Beclin-1 Research Articles

Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

Effect of 1,25-dihydroxyvitamin D3 on the expression of mannose receptor, DC-SIGN and autophagy genes in pulmonary tuberculosis

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a powerful immuno-modulator, which enhances expression of antimicrobial peptides and induces autophagy in monocytes/macrophages. Since 1,25(OH)2D3 increases the phagocytic potential of monocytes/macrophages, we have explored the effect of 1,25(OH)2D3 on the expression of receptors such as mannose receptor (CD206) and DC-SIGN (CD209) as well as autophagy genes such as ATG5 and Beclin-1 (BECN1) in monocytes/macrophages of healthy controls (HCs) and pulmonary tuberculosis (PTB) patients with and without cavitary disease. Peripheral blood mononuclear cells (PBMCs) were isolated from 40 HCs and 40 PTB patients and were cultured for 72h with Mtb in the presence or absence of 1,25(OH)2D3 at 10(-7)M concentration. 1,25(OH)2D3 significantly upregulated the expression of mannose receptor, ATG5 and BECN1; whereas DC-SIGN expression was suppressed in Mtb infected cells of both study groups (p<0.05). The 1,25(OH)2D3-induced expression of CD206, ATG5 and BECN1 genes was lower in PTB patients compared to HCs, whereas expression of these genes was impaired in PTB patients with cavitary disease. Moreover, the relative expression of ATG5 and BECN1 was positively correlated with monocyte/macrophage phagocytosis and cathelicidin antimicrobial peptide gene expression in HCs and PTB patients (p<0.05). Our study results suggest that vitamin D supplementation in PTB patients without cavitary disease could enhance innate immune functions and may help to control intracellular growth of mycobacteria in macrophages.

Dietary fat may modulate adipose tissue homeostasis through the processes of autophagy and apoptosis.

Obesity increases the risk of cardiovascular disease, type 2 diabetes mellitus and cancer development. Autophagy and apoptosis are critical processes for development and homeostasis in multicellular organisms and have been linked to a variety of disorders. We aimed to investigate whether the quantity and quality of dietary fat can influence these processes in the adipose tissue of obese people. A randomized, controlled trial within the LIPGENE study assigned 39 obese people with metabolic syndrome to 1 of 4 diets: (a) a high-saturated fatty acid diet, (b) a high-monounsaturated fatty acid (HMUFA) diet, and (c, d) two low-fat, high-complex carbohydrate diets supplemented with long-chain n-3 polyunsaturated fatty acids (LFHCC n-3) or placebo (LFHCC), for 12weeks each. We found an increase in the expression of autophagy-related BECN1 and ATG7 genes after the long-term consumption of the HMUFA diet (p=0.001 and p=0.004, respectively) and an increase in the expression of the apoptosis-related CASP3 gene after the long-term consumption of the LFHCC and LFHCC n-3 diets (p=0.001 and p=0.029, respectively). CASP3 and CASP7 gene expression changes correlated with HOMA index. Our results suggest that the processes of autophagy and apoptosis in adipose tissue may be modified by diet and that the consumption of a diet rich in monounsaturated fat may contribute to adipose tissue homeostasis by increasing autophagy. They also reinforce the notion that apoptosis in adipose tissue is linked to insulin resistance. ClinicalTrials.gov NCT00429195.

Effect of ischemia-reperfusion on the autophagy and interferon regulatory factor 1 expression in mouse livers

Objective To explore the effect of ischemia-reperfusion (IR) on autophagy and interferon regulatory factor 1(IRF-1) expression in mouse liver. Methods Hepatic IR injury (IRI) model was established in C57BL/6 mice, and the serum AST and ALT levels were tested in all groups (Sham and IR) at 2 h, 6 h, 12 h and 24 h, respectively. Histopathological feature of IRI was observed, and autophagosomes changes were observed by transmission electron microscope. LC3 expression was detected by immunofluorescence and cell apoptosis was detected by TUNEL assay. IRF-1 mRNA and IRF-1, LC3 and Beclin1 protein were detected by RT-PCR and western blot, respectively. Results As the reperfusion progressed, serum AST and ALT levels in the mice were increased and peaked at 12 h. Hepatocyte swelling, hepatic sinusoids narrowing, inflammatory cell infiltration and hepatocyte necrosis were observed in the livers from IR group. Compared with Sham group, there was a significant increase on the number of autophagosomes in IR group. In addition, apoptotic cells were also significantly increased. The expression of IRF-1, Beclin1, and LC3 levels were up-regulated in IR group compared to Sham group. Conclusion IR could activate autopha-gy to cause hepatocyte damage, which may be related to the up-regulation of IRF-1. Key words: Ischemia-reperfusion; Hepatocytes; Autophagy; Interferon regulatory factor 1

Effect of sufentanil on autophagy during myocardial ischemia-reperfusion in rats

Objective To evaluate the effect of sufentanil on autophagy during myocardial ischemia-reperfusion(I/R)in rats. Methods Eighteen adult male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 3 groups(n=6 each)using a random number table: sham operation group(group SH), I/R group, and sufentanil group(group S). Myocardial I/R injury was produced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120 min reperfusion in the rats anesthetized with chloral hydrate in I/R and S groups.Sufentanil 3 μg/kg was injected via the caudal vein at 30 min prior to ischemia in group S. The equal volume of normal saline was given in SH and I/R groups.At 120 min of reperfusion, arterial blood samples were collected for determination of serum creatine kinase-MB(CK-MB)and lactic dehydrogenase(LDH)concentrations, and myocardial specimens were obtained for examination of the ultrastructure of myocardial cells(using transmission electron microscopy)and for determination of autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ(LC3Ⅱ), Beclin-1 and Bcl-2 expression(by Western blot). Results Compared with group SH, the serum CK-MB and LDH concentrations were significantly increased, the expression of LC3Ⅱ and Beclin-1 was significantly up-regulated, and Bcl-2 expression was significantly down-regulated in group I/R, and the serum CK-MB concentration was significantly increased, the expression of Beclin-1 and Bcl-2 was significantly down-regulated in group S(P 0.05), and the pathological changes were reduced in group S. Conclusion The mechanism by which sufentanil reduces myocardial I/R injury may be related to decreased autophagy in rats. Key words: Sufentanil; Myocardial reperfusion injury; Autophagy

Hydrogen-rich liquid down-regulates the expressions of inflammatory factors by ultraviolet B-induced human HaCaT keratinocytes through the autophagy pathway

Objective To investigate whether hydrogen can regulate the expressions of inflammatory factors by ultraviolet B (UVB) -induced human HaCaT keratinocytes through the autophagy pathway. Methods Cultured HaCaT keratinocytes were divided into several groups: blank control group receiving no treatment, hydrogen group cultured in hydrogen-rich medium, three UVB groups irradiated with UVB at 1, 10, 50 mJ/cm2 respectively, three UVB + hydrogen groups irradiated with UVB at 1, 10, 50 mJ/cm2 respectively followed by culture in hydrogen-rich medium, UVB + 3MA group pretreated with the autophagy inhibitor 3MA for 1 hour followed by UVB radiation at 50 mJ/cm2, UVB + rapamycin group pretreated with the autophagy activator rapamycin for 1 hour followed by UVB radiation at 50 mJ/cm2, UVB + 3MA+ hydrogen group pretreated with 3MA for 1 hour followed by UVB radiation at 50 mJ/cm2 and culture in hydrogen-rich medium, UVB + rapamycin + hydrogen group pretreated with rapamycin for 1 hour followed by UVB radiation at 50 mJ/cm2 and culture in hydrogen-rich medium. After additional culture with or without hydrogen for 12 hours, methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity, Western-blot analysis to measure the expressions of autophagy-associated protein 1 light chain 3 (LC3) and Beclin1, and enzyme-linked immunosorbent assay (ELISA) to measure the supernatant levels of inflammatory factors including tumor necrosis factor (TNF) -α, interleukin (IL) -1β, IL-6 and high mobility group protein B1 (HMGB1) , and a test kit was used to determine the level of lactate dehydrogenase (LDH) . Results Compared with the blank control group, the 10- and 50-mJ/cm2 UVB groups showed significantly increased release of LDH, expressions of LC3 and Beclin1 and supernatant levels of TNF-α, IL-1β, IL-6 and HMGB1, but decreased cellular proliferative activity (all P < 0.05) . Hydrogen significantly attenuated the release of LDH, down-regulated the supernatant levels of TNF-α, IL-1β, IL-6 and HMGB1, but up-regulated cellular proliferative activity as well as LC3 and Beclin1 expressions in the 10- and 50-mJ/cm2 UVB + hydrogen groups compared with the 10- and 50-mJ/cm2 UVB groups respectively (all P < 0.05) . In addition, the levels of TNF-α, IL-1β, IL-6 and HMGB1 were significantly higher in the 50-mJ/cm2 UVB + 3MA group than in the 50-mJ/cm2 UVB group, and higher in the 50-mJ/cm2 UVB + 3MA + hydrogen group than in the 50-mJ/cm2 UVB + hydrogen group, but lower in the 50-mJ/cm2 UVB + rapamycin group than in the 50-mJ/cm2 UVB group (all P < 0.05) . Conclusion UVB radiation can increase the expressions of autophagy-associated proteins, and hydrogen-rich medium can down-regulate the expressions of inflammatory factors by UVB-induced HaCaT cells through the autophagy pathway. Key words: Ultraviolet rays; Autophagy; Chemokines; Hydrogen; HaCaT cells

Effect of mild hypothermia on expression of autophagy-related proteins in hippocampus after traumatic brain injury in rats

Objective To investigate the effect of mild hypothermia on expression of autophagy-related proteins LC3 and Beclin-1 in the hippocampus of rats with traumatic brain injury (TBI). Methods Sixty healthy male adult SD rats were divided into sham group, TBI group, hypothermia-treated group (31-33℃) according to the random number, with 20 rats per group. Rat models of TBI were established using the Marmarou's method. After 4 h of normothermia or hypothermia, the rats were killed 6, 12, 24, and 48 h later. Co-localization of LC3 with neuron specific protein (NeuN) or mammalian target of rapamycinm (mTOR) was determined by double immunofluorescence. Levels of LC3, Beclin-1 and p-mTOR proteins were detected by Western blot analysis. Results LC3 co-localization with NeuN (or mTOR) was observed under confocal laser scanning microscope. In TBI group LC3 and Beclin-1 levels were seen to rise at 6 h, peaked at 24 h and fell slightly at 48 h, but the levels were still higher than these in sham group (P<0.05). In TBI group p-mTOR level began to decrease at 6 h, reached the lowest point at 12 h, and then ascended at 24 and 48 h, but the level was still lower than that in sham group (P<0.05). Mild hypothermia contributed to remarkable decreases in LC3 and Beclin-1 expressions, while up-regulating p-mTOR after injury(P<0.05). Conclusion Mild hypothermia appears to be neuroprotective as it inhibits autophagy in hippocampal neurons after TBI in rats through activation of mTOR signaling pathway. Key words: Traumatic brain injury; Mild hypothermia; Autophagy; Hippocampus

Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium

ABSTRACTEnvironmental ultrafine particulate matter (PM) is capable of inducing airway injury, while the detailed molecular mechanisms remain largely unclear. Here, we demonstrate pivotal roles of autophagy in regulation of inflammation and mucus hyperproduction induced by PM containing environmentally persistent free radicals in human bronchial epithelial (HBE) cells and in mouse airways. PM was endocytosed by HBE cells and simultaneously triggered autophagosomes, which then engulfed the invading particles to form amphisomes and subsequent autolysosomes. Genetic blockage of autophagy markedly reduced PM-induced expression of inflammatory cytokines, e.g. IL8 and IL6, and MUC5AC in HBE cells. Mice with impaired autophagy due to knockdown of autophagy-related gene Becn1 or Lc3b displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Interference of the autophagic flux by lysosomal inhibition resulted in accumulated autophagosomes/amphisomes, and intriguingly, this process significantly aggravated the IL8 production through NFKB1, and markedly attenuated MUC5AC expression via activator protein 1. These data indicate that autophagy is required for PM-induced airway epithelial injury, and that inhibition of autophagy exerts therapeutic benefits for PM-induced airway inflammation and mucus hyperproduction, although they are differentially orchestrated by the autophagic flux.

Effect of deferoxamine on autophagy in hippocampal neurons after exploratory laparotomy in aged rats

Objective To investigate the effect of deferoxamine on autophagy in hippocampal neurons after exploratory laparotomy in aged rats. Methods Forty-four healthy male Sprague-Dawley rats, aged 18 months, weighing 500-600 g, were randomly divided into 2 groups(n=22 each)using a random number table: operation group(group O)and deferoxamine group(group D). In group D, deferoxamine 50 mg/kg was injected intraperitoneally once a day for 14 consecutive days, while the equal volume of normal saline was given instead in group O. Exploratory laparotomy was performed at 2 h after administration on 7th day.Learning and memory function was assessed using Morris water maze test which was performed before operation and at 1, 3, and 7 days after operation.The escape latency and swimming distance were recorded.The rats were sacrificed, and the hippocampi were removed for determination of the expression of Beclin-1, ferritin H and ferritin L by Western blot. Results Compared with the value before operation, the escape latency and swimming distance were significantly prolonged at each time point after operation in group O(P 0.05). Compared with group O, the escape latency and swimming distance were significantly shortened, the expression of Beclin-1 was up-regulated, and the expression of ferritin H and ferritin L was down-regulated at each time point after operation in group D(P<0.05). Conclusion Deferoxamine reduces postoperative cognitive dysfunction probably through inducing autophagy in hippocampal neurons, degrading ferritin and inhibiting iron overload in the brains of aged rats. Key words: Deferoxamine; Cognitive disorders; Hippocampus; Autophagy

BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells

Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer – both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.

Lack of Altered BECN1 Gene Expression in Iranian Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML), one of the most prevalent leukemia types in adults, demonstrates great heterogeneity in molecular and clinical terms. Hence, there is a necessity to the mechanisms involved in AML generation in order to determine optimal treatment. This cross sectional study aimed to assess changes in BECN1 gene expression in with blood samples from 30 AML patients, compared with samples from 15 healthy persons. RNA was extracted and cDNA was synthesized and Real Time PCR applied to determine BECN1 gene expression. The results showed no significant differences in BECN1 gene expression between patients with AML and normal controls (P > 0.05). It appears that expression of BECN1 does not play a significant role in genesis of AML leukemia.

AntimiR-30b Inhibits TNF-α Mediated Apoptosis and Attenuated Cartilage Degradation through Enhancing Autophagy

Objective: Cell death plays an important role in the pathology associated with inflammatory diseases such as osteoarthritis. It has been reported that autophagy can protect cells against tumour necrosis factor-α (TNF-α)-induced apoptosis. This study aimed to determine the potential role of microRNA-30b (miR-30b) in TNF-α-induced apoptosis, autophagy and differentiation in the chondrogenic ADTC5 cell line. Methods: To analyse the effect of TNF-α on the viability of ADTC5 cells, cell counting kit-8 and Hoechst 33342 staining were employed and the expression levels of caspase-3 and -9 were assessed. Autophagy was examined by analysing the levels of LC3B-II and p62 and quantitating GFP-LC3B by fluorescence microscopy. A luciferase reporter assay investigated the putative binding sites of miR-30b. The effects of miR-30b and antimiR-30b on autophagy, apoptosis and osteogenic differentiation of TNF-α-treated cells were determined by autophagosome, apoptosis and alkaline phosphatase assays, respectively. Results: TNF-α exposure decreased cell viability, increased apoptosis and positively regulated autophagy in ADTC5 cells. A direct interaction was detected between miR-30b and the mRNA 3ʹ-UTRs of autophagy genes BECN1 and ATG5. Overexpression of miR-30b downregulated autophagy genes and upregulated pro-apoptotic gene expression in TNF-α-treated cells, while treatment with antimiR-30b had the inverse effect. Overexpression of miR-30b also downregulated ECM degradation and anti-miR-30b reverse TNF-α-induced ECM degradation. Conclusions: Anti-miR-30b enhanced autophagy and attenuated cartilage degradation and played a protective role in TNF-α-induced apoptosis of ATDC5 cells. Anti-miR-30b may therefore elevate cellular survival during inflammation and has therapeutic potential for inflammatory diseases such as osteoarthritis.

MEK1 Inhibitor Selumetinib Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia Cells (B-ALL) to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy

Glucocorticoids (GC) have been used for decades in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) in children and adults. Induction of apoptosis is thought to be the principal effector mechanism of GC's action, but recent studies highlight the role of autophagy upstream of apoptotic cell death (Laane et al 2009). Resistance to GCs is a major adverse prognostic factor, however the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular mechanisms driving GC-resistance in precursor B-cell acute lymphoblastic leukemia cells and in vitro characterize the therapeutic potential of targeted intervention in these mechanisms.To identify molecular mechanisms involved in GC resistance, we performed gene set enrichment analysis of gene expression profiles GC-sensitive and -resistant B-ALL blasts using publicly available datasets and GenePattern program. Resistant cells exhibited significantly higher expression of MAPK/ERK pathway components (p<.002, FDR=0.13). To validate these findings, we assessed DEX sensitivity in ALL cells with high (SEMK2) or undetectable (RS4;11) activity of MAPK/ERK pathway. SEMK2 cells were resistant to DEX, whereas RS4;11 were highly sensitive to this drug. In GC-resistant cell line SEMK2, inhibition of MEK1 kinase with SEL completely abrogated ERK and p90RSK phosphorylation and increased sensitivity to GC by 1.8-2.6-fold. Similar pattern was observed in primary ALL blasts from 19 of 23 tested patients. Overexpression of a constitutively active MEK mutant in GC-sensitive cells (RS4;11) reversed sensitivity of these cells to DEX. Since GC in leukemic cells induce autophagic cell death, we assessed LC3 processing, MDC staining (a dye of autophagolysosomes) and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Either drug alone caused only marginal change in the level of these markers, but their combination markedly increased autophagic flux. Since mTORC1 is the critical regulator of autophagy, we assessed the activity of mTORC1 following DEX/SEL co-treatment and found that the combination resulted in a marked decrease of p4E-BP1, an mTORC1 substrate. Finally, to assess whether induction of autophagy is required for the observed synergy between SEL and DEX we used an shRNA approach to silence beclin-1 (BCN1), a gene required for autophagosome formation, and assessed cellular responses to DEX/SEL co-treatment. In control cells transduced with non-targeting shRNA, SEL sensitized cells to DEX, but in BCN1-deficient cells, the synergy of DEX and SEL was markedly decreased. Taken together, we show that MEK1 inhibitor selumetinib enhances DEX toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTORC1 signaling pathway and induction of autophagy that leads to apoptotic cell death. DisclosuresNo relevant conflicts of interest to declare.

Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary

In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P < 0.001), the later period being the peak of apoptosis of the follicular cells. The immunostaining for cathepsin-D was more elevated until 2 to 3 dps and decreased significantly at 4 to 5 dps, when the POFs were in late stage of regression. Double labeling for BECN1 and caspase-3 indicated a shift in the relationship between autophagy and apoptosis at 2 to 3 dps, a critical period in determining the fate of follicular cells in POFs. Together, these results indicate that the bcl-2 family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish.

Effect of glucose regulated protein 78 on autophagy and apoptosis in ovarian epithelial carcinoma

To explore the effect and mechanism of glucose regulated protein 78 (GRP78) on autophagy and apoptosis in ovarian carcinoma, and to investigate the influence on the growth and sensitivity to cisplatin on the ovarian cancer cells. The human ovarian cancer cell line SKOV3 were treated by the GRP78 regulator BAPTA-AM and A23187, which were used to decrease or increase the expression levels of GRP78, respectively. The experiment were divided into three groups. Cells in the group of BAPTA-AM were treated by BAPTA-AM at the final concerntration of 40 µmol/L for 1 hour. Cells in the group of A23187 were treated by A23187 at the final concerntration of 4 µmol/L for 24 hours. While, cells in the control group were treated by culture medium without any GRP78 regulator for 24 hours. The expressions of GRP78, beclin1, Bcl-2 and CHOP mRNA and protein were detected by reverse transcription (RT)-PCR and western blot. The autophagy levels was observed by green fluorescent protein-microtubule-associated protein 1 light chain 3-II (GFP-LC3-II) fluorescence staining. The flow cytometry was used to analyse the apoptosis rates of cells. The effect on cell growth and the sensitivity to cisplatin of SKOV3 were accessed by methyl thiazolyl tetrazolium (MTT). (1) The mRNA expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of BAPTA-AM were 0.583±0.025, 0.860±0.055, 0.714±0.032 and 0.811±0.004, respectively. The mRNA expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of A23187 were 0.840±0.044, 0.654±0.065, 0.908±0.047 and 0.620±0.062, respectively. The mRNA expressions of GRP78, beclin1, Bcl-2 and CHOP in the control group were 0.687±0.032, 0.772±0.029, 0.845±0.018, 0.712±0.077, respectively. While the protein expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of BAPTA-AM were 0.423±0.035, 0.952±0.022, 0.385±0.032, 0.681±0.095, respectively. The protein expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of A23187 were 0.743±0.032, 0.638±0.025, 0.596±0.029, 0.431±0.095, respectively. The protein expressions of GRP78, beclin1, Bcl-2 and CHOP in the control group were 0.617±0.031, 0.789±0.083, 0.492±0.036, 0.531±0.003, respectively. The mRNA and protein expressions of beclin and CHOP in the group of BAPTA-AM were both higher than those in the control group (P<0.05). While, the mRNA and protein expressions of beclin and CHOP in the group of A23187 were both lower than those in the control group (P< 0.05). (2) The autophagy fluorescence of SKOV3 in the group of BAPTA-AM, A23187 and the control group were 706±117, 473±128, 595±126, respectively, in which there were significant differences among three groups (P<0.05). (3) The apoptosis rate of SKOV3 in the group of BAPTA-AM was (27.4±2.2)%, which was higher than that in the control group [(19.6±1.4)%, P<0.05]. The apoptosis rate of SKOV3 in the group of A23187 was (12.2±1.9)%, which was lower than that in the control group (P<0.05). (4) The comparison of the sensitivity to cisplatin in 3 groups of SKOV3. The 50% inhibition concentration (IC50) of SKOV3 to cisplatin was (3.02±0.62) mg/L. After treated by BAPTA-AM + cisplatin, the IC50 was (2.00±0.17) mg/L and the sensitivity of SKOV3 to cisplatin was increased by 33.8%, and there was significant difference (P<0.05), compared with the control group. And after treated by A23187 + cisplatin, the IC50 was (4.91±2.52) mg/L and the sensitivity of SKOV3 to cisplatin was decreasd by 62.6%; and there was significant difference (P<0.05), compared with the control group. GRP78 could regulate autophagy and apoptosis of ovarian cancer cells by regulating the expressions of beclin1, Bcl-2 and CHOP, thereby affecting the sensitivity to cisplatin in ovarian carcinoma, which may be a new method for the treatment and improvement of the sensitivity to cisplatin in ovarian carcinoma.

