Beclin-1 Research Articles

MicroRNA expression profiles in non‑epithelial ovarian tumors.

Ovarian germ cell tumors (OGCTs) and sex cord stromal tumors (SCSTs) are rare gynecologic tumors that are derived from germ and stromal cells, respectively. Unlike their epithelial counterparts, molecular pathogenesis of these tumor types is still poorly understood. Here, we characterized microRNA (miRNA) expression profiles of 9 OGCTs (2 malignant and 7 benign) and 3 SCSTs using small RNA sequencing. We observed significant miRNA expression variations among the three tumor groups. To further demonstrate the biological relevance of our findings, we selected 12 miRNAs for validation in an extended cohort of 16 OGCTs (9 benign and 7 malignant) and 7 SCSTs by reverse transcription-quantitative polymerase chain reaction. Higher expression of miR-373-3p, miR-372-3p and miR-302c-3p and lower expression of miR-199a-5p, miR-214-5p and miR-202-3p were reproducibly observed in malignant OGCTs as compared to benign OGCTs or SCSTs. Comparing with benign OGCTs, miR-202c-3p and miR-513c-5p were more abundant in SCSTs. Additionally, we examined Beclin 1 (BECN1), a target of miR-199a-5p, in the clinical samples using western blot analysis. Our results show that BECN1 expression was higher in malignant OGCTs than benign OGCTs, which is concordant with their lower miR-199a-5p expression. This study suggests that these miRNAs may have potential value as tumor markers and implications for further understanding the molecular basis of these tumor types.

Loss of beclin 1 expression in ovarian cancer: A potential biomarker for predicting unfavorable outcomes.

The clinicopathological significance and prognostic value of the expression of proteins associated with autophagy, beclin 1 (BECN1), 1A/1B-light chain 3 (LC3) and high mobility group box-1 protein (HMGB-1), were investigated in patients with ovarian carcinoma, receiving combination chemotherapy with a platinum agent and a taxane. Immunohistochemical staining was performed for autophagy-associated proteins in tumor tissues from 141 patients with ovarian carcinoma. Clinical data were collected retrospectively by reviewing medical charts, and the association between protein expression, clinicopathological features and survival was investigated. Amongst 141 ovarian carcinoma samples, the loss of BECN1, LC3, and HMGB-1 expression was identified in 59 (41.8%), 35 (24.8%), and 66 (46.8%) samples, respectively. Clinicopathological factors were not significantly associated with the loss of BECN1 expression. However, significant associations were demonstrated between the expression of BECN1, LC3, and HMGB-1. In addition, loss of BECN1 expression demonstrated a significant association with poor progression-free and poor overall survival. Multivariate analysis demonstrated that loss of BECN1 expression and postoperative residual tumor were significant independent predictors of poor progression-free survival and poor overall survival. These results indicated that loss of BECN1 expression in ovarian carcinoma is a negative prognosticator in patients receiving platinum-based chemotherapy. Assessment of BECN1 expression may be useful for predicting an unfavorable response to platinum-based chemotherapy in ovarian carcinoma.

Effect of tetramethylpyrazine on autophagy in hippocampal neurons of rats with sepsis-associated en-cephalopathy

Objective To evaluate the effect of tetramethylpyrazine on autophagy in the hippocampal neurons of rats with sepsis-associated encephalopathy. Methods Sixty SPF healthy male Sprague-Dawley rats, aged 11-14 weeks, weighing 200-240 g, were divided into 3 groups(n=20 each)using a random number table: sham operation group(group Sham), sepsis group(group Sep)and tetramethylpyrazine group(group TMP). Sepsis was induced by cecal ligation and puncture(CLP), and group Sham only underwent simple laparotomy.Tetramethylpyrazine 10 mg/kg was injected intraperitoneally at 1 h before CLP in group TMP.Morris water maze test was performed in 10 rats randomly selected at 12 and 36 h after CLP.Then the rats were sacrificed, and hippocampi were isolated for determination of the expression of microtubule-associated protein 1 light chain 3Ⅰ(LC3Ⅰ), LC3Ⅱ, Beclin-1 and p62 in hippocampal tissues by Western blot, and the LC3Ⅱ/LC3Ⅰratio was calculated. Results Compared with group Sham, the escape latency was significantly prolonged, the rate of time spent in the target quadrant was decreased, the LC3Ⅱ/LC3Ⅰratio was increased, the expression of Beclin-1 was up-regulated, and the expression of p62 was down-regulated at 12 and 36 h after CLP in group Sep and group TMP(P<0.05). Compared with group Sep, the escape latency was significantly shortened, the rate of time spent in the target quadrant was increased, the LC3Ⅱ/LC3Ⅰratio was decreased, the expression of Beclin-1 was down-regulated, and the expression of p62 was up-regulated at 12 and 36 h after CLP in group TMP(P<0.05). Conclusion The mechanism by which tetramethylpyrazine reduces sepsis-associated encephalopathy is related to inhibiting autophagy in the hippocampal neurons of rats. Key words: TETRAMETHYLPYRAZINE; Sepsis; Brain diseases; Autophagy; Hippocampus

