Expression Of Autophagy-related Gene Research Articles

Inhibition of ROS/NUPR1-dependent autophagy antagonises repeated cadmium exposure -induced oral squamous cell carcinoma cell migration and invasion

Cadmium (Cd), an established carcinogen, is a risk factor for oral squamous cell carcinoma (OSCC). Macroautophagy/autophagy is proposed to play a pivotal role in Cd-mediated carcinogenic activity. However, the mechanisms underlying Cd-induced autophagy are poorly understood. In the present study, a CAL27 OSCC cell line exposed to 10−6 M Cd for 8 weeks was used as a model system. Repeated Cd exposure induced significant migration and invasion of CAL27 cells. Furthermore, we showed that Cd increased the autophagic flux in CAL27 cells, as evidenced by the upregulation of LC3-II and the downregulation of P62/SQSTM1. The genetic blocking of autophagy inhibited Cd-induced migration and invasion, indicating a carcinogenic role of autophagy in Cd-treated CAL27 cells. Cd-induced NUPR1 expression, which contributes to lysosomal biogenesis and expression of autophagy-related gene, was found to mechanistically initiate autophagy in CAL27 cells. Of note, NUPR1 shRNA abolished Cd-induced autophagy both in vitro and in vivo. We also found that Cd triggered the generation of MDA in a xenograft tumour model and that N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, abrogated the effects of Cd on NUPR1-dependent autophagy in vivo. Taken together, these results demonstrate that ROS-dependent NUPR1-mediated autophagy plays an important role in repeated Cd exposure -induced cell growth, migration and invasion in OSCC cells.

RNA interference in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development.

BOS-93, a novel bromophenol derivative, induces apoptosis and autophagy in human A549 lung cancer cells via PI3K/Akt/mTOR and MAPK signaling pathway.

The novel bromophenol derivative, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (BOS-93), was synthesized in the CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (Qingdao, China). Experimental studies have demonstrated that it could induce apoptosis and autophagy in human A549 lung cancer cells, and it could also inhibit tumor growth in human A549 lung cancer xenograft models. In the present study, the molecular pathways underlying these effects were identified. The results demonstrated that BOS-93 could inhibit cell proliferation in A549 cells and block A549 cells at the G0/G1 phase. Furthermore, BOS-93 could induce apoptosis, activate caspase-3 and poly ADP ribose polymerase, and increase the B cell lymphoma (Bcl)-2 associated X protein/Bcl-2 ratio. Notably, BOS-93 could also induce autophagy in A549 cells. BOS-93-induced autophagy was confirmed by detecting light chain 3 (LC3)-I/LC3-II conversion and increasing expression of beclin1 and autophagy-related gene 14. Notably, BOS-93-induced autophagy could be inhibited by the autophagy inhibitor 3-MA. Flow cytometry, transmission electron microscopy (TEM) and western blot analysis indicated that BOS-93 induced apoptosis and autophagy activities by deactivating phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin and activating the mitogen-activated protein kinase signaling pathway. The present findings indicated that BOS-93 might be a novel anti-cancer agent for treatment of human lung cancer.

RNA Binding Protein HuR Promotes Autophagosome Formation by Regulating Expression of Autophagy-Related Proteins 5, 12, and 16 in Human Hepatocellular Carcinoma Cells.

Autophagy is a process of lysosomal self-degradation of cellular components by forming autophagosomes. Autophagosome formation is an essential process in autophagy and is fine-tuned by various autophagy-related gene (ATG) products, including ATG5, ATG12, and ATG16. Although several reports have shown that numerous factors affect multiple levels of gene regulation to orchestrate cellular autophagy, the detailed mechanism of autophagosome formation still needs further investigation. In this study, we demonstrate that the RNA binding protein HuR (human antigen R) performs an essential function in autophagosome formation. We observe that HuR silencing leads to inhibition of autophagosome formation and autophagic flux in liver cells. Ribonucleoprotein immunoprecipitation (RIP) assay allows the identification of ATG5, ATG12, and ATG16 mRNAs as the direct targets of HuR. We further show that HuR mediates the translation of ATG5, ATG12, and ATG16 mRNAs by binding to their 3' untranslated regions (UTRs). In addition, we show that HuR expression positively correlates with the levels of ATG5 and ATG12 in hepatocellular carcinoma (HCC) cells. Collectively, our results suggest that HuR functions as a pivotal regulator of autophagosome formation by enhancing the translation of ATG5, ATG12, and ATG16 mRNAs and that augmented expression of HuR and ATGs may participate in the malfunction of autophagy in HCC cells.

Betulinic acid attenuates liver fibrosis by inducing autophagy via the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.

The present study was designed to investigate the effects of betulinic acid on human hepatic stellate cells in vitro and C57BL/6 mice in vivo, as well as the signaling pathways involved. In this study, we explored the effects of betulinic acid on expression of alpha smooth muscle actin and autophagy-related proteins. Betulinic acid reduced pathological damage associated with liver fibrosis, as well as serum platelet-derived growth factor and serum hydroxyproline levels. Furthermore, betulinic acid downregulated the expression of alpha smooth muscle actin and type I collagen in mouse liver and upregulated the expression of microtubule-associated protein light chain 3B and autophagy-related gene 7 at the gene and protein levels. LC3II expression was increased and alpha smooth muscle actin expression was decreased in betulinic acid-treated hepatic stellate cells. Interventions with bafilomycin A1 and mCherry-GFP-LC3 adenoviruses promoted the formation of autophagosomes in hepatic stellate cells and the development of autophagic flow. Our study found that mitogen-activated protein kinase/extracellular signal-regulated kinase may be involved in the effects of betulinic acid on liver fibrosis. The present study suggests that betulinic acid has anti-hepatic fibrosis activity by inducing autophagy and could serve as a promising new agent for treating hepatic fibrosis.

Organosulfur compounds induce cytoprotective autophagy against apoptosis by inhibiting mTOR phosphorylation activity in macrophages.

Organosulfur compounds (OSCs) are the bioactive components of garlic. Some OSCs have apoptotic or autophagy-inducing effects. Autophagy plays roles in both cytoprotection and apoptosis-related cell death, and the interaction between autophagy and apoptosis is important in the modulation of immune responses. The mechanism of an OSC-mediated effect via the interaction of autophagy and apoptosis is unknown. In this study, the effects of five OSC compounds on autophagy in the macrophage cell line RAW264.7 and primary macrophages were investigated. We found that S-allylcysteine (SAC), diallyl disulde (DADS) and diallyl tetrasulfide (DTS) treatment increased the number of autophagosomes of RAW264.7 cells, inhibited the phosphorylation of ribosomal protein S6 kinase beta-1 (p70S6K/S6K1) which is a substrate of mammalian target of rapamycin (mTOR), and significantly enhanced autophagy flux. The induction of autophagy by SAC, DADS and DTS was inhibited by stably knocking down the expression of autophagy-related gene 5 (ATG5) with short hairpin RNA (shRNA). Further experiments confirmed that SAC, DADS and DTS also induced apoptosis in RAW264.7 cells. The induction of apoptosis and Caspase 3 activity by SAC, DADS and DTS were increased by stably knocking down of ATG5 expression with shRNA in RAW264.7 cells or treating with 5 mM 3-MA in primary macrophages. Our results suggest that SAC, DADS and DTS induce both autophagy and apoptosis. The autophagy induction protects macrophages from apoptosis by inhibiting mTOR phosphorylation activity to maintain the mass of immune cells.

RNA interference of long noncoding RNA HOTAIR suppresses autophagy and promotes apoptosis and sensitivity to cisplatin in oral squamous cell carcinoma.

Long noncoding RNA HOX transcript antisense RNA (lncRNA HOTAIR) is overexpressed in many types of human cancers and is correlated with clinical stage and lymph node metastasis in oral squamous cell carcinoma (OSCC). Autophagy, an important mechanism of self-protection, plays vital roles in adapting to hypoxia, tolerating external stimulation, and inducing chemotherapy resistance in OSCC cells. This study aims to investigate the effect of HOTAIR on autophagy, apoptosis, and invasion of OSCC cells. HOTAIR expression in OSCC cells was knocked down by small RNA interference. Transmission electron microscope, Western blot, and flow cytometry assay were used to detect the level of autophagy and apoptosis. OSCC cells were medicated with cisplatin, and median lethal dose (LD50) was performed to evaluate the effect on chemosensitivity of HOTAIR. After HOTAIR silence, autophagy was inhibited with the downregulated expression of MAP1LC3B (microtubule-associated protein 1 light chain 3B), beclin1, and autophagy-related gene (ATG) 3 and ATG7. The expressions of mTOR increased. Proliferation, migration, and invasion of OSCC cells were suppressed. Furthermore, apoptosis rate was enhanced, and the sensitivity to cisplatin was promoted when compared with the negative control group. HOTAIR acts as an oncogene in OSCC cells, and HOTAIR silence may be a potential therapeutic target for OSCC.