Effect of hydrogen-rich saline on acute kidney injury after orthotopic liver transplantation and the role of autophagy

Objective To investigate the effects of hydrogen-rich saline on acute kidney injury after orthotopic liver transplantation(OLT)in rats and the role of autophagy. Methods Fifty-six healthy adult male Sprague-Dawley rats, weighing 220–250 g, served as donors and recipients.Thirty-two recipient rats were randomly assigned into 4 groups using a random number table: sham operation group(group S), OLT group, hydrogen-rich saline group(group HS), and autophagy inhibitor chloroquine group(group CQ). In group S, laparotomy was performed, and the related blood vessels were isolated.The model of OLT was established in OLT, HS and CQ groups.In OLT and HS groups, normal saline and hydrogen-rich saline 6 ml/kg were injected, respectively, through the inferior vena cava at 5 min before anhepatic phase.In group CQ, chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model, and the other treatments were similar to those previously described in group HS.Blood samples were obtained at 6 h of neohepatic phase for determination of serum blood urea nitrogen(BUN)and creatinine(Cr)concentrations.After the end of blood sampling, the left kidneys were removed for determination of malondialdehyde(MDA)content(using thiobarbituric acid method), superoxide dismutase(SOD)activity(xanthine oxidase method), and cell apoptosis(using TUNEL), and for examination of the pathological changes of renal tissues.The damage to the renal tubules was scored.Apoptosis index(AI)was calculated.The expression of phosphorylated p53(p-p53), activated caspase-3, cytochrome c(Cyt c), microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ), and Beclin-1 was detected by Western blot analysis. Results Compared with group S, the levels of BUN, Cr, and MDA, AI, and renal tubular damage score were significantly increased, the SOD activity was decreased, and the expression of activated caspase-3, Cyt c and LC3Ⅱ was up-regulated(P 0.05). Compared with group OLT, the levels of BUN, Cr, and MDA, AI, and renal tubular damage score were significantly decreased, the SOD activity was increased, the expression of activated caspase-3, Cyt c was down-regulated, and the expression of LC3Ⅱ, Beclin-1 and p-p53 was up-regulated in group HS(P 0.05). Conclusion Hydrogen-rich saline can attenuate acute kidney injury after OLT, and the mechanism is associated with activation of p53, enhancement of autophagy and inhibition of cell apoptosis in rats. Key words: Hydrogen; Liver transplantation; Kidney injury; Autophagy; Apoptosis

Role of autophagy in hippocampal neurons in cognitive dysfunction caused by sevoflurane anesthesia in juvenile rats

Objective To evaluate the role of autophagy in hippocampal neurons in cognitive dysfunction caused by sevoflurane anesthesia in juvenile rats. Methods One hundred four male Sprague-Dawley rats, aged 7 days, weighing 10-16 g, were randomly assigned into 3 groups using a random number table: control group(group C, n=8), sevoflurane anesthesia group(group S, n=48), and sevoflurane anesthesia + rapamycin group(group SR, n=48). Group C inhaled 60% oxygen for 6 h. S and SR groups inhaled 3.6% sevoflurane anesthesia for 6 h, and in addition, rapamycin 2 mg/kg was injected intraperitoneally at 1 h before of sevoflurane inhalation in group SR.The equal volume of phosphate buffer solution was given in C and S groups.At 1 h before sevoflurane anesthesia(T0), immediately after the end of anesthesia(T1), and at 12, 24 and 48 h after the end of anesthesia(T2-4), 8 rats were randomly selected from S and SR groups to determine the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3(LC3)Ⅰ, LC3Ⅱ and Beclin-1 in hippocampal neurons(by Western blot). The ratio of LC3Ⅱ/LC3Ⅰ was calculated.Morris water maze test was performed at 5 weeks after the end of anesthesia to assess the cognitive function.The escape latency, frequency of crossing the original platform, and duration of swimming spent at the target quadrant were recorded. Results Compared with the values at T0, the expression of LC3Ⅱ and Beclin-1 was significantly down-regulated at T1-3, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in group S, and the expression of LC3Ⅱ and Beclin-1 was significantly up-regulated at T1-4, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group SR(P 0.05). Compared with group S, the escape latency was significantly shortened, the frequency of crossing the original platform was increased, and the duration of swimming spent at the target quadrant was prolonged at 3-5 days, the expression of LC3Ⅱ and Beclin-1 was up-regulated at T1-4, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group SR(P<0.05). Conclusion Weakened autophagy in hippocampal neurons is involved in cognitive dysfunction caused by sevoflurane anesthesia in the juvenile rats. Key words: Anesthetics, inhalation; Cognition disorders; Autophagy; Hippocampus; Child

Effects of sevoflurane postconditioning on mitophagy during ischemia-reperfusion in rats

Objective To evaluate the effects of sevoflurane postconditioning on mitophagy during myocardial ischemia-reperfusion (I/R) in rats. Methods Forty-two pathogen-free adult male Sprague-Dawley rats, weighing 250-300 g, were randomly divided into 3 groups (n= 14 each) using a random number table: sham operation group (group S), I/R group and sevoflurane postconditioning group (group SP). Myocardial I/R was induced by 30 min ligation of the left anterior descending branch of the coronary artery followed by 2 h of reperfusion.In group SP, 2.4% sevoflurane was inhaled for 15 min starting from the onset of reperfusion, while 33% oxygen was inhaled in group I/R.The rats were sacrificed at the end of reperfusion, and the hearts were removed for measurement of myocardial infarct size (by 1% 2, 3, 5 triphenyltetrazolium chloride), expression of LC3Ⅱ/LC3Ⅰ, Beclin-1, p62 and Parkin (by Western blot), and mitochondrial membrane potential (by using JC-1 probe), and for examination of the ultrastructure of cardiomyocytes (with transmission electron microscope). Results Compared with group S, the myocardial infarct size was significantly increased, mitochondrial membrane potential was decreased, the expression of LC3Ⅱ/LC3Ⅰ, Beclin-1 and Parkin was up-regulated, and the expression of p62 was down-regulated in group I/R.Compared with group I/R, the myocardial infarct size was significantly decreased, the mitochondrial membrane potential was increased, and the expression of LC3Ⅱ/LC3Ⅰ, Beclin-1, p62 and Parkin was down-regulated in group SP. Conclusion Sevoflurane postconditioning can mitigate I/R injury in rats, and inhibition of excessive activation of mitophagy may be involved in the mechanism. Key words: Anesthetics, inhalation; Myocardial reperfusion injury; Mitochondria; Autophagy; Postconditioning

Beclin-1 expression is retained in high-grade serous ovarian cancer yet is not essential for autophagy induction in vitro.

