Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.     

Clostridium difficile toxin B induces autophagic cell death in colonocytes

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3⁺ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.     

Autophagy protects against hypoxic injury in C. elegans

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after an hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell death mechanisms is inhibited.     

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.     

Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) cells in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

Photodynamic therapy (PDT) uses a photosensitizer, light, and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7+ cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

Inhibition of macroautophagy triggers apoptosis

Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O₂⁻) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.     

Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A₁, Atg7 knockdown or in atg5^?/? MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.     

Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium

Calcium (Ca²⁺) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca²⁺ level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca²⁺ triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca²⁺ agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.     

The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells

In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.     

Cyclosporine A Induces Apoptotic and Autophagic Cell Death in Rat Pituitary GH3 Cells

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.     

Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine

We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.     

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.     

Inhibition of mTOR-Dependent Autophagy Sensitizes Leukemic Cells to Cytarabine-Induced Apoptotic Death

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.     

Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury

Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.