Beclin-1 Research Articles

Abstract 4121: Autophagy influences the development of the pre-metastatic niche

Abstract Background: Autophagy is a lysosomal degradation process that is characterized by cellular self-consumption. Autophagy allows for the recycling and removal of damaged cytoplasmic material. Many types of cancer have elevated autophagic activity in order to sustain a high rate of metabolism and circumvent stress-induced cell death. However, little is known of autophagy's role in metastasis. Therefore, we tested the effect of autophagy modulation in different mouse models and cell based assays that reflect different points along the metastatic cascade. Methods: Pharmacologic autophagy inhibition in the murine mammary carcinoma cell line 4T1 was achieved with 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1). Knockdown of autophagy critical genes Becn1 or Atg7 by lentiviral delivery of shRNA was also used as a means of inhibition. Proliferation, invasion, migration, and anchorage independent growth of cells were assessed in the presence or absence of active autophagy. For an experimental model of metastasis, Balb/c mice were challenged with 4T1 cells expressing luciferase (4T1-luc) injected via the tail vein. Mice were pre-treated with CQ (60 mg/kg, qd, i.p.) and monitored until the development of luciferase positive metastases. Mice were also challenged with 4T1 Becn1 knockdowns or cells pre-treated with CQ for 2h to assess effect of cellular autophagy impairment. An orthotopic model was also utilized by implanting 4T1-luc cells into the mammary fat pad, with tumors resected at 100-200 mm3 and mice followed until metastasis development. CQ was given either adjuvantly, 24h after surgery, or neoadjuvantly, 24h after implantation. Autophagy stimulation, 2% Trehalose in drinking water, was also given as a neoadjuvant therapy. Bone marrow derived cells (BMDCs), mediators of pre-metastatic niche formation, were measured by flow cytometry in the lungs and blood of treated mice prior to the arrival of tumor cells. Results: Autophagy inhibition was able to reduce cell proliferation, but did not significantly delay metastasis in the experimentally induced model. Similarly, CQ was not effective as an adjuvant therapy. Yet, as a neoadjuvant therapy both CQ and Becn1 knockdown were able to significantly delay metastases. Autophagy stimulation actually sped up metastasis development. Autophagy modulation had no effect on invasion, migration, or anchorage independent growth. Conversely, altering autophagy did impact the number of BMDCs present in the lung and blood. Trehalose treated mice had more than either vehicle or CQ. In the blood, CQ treated mice had even less BMDCs present than vehicle treated mice. Conclusions: Autophagy does not seem to be required for metastatic colonization and survival. Nor does it seem to affect the metastatic capability of cells. Rather, autophagy may be influencing the pre-metastatic microenvironment by impacting BMDCs. Thus autophagy appears to be most essential before the arrival of tumor cells to the site of metastasis. Citation Format: Rebecca A. Barnard, Daniel Regan, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Autophagy influences the development of the pre-metastatic niche. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2015-4121

Age-regulated function of autophagy in the mouse inner ear

Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles during early development in the chicken embryo and in the response to otic injury in the adult mouse. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation.We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the autophagy machinery expression in the inner ear is regulated with age but is not compromised by the chronic absence of IGF-1. Our data also strongly support that the up-regulation of autophagy machinery genes is concomitant with the functional maturation of the inner ear.This article is part of a Special Issue entitled <IEB Kyoto>.

Changes of autophagy in injured cortex after trauma and radiation injury of brain