BackgroundAutophagy is a conserved cellular self-digestion mechanism that can either suppress or promote cancer in a context-dependent manner. In ovarian cancer, prevalent mono-allelic deletion of BECN1 (a canonical autophagy-inducer) suggests that autophagy is impaired to promote carcinogenesis and that Beclin-1 is a haploinsufficient tumor suppressor. Nonetheless, autophagy is known to be readily inducible in ovarian cancer cells. We sought to clarify whether Beclin-1 expression is in fact disrupted in ovarian cancer and whether this impacts autophagy regulation.MethodsBECN1 expression levels were assessed using The Cancer Genome Atlas (TCGA) datasets from 398 ovarian high-grade serous cystadenocarcinomas (HGSC) and protein immunoblot data from HGSC samples obtained at our institution. Knockdown of BECN1 and other autophagy-related gene expression was achieved using siRNA in established human ovarian cancer cell lines (CaOV3, OVCAR8, SKOV3, and HeyA8) and a novel early-passage, ascites-derived cell line (iOvCa147-E2). LC3 immunoblot, autophagic flux assays, transmission electron microscopy and fluorescence microscopy were used to assess autophagy.ResultsWe observed prevalent mono-allelic BECN1 gene deletion (76 %) in TCGA tumors, yet demonstrate for the first time that Beclin-1 protein expression remains relatively unaltered in these and additional samples generated at our institution. Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process. Beclin-1 knockdown instead decreased cell viability without inducing apoptosis.ConclusionsTaken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation. Overall, this work makes novel observations about tumor expression of Beclin-1 and challenges the accepted understanding of its role in regulating autophagy in ovarian cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-015-0182-y) contains supplementary material, which is available to authorized users.

Abstract 2905: HSP27 as a potential factor to determine the fate of Gemcitabine- induced autophagy in osteosarcoma: Survival vs. death

Abstract Lung metastasis constitutes the main cause of death in osteosarcoma (OS) patients. Survival has not improved in the past ten years. Therefore, new therapeutic strategies are needed. We previously reported that aerosol Gemcitabine (GCB) has a significant therapeutic effect against OS lung metastases. There was however, a subset of cells that fail to respond to GCB treatment resulting in persistent small isolated lung metastases that led to relapse and death. The mechanisms underlying OS resistance to GCB have remained elusive. Autophagy, a catabolic process involved in cellular homeostasis, has emerged as an important mechanism involved in cancer cells response to treatment. However, whether autophagy plays a role in OS resistance to GCB is unknown. Thus, the purpose of this study is to investigate the role of autophagy in OS lung metastases resistance to GCB. In this study, LM7 and CCH-OS-D human OS cells were treated with GCB and induction of autophagy was evaluated. GCB-induced autophagy was demonstrated by formation of acidic vesicular organelles (AVOs), conversion of microtubule-associated light chain 3 (LC3I/LC3II), increase in Beclin 1 (BECN1) and decrease in p62/SQSTM1 protein expression. Additionally, formation of autophagic vacuoles was confirmed by electron microscopy. GCB-induced autophagy also resulted in the inhibition of AKT /mTOR phosphorylation, suggesting this signaling pathway as a possible underlying mechanism responsible for GCB-induced autophagy in OS. To test whether autophagy contributes to OS cells survival to GCB, we determined the impact of autophagy inhibition on the sensitivity of OS cells to GCB. Sensitivity of LM7 cells to GCB was greatly enhanced after autophagy inhibition by the pharmacologic inhibitor hydroxychloroquine (HCQ) and shRNA targeting BECN1, suggesting autophagy as a pro-survival mechanism. Interestingly, inhibition of autophagy in CCH-OS-D cells decreased cell sensitivity to GCB, suggesting that GCB-induced autophagy contributed to cell death. Better understanding of the molecular pathways that govern the process of autophagy will allow identification of new strategies to enhance chemotherapeutic efficacy. Our preliminary work showed that the phosphorylation of Heat Shock protein 27 (HSP27) was significantly higher in GCB-treated LM7 cells when compared to CCH-OS-D GCB-treated cells, indicating a potential role of HSP27 in outlining the role of autophagy in osteosarcoma cells. Taken together, these results suggest that autophagy can be induced by GCB in OS cells and that inhibition of autophagy can lead to either increase or decrease OS cells sensitivity to Gemcitabine. Citation Format: Janice M. Santiago-O'Farrill, Mario Hollomon, Eugenie Kleinerman, Nancy Gordon. HSP27 as a potential factor to determine the fate of Gemcitabine- induced autophagy in osteosarcoma: Survival vs. death. [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 2905. doi:10.1158/1538-7445.AM2015-2905