Increase In LC3-II Research Articles

Neuroprotective Effects of Autophagy Induced by Rapamycin in Rat Acute Spinal Cord Injury Model

Background/Aims: To explore the effects of rapamycin-induced autophagy on apoptosis in a rat model of acute spinal cord injury (SCI), and to explore the effect of rapamycin on apoptosis in primary spinal cord cell culture. Methods: SCI was induced at T10 in female adult Sprague-Dawley rats. After injury was induced, the rats were injected with rapamycin and/or methylprednisolone and were sacrificed at various days after injury. Apoptosis and autophagy were examined with TUNEL staining and electron microscopy. Hind limb function was assessed by the Gale scale. Results: The expression of the apoptosis-related protein caspase-3 did not significantly increase until 21 days following injury, while increases in LC3II and LC3I began 10 days after injury, but then declined. TUNEL staining and electron microscopy confirmed that following injury autophagy occurred before apoptosis, but by 14 days after the injury, the level of autophagy had decreased significantly while the level of apoptosis showed a continued increase. Following treatment with rapamycin, apoptosis was significantly higher than in the vehicle control group, but significantly lower than in the sham-operated group, showing a protective effect of rapamycin. Gale scale grades in rats treated with rapamycin were significantly higher compared with the vehicle control group, suggesting a functional effect of rapamycin-induced inhibition of apoptosis. Conclusions: The results indicate that rapamycin significantly improved the prognosis of acute SCI in rats by inhibiting cell apoptosis. Rapamycin might be useful as a therapeutic agent for acute SCI.

Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke

Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model. However, the involvement of mitophagy in this process and its contribution to the protection of mitochondrial function remains unknown. The present study explored the characteristics of mitophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function. Male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO). Neurological deficits scores; infarct volumes; mitophagy morphology; and the levels of malondialdehyde (MDA), adenosine triphosphate (ATP) and mitochondrial membrane potentials (Δψm) were examined. The expression of LC3, Beclin-1 and p62 in the mitochondrial fraction combined with transmission electronic microscopy were used to explore mitophagic activity after ischemia. We also blocked autophagosome formation using 3-methyladenine (3-MA) to check the linkage between the mitochondrial protective effect of rapamycin and enhanced mitophagy. We observed that rapamycin significantly enhanced mitophagy, as evidenced by the increase in LC3-II and Beclin-1 expression in the mitochondria and p62 translocation to the mitochondria. Rapamycin reduced infarct volume, improved neurological outcomes and inhibited mitochondrial dysfunction compared with the control animals (p<0.05). However, these protective effects were reversed by 3-methyladenine treatment after rapamycin. The present study indicates that rapamycin treatment attenuates mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced mitophagy.

Abstract B161: SLC1A5 inactivation induces apoptosis-mediated cells death in non-small cell lung cancer.

Abstract Introduction: We recently reported that solute carrier family A1 member 5 (SLC1A5) controls glutamine (GLN) uptake and modulates cell growth, oxidative stress and mTOR signalling pathway. SLC1A5 is a transmembrane, high affinity glutamine transporter that is overexpressed and located at the plasma membrane in non-small cell lung cancer (NSCLC). GLN deprivation has been shown to induce cell death in several types of cancers, however the exact mechanism by which SLC1A5 targeting affects NSCLC survival remains unknown. We hypothesize that inhibition of SLC1A5 deprives cells from GLN which induces autophagy which eventually leads to cell starvation and ultimately apoptotic cell death. Methods: To test our hypothesis we targeted SLC1A5 by siRNA or by its specific inhibitor, GPNA, in a panel of 6 NSCLC lung cancer cell lines (express high level of SLC1A5). For comparison 2 human bronchial epithelial cell lines (HBEs) (express low level of SLC1A5) were used. The effects of targeting SLC1A5 on growth, GLN uptake, ATP level, autophagy, and cell death were examined. Markers of autophagy and cell death were analysed using western blot, nuclear staining and cell cycle analysis. Results: SLC1A5 inactivation by GPNA or by siRNA resulted in a significant decrease in cellular GLN uptake in NSCLC cell lines, while HBE cell lines were unaffected. Decrease in GLN uptake was accompanied by an increase in autophagy as evident by decrease in cell size, increase of LC3-II and decrease if LC3-I markers of autophagy. In addition, we observed a significant drop in ATP levels by 2 folds with increasing doses of GPNA. Markers of internal apoptotic pathway that include decrease of mitochondrial potential and cleaved caspases-3 and 9 were detected in response to increasing doses of GPNA. No change was observed in external apoptotic markers, caspases 8 or 1. In addition a 2-10 fold increase (based on the cell line) of sub-G1 was observed in NSCLC cell lines but not in HBE cell lines. Conclusion: Our results show that targeting SLC1A5 in NSCLC cells induces apoptotic cell death. Cells starve due to GLN deprivation and undergo autophagy which leads to a decrease in ATP level resulting in apoptotic cell death. These findings suggest that targeting SLC1A5 may have therapeutic implications in NSCLC. Grant funding: Lung Cancer Research Foundation Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B161. Citation Format: Mohamed Hassanein, Jun Qian, Megan Hoeksema, Marie Jacobovitz, Fredrick T. Harris, Bradford Harris, Pierre P. Massion. SLC1A5 inactivation induces apoptosis-mediated cells death in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B161.