Objective To clarify the expression of autophagy related proteins in cortical neurons following trauma combined with radiation injury of brain (TRI) and the underlying clinical significance. Methods Fifty-eight male SD adult rats were divided into sham group (n=15), traumatic brain injury group (TBI group, n=15), radiation-induced brain injury group (RBI group, n=14), and TRI group (n=14), according to the random number table. Rats in TBI group were subjected to lateral fluid percussion injury in the cortex, in RBI group whole body 4.5 Gy single dose irradiation to 60Co, and in TRI group whole body 4.5 Gy single dose irradiation to 60Co within 30 minutes after the lateral fluid percussion injury in the cortex. Six weeks after the modeling in each group, neuronal injury was detected with HE staining and expressions of autophagy-related proteins, LC3- II and Beclin-1, were detected through Western blotting. Results TBI group cortical neurons was reduced compared to sham group[(68.7±7.8) vs (96.9±9.6)] (P 0.05), vacuoles, pyknosis and dissolution were not obvious, and expressions of Beclin-1 and LC3-Ⅱ were significantly increased in contrast with sham and TBI groups (P<0. 01). In TRI group number of cortical was grossly reduced to 45.8±7.8 in contrast with TBI and RBI groups (P<0. 05), vacuoles, pyknosis and dissolution were obvious, expressions of Beclin-1 and LC3-Ⅱ decreased significantly as compared to TBI and RBI groups ( P<0. 01). Conclusion Autophagy in the injured cortex is increased after the separate TBI or RBI, but the combined injury of TRI results in significantly reduced autophagy in the injured cortex along with increased neuron degeneration which suggests a protective role of autophagy in brain injuries. Key words: Brain injuries; Radiation injuries; Autophagy

Impacts of chronic sleep deprivation on learning and memory, autophagy and neuronal apoptosis in mice

Objective To establish chronic sleep deprivation mouse model, evaluate the learning and memory ability of mice and observe autophagy and apoptosis levels in mouse brain. Methods C57BL/6 mice (n=20) were randomly separated into sleep deprivation group and control group. After 2-month sleep deprivation by using an adapted multiple platform method, the behavioral performance of mice was measured by IntelliCage system. The expression of microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) and Beclin-1 was detected by Western blotting. Confocol microscopy was used to observe autophagosome. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to detect neuronal apoptosis level in mouse brain. Results The results of behavioral test showed that the incorrect visit ratio was much higher in sleep deprivation group than that in control group. Moreover, the expression of LC3-Ⅱ(sleep deprivation group 1.681±0.186, control group 1.125±0.048, t=2.892, P=0.027 6) and Beclin-1(sleep deprivation group 1.144±0.048, control group 1.006±0.017, t=2.721, P=0.018 6) in mouse hippocampus and cortex was significantly elevated in sleep deprivation group than those in control group. Accordingly, the confocal microscopy observation also revealed an increased nuclear LC3-positive puncta in hippocampus and cortex of sleep deprived mice (hippocampus in sleep deprivation group 1.665±0.153, in control group 0.819±0.072, t=5.024, P=0.002 4; cortex in sleep deprivation group 1.925±0.175, in control group 1.195±0.111, t=3.521, P=0.012 5). In addition, TUNEL staining showed a much higher percentage of TUNEL-positive nuclei in these brain regions (hippocampus in sleep deprivation group 47.24±4.15, in control group 19.26±3.72, t=5.025, P=0.007 4; cortex in sleep deprivation group 42.25±1.25, in control group 27.50±3.23, t=4.262, P=0.005 3). Conclusions Chronic sleep deprivation can impair the learning and memory, increase the expression of LC3-Ⅱ and Beclin-1, elevate the formation of autophagosome, and promote apoptosis in mouse brain. These findings suggest that autophagy and apoptosis might be involved in the cognitive impairment induced by chronic sleep deprivation. Key words: Sleep deprivation; Neurons; Autophagy; Apoptosis; Memory disorders; Disease models, animal

Activated peroxisome proliferator-activated receptor α inhibiting the lipopolysaccharide-induced macrophage-mediated inflammatory responses by promoting the autophagy

Objective To investigate the effects of peroxisome proliferator-activated receptor α (PPARα) on macrophage-mediated inflammatory responses with the interference of lipopolysaccharide and the possible mechanism. Methods The bone marrow stem cells were isolated from the femora of mice. The granulocyte-macrophage colony stimulating factor (GM-CSF) was used to stimulate the in vitro differentiation from bone marrow stem cells into primary macrophages. An in vitro model with cultured cells expressing inflammatory cytokines was established by treating the primary macrophages with lipopolysaccharide (LPS). A specific chemical agonist, Wy-14643, was used to activate PPARα. Autophagy inhibitors including 3-methyladenine (3-MA) and small interfering RNA against Atg7 (Atg7 siRNA) were used to inhibit the autophagy. Western blot assay was performed to detect the expression of autophagy-related proteins (Atg5, Atg7, Beclin-1 and LC3). The transcriptional levels of TNF-α, IL-1β, IL-6, Atg5, Atg7 and Beclin-1 were analyzed by qRT-PCR. Results Compared with the macrophages treated with LPS alone, those pretreated with various concentrations of Wy-14643 (10 μmol/L, 25 μmol/L and 50 μmol/L) showed inhibited expression of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and enhanced expression of autophagy-related proteins (Atg5, Atg7 and Beclin-1) at mRNA level in a dose-dependent manner. The expression of autophagy-related proteins (Atg5, Atg7, Beclin-1 and LC3) by macrophages was promoted with the pretreatment of Wy-14643 as indicated by Western blot assay. The transcriptional levels of TNF-α, IL-1β and IL-6 were increased in Wy-14643 pretreated-macrophages after stimulation with 3-MA or Atg7 siRNA. Conclusion PPARα suppressed the macrophage-mediated inflammatory responses by promoting autophagy, suggesting that the PPARα-autophagy pathway might be one of the signaling pathways regulating LPS induced-inflammatory responses. Key words: Peroxisome proliferator-activated receptor α; Autophagy; Inflammatory response; Macrophages; Lipopolysaccharide

Human papillomavirus 16 E6 inducing autophagy of the cervical cancer C33A cells by endoplasmic reticulum stress pathway

Objective To investigate the effects of human papilloma virus 16 E6 (HPV16 E6) on endoplasmic reticulum (ER) stress-autophagic response in the cervical cancer C33A cells. Methods Polymerase chain reaction was used for detecting the integration of HPV DNA. The eukaryotic expression vector of HPV16 E6 was constructed and transfected via lipofectamine into C33A cells. Experimental cells were classified into 3 groups: pcDNA3.1--HPV16 E6 group, pcDNA 3.1- group and C33A group. Western blot was used to measure expression of protein of HPV16 E6, Beclin 1, LC3Ⅱ, IRE1, PERK and ATF6 in transfected cells. Results here was no HPV DNA integration in C33A cells that were confirmed as the intervention cells. Eukaryotic expression vector pcDNA3.1--HPV16 E6 was constructed successfully. The eukaryotic expression vector pcDNA3.1--HPV16 E6 significantly improved the expression of protein of HPV16 E6 in C33A cells. The protein expression of Beclin 1, LC3 Ⅱ, IRE1, PERK and ATF6 were significantly improved after transfection with vector pcDNA3.1+-HPV16 E6 (P<0.05). Furthermore, LC3Ⅱ protein level was reduced by treatment with ER stress inhibitor. Conclusion HPV16 E6 can improve autophagy through the ER stress pathway, and this response may play an important role in the process of HPV16 E6 inducing cervical cancer, providing one of the new strategies for gene therapy of cervical carcinoma. Key words: Uterine cervical neoplasms; Human papillomavirus 16 E6; Endoplasmic reticulum stress; Autophagy

Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration

HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

Effect of omega-3 polyunsaturated fatty acids supplementation on traumatic brain injury in a rat model

Objective To investigate the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFA) supplementation on brain edema, autophagy response and neurobehavioral outcome after traumatic brain injury (TBI) in rats and the related mechanisms. Methods TBI rat models were established using Feeney's method. Seventy-two SD rats were divided into 4 groups using random number table: sham operation group, TBI group, ω-3 PUFA supplementation group (TBI+ ω-3 group) and autophagy inhibitor 3-methyladenine group (TBI+ 3-MA group) (all n=18), each group was further divided into 3 sub-groups (n=6) corresponding to 3 time points (days 1, 3, and 7 after TBI). On each of the 3 time points, we measured rat behavioral outcomes with modified neurologic severity score (mNSS) tests; brain water content was measured with wet-dry weight method. The mRNA and protein expressions of autophagy-related factors (LC3-Ⅱ and Beclin-1) in TBI cerebral cortex were determined by immunohistochemistry staining, reverse transcription-polymerase chain reaction and Western blot on day 3 after TBI. Results Compared with the sham group, on days 1, 3, and 7 after injuary, the TBI group, the TBI+ ω-3 group, and the TBI+ 3-MA group had significantly higher mNSS scores (TBI group: 12.42±0.27 vs.1.34±0.32, 12.07±0.27 vs. 1.16±0.29, 10.22±0.39 vs. 1.22±0.30; TBI+ ω-3 group: 12.05±0.23 vs. 1.34±0.32, 11.38±0.21 vs. 1.16±0.29, 8.20±0.21 vs. 1.22±0.30; TBI+ 3-MA group: 11.93±0.20 vs. 1.34±0.32, 11.09±0.19 vs. 1.16±0.29, 7.93±0.17 vs. 1.22±0.30; all P=0.00) and brain water content [TBI group: (79.82±0.61)% vs. (71.87±0.43)%, (83.04±0.42)% vs. (72.13±0.53)%, (75.12±0.72)% vs. (71.78±0.38)%; TBI+ ω-3 group: (76.81±0.63)% vs. (71.87±0.43)%, (79.39±0.59)% vs. (72.13±0.53)%, (73.86±0.38)% vs. (71.78±0.38)%; TBI+ 3-MA group: (75.98±0.49)% vs. (71.87±0.43)%, (77.14±0.46)% vs. (72.13±0.53)%, (72.24±0.37)% vs. (71.78±0.38)%; all P=0.00]. The mRNA and protein expressions of LC3-Ⅱ and Beclin-1 in the brain were also significantly higher on day 3 in the TBI group, the TBI+ ω-3 group, and the TBI+ 3-MA group (all P=0.00). Compared with the TBl group, on day 3 and day 7 after injury, the TBI+ ω-3 group and the TBI+ 3-MA group had significantly lower mNSS scores (TBI+ ω-3 group: 11.38±0.21 vs. 12.07±0.27, P=0.04, 8.20±0.21 vs. 10.22±0.39, P=0.01; TBI+ 3-MA group: 11.09±0.19 vs. 12.07±0.27, P=0.01, 7.93±0.17 vs. 10.22±0.39, P=0.00). On days 1, 3, and 7, compared with the TBI group, the TBI+ ω-3 group and the TBI+ 3-MA group had significantly lower brain water content [TBI+ ω-3 group: (76.81±0.63)% vs. (79.82±0.61)%, P=0.04, (79.39±0.59)% vs. (83.04±0.42)%, P=0.01, (73.86±0.38)% vs. (75.12±0.72)%, P=0.03; TBI+ 3-MA group: (75.98±0.49)% vs. (79.82±0.61)%, P=0.01, (77.14±0.46)% vs. (83.04±0.42)%, P=0.00, (72.24±0.37)% vs. (75.12±0.72)%, P=0.02]. On day 3, the TBI+ ω-3 group and the TBI+ 3-MA group had significantly reduced LC3-Ⅱ and Beclin-1 mRNA expression compared with the TBI group (TBI+ ω-3 group: P=0.04, P=0.01; TBI+ 3-MA group: P=0.01, P=0.00) and protein expression (TBI+ ω-3 group: P=0.01, P=0.03; TBI+ 3-MA group: both P=0.00). Conclusion ω-3 PUFA supplementation could markedly reduce brain edema and improve neurological functions after TBI, showing a neuroprotective effect, possibly through inhibiting TBI-induced autophagy responses. Key words: Traumatic brain injury; Omega-3 polyunsaturated fatty acids; Autophagy; Neuroprotection

Effects of sevoflurane on invasion and migration of mouse lung cancer cells induced by hypoxia

Objective To evaluate the effects of sevoflurane on invasion and migration of mouse lung cancer cells induced by hypoxia. Methods Mouse Lewis lung cancer cells were inoculated in the culture plate.After being cultured for 24 h, the cells were randomly divided into 3 groups(n=18 each)using a random number table: control group(group C), hypoxia group(group H)and hypoxia+ 2% sevoflurane group(group HS). Cells were exposed to 95% air-5%CO2(2 L/min)for 4 h in group C. Cells were exposed to 94% N2-5%CO2-1% O2 for 4 h in group H. In group HS, cells were exposed to 2% sevoflurane and 94% N2(2 L/min)for 4 h. The invasion of cells was determined by Transwell assay, and the invaded cells were counted.The migration of cells was evaluated by wound healing assay, and cell migration rates were calculated.The expression of Beclin 1 and LC3Ⅱ protein in cells was detected by Western blot. Results Compared with group C, the number of invaded cells and cell migration rates were significantly increased, and the expression of Beclin 1 and LC3Ⅱ was up-regulated in H and HS groups.Compared with group H, the number of invaded cells and cell migration rates were significantly decreased, and the expression of Beclin 1 and LC3Ⅱ was down-regulated in group HS. Conclusion Sevoflurane can inhibit the invasion and migration of mouse lung cancer cells induced by hypoxia, and inhibition of autophagy is involved in the mechanism. Key words: Anesthetics, inhalation; Lung neoplasms; Cell hypoxia; Neoplasm invasiveness; Neoplasm metastasis

Activated autophagy in spinal cord contributes to maintaining diabetic neuropathic pain in rats model

To observe the role of autophagy in maintaining diabetic neuropathic pain in rats model. A total of 44 male Sprague-Dawley rats were randomly divided into diabetic neuropathic (n = 36) and normal control (n = 8) groups. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg body weight, i.p) freshly dissolved in citrate buffer (pH = 4.5). For assessing the presence of mechanical hyperalgesia in diabetic rats, mechanical paw-withdrawal threshold (MWT) in response to punctuate mechanical stimuli was measured. At Week 4 post-injection, the rats with mechanical pain threshold decreasing over 50% as compared to baseline were designated as diabetic neuropathic pain rats. They were randomly divided into three groups of neuropathic pain (DP), neuropathic pain plus rapamycin (DR) and neuropathic pain plus 3-methyladenine (3-MA) (DA). The DR group received an intraperitoneal injection of rapamycin (1 mg/kg body weight) for Day 1 to Day 14 after grouping. At the same timepoint, the DA group had an intraperitoneal injection of 3-MA (2 mg/kg body weight) and the other group phosphate buffered saline (PBS) (1 ml/kg body weight). MWT was measured at week 1, 2, 3, 4 after STZ injection and at day 1, 3, 5, 7, 9, 14 after rapamycin, 3-MA or PBS injections. Spinal cord tissues were used to examine the expression of LC3, Beclin-1 and P62 protein by Western blot at Day 14 after medication. The mechanical threshold of group DR decreased further from Day 3 to Day 14 after rapamycin injection compared to baseline [(4.8±0.8), (4.3±0.7), (4.1±0.6), (3.6±0.5), (3.3±0.6) vs (5.3±0.9) g, P<0.05]. The mechanical threshold of group DA began to increase from Day 5 to Day 14 after 3-MA injection [(7.0±0.8), (7.7±1.0), (9.1±0.9), (9.6±1.1) vs (5.3±0.6) g, P<0.05]. The expressions of LC3-II and Beclin-1 protein in spinal cord of rapamycin-treated rats was significantly higher than those of non-supplemented diabetics (1.32±0.12 vs 1.02±0.11; 1.03±0.08 vs 0.80±0.06, P<0.05). Otherwise the expressions of these protein in spinal cord of 3-MA-treated rats were significantly lower than those of non-supplemented diabetics (0.70±0.09 vs 1.02±0.11; 0.55±0.05 vs 0.80±0.06, P<0.05). Up-regulated autophagy in spinal cord partially contributes to the maintenance of diabetic neuropathic pain.

Interferon regulatory factor-1 signaling regulates the switch between autophagy and apoptosis to determine breast cancer cell fate.

Interferon regulatory factor-1 (IRF1) is a tumor suppressor that regulates cell fate in several cell types. Here, we report an inverse correlation in expression of nuclear IRF1 and the autophagy regulator ATG7 in human breast cancer cells that directly affects their cell fate. In mice harboring mutant Atg7, nuclear IRF1 was increased in mammary tumors, spleen, and kidney. Mechanistic investigations identified ATG7 and the cell death modulator beclin-1 (BECN1) as negative regulators of IRF1. Silencing ATG7 or BECN1 caused estrogen receptor-α to exit the nucleus at the time when IRF1 nuclear localization occurred. Conversely, silencing IRF1 promoted autophagy by increasing BECN1 and blunting IGF1 receptor and mTOR survival signaling. Loss of IRF1 promoted resistance to antiestrogens, whereas combined silencing of ATG7 and IRF1 restored sensitivity to these agents. Using a mathematical model to prompt signaling hypotheses, we developed evidence that ATG7 silencing could resensitize IRF1-attenuated cells to apoptosis through mechanisms that involve other estrogen-regulated genes. Overall, our work shows how inhibiting the autophagy proteins ATG7 and BECN1 can regulate IRF1-dependent and -independent signaling pathways in ways that engender a new therapeutic strategy to attack breast cancer.

Enhancement of hydroxycamptothecin to human Tenon capsule fibroblasts autophagy via PERK pathway

Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction, so they remain a close association.However, the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear. Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs. Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours, and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells, including autophagy related gene 5(ATG-5), Beclin-1, light chain 3(LC-3). Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups. Results The gray scales for the expressions of Beclin 1, ATG-5, LC-3-Ⅰand LC-3-Ⅱ proteins in HTFs were 0.365±0.045, 0.765±0.055, 0.120±0.030 and 0.215±0.035 in the control group, and those in the hydroxycamptothecin treated group were 0.980±0.070, 1.495±0.095, 0.585±0.025 and 0.785±0.055, showing a significant decline in the hydroxycamptothecin treated group(P=0.018, 0.022, 0.007, 0.013). The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group, with a significant reduce in comparison with 0.765±0.055 of the control group (P=0.010). However, no obvious distinctions were seen in the band intensities of the expressions of Beclin-1, ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790±0.060 in the 0.10 g/L hydroxycamptothecin group, which was significantly higher than 0.880±0.070 of the control group (P=0.010). Expression levels (gray scales) of Beclin-1, ATG-5, LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475±0.045, 0.390±0.040, 0.055±0.015 and 0.075±0.025, which were significantly lowed in comparison with 0.955±0.065, 0.765±0.055, 0.155±0.015 and 0.280±0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P=0.026, 0.031, 0.042, 0.034). In addition, the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group compared with the control+ 0.10 g/L hydroxycamptothecin group. Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway. Key words: Autophagy; Hydroxycamptothecin; Endoplasmic reticulum stress; Signal transduction; Humans, Tenon capsule; Fibroblasts; Gene transfection; Gene knockout

High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown.In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies on the role of autophagy in CLL, and they may further represent targets of treatment.

Regulation of autophagy on dendritic cells during rat liver regeneration by IPA

To understand the mechanism underlying autophagy in regulating dendritic cells during rat liver regeneration, we used the method of percoll density gradient centrifugation combined with immunomagnetic bead to isolate dendritic cells, the Rat Genome 230 2.0 Array to determine the expression changes of autophagy-related genes, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the autophagy activities. The results indicated that LC3, BECN1, ATG7 and SQSTM1 genes had significant expression changes during rat liver regeneration. There were 593 genes related to autophagy, among which 210 genes were identified as significant. We also showed that the activity of autophagy was enhanced in the priming phase and teminal phase of liver regeneration, weakened in the proliferative stage by comparative analysis method of IPA. The autophagy-related physiological activities mainly included RNA expression, RNA transcription, cell differentiation and proliferation, involving in PPARα/RXRα activation, acute phase response signaling, TREM1 signaling, IL-6 signaling, IL-8 signaling and IL-1 signaling, whose activities were increased or decreased in liver regeneration. Cluster analysis found that P53 and AMPK signaling participated in the regulation of dendritic cells autophagy, with AMPK signaling in the priming phase of liver regeneration, and both signaling pathways in the terminal phase. We conclude that dendritic cells autophagy played an important role in initiation of the immune response in priming phase and depletion of dendritic cells in late phase during rat liver regeneration.

