncRNA expression profiles and clinical information of 499 HNSCC samples were available in The Cancer Genome Atlas. Autophagic LncRNAs were analyzed using Pearson correlation. A co-expression network showed the interactions between autophagic genes and LncRNAs. An autophagic LncRNAs prognostic signature, consisting of MYOSLID, AL139287.1, AC068580.1, AL022328.2, AC104083.1, AL160006.1, AC116914.2, LINC00958, and AL450992.2, was developed through uni- and multivariate Cox regressions. High- and low-risk groups were classified based on the median risk scores. The high-risk group had significantly worse overall survival according to Kaplan–Meier curve analysis. Multivariate Cox regression demonstrated that risk scores were a significant independent prognostic factor (hazard ratio = 1.739, 95% confidence interval: 1.460–2.072), with an area under the curve of 0.735. Principal component analysis distinguished two categories based on the nine-LncRNA prognostic signature. In conclusion, this novel autophagic LncRNA signature is an independent prognostic factor and may suggest novel therapeutic targets for HNSCC.

Methamphetamine-Triggered Neurotoxicity in Human Dorsolateral Prefrontal Cortex.

Background:Methamphetamine (MA), is an extremely addictive stimulant that adversely affects the central nervous system. Accumulating evidence indicates that molecular mechanisms such as oxidative stress, apoptosis, and autophagy are involved in the toxicity of MA. Considering experimental animal studies exhibiting MA-induced neurotoxicity, the relevance of these findings needs to be evidently elucidated in human MA users. It is generally assumed that multiple chemical substances released in the brain following MA-induced metabolic activation are primary factors underlying damage of neural cells. Hence, this study aimed to investigate the role of autophagy and apoptosis as well as oxidative stress in the brain of postmortem MA-induced toxicity.Materials and Methods:In this study, we determine the gene expression of autophagy and apoptosis, including BECN1, MAP1ALC3, CASP8, TP53, and BAX genes in ten healthy controls and ten chronic users of MA postmortem dorsolateral prefrontal cortex (DLPFC) by real-time polymerase chain reaction. Also, we applied immunohistochemistry in formalin-fixed and paraffin-embedded human brain samples to analyze brain-derived neurotrophic factor (BDNF). Also, spectrophotometry was performed to measure glutathione (GSH) content.Results:The expression level of apoptotic and autophagic genes (BECN1, MAP1ALC3, CASP8, TP53, and BAX) were significantly elevated, while GSH content and BDNF showed substantial reductions in DLPFC of chronic MA users. Discussion: Our data showed that MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. Also, MA induces multiple functional and structural perturbations in the brain, determining its toxicity and possibly contributing to neurotoxicity. Discussion:Our study showed BDNF-positive cells as well as GSH amount, displayed significant declines in DLPFC of MA user. MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. However, further investigations are needed to throw light on the cellular and molecular mechanisms that act in the various regions of the addicted brain, especially in DLPFC.