Abstract B50: Preclinical efficacy of combined MEK and Src inhibition: MEK inhibitor reverses resistance to Src inhibitor therapy in ovarian cancer cells

Abstract Introduction: More effective, less toxic treatments for recurrent ovarian cancer are needed. Up to 80% of ovarian cancers show activation of the Src kinase and up to 40% show MEK/MAPK pathway activation. Therapies targeting signaling pathways that drive proliferation have shown promise in ovarian cancers but the development of drug resistance presents a major problem. Saracatinib, is a potent inhibitor of Abl and Src family kinases and selumetinib, is a potent, selective, ATP-uncompetitive inhibitor of MEK 1/2 kinases. Our earlier work has shown that resistance to saracatinib monotherapy emerges within weeks in cell culture and within months in xenograft models. Since oncogenic activation of both Src and MEK pathways has been observed in ovarian cancers and because resistance to Src inhibition rapidly emerges with monotherapy, we hypothesized that blocking both kinases with dual therapy might more effectively decrease ovarian cancer cell proliferation than either drug alone. Experimental Design: Dual Src and MEK blockade effects on cell cycle and survival were assayed in ovarian cancer lines, and a primary human ovarian cancer tumor cultured immediately in vitro. Cells enriched for the stem cell marker, aldeflour dehydrogenase 1 (ALDH1+) were sorted by flow cytometry. Drug effects on tumor sphere formation and on cell cycle profiles of ALDH+ cells and those lacking this activity were analyzed. In vivo experiments are ongoing. Results: Src activity is greater in ovarian cancer lines than normal human ovarian epithelial cells (HOSE). In saracatinib naïve ovarian cancer cells, Src inhibition led to partial growth arrest after 48 hrs of treatment. However, resistance of ovarian cancer cells to saracatinib monotherapy emerged rapidly, over a period of weeks of continuous drug exposure in vitro and in vivo. Activation of the MEK and Akt pathways developed with prolonged saracatinib treatment. While each drug alone caused a partial growth arrest in saracatinib naïve lines, treatment with both saracatinib and the MEK inhibitor drug, selumetinib, caused synergistic cell cycle arrest which was mediated by p27 binding to cylin E/cdk2. Combination treatment decreased Src, Akt and MAPK activities. Combination treatment was required for cell cycle arrest in saracatinib resistant cells. Combination treatment also induced apoptosis by an increase in cleaved PARP and induced autophagy as shown by an increase in LC3-II. Src and MEK activities were increased in ALDH1+ sorted populations compared to the ALDH- population and combination treatment decreased the % of ALDH1+cells and the % of sphere-generating cells. Conclusions: MEK inhibition augments the effects of saracatinib by blocking bypass activation of MEK and Akt pathways, increasing cell cycle arrest, impairing survival and targeting self-renewing subpopulations. These data support further pre-clinical and clinical evaluation of combined saracatinib and MEK inhibition in ovarian cancer. Citation Format: Fiona Simpkins, Diana Azzam, Joyce M. Slingerland. Preclinical efficacy of combined MEK and Src inhibition: MEK inhibitor reverses resistance to Src inhibitor therapy in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B50.

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-xL counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-xL observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.

DRAM1 regulates autophagy flux through lysosomes.

We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes.