Chemoresistance is associated with Beclin-1 and PTEN expression in epithelial ovarian cancers.

The aim of the present study was to investigate the protein expression of the autophagy-related genes, BECN1 and PTEN, and the association with drug resistance in epithelial ovarian cancers. In total, 40 patients with pathologically diagnosed epithelial ovarian cancer were divided into a chemotherapy-sensitive group (n=20) and a chemotherapy-resistant group (n=20), according to the results of the pre- or post-operative normative chemotherapy and the post-operative follow-up. The protein expression of the phosphatase and tensin homolog (PTEN) and the BECN1 gene product, Beclin-1, was analyzed using immunohistochemistry in the 40 patients with ovarian carcinoma. The positive rate of Beclin-1 expression was significantly lower in the resistant group (35.0%) compared with the sensitive group (50.0%). The positive rate of PTEN expression was also significantly lower in the resistant group (30.0%) compared with the sensitive group (65.0%). Furthermore, the differences in the expression rates were revealed to be significant (P<0.05). The expression of Beclin-1 was identified to be positively correlated with the expression of PTEN (rs=0.816; P<0.0001). The low expression of the Beclin-1 and PTEN proteins in the ovarian cancer tissues was revealed to be closely associated with drug resistance. Therefore, Beclin-1 may interact with PTEN to participate in the mechanism of drug resistance and the changes in macrophage activity observed in cases of drug-resistant ovarian cancer.

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.

Decreased BECN1 mRNA Expression in Human Breast Cancer is Associated With Estrogen Receptor-Negative Subtypes and Poor Prognosis

Both BRCA1 and Beclin 1 (BECN1) are tumor suppressor genes, which are in close proximity on the human chromosome 17q21 breast cancer tumor susceptibility locus and are often concurrently deleted. However, their importance in sporadic human breast cancer is not known. To interrogate the effects of BECN1 and BRCA1 in breast cancer, we studied their mRNA expression patterns in breast cancer patients from two large datasets: The Cancer Genome Atlas (TCGA) (n=1067) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n=1992). In both datasets, low expression of BECN1 was more common in HER2-enriched and basal-like (mostly triple-negative) breast cancers compared to luminal A/B intrinsic tumor subtypes, and was also strongly associated with TP53 mutations and advanced tumor grade. In contrast, there was no significant association between low BRCA1 expression and HER2-enriched or basal-like subtypes, TP53 mutations or tumor grade. In addition, low expression of BECN1 (but not low BRCA1) was associated with poor prognosis, and BECN1 (but not BRCA1) expression was an independent predictor of survival. These findings suggest that decreased mRNA expression of the autophagy gene BECN1 may contribute to the pathogenesis and progression of HER2-enriched, basal-like, and TP53 mutant breast cancers.

Changes and its significance of autophagy in rats with acute necrosis pancreatitis

Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis (ANP). Methods According to method of random number, 18 rats were randomly divided into control group, ANP group, ANP+ rapamycin (RAP) group. The ANP rat model was established by intraperitoneal injection of 20% L–arginine. The rats of ANP+ RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling. The rats of control group were intraperitoneal injected with 0.9% NaCl solution. The blood was drawed from the hearts nine hours after modeling for subsequent experiments. Serum levels of trypsinogen activation peptide (TAP), interleukin (IL–1), IL–6 and tumor necrosis factor (TNF)α were measured with enzyme–linked immunosorbent assay. The pancreatic tissues were pathologically scored. Autophagy–related structures in rat pancreatic acinar cells were observed by transmition electron microscopy. The expression of autophagy marker microtuble assciated protein 1 light chain 3 (LC3)–Ⅱ and Beclin–1 at mRNA and protein level were measured by quantitative real–time polymerase chain reaction (qRT–PCR), Western bloting and immunohistochemistry. The single factor analysis of variance was used for mean comparison among groups. Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP+ RAP group (2.19±1.38) was higher than that of ANP group (0.97±0.68), and the difference was statistically significant(F=33.75, P 0.05). Conclusions Autophagy increased in rats with ANP. Promoting autophagy could significantly activate trypsinogen, aggravate pancreatic injury and increase inflammation reaction, which indicated that autophagy might involve in the pathogenesis of ANP through trypsinogen activation. Key words: Pancreatitis acute necrotizing; Autophagy; Trypsinogen