Induction and molecular mechanism of placental trophoblast cell autophagy in preeclampsia

Objective To investigate the induction and regulatory mechanism of placental trophoblast cell autophagy in women with preeclampsia (PE). Methods Twenty gravidas with severe PE who underwent cesarean section in the Department of Obstetrics and Gynecology of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University from August 2016 to November 2016 were enrolled in PE group. An equal number of normotensive gravidas without proteinuria who also underwent cesarean section during the same period were randomly selected as control group. Placental tissue samples were collected from all gravidas. Ultrastructure of placental trophoblast cells and changes in autophagosome formation were observed by transmission electron microscope. Expressions of microtubule associated protein 1 light chain 3B (MAP1LC3B, or LC3B) and Beclin 1 in placental tissue samples were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot. Activities of protein kinase B (PKB, also known as Akt)/mammalian target of rapamycin (mTOR) pathway in placental tissue samples were detected by Western blot. Two independent samples t-test or Mann-Whitney U test was used for statistical analysis. Results Sparse and disordered villi and many typical autophagosomes were observed in placental trophoblast cells from patients with severe PE. Significantly enhanced expression of LC3B at mRNA and protein levels and increased ratio of LC3-II/LC3-I were observed in the PE group as compared with the control group [3.37 (2.37-6.11) vs 0.62 (0.25-4.15), 1.40±0.17 vs 1.00±0.13, 1.57±0.25 vs 1.00±0.31, Z or t=-4.440, 3.274 and 3.113, all P 0.05). Furthermore, Akt and mTOR phosphorylation in the PE group was significantly suppressed as compared with that in the control group (1.00±0.29 vs 0.64±0.21, 1.00±0.32 vs 0.60±0.22, t=-3.672 and -2.895, both P 0.05). Conclusions Suppressed activity of Akt/mTOR pathway and enhanced induction of trophoblast cell autophagy are detected in placental tissues of patients with severe PE, indicating that excessive trophoblast cell autophagy, induced by decreased activity of Akt/mTOR pathway, may be the pathogenesis for PE. Key words: Pre-eclampsia; Trophoblasts; Autophagy; Placenta; Proto-oncogene proteins c-akt; TOR serine-threonine kinases

Effect and mechanism of SS31 peptide on autophagy after spinal cord injury in rats

Objective To explore the effect of SS31 peptide on autophagy after spinal cord injury (SCI) and possible mechanism. Methods Allen method was used to construct the spinal cord injury model in rats. Sprague-Dawley rats were randomly divided into sham surgery group (sham group), SCI group and SS31 peptide group, with 30 rats in each group. The sham group only received laminectomy. The rats in SCI group were sustained SCI and were given no intervention. The rats in SS31 group received SS31 peptide injection after SCI. Scores of Basso Beattie Bresnahan (BBB) motor functions were assessed at 6 h, 1, 3, 7 and 14 d after the injury. The changes in related proteins of Beclin-1 and LC3-II were also detected. Results Scores of BBB scale at 6 h and at days 1, 3, 7 and 14 after injury in SCI group (0, 1.7±0.4, 3.5±0.6, 6.1±0.7, 10.1±0.6) and SS31 peptide group (0, 2.5±0.7, 4.1±0.7, 9.3±0.6, 13.4±0.6) were lower than that in sham group (21 at all time points) (P 0.05). Compared with sham group, the expression of Beclin-1 in SCI group and SS31 peptide group was increased, reached a peak at day 3 (1.478±0.030, 1.841±0.051), remained high level at day 7 (1.302±0.049, 1.551±0.032) and showed high expression at day 14 (1.252±0.048, 1.471±0.062) (P 0.05). Conclusion SS31 peptide can improve motor function and enhance the autophagy of nerve cells after SCI in rats, which may be one of the mechanisms for SS31 peptide treating spinal cord injury. Key words: Spinal cord injuries; Autophagy; Beclin-1

GSK3β to adjust the role of autophagy in Parkinson's disease nerve cells and mechanism research

Objective To screen stable express A30P a-synuclein PC12 cell as the research object, and to preliminarily investigate the activity of different glycogen systhesis kinase 3β (GSK3β) for the regulation of autophagy pathway causes a-synuclein autophagy level change and its effects on cell growth. Methods The vector of A30P a-synuclein was used to transfect to PC12 cells. G418 was used to screen stable expressed cells. Liposome method was used to transfect GSK3β-expressed plasmid, GSK3β silence plasmid, and blank plasmid into these cell lines, and stable expressed activity of GSK3β cell lines were screened. Western blot was used to detect the expression of GSK3β after transfection and verify the transfection efficiency. Western blot was used to test groups of cells in LC-3Ⅱ, Beclin1, and a-synuclein expressions. Methyl thiazolyl tetrazolium (MTT) method was used to detect the change of cell proliferation. Terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) method was used to detect apoptosis. Results ⑴ When GSK3β expressed group was compared to GSK3β silence group, blank plasmidgroup, and blank control group, the expressions of LC-3Ⅱ and Beclin1 were significantly increased (P<0.05), the expression of a-synuclein was significantly reduced (P<0.05), the results of MTT and TUNEL showed that the cells were the decrease of apoptosis and increase of cell proliferation, with a statistically significant difference (P<0.05). ⑵ When GSK3β silence group was compared to GSK3β express group, blank plasmidgroup, and blank control group, the expressions of LC-3Ⅱ and Beclin1 were significantly reduced (P<0.05), the expression of a-synuclein was significantly increased (P<0.05), the results of MTT and TUNEL showed that the cells were the increase of apoptosis and decrease of cell proliferation, with a statistically significant difference (P<0.05). Conclusions ⑴ Activation of GSK3β activity can improve the level of cell autophagy, enhance the ability of alpha-synuclein degradation, and promote cell survival. ⑵ Inhibition of GSK3β activity can reduce the level of cell autophagy, weaken the ability of alpha-synucleindegradation, and reduce cell survival. ⑶ Autophagy is closely related to the pathogenesis of Parkinson's disease (PD). The improvement of the level of autophagy and enhancing of the degradation of a-synuclein is a new way of the future treatment of PD. Key words: Glycogen synthase kinase 3; Autophagy; alpha-synuclein; Parkinson disease

Relationship between heme oxygenase-1 expression and autophagy during acute lung injury in septic mice

Objective To evaluate the relationship between heme oxygenase-1 (HO-1) expression and autophagy during acute lung injury in septic mice. Methods Forty-eight male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were randomly divided into 4 groups (n=12 each) using a random number table: sham operation group (group SH), sepsis group (group CLP), HO-1 agonist hemin plus sepsis group (group Hemin+ CLP) and HO-1 inhibitor SnPP plus sepsis group (group SnPP+ CLP). Sepsis was induced by cecal ligation and puncture in CLP, Hemin+ CLP and SnPP+ CLP groups.Hemin 50 μmol/kg and SnPP 50 μmol/kg were intraperitoneally injected at 2 h before operation in Hemin+ CLP group and SnPP+ CLP group, respectively.Arterial blood samples were taken at 24 h after operation for blood gas analysis, PaO2 was recorded, and oxygenation index was calculated.Lungs were removed for examination of pathological changes which were scored and for determination of autophagosome count (with an electron microscope), weight/dry weight ratio (W/D ratio) and expression of HO-1, Becling-1 and microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) in lung tissues (by Western blot). Results Compared with group SH, oxygenation index was significantly decreased, and W/D ratio, autophagosome count and pathological scores were increased in CLP, Hemin+ CLP and SnPP+ CLP groups, and the expression of HO-1, Beclin-1 and LC3 Ⅱ was significantly up-regulated in CLP and Hemin+ CLP groups (P<0.05). Compared with group CLP, oxygenation index and autophagosome count were significantly increased, W/D ratio and pathological scores were decreased, and the expression of HO-1, Beclin-1 and LC3 Ⅱ was up-regulated in group Hemin+ CLP, and oxygenation index and autophagosome count were significantly decreased, W/D ratio and pathological scores were increased, and the expression of HO-1, Beclin-1 and LC3 Ⅱ was down-regulated in group SnPP+ CLP (P<0.05). Conclusion Up-regulated expression of HO-1 mediates autophagy, which is involved in the endogenous protective mechanism underlying acute lung injury in septic mice. Key words: Heme oxygenase-l; Autophagy; Sepsis; Respiratory distress syndrome, adult

Regulation of autophagy, NF-κB signaling, and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells.

KRAS mutant non-small cell lung cancer (NSCLC) may be classified into epithelial or mesenchymal subtypes. Despite having the same "driver" mutation, mesenchymal NSCLCs are less responsive than are epithelial NSCLCs to inhibition of the RAS pathway. Identifying alternative networks that promote survival specifically in mesenchymal NSCLC may lead to more effective treatments for this subtype. Through their numerous targets in cellular signaling pathways, noncoding microRNAs (miRNAs) often function as tumor suppressors or oncogenes. In particular, some miRNAs regulate the epithelial-mesenchymal transition (EMT). We derived an EMT-related miRNA signature by profiling the abundance of miRNAs in a panel of epithelial (KE) or mesenchymal (KM) KRAS mutant NSCLC cell lines. This signature revealed a number of suppressed miRNAs in KM cell lines, including members of the miR-200 family, which can suppress tumor progression by inhibiting EMT. Reconstituting KM cells with one of these miRNAs, miR-124, disrupted autophagy and decreased cell survival by reducing the abundance of p62, which is both an adaptor for selective autophagy and a regulator of the transcription factor nuclear factor κB (NF-κB). Suppression of p62 by miR-124 correlated with reduced abundance of the autophagy activator beclin 1 (BECN1), the ubiquitin ligase TRAF6, and the NF-κB subunit RELA/p65. The abundance of miR-124 inversely correlated with the expression of BECN1 and TRAF6 in patient NSCLC samples. These findings reveal how the loss of miR-124 promotes cell survival networks in the aggressive mesenchymal subtype of KRAS mutant NSCLC, which might lead to improved subtype-selective therapeutic strategies for patients.

Mild hypothermia alleviates cerebral ischemia reperfusion injury via promoting neurons autophagy in mice

Objective To investigate the neurons autophagy and apoptosis after cerebral ischemia reperfusion (I/R)injury in mice, and to explore the effect of mild hypothermia on neurons autophagy and apoptosis. Methods The global cerebral ischemia-reperfusion injury model in C57 mice was established with carotid artery ligation method. Ninety-six C57 mice were randomly(random number) divided into 8 groups(n=12 in each group), namely control group (C0), sham group, normal body temperature group (NT, 37 ℃) after I/R 6 h (C6 h), normal body temperature group after I/R 12 h (C12 h), normal body temperature group after I/R 72 h(C72 h), mild hypothermia group (MH, 34 ℃) after I/R6 h (C6 h+ MH), mild hypothermia group after I/R12 h (C12 h+ MH), mild hypothermia group after I/R 72 h (C72 h+ MH). The protein expressions of Sirt1, P-FoxO1, Rab7, P53 and autophagy related genes such as Beclin1, LC3 were detected by Western blot at given intervals. The LC3 granules were assayed by immunofluorescence. The neurons apoptosis was detected by TUNEL. The software of SPSS13.0 was used for statistical analysis. Measurement data was expressed with mean±SD, and comparison between two groups was carried out with Student’s t test, One-way ANOVA was used for comparisons among groups, and P<0.05 was considered statistically significant. Results The global cerebral ischemia-reperfusion injury model in C57 mice was established successfully with bilateral carotid arteries ligation method. Compared with the control group, the protein expressions of Sirt1, P-FoxO1, Rab7, Beclin1 and LC3Ⅱ/Ⅰwere gradually reduced, especially at 12 h after I/R in NT group (P<0.05), while the expression of P53 was obviously increased (P<0.05). In MH group, the expressions of Sirt1, P-FoxO1, Rab7, Beclin1, LC3Ⅱ/Ⅰwere higher than those in NT group (P< 0.05). And the expression of P53 was lower than that in NT group (P<0.05). Immuno-fluorescence test showed that compared with the control group, the LC3 particles of neurons cells were decreased significantly in group C12(at 12 h after I/R 6.0±1.5 vs. 18.1±2.5, P<0.05). Nevertheless, LC3 particles were increased in MH group compared with NT group(36.1±4.5 vs. 6.0±1.5, P<0.05). The results of TUNEL test showed that compared with the control group, neurons cells apoptosis were significantly increased in C72 group(at 72 h after I/R, 54.8%±7.5% vs. 5.5%±1.2%, P<0.05). However, compared with NT group, neurons apoptosis were decreased in MH group(28.8%±4.5% vs. 54.8%±7.5%, P<0.05). Conclusions The neuron autophagy was significantly reduced and apoptosis was significantly increased after ischemia reperfusion injury (I/R) in mice. However, mild hypothermia could increase the expression of Sirt1, FoxO1, beclin1 and LC3, so as to promote neurons autophagy and reduce apoptosis, which would provide therapy target for neurons injury after hypoxia and provide soundly theoretical basis for mild hypothermia for clinical application. Key words: Ischemia reperfusion; Mild hypothermia; Neurons; Autophagy; Apoptosis

Structural transitions in conserved, ordered Beclin 1 domains essential to regulating autophagy

Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositol-3-kinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiled-coil domain (CCD). However, the last 16 CCD residues, composing an "overlap helix" (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the C-terminal β-α repeated, autophagy-specific domain (BARAD). Here, using CD spectroscopy, isothermal titration calorimetry, and small-angle X-ray scattering, we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the two-domain CCD-BARAD. Last, we used cellular assays to demonstrate that mutation of these OH-interface residues abrogates starvation-induced up-regulation of autophagy but does not affect basal autophagy. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains (i.e. the CCD or the BARAD). Our findings have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes.

Relation of autophagy and postherpetic neuralgia in older adults

Objective To investigate the relation of autophagy and postherpetic neuralgia in elderly patients, and provide new methods for prevention and treatment of postherpetic neuralgia. Methods One hundred thirty-five specimens were obtained from elderly patients with herpes zoster, containing 83 patients with herpes zoster plus neuralgia and 52 cases with non-neuralgia herpes zoster.The level of autophagy was determined by acridine orange-stain.The protein expressions of autophagy marker protein LC3 and autophagy related protein Beclin1 were detected by Western blot. Results Flow cytometer detection after acridine orange-stain showed that mean fluorescence intensity (MFI) of herpes zoster plus neuralgia patients was 0.775±0.068 and non-neuralgia herpes zoster patients was 0.342±0.025.The level of autophagy of non-neuralgia herpes zoster patients was significant lower than herpes zoster plus neuralgia patients (P<0.05). The expressions of autophagy marker protein LC3 and autophagy related protein Beclin1 detected by Western blot also showed the level of autophagy of non-neuralgia herpes zoster patients was decreased compared with herpes zoster plus neuralgia patients. Conclusions High level autophagy is risk factor for postherpetic neuralgia in elderly patients. Key words: Autophagy; Herpes, zoster; Neuralgia

Promoter Variant Alters Expression of the Autophagic BECN1 Gene: Implications for Clinical Manifestations of Machado-Joseph Disease.

Autophagy is especially important in disorders where accumulation of the mutant protein is a hallmark, such as the Machado-Joseph disease/spinocerebellar ataxia type 3 (MJD/SCA3). We analyzed the promoter of the BECN1 gene, whose overexpression has been reported to exert neuroprotective effects in MJD, with the aim of finding variants that could be associated with expression levels of beclin-1 and could be tested as modifiers of onset and disease severity. A fragment encompassing the BECN1 promoter was sequenced in 95 MJD subjects and 120 controls. The impact of the variation detected on transcription factors (TFs) binding affinity was evaluated in silico and inferences concerning levels of expression were confirmed by fluorescence-based quantitative real-time PCR in a subset of 28 MJD subjects and 26 controls. Four previously described (rs60221525, rs116943570, rs34882610, and rs34037822) and one novel (c.-933delG) variants were identified. In silico analysis performed for the most frequent variants-rs60221525 C allele and rs116943570 T allele-predicted an impact of the presence of these alleles on TF binding affinity. BECN1 expression levels were in agreement with the in silico predictions, showing a tendency for decreased levels in samples with the rs60221525 C allele and for increased levels in samples with the rs116943570 T allele. MJD patients carrying the rs60221525 C allele presented a tendency for earlier estimated age at onset. Moreover, patients with the rs60221525 C allele presented a more severe clinical picture, compared to patients without this allele. The analysis of a larger number of patients from different cohorts, currently unavailable, would be required to confirm these results.

Survivin inhibitor YM155 inhibits proliferation of hepatocellular carcinoma cells by inducing autophagy

Objective To investigate the effects of YM155, a small molecule Survivin inhibitor, on the proliferation of hepatocellular carcinoma (HCC) cells and to explore its molecular mechanism. Methods After HCC cell line SK-Hep-1 was treated with different concentrations of YM155 (0-60 nmol/L), cell proliferation rate of 24, 48 h was assessed by methyl thiazol tetrazolium (MTT), dead cells of 60 h were examined by trypan blue exclusion. SK-Hep-1 cells were treated with 20 nmol/L YM155 for 12 h and 24 h, the expression of Beclin 1, LC3 Ⅰ/Ⅱ, B cell lymphoma/leukemia-2 (bcl-2) and myeloid cell leukemia-1 (Mcl-1) were detected by Western blotting. Results MTT showed that YM155 (0-60 nmol/L)inhibit the proliferation of HCC cells in time and concentration-dependent manner. After treatment with 2 nmol/L and 4 nmol/L YM155 for 60 h, trypan blue exclusion assay showed, dead cells rates were 45% and 90% respectively (P=0.025) relative to the control group [dimethylsurfoxide (DMSO)]; Western blotting results showed that, after SK-Hep-1 cells were treated with 20 nmol/L YM155 for 12 h and 24 h, Beclin 1 and LC3 Ⅱ protein expression levels were increased, while Mcl-1 expession was downregulated, but bcl-2 expression was not altered. Conclusion YM155 significantly suppresses HCC cell proliferation and induced autophagy. Anti-cancer activities of YM155 may be associated with downregulation of Mcl-1 and induction of autophagic cell death. Key words: YM155; Hepatocellular carcinoma; Autophagy; Myeloid cell leukemia-1 gene

Inhibitor of growth protein 4 interacts with Beclin 1 and represses autophagy.

Beclin 1 (BECN1) is a multifunctional protein that activates the pro-autophagic class III phosphatidylinositol 3-kinase (PIK3C3, best known as VPS34), yet also interacts with multiple negative regulators. Here we report that BECN1 interacts with inhibitor of growth family member 4 (ING4), a tumor suppressor protein that is best known for its capacity to interact with the tumor suppressor protein p53 (TP53) and the acetyltransferase E1A binding protein p300 (EP300). Removal of TP53 or EP300 did not affect the BECN1/ING4 interaction, which however was lost upon culture of cells in autophagy-inducing, nutrient free conditions. Depletion of ING4 stimulated the enzymatic activity of PIK3C3, as visualized by means of a red fluorescent protein-tagged short peptide (FYVE) that specifically binds to phosphatidylinositol-3-phosphate (PI3P)-containing subcellular vesicles and enhanced autophagy, as indicated by an enhanced lipidation of microtubule-associated proteins 1A/1B light chain 3 beta (LC3B) and the redistribution of a green-fluorescent protein (GFP)-LC3B fusion protein to cytoplasmic puncta. The generation of GFP-LC3B puncta stimulated by ING4 depletion was reduced by simultaneous depletion, or pharmacological inhibition, of PIK3C3/VPS34. In conclusion, ING4 acts as a negative regulator of the lipid kinase activity of the BECN1 complex, and starvation-induced autophagy is accompanied by the dissociation of the ING4/BECN1 interaction.

Catalase ameliorates diabetes-induced cardiac injury through reduced p65/RelA- mediated transcription of BECN1.

Catalase is an antioxidative enzyme that converts hydrogen peroxide (H2O2) produced by superoxide dismutase from highly reactive superoxide (O2 −) to water and oxygen molecules. Although recent findings demonstrate that catalase, autophagy and the nuclear factor κB (NF‐κB) signalling pathway are centrally involved in diabetic cardiomyopathy (DCM), the interplay between the three has not been fully characterized. Thus, the mechanism responsible for catalase‐mediated protection against heart injury in diabetic mice was investigated in this study, as well as the role of NF‐κB‐p65 in the regulation of autophagic flux was investigated in this study. Western blot analysis revealed that catalase inhibited NF‐κB activity and decreased LC3‐II (microtubule‐associated protein 1 light chain 3) and beclin‐1 (Atg6) expression. Furthermore, up‐regulation of autophagy was detrimental for cardiac function in diabetic mice. Catalase overexpression reduced the level of NF‐κB subunit in the nucleus, where it initiates autophagy through activation of the key autophagy gene BECN1. To evaluate the role of the NF‐κB pathway in diabetes‐induced autophagy, Bay11‐7082, an NF‐κB inhibitor, was injected into diabetic mice, which suppressed NF‐κB and attenuated diabetes‐induced autophagy and myocardial apoptosis. In agreement with the in vivo results, Bay11‐7082 also inhibited high‐glucose‐induced activation of NF‐κB and the up‐regulation of LC3‐II and beclin‐1 expression in H9c2 cells. In addition, high‐glucose‐induced activation of autophagic flux and apoptosis were largely attenuated by p65 siRNA, suggesting that catalase ameliorates diabetes‐induced autophagy, at least in part by increasing the activity of the NF‐κB pathway and p65‐mediated transcription of BECN1.

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma.

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex™. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.

Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia.

Glucocorticoids (GCs) and topoisomerase II inhibitors are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoid cells through the glucocorticoid receptor (GR) and p53 respectively. Mechanisms underlying ALL cell death and the contribution of the bone marrow microenvironment to drug response/resistance remain unclear. The role of the microenvironment and the identification of chemoresistance determinants were studied by transcriptomic analysis in ALL cells treated with Dexamethasone (Dex), and Etoposide (Etop) grown in the presence or absence of bone marrow conditioned media (CM). The necroptotic (RIPK1) and the apoptotic (caspase-8/3) markers were downregulated by CM, whereas the inhibitory effects of chemotherapy on the autophagy marker Beclin-1 (BECN1) were reduced suggesting CM exerts cytoprotective effects. GCs upregulated the RIPK1 ubiquitinating factor BIRC3 (cIAP2), in GC-sensitive (CEM-C7-14) but not in resistant (CEM-C1-15) cells. In addition, CM selectively affected GR phosphorylation in a site and cell-specific manner. GR is recruited to RIPK1, BECN1 and BIRC3 promoters in the sensitive but not in the resistant cells with phosphorylated GR forms being generally less recruited in the presence of hormone. FACS analysis and caspase-8 assays demonstrated that CM promoted a pro-survival trend. High molecular weight proteins reacting with the RIPK1 antibody were modified upon incubation with the BIRC3 inhibitor AT406 in CEM-C7-14 cells suggesting that they represent ubiquitinated forms of RIPK1. Our data suggest that there is a correlation between microenvironment-induced ALL proliferation and altered response to chemotherapy.

Autophagy in the Vertebrate Inner Ear.

Autophagy is a conserved catabolic process that results in the lysosomal degradation of cell components. During development, autophagy is associated with tissue and organ remodeling, and under physiological conditions it is tightly regulated as it plays a housekeeping role in removing misfolded proteins and damaged organelles. The vertebrate inner ear is a complex sensory organ responsible for the perception of sound and for balance. Cell survival, death and proliferation, as well as cell fate specification and differentiation, are processes that are strictly coordinated during the development of the inner ear in order to generate the more than a dozen specialized cell types that constitute this structure. Here, we review the existing evidence that implicates autophagy in the generation of the vertebrate inner ear. At early stages of chicken otic development, inhibiting autophagy impairs neurogenesis and causes aberrant otocyst morphogenesis. Autophagy provides energy for the clearing of dying cells and it favors neuronal differentiation. Moreover, autophagy is required for proper vestibular development in the mouse inner ear. The autophagy-related genes Becn1, Atg4g, Atg5, and Atg9, are expressed in the inner ear from late developmental stages to adulthood, and Atg4b mutants show impaired vestibular behavior associated to defects in otoconial biogenesis that are also common to Atg5 mutants. Autophagic flux appears to be age-regulated, augmenting from perinatal stages to young adulthood in mice. This up-regulation is concomitant with the functional maturation of the hearing receptor. Hence, autophagy can be considered an intracellular pathway fundamental for in vertebrate inner ear development and maturation.

Effect of emulsified isoflurane postconditioning on mitophagy during myocardial ischemia-reperfusion in rats

Objective To evaluate the effect of emulsified isoflurane postconditioning on mitophagy during myocardial ischemia-reperfusion(I/R)in rats. Methods Forty-eight pathogen-free healthy male Sprague-Dawley rats, aged 4-5 months, weighing 250-300 g, were divided into 4 groups(n=12 each)using a random number table: sham operation group(group S), group I/R, fat emulsion group(group F)and emulsified isoflurane postconditioning group(group EIP). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120 min of reperfusion in pentobarbital sodium-anesthetized rats.Starting from 3 min before reperfusion, 8% emulsified isoflurane 2 ml/kg was intravenously infused over 8 min in group EIP, while 30% fat emulsion 2 ml/kg was intravenously infused over 8 min in group F. Rats were sacrificed at the end of reperfusion, and hearts were removed for measurement of the myocardial infarct size(by 2, 3, 5-triphenyltetrazolium chloride staining), cell apoptosis(by TUNEL), mitochondrial membrane potential and expression of microtubule-associated protein 1 light chain 3(LC3), Beclin1, P62, PINK1 and Parkin in cardiomyocytes(by using Western blot). Apoptosis index(AI)was calculated. Results Compared with group S, the myocardial infarct size and AI were significantly increased, the mitochondrial membrane potential was decreased, the expression of LC3, Beclin1, PINK1 and Parkin was up-regulated, and the expression of P62 was down-regulated in I/R, F and EIP groups(P<0.05). Compared with group I/R, the myocardial infarct size and AI were significantly decreased, the mitochondrial membrane potential was increased, the expression of LC3, Beclin1, PINK1 and Parkin was down-regulated, and the expression of P62 was up-regulated in group EIP(P<0.05). Compared with group F, the myocardial infarct size and AI were significantly decreased, the mitochondrial membrane potential was increased, the expression of LC3, Beclin1, PINK1 and Parkin was down-regulated, and the expression of P62 was up-regulated in group EIP(P<0.05). Conclusion The mechanism by which emulsified isoflurane postconditioning reduces myocardial I/R injury is related to inhibition of mitophagy in rats. Key words: Isoflurane; Fat emulsions, intravenous; Myocardial reperfusion injury; Mitochondria; Autophagy; Ischemic postconditioning

Effects of mild hypothermia on autophagy in hippocampal CA1 neurons in rats after CPR

Objective To establish the cardiac arrest-cardiopulmonary resuscitation model in rats, and to observe the effect of mild hypothermia on autophagy in hippocampal CA1 neurons after ROSC. Methods A total of 36 Wistar rats were randomly divided into two groups: normal temperature treatment group(NT group) and mild hypothermia treatment group(HT group). To establish the cardiac arrest-cardiopulmonary resuscitation(CA-CPR) model in rats by epicardial electrical stimulation induced ventricular fibrillation, and to sacrifice 3 animals in each group to obtain the brain cortex in 2nd and 4th hours after ROSC in order to observe the expression of p-AMPK by electron microscope and LC3 granules through Western blot. The neurological deficit score(NDS) was assessed in 24、48、72 hours respectively after ROSC. To sacrifice the animals so as to take the cerebrum in 72 hours after ROSC, then calculate the apoptotic index of the hippocampal CA1 neurons, which were dyed through TUNEL method. Results The expression of p-AMPK、Beclin-1 and LC3-Ⅱ/LC3-Ⅰratio in Normothermia group were all lower than the Mild hypothermia group(P<0.05), the neurons plasma of hippocampal CA1 area in the Hypothermia group demonstrated obvious LC3 granules formation, the NDS score of the Normothermia group and the Mild hypothermia group in ROSC24h、ROSC48h、ROSC72h were 320vs205、285vs140、266vs120, respectively. The apoptotic index of the hippocampal CA1 area in the Normothemia group in ROSC72h was higher than the Mild hypothermia group, (P<0.05). Conclusions Mild hypothermia after cardiopulmonary resuscitation promotes autophagy of the hippocampal CA1 area neurons in rats and reduce neuronal apoptosis. Key words: Cardiac arrest; CPR; Mild hypothermia; Rat; Autophagy