RLIP76 Depletion Enhances Autophagic Flux in U251 Cells

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.     

Retracted: TNF-α–TNFR signal pathway inhibits autophagy and promotes apoptosis of alveolar macrophages in coal worker's pneumoconiosis

Objective : Exposure to coal dust causes the development of coal worker's pneumoconiosis (CWP), which is associated with accumulating macrophages in the lower respiratory tract. This study was performed to investigate the effect of tumor necrosis factor-α (TNF-α)–tumor necrosis factor receptor (TNFR) signal pathway on autophagy and apoptosis of alveolar macrophages (AMs) in CWP. Methods: AMs from controls exposed to coal dust and CWP patients were collected, in which expressions of TNF-α and TNFR1 were determined. Autophagy was observed by transmission electron microscopy, and apoptosis by light microscope and using terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. AMs in CWP patients were treated with TNF-α or anti-TNF-α antibody. Besides, expressions of autophagy marker proteins, apoptosis-related factors, FAS, caspase-8, and receptor-interacting serine–threonine-protein kinase 3 (RIPK3) were determined by western Blot. Activities of caspase-3 and caspase-8 were determined by a fluorescence kit. Flow cytometry was applied to measure the expression of TNFR1 on the surface of the AM. Results: TNF-α expression and TNFR1 expression on the surface of AM, as well as autophagy and apoptotic index were significantly increased in AMs of CWP patients. In response to the treatment of TNF-α, TNF-α expression and TNFR1 expression on the surface of AM as well as LC3I expression were increased, autophagy was decreased, and LC3, LC3II, Beclin1 and B-cell lymphoma 2 expressions decreased, whereas FAS expression and activity and expression of caspase-3 and caspase-8 increased, and apoptotic index increased. Moreover, the situations were reversed with the treatment of anti-TNF-α antibody. Conclusion: TNF-α–TNFR signal pathway was involved in the occurrence and development of CWP by activating FAS–caspase-8 and thus inhibiting autophagy while promoting apoptosis of AM.     

Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells

We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.     

神经胶质瘤U251细胞氧化损伤中自噬和凋亡过程的时间顺序

目的：探讨过氧化氢（H2O2）诱导神经胶质瘤U251细胞损伤中自噬和凋亡发生的时间顺序。方法：实验分为4组：正常对照组、1mmol／L H2O2作用（6h、12h、24h）组。应用MTF法检测H202对神经胶质瘤U251细胞生存率的影响;MDC染色检测自噬空泡的变化;流式细胞仪检测细胞凋亡率变化。Western blot检测Beclin1和胞浆cyt c蛋白的表达。结果：与对照组相比,1mmol／L H2O2作用下,U251细胞存活率明显降低,并呈时间依赖性。与对照组相比,1mmol／L H2O2作用后,6h时U251细胞自噬空泡明显增加,自噬相关蛋白Beclin1表达明显增加,12h、24h细胞自噬水平逐渐增强;而6h时未见细胞凋亡率明显变化及cyt c由线粒体向胞浆的释放,12h、24h时细胞凋亡率明显增加,胞浆中cyt c蛋白表达明显增强（P〈0．05）。结论：氧化损伤能够诱导神经胶质瘤U251细胞发生自噬和凋亡,并且自噬发生于凋亡之前。     

Fish oil up-regulates hepatic autophagy in rats with chronic ethanol consumption

In this study, we examined the regulation of autophagy by fish oil in rats under ethanol-containing diets. Thirty male Wistar rats (8-week-old) were divided into six groups and fed a control diet or an ethanol-containing diet, which was adjusted with fish oil to replace 25% or 57% of the olive oil. After 8 weeks, rats in the E (ethanol diet) group showed the significantly higher plasma aspartate transaminase (AST) and alanine transaminase (ALT) activities, protein expression of cytochrome P450 2E1 (CYP2E1), and levels of hepatic inflammatory cytokines. However, all of those items had significantly decreased in the EF25 (ethanol with 25% fish oil) and EF57 (ethanol with 57% fish oil) groups. As to autophagic indicators, protein expressions of mammalian target of rapamycin (mTOR), Unc-51-like autophagy activating kinase 1 (ULK1) and p62 were significantly increased in the E group. Conversely, the protein expressions of light chain 3II (LC3II)/LC3I and Beclin1 were significantly decreased in the E group. On the other hand, protein expressions of phosphorylated Akt, mTOR, ULK1, and p62 were down-regulated, protein expressions of LC3II/LC3I and Beclin1 were conversely up-regulated in the EF25 and EF57 groups. Fish oil activated hepatic autophagy via inhibiting the Akt signaling pathway, which exerted protective effects against ethanol-induced liver injury in rats.     

MiR-9 enhances the sensitivity of A549 cells to cisplatin by inhibiting autophagy

Objectives

To demonstrate that miR-9 inhibits autophagy by down-regulating Beclin1 and thus enhances the sensitivity of A549 cells to cisplatin.

Results

MiR-9 inhibited Beclin1 expression by binding to its 3′UTR. The inhibition decreased the cisplatin-induced autophagy in A549 cells, evidenced by the decreased expression of LC3II and GFP-LC3 puncta and the increased expression of P62. Upregulation of miR-9 level enhanced the sensibility of A549 cells to cisplatin and increased the cisplatin-induced apoptosis. Overexpression of Beclin1 reversed above effects of miR-9 mimics, cisplatin-induced autophagy was increased and apoptosis was decreased.

Conclusions

MiR-9 inhibits autophagy via targeting Beclin1 3′UTR and thus enhances cisplatin sensitivity in A549 cells.

     

电针对快速衰老小鼠肝脏自噬及脂质代谢的影响

目的: 研究电针对快衰老小鼠(SAMP8)肝脏中自噬相关因子LC3-Ⅱ、Beclin1、Atg7、P62表达的影响,探讨电针改善衰老小鼠肝脏脂质代谢的机制。方法: 30周龄雄性SAMP8小鼠随机分为模型组、药物组、电针组,每组7只,以同周龄抗快速老化SAMR1小鼠7只作为对照组。对照组和模型组动物常规饲养4周,不进行任何干预;药物组采用雷帕霉素按(10 mg·kg^-1·d^-1)腹腔注射干预(1次/日,连续每周6 d);电针组予双侧“肾俞”“太冲”穴电针治疗(15 min/次,1次/日,连续每周6 d)。检测小鼠的血清脂质代谢情况、肝脏脂质沉积情况、肝脏自噬体的分布、肝脏LC3-Ⅱ、Beclin1、Atg7、P62蛋白表达与mRNA表达的变化。结果: 与对照组比较,模型组小鼠血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)显著升高(P＜0.01);模型组肝脏脂滴沉积明显,自噬体减少,自噬相关因子LC3-Ⅱ、Beclin1、Atg7蛋白及mRNA表达水平明显下降(P＜0.01),P62蛋白及mRNA表达水平明显增高(P＜0.01)。与模型组相比,电针组与药物组小鼠血清TG、TC、LDL含量明显降低(P＜0.01);肝脏脂滴沉积减轻,自噬体增多,LC3-Ⅱ、Beclin1、Atg7的蛋白及mRNA表达水平均显著升高(P＜0.01),P62蛋白及mRNA表达水平显著下降(P＜0.01)。电针组肝脏Beclin1、Atg7蛋白及mRNA表达水平均与药物组无明显差异(P＞0.05)。结论: 电针能够减轻肝脏脂质代谢紊乱,可能与调节肝脏自噬相关因子LC3-Ⅱ、Beclin1、Atg7、P62的表达变化,从而促进SAMP8小鼠肝脏自噬有关。     

Plantago asiatica L. seeds extract protects against cardiomyocyte injury in isoproterenol- induced cardiac hypertrophy by inhibiting excessive autophagy and apoptosis in mice

BackgroundCardiac hypertrophy is the early stage of many heart diseases, such as coronary heart disease, hypertension, valvular dysfunction and cardiomyopathy. Cardiomyocyte autophagy and apoptosis play an important role in the process of cardiac hypertrophic response. Plantago asiatica L. seeds extract (PASE) is prepared from a traditional herbal medicine in Asia with tremendous pharmacological activities. However, whether PASE could relieve cardiac hypertrophy has not been elucidated. The present study is aimed to investigate the effect of PASE on cardiac hypertrophy and explore its potential underlying mechanism.MethodsCardiac hypertrophy was induced in C57BL/6 mice by subcutaneous injection of isoproterenol (ISO) for two weeks. Meanwhile, the mice were intraperitoneally injected with PASE at dosages of 20, 40 and 80 mg/kg/day. Cardiac hypertrophy was evaluated by echocardiographic examination, haematoxylin and eosin staining and quantitative real-time polymerase chain reaction. Expressions of proteins involved in autophagy and apoptosis such as Beclin1, p62, LC3II, Bax, Bcl-2 and Cleaved-caspase-3 were detected by western blot analysis. Western blot, transient transfection, acridine orange staining, TUNEL staining and autophagy inducer were used to observe the effect and explore the mechanism of PASE on cardiomyocyte and H9c2 cells with excessive autophagy and apoptosis induced by ISO.ResultsISO induction for two weeks disturbed the myocardial contractility and cardiac function of left ventricles of mice. PASE treated mice showed significantly improved cardiac function indexes, including EF, FS, SV and CO, compared with the ISO group. Treatment with PASE also decreased the heart weight/body weight ratio and cardiomyocyte size, and downregulated the mRNA and protein expressions of hypertrophic markers ANP, BNP, and β-MHC. Furthermore, the changes of autophagy and apoptosis markers, such as LC3II, Beclin1, p62, Bcl-2, Bax and Cleaved-caspase-3 induced by ISO were resumed by PASE treatment. Consistently, PASE demonstrated similar effects on ISO-induced H9c2 cells as it did in vivo. In addition, PASE could counteract the increased autophagy induced by the autophagy inducer, rapamycin.ConclusionPASE attenuated ISO-induced cardiac hypertrophy in mice by inhibiting excessive autophagy and apoptosis in cardiomyocytes. The novel findings may pave the way for the clinical usage of PASE for the prevention of heart diseases related with cardiac hypertrophy.     

氧化低密度脂蛋白通过Wnt5a/PKCδ信号通路诱导大鼠肝星状细胞自噬

该文旨在研究氧化低密度脂蛋白(ox-LDL)对大鼠肝星状细胞(HSC-T6)自噬的影响及机制,探讨非酒精性脂肪肝炎的发病机理。体外培养的HSC-T6细胞以不同质量浓度(0、10、20、40、60μg/mL)的ox-LDL分别处理不同时间(0、3、6、12、24 h)后,用Western blot检测LC3 II、Beclin1、p62的含量。不同质量浓度(0、10、20、40、60μg/mL)的ox-LDL处理HSC-T6细胞12 h后,Western blot检测Wnt5a、p-PKCδ、p-STAT3的含量。将HSC-T6细胞分为对照组(Control)、ox-LDL组、ox-LDL+si-NC组和ox-LDL+si-Wnt5a组,经相应处理后用Western blot、qRT-PCR分别检测LC3 II、Beclin1、p62、p-PKCδ、p-STAT3的蛋白和mRNA含量变化;免疫荧光检测LC3 II的变化;油红O染色观察HSC-T6脂滴含量变化;比色法检测各组细胞培养上清中羟脯氨酸(Hyp)含量;ELISA检测细胞培养上清中透明质酸(HA)和层黏连蛋白(LN)含量。PKCδ抑制剂Rottlerin预处理细胞:将HSC-T6细胞分为对照组(Control)、ox-LDL组、ox-LDL+DMSO组和ox-LDL+Rottlerin组,检测方法与敲低Wnt5a一致。经ox-LDL处理后,HSC-T6细胞中LC3 II、Beclin1含量增加(P<0.05),p62含量减少(P<0.01),且在ox-LDL质量浓度为20μg/mL、作用12 h时达到峰值。ox-LDL质量浓度为20μg/mL、作用12 h时,HSC-T6细胞中Wnt5a、p-PKCδ、p-STAT3蛋白表达显著升高(P<0.01)。敲低Wnt5a后,HSC-T6细胞中Wnt5a、LC3 II、Beclin1 mRNA和蛋白表达水平显著降低(P<0.001),p62蛋白表达增多(P<0.01),p-PKCδ、p-STAT3蛋白表达减少(P<0.05),细胞内LC3 II点状聚集减少,脂滴含量减少,细胞培养上清中Hyp、HA、LN含量也减少(P<0.05)。抑制PKCδ后,结果与敲低Wnt5a一致。ox-LDL可通过增强Wnt5a/PKCδ通路诱导HSC-T6细胞自噬。     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

Transduction of Apaf-1 or caspase-9 induces apoptosis in A-172 cells that are resistant to p53-mediated apoptosis

p53 replacement gene therapy has been carried out clinically for cancers with p53 mutations; however, some cancers are resistant to p53 gene therapy. In this study, we transduced A-172 and U251 cells harboring p53 mutations with wild-type p53 using adenovirus vectors to induce wild-type p53 protein at similar expression levels. A-172 cells did not undergo apoptosis after p53 transduction, whereas U251 cells were markedly sensitive to p53-mediated apoptosis. A-172 cells showed higher endogenous expression of Bcl-X(L) than U251, and transduction of Bcl-X(L) repressed p53-mediated apoptosis in U251 cells, suggesting that high endogenous expression of Bcl-X(L) renders A-172 cells, at least in part, resistant to p53-mediated apoptosis. We transduced A-172 cells and U251 cells with the Apaf-1 or caspase-9 genes; both are downstream components of p53-mediated apoptosis. We found that A-172 cells were highly sensitive to Apaf-1- and caspase-9-mediated apoptosis. The results indicate that A-172 cells harboring mutant p53 were not susceptible to p53-mediated apoptosis, possibly due to high endogenous expression of Bcl-X(L). Transduction of Apaf-1 or caspase-9 would override the resistance mechanism of apoptosis in A-172 cells. These findings provide potentially a novel approach in killing cancers that are resistant to p53 replacement gene therapy.     

Aristolochic acid I induced autophagy extenuates cell apoptosis via ERK 1/2 pathway in renal tubular epithelial cells

Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. However, limited information is available about autophagy in aristolochic acid (AA) nephropathy. In this study, we investigated the role of autophagy and related signaling pathway during progression of AAI-induced injury to renal tubular epithelial cells (NRK52E cells). The results showed that autophagy in NRK52E cells was detected as early as 3-6 hrs after low dose of AAI (10 μM) exposure as indicated by an up-regulated expression of LC3-II and Beclin 1 proteins. The appearance of AAI-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in NRK52E cells provided further evidence for autophagy. However, cell apoptosis was not detected until 12 hrs after AAI treatment. Blockade of autophagy with Wortmannin or 3-Methyladenine (two inhibitors of phosphoinositede 3-kinases) or small-interfering RNA knockdown of Beclin 1 or Atg7 sensitized the tubular cells to apoptosis. Treatment of NRK52E cells with AAI caused a time-dependent increase in extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity, but not c-Jun N-terminal kinase (JNK) and p38. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased AAI-induced autophagy that was accompanied by an increased apoptosis. Taken together, our study demonstrated for the first time that autophagy occurred earlier than apoptosis during AAI-induced tubular epithelial cell injury. Autophagy induced by AAI via ERK1/2 pathway might attenuate apoptosis, which may provide a protective mechanism for cell survival under AAI-induced pathological condition.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.     

PLCγ1 inhibition-driven autophagy of IL-1β-treated chondrocyte confers cartilage protection against osteoarthritis,involving AMPK,Erk and Akt

Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1β for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.     

Retracted: Long noncoding RNA MEG3 inhibits proliferation and migration but induces autophagy by regulation of Sirt7 and PI3K/AKT/mTOR pathway in glioma cells

Glioma is a common primary brain tumor with high mortality rate and poor prognosis. Long noncoding RNA maternally expressed gene 3 (MEG3) is a tumor suppressor in diverse cancer types. However, the role of MEG3 in glioma remains unclear. We aimed to explore the effects of MEG3 on U251 cells as well as the underlying mechanisms. U251 cells were stably transfected with different recombined plasmids to overexpress or silence MEG3. Effects of aberrantly expressed MEG3 on cell viability, migration, apoptosis, expressions of apoptosis-associated and autophagy-associated proteins, and phosphorylated levels of key kinases in the PI3K/AKT/mTOR pathway were all evaluated. Then, messenger RNA (mRNA) and protein expression of Sirt7 in cells abnormally expressing MEG3 were estimated. In addition, effects of abnormally expressed MEG3 and Sirt7 on U251 cells were determined to reveal the underlying mechanism of MEG3-associated modulation. Cell viability and migration were significantly reduced by MEG3 overexpression whereas cell apoptosis as well as Bax and cleaved caspase-3/-9 proteins were obviously induced. Beclin-1 and LC3-II/LC3-I were upregulated and p62 was downregulated in MEG3 overexpressed cells. In addition, the autophagy pharmacological inhibitor (3-methyladenine, 3-MA) affected the effect of MEG3 overexpression on cell proliferation. Furthermore, the phosphorylated levels of key kinases in the PI3K/AKT/mTOR pathway were all reduced by MEG3 overexpression. Sirt7 was positively regulated by MEG3 expression, and effects of MEG3 overexpression on U251 cells were ameliorated by Sirt7 silence. MEG3 suppressed cell proliferation and migration but promoted autophagy in U251 cells through positively regulating Sirt7, involving in the inhibition of the PI3K/AKT/mTOR pathway.     

The effect of transforming growth factor β1 on the crosstalk between autophagy and apoptosis in the annulus fibrosus cells under serum deprivation

Autophagy and apoptosis are important in maintaining the metabolic homeostasis of intervertebral disc cells, and transforming growth factor-β1 (TGF-β1) is able to delay intervertebral disc degeneration. This study determined the effect of TGF-β1 on the crosstalk between autophagy and apoptosis in the disc cells, with the aim to provide molecular mechanism support for the prevention and treatment of disc degeneration. Annulus fibrosus (AF) cells were isolated and cultured under serum starvation. 10 ng/mL TGF-β1 reduced the apoptosis incidence in the cells under serum starvation for 48 h, down-regulated the autophagy incidence in the cells pretreated with 3-methyladenine (3-MA) or Bafilomycin A (Baf A), partly rescued the increased apoptosis incidence in the cells pretreated with 3-MA, while further reduced the decreased apoptosis incidence in the cells pretreated with Baf A. Meanwhile, TGF-β1 down-regulated the expressions of autophagic and apoptotic markers in the cells under starvation, partly down-regulated the expressions of Beclin-1, LC3 II/I and cleaved caspase-3 in the cells pretreated with 3-MA or Baf A, while significantly decreased the expression of Bax/Bcl-2 in the cells pretreated with Baf A. 3-MA blocked the phosphorylation of both AKT and mTOR and partly reduced the inhibitory effect of TGF-β1 on the expression of LC3 II/I and cleaved caspase-3. TGF-β1 enhanced the expression of p-ERK1/2 and down-regulated the expressions of LC3 II/I and cleaved caspase-3. U0126 partly reversed this inhibitory effect of TGF-β1. In conclusion, TGF-β1 protected against apoptosis of AF cells under starvation through down-regulating excessive autophagy. PI3K–AKT–mTOR and MAPK–ERK1/2 were the possible signaling pathways involved in this process.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.