Is BECLIN-1 Immunoreactivity More Effective than HBME-1 in Diagnosis of Papillary Thyroid Cancer ?

Background: Even though morphologic findings are generally important in the diagnosis of thyroid tumors, in some cases, morphologic similarities between benign and malignant lesions lead to noticeable differences in evaluation and different diagnoses in the same cases. In our study, we researched whether the autophagy-related protein Beclin-1 (BECN1) is a diagnostic marker in thyroid tumors, as well as its correlation with HBME-1, which has high sensitivity and specificity in distinguishing malignant thyroid lesions.Methods: Samples from 136 patients that received a thyroidectomy were fixed in 10% formalin. All cases had hematoxylin & eosin (H&E) stains available for review and paraffin blocks for immunohistochemical staining. In immunochemistry tests, BECN1, HBME-1, and Ki-67 were studied.Results: BECN1 immunoreactivity rates were found to be 98.9% in papillary thyroid carcinoma (PTC), 57.1% in follicular carcinoma (FC), and 21.4% in follicular adenoma (FA). HBME-1 immunoreactivity was 100% in PTC, 85.7% in FC, and 64% in FA. In thyroid carcinomas, BECN1 was as effective as HBME-1 as a marker for the diagnosis of malignancy.Conclusions: We revealed an important role of autophagy in thyroid carcinogenesis, as evidenced by the high rate of BECN1 immunoreactivity in PTC and FC. Moreover, we found that autophagy plays a more important role in PTC, as evidenced by the high immunoreactivity rates. According to our results, BECN1 is a more specific marker than HBME-1 in PTC and has a higher correlation with Ki-67. In routine studies, BECN1 will be more helpful than HBME-1 in the diagnosis of PTC.

Corresponding erdosteine changes autophagy genes expression in hippocampus on Rhinitis medicamentosa model

In our study, rats were subjected to Oxymetazoline hydrochloride treatment and Rhinitis medicamentosa (RM) was formed and then autophagy gene expression levels were determined after the application of an antioxidant agent erdosteine (ED). The rats were divided into three groups; Group 1 was the control group. Group 2 (RM) and group 3 (RM+ED) rats received two spray puffs of 0.05% oxymetazoline into the nasal cavities three times daily for eight weeks. After determination of RM in the rats, the RM group were killed. The ED+RM group received 10 mg/kg of an ED suspension. At the end of seven days, these rats were also killed. All groups? hippocampus tissues were obtained for the measurement of autophagy gene expressions. In rhinitis medicamentosa group Atg5, Atg7 and Atg10 gene expressions in the left hippocampus were reduced as compared to control group (p=0.01, p&gt;0.05, p=0.01, respectively). Also, erdosteine treatments were restored mRNA expression of autophagy genes. In right hippocampus of rhinitis medicamentosa group, Atg5 and Atg10 gene expressions was found to be down-regulated as compared to control group (p&gt;0.05, p&lt;0.05, respectively). Both BECN1 and ULK genes expression were found to be reduced in left hippocampus of rhinitis medicamentosa group. Erdosteine applications was restored the expression of these genes (p=0.03, p=0.03, respectively). Additionally, in right hippocampus, Erdosteine application was restored the expression of ULK gene (p=0.01). This is the first report that evaluated the expression autophagy genes in RM rat models and the changes observed after erdosteine applications.