Interferon-α Triggers Autoimmune Thyroid Diseases via Lysosomal-Dependent Degradation of Thyroglobulin.

Autoimmune thyroid diseases (AITDs) arise from complex interactions among genetic, epigenetic, and environmental factors. Thyroglobulin (TG) is a major susceptibility gene for both Graves disease and Hashimoto thyroiditis. Interferon-α (IFNα), a cytokine secreted during viral infections, has emerged as a key trigger of AITD. We have shown that IFNα upregulates TG transcription; however, how the upregulation of TG transcription by IFNα triggers AITD is still unknown. To evaluate how IFNα triggers AITD by testing its effects on TG processing. We exposed human thyroid cells to IFNα and evaluated its effects on TG expression and processing. Human thyroid cells exposed to INFα had increased levels of TG mRNA but reduced TG protein levels, indicating TG protein degradation. IFNα induced endoplasmic reticulum stress, but surprisingly, neither the use of chemical chaperones nor proteasome inhibitor prevented IFNα-induced TG degradation. IFNα also increased LysoTracker staining and autophagy flux measured by net light chain 3 (LC3)-II and p62 fluxes. In addition, expression of autophagy markers LC3 and autophagy-related gene 5 was higher in thyroid tissues from patients with AITD. Finally, blocking lysosomal degradation prevented IFNα-induced degradation of TG. We have shown in this study IFNα-induced lysosomal-dependent degradation of TG in human thyroid cells. Our findings suggest that during viral infections, local thyroidal IFNα production can lead to lysosomal TG degradation, releasing pathogenic TG peptides that can trigger AITD.

Blockade of histone deacetylase 6 protects against cisplatin-induced acute kidney injury.

Histone deacetylase 6 (HDAC6) has been shown to be involved in various pathological conditions, including cancer, neurodegenerative disorders and inflammatory diseases. Nonetheless, its specific role in drug-induced nephrotoxicity is poorly understood. Cisplatin (dichlorodiamino platinum) belongs to an inorganic platinum - fundamental chemotherapeutic drug utilized in the therapy of various solid malignant tumors. However, the use of cisplatin is extremely limited by obvious side effects, for instance bone marrow suppression and nephrotoxicity. In the present study, we utilized a murine model of cisplatin-induced acute kidney injury (AKI) and a highly selective inhibitor of HDAC6, tubastatin A (TA), to assess the role of HDAC6 in nephrotoxicity and its associated mechanisms. Cisplatin-induced AKI was accompanied by increased expression and activation of HDAC6; blocking HDAC6 with TA lessened renal dysfunction, attenuated renal pathological changes, reduced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule 1, and decreased tubular cell apoptosis. In cultured human epithelial cells, TA or HDAC6 siRNA treatment also inhibited cisplatin-induced apoptosis. Mechanistic studies demonstrated that cisplatin treatment induced phosphorylation of AKT and loss of E-cadherin in the nephrotoxic kidney, and administration of TA enhanced AKT phosphorylation and preserved E-cadherin expression. HDAC6 inhibition also potentiated autophagy as evidenced by increased expression of autophagy-related gene (Atg) 7 (Atg7), Beclin-1, and decreased renal oxidative stress as demonstrated by up-regulation of superoxide dismutase (SOD) activity and down-regulation of malondialdehyde levels. Moreover, TA was effective in inhibiting nuclear factor-κ B (NF-κB) phosphorylation and suppressing the expression of tumor necrosis factor-α (TNF-α)and interleukin-6 (IL-6). Collectively, these data provide strong evidence that HDAC6 inhibition is protective against cisplatin-induced AKI and suggest that HDAC6 may be a potential therapeutic target for AKI treatment.

Amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in weaning piglets derived from sows fed low-protein diet during gestation and lactation : Autophagy is involved in reduced fat deposition in maternal low-protein piglets.

The study aimed to determine the effects of maternal low-protein (LP) diet on subcutaneous fat deposition of weaning piglets and the potential mechanism. Sows were fed either a standard protein (SP, 15 and 18% crude protein) or a LP diet (50% protein levels of SP) throughout pregnancy and lactation. Subcutaneous fat and blood were sampled from male piglets at 28 days of age. Serum biochemical metabolites and hormone concentrations were detected with the enzymatic colorimetric methods. Serum-free amino acid (FAA) levels were measured by amino acid auto-analyzer. The mRNA and protein were measured by qRT-PCR and Western blot. Body weight, back fat thickness, triglycerides concentrations in subcutaneous fat tissue, and serum, as well as FFA concentrations were significantly reduced in LP piglets when compared with SP piglets. Further studies showed that mRNA and protein expression of acetyl-CoA carboxylase and fatty acid synthetase, two key enzymes of de novo lipogenesis, were significantly reduced in LP piglets, while mRNA expression and the lipolytic enzymes activities of lipolysis genes, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased. Furthermore, expression of autophagy-related gene 7 and autophagy maker gene microtubule-associated protein 1A/1B-light chain 3 (LC 3) as well as the conversion of LC3I to LC3II were significantly elevated, along with the expression of activating transcription factor-4 and eukaryotic translation initiation factor-2a. These results indicate that amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in maternal LP weaning piglets, demonstrating links between maternal protein restriction and offspring fat deposition.

MiR-224-3p inhibits autophagy in cervical cancer cells by targeting FIP200.

Cervical cancer (CC) is a malignant solid tumor, which is one of the main causes of morbidity and mortality in women. Persistent High-risk human papillomavirus (hrHPV) infection is closely related to cervical cancer and autophagy has been suggested to inhibit viral infections. miRNAs have been reported to regulate autophagy in many solid tumors with many studies implicating miR-224-3p in the regulation of autophagy. In this study, we performed a miRNA microarray analysis on CC tissues and found that a large number of miRNAs with differential expressions in hrHPV-infected tissues. We identified miR-224-3p as a candidate miRNA selectively up regulated in HPV-infected tissues and cell lines. Further analysis revealed that miR-224-3p regulates autophagy in cervical cancer tissues and cell lines. While the overexpression of miR-224-3p inhibits autophagy in HPV-infected cells, knocking down endogenous miR-224-3p increases autophagy activity in the same cells. In addition, we found that miR-224-3p directly inhibits the expression of autophagy related gene, FAK family-interacting protein of 200 kDa (FIP200). In summary, we found that miR-224-3p regulates autophagy in hrHPV-induced cervical cancer cells through targeting FIP200 expression.

Endoplasmic reticulum stress intolerance in EIF2B3 mutant oligodendrocytes is modulated by depressed autophagy

ObjectiveEukaryotic translation initiation factor 2B (eIF2B) is an essential factor for the initiation of protein synthesis. Mutations in eIF2B encoded by EIF2B1-5 cause a lethal leukoencephalopathy—vanishing white matter disease (VWM). Previous studies have suggested that an improper activated unfolded protein response (UPR) after endoplasmic reticulum stress (ERS) contributed to the pathogenesis of the disease. Autophagy, an important compensatory pathway after ERS, was analyzed in this study. MethodsTo determine the tolerance differences to ERS, cell viability and apoptosis rates were detected in oligodendrocyte cell lines transfected with EIF2B3-c.1037T>C or the wild type. Autophagy flux was measured between groups. Autophagy inducers and inhibitors were used to identify the role of autophagy in the mutant oligodendrocytes. ResultsWe confirmed that oligodendrocytes with mutant EIF2B3 was less tolerant to ERS than the wild type, with decreased cell viability and increased apoptosis rates. Autophagy flux was depressed in mutant oligodendrocytes under baseline condition and after ERS stimulation. Reduced expression of autophagy related gene (Atg) 3 and Atg 7 were involved in the depression of autophagy flux. The mutant oligodendrocytes pretreated with autophagy inducers showed stable cell viability and decreased apoptosis despite ERS induction, whereas the autophagy inhibitors aggravated cell apoptosis and viability declination. ConclusionsOligodendrocytes transfected with mutant EIF2B3 was less tolerant to ERS than the wild type. Depressed autophagy flux was observed in the mutant cells at baseline and after ERS stimulation. Improperly depressed autophagy played a role in the susceptibility to ERS in EIF2B3 mutant oligodendrocytes.

Blockage of cisplatin-induced autophagy sensitizes cervical cancer cells to cisplatin

Development of chemoresistance is a major obstacle that leads to the recurrence and progression of cervical cancer (CC). Autophagy, meaning, "eating of self", has shown paradoxical functions in tumors. In this study, we first investigated the process of autophagy induction by cisplatin in CC cells. Next, we investigated the role of autophagy in cisplatin-sensitivity of CC cells via blockage of cisplatin-induced autophagy. The results demonstrated that cisplatin induces autophagy in CC HeLa cells via upregulating the formation of autophagic vesicle, promoting the conversion of LC-I to LC-II, and increasing the expression of autophagy-related gene 7 (Atg-7). On the other hand, the autophagy inhibitor, 3MA, downregulated cisplatin-induced formation of autophagic vesicles, reduced the conversion of LC-I to LC-II, and decreased Atg-7 expression. Moreover, 3MA reversed the reduction in cellular viability and induction of apoptosis by cisplatin in HeLa cells. Our results imply that autophagy blockage may play a key role in the chemosensitivity of cervical cancers.

The role of autophagy in U87 cell death induced by resveratrol

Objective To study the effect of resveratrol (Res) on the expression of autophagy related gene in U87 glioma cell line.Methods U87 cells were treated with different concentrations of Res (0,12.5,25.0,50.0,100.0,150.0,and 200.0 μmol/L).Cell growth inhibiotin was detected by typan blue exclusion assay.The mRNA expression level of autophagy related genes microtubule-associated protein 1 light chain 3 beta (LC-3),Beclin-1 and VPS34 was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (Real-time PCR),and the protein expression level of them by Western blotting.Results The cell number in the control group was in a upward trend,but that in the experimental group was decreased significantly.There was increasement of mRNA expression of autophagy related genes LC-3,Beclin-1 and VPS34.The expression of LC-3 gene was increased to 1.71 ±0.28,2.65 ±0.54 and 3.03 ±0.61 fold respectively.The expression of Beclin-1 gene was increased to 1.96 ± 0.07,3.22 ± 0.19 and 6.15 ± 0.10 respectively.And the expression of VPS34 gene was increased to 2.14 ± 0.23,4.13 ± 0.27 and 6.21 ± 0.45 respectively.Western blotting showed that the protein expression of these genes was also increased.Conclusion Res can inhibit the growth of U87 cells.Autophagy is involved in Res-induced U87 cell death.The up-expression of autophagy related genes LC-3,Beclin-1 and VPS34 may mediate this process. Key words: Resveratrol ; Glioma; Autophagy

No evidence for activated autophagy in left ventricular myocardium at early reperfusion with protection by remote ischemic preconditioning in patients undergoing coronary artery bypass grafting.

ObjectiveRemote ischemic preconditioning (RIPC) by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG). Activation of signal transducer and activator of transcription 5 (STAT5) in left ventricular (LV) myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.MethodsCABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation) or placebo (cuff deflated) before skin incision (n = 10/10). Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline) and at early (5–10 min) reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p = 0.002). Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5–12 (ATG5-12) complex, light chain 3 (LC3), parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2)-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.ResultsSignals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p = 0.02). STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (≥2.7-fold versus baseline, p≤0.05).ConclusionProtection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.

Abstract WP102: Interleukin-21 Promotes Neuronal Autophagy Following Cerebral Ischemia

Currently there is no treatment for the inflammatory responses that develop following cerebral ischemia. We have previously shown that the CD4+ T cell derived inflammatory cytokine IL-21 is highly upregulated in ischemic mouse brain following transient middle cerebral artery occlusion (tMCAO) and promotes neuronal tissue injury. We also found IL-21+ CD4+ cells in perivascular spaces of ischemic human brain tissue; however, the primary effects and cellular targets of IL-21 remain unknown. Thus, we compared genome-wide whole brain gene expression in wild-type (WT) and IL-21 -/- mice following 1 hour tMCAO (n=3-5). RNA from whole brain was analyzed by Mouse Gene 1.0 ST microarray (Affymetrix) revealing that 2019 gene fragments representing 1505 genes had >3 fold higher expression in WT brain compared to IL-21 -/- after 24 hours reperfusion. Gene score resampling of annotated probe scores identified biological process gene ontology (GO) terms with 3-8 member genes most significantly over-represented including: GO:0010507 Negative Regulation of Autophagy, GO:0090174 Organelle Membrane Fusion, GO:0000046 Autophagic vacuole fusion, and GO:0016242 Negative regulation of macroautophagy (p <0.001). These findings suggested a role for IL-21 in neuronal autophagy_consistent with our earlier reports of reduced expression of autophagy related gene 6 (ATG6) in infarcted brain tissue of IL-21 -/- mouse compared to WT mice. Thus, we hypothesized that IL-21 could directly induce autophagy in hypoxic neuronal cells. IL-21 receptor was expressed on 10% of CD45- cells isolated from healthy mouse brain. Furthermore, using RT-PCR we detected Il21r mRNA expression on neuronal (Neura2A), astrocytic, and endothelial cell lines (MB114) with highest expression levels on Neuro2A cells. Surface expression on Neuro2A cells and primary neurons was confirmed by immunofluorescence. In preliminary experiments primary neurons treated with 256 ng/mL rIL-21 for 4 hours following 1-2 hour oxygen glucose deprivation showed increased expression of ATG6 compared to control treated cells (p<.05). In conclusion, these data suggest that IL-21 could directly promote neuronal autophagy following cerebral ischemia.

Autophagy-related Gene 7 (ATG7) and Reactive Oxygen Species/Extracellular Signal-regulated Kinase Regulate Tetrandrine-induced Autophagy in Human Hepatocellular Carcinoma

Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the broadly used Chinese medicinal herb Stephaniae tetrandrae, exhibits potent antitumor effects and has the potential to be used as a cancer chemotherapeutic agent. We previously reported that high concentrations of tetrandrine induce apoptosis in liver cancer cells. Here, we found that in human hepatocellular carcinoma (HCC) cells, a low dose of tetrandrine (5 μm) induced the expression of LC3-II, resulted in the formation of acidic autophagolysosome vacuoles (AVOs), and caused a punctate fluorescence pattern with the GFP-LC3 protein, which all are markers for cellular autophagy. Tetrandrine induced the production of intracellular reactive oxygen species (ROS), and treatment with ROS scavengers significantly abrogated the tetrandrine-induced autophagy. These results suggest that the generation of ROS plays an important role in promoting tetrandrine-induced autophagy. Tetrandrine-induced mitochondrial dysfunction resulted in ROS accumulation and autophagy. ROS generation activated the ERK MAP kinase, and the ERK signaling pathway at least partially contributed to tetrandrine-induced autophagy in HCC cells. Moreover, we found that tetrandrine transcriptionally regulated the expression of autophagy related gene 7 (ATG7), which promoted tetrandrine-induced autophagy. In addition to in vitro studies, similar results were also observed in vivo, where tetrandrine caused the accumulation of ROS and induced cell autophagy in a tumor xenograft model. Interestingly, tetrandrine treatment also induced autophagy in a ROS-dependent manner in C. elegans muscle cells. Therefore, these findings suggest that tetrandrine is a potent autophagy agonist and may be a promising clinical chemotherapeutic agent.

Expression of Beclinl in non small cell lung cancer and its clinical significance

Objective To investigate the expression of autophagy related gene; Beclinl in human non-small cell lung cancer(NSCLC) tissues. Methods Protein expression of Beclinl was determined by immunofluorescence staining and Western Blot, mRNA expression was analyzed by RT-PCR. Results Immunofluorescence staining revealed that the level of Beclinl expression in lung cancer was significantly lower than that in adjacent noncancerous tissues and normal tissues(expression rate 8.3%, P=0.000). The Beclinl mRNA expression in lung cancer, adjacent noncancerous tissues and normal tissues was 1.372 (+ 0.475) 1.721 (+ 0.521) and 1.553(+ 0.554) when F = 15.0, P < 0.01. Beclinl protein expression in lung cancer, adjacent noncancerous tissues and normal tissues was 3.453(+ 0.852) 5.423(+ 1.351) and 6.878(+0.997) F= 11.2, P < 0.01. Conclusion The protein and mRNA expression of Beclinl in lung cancer was much lower than those in and around cancer tissues and normal tissues, those differences having statistical significance. However in adjacent noncancerous tissues and normal tissues, the expression showed no difference.