Transmembrane Protein 208: A Novel ER-Localized Protein That Regulates Autophagy and ER Stress

Autophagy and endoplasmic reticulum (ER) stress are both tightly regulated cellular processes that play central roles in various physiological and pathological conditions. Recent reports have indicated that ER stress is a potent inducer of autophagy. However, little is known about the underlying molecular link between the two processes. Here we report a novel human protein, transmembrane protein 208 (TMEM208) that can regulate both autophagy and ER stress. When overexpressed, TMEM208 impaired autophagy as characterized by the decrease of the accumulation of LC3-II, decreased degradation of autophagic substrates, and reduced expression of critical effectors and vital molecules of the ER stress and autophagy processes. In contrast, knockdown of the TMEM208 gene promoted autophagy, as demonstrated by the increase of LC3-II, increased degradation of autophagic substrates, and enhanced expression levels for genes key in the ER stress and autophagic processes. Taken together, our results reveal that this novel ER-located protein regulates both ER stress and autophagy, and represents a possible link between the two different cellular processes.

Induction of autophagy by Type I interferon in human cancer cells (P6217)

Abstract Type I IFNs (interferons) are cytokines that induce an antiviral state in cells and have antiproliferative, pro-apoptotic, and immunomodulatory activities. Autophagy is a basic cellular mechanism that involves degradation of cellular components via lysosomes. While induction of autophagy is well documented for Type II IFN, induction of autophagy by Type I IFN has not been previously reported. We determined that Type I IFN can induce autophagy in multiple human cancer cell lines. IFN-α2c and IFN-β induces autophagy in Daudi B cells, as indicated by an increase in autophagy markers LC3-II, Atg5-Atg12 complexes, and a decrease in p62. An increase in LC3-II was also detected post IFN-α2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines was confirmed by electron microscopy analysis. An increase in autophagy markers correlated with inhibition of mTORC1 activity and cell proliferation as well as changes in the cell cycle progression in Daudi cells. Treatment of Daudi and T98G cells with IFN-α2c and rapamycin (mTOR inhibitor), or LY294002 (PI3K inhibitor) increased the level of LC3-II, indicating that the PI3K/Akt/mTORC1 signaling pathway may affect IFN-induced autophagy in Daudi and T98G cells. The role of mTOR in Type I IFN-induced autophagy was confirmed by siRNA silencing experiments. Taken together, our findings demonstrate a novel function for Type I IFN as inducer of autophagy in multiple cancer cell lines.

PD-1 Couples Glucose Starvation with Autophagy and Survival Through AMPK-Mediated Phosphorylation of Ulk1

Abstract 836PD-1 is a potent inhibitor of T cell proliferation and can compromise anti-viral and anti-tumor T cell responses. PD-1 inhibits expression of Glut1 via which T cells uptake glucose, which is utilized to generate energy by glycolysis. Recently, we determined that CD4+ primary human T cells receiving PD-1 signals are blocked at the G1 phase and do not progress to the S and G2/M phases. Although PD-1-stimulated T cells did not proliferate, they remained viable at levels comparable or even better than T cells stimulated by CD3 and CD28. Here, we sought to determine whether T cells receiving PD-1 signals remain metabolically active and to identify the mechanisms via which PD-1 might lead to generation of energy and promotion of cell survival. We used primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies (mAbs) against CD3 and CD28 with or without agonist mAb against PD-1. Stimulation of T cells via TCR/CD3 and CD28 resulted in prominent upregulation of Glut1 by 48 hours, which remained elevated for five days of culture and gradually declined. In contrast, T cells receiving signals via TCR/CD3, CD28 and PD-1 did not upregulate Glut1 at any time point. T cells stimulated via TCR/CD3 and CD28 but not those receiving PD-1 signals displayed glucose uptake as determined by incorporation of 14C(U)}-deoxy-D-glucose. To determine whether T cells receiving PD-1 signals were metabolically active, we examined mitochondrial homeostasis by assessing membrane potential using the potentiometric dye TMRE. At all time intervals of culture, mitochondrial membrane potential was comparable between T cells stimulated via TCR/CD3/CD28 and T cells stimulated via TCR/CD3/CD28 and PD-1. Thus, despite the lack of glucose uptake, T cells receiving PD-1 signals were metabolically active. In other cell types glucose deprivation activates AMPK (AMP activated protein kinase), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. The substrate of AMPK that connects energy sensing to cell survival during starvation is Ulk1, the mammalian homologue of the yeast kinase ATG1, which has an essential role in autophagy, survival and longevity under limited nutrient supplies. AMPK-mediated phosphorylation of Ulk1 is required for Ulk1 activation and initiation of autophagy, thereby maintaining mitochondrial homeostasis and cell survival during starvation. Because by suppressing glucose uptake PD-1 induces a state of glucose starvation in T cells, we examined whether PD-1 might induce AMPK activation. In CD4+ T cells activated via TCR/CD3 and CD28, AMPK activation was transient and insignificant. In contrast, T cells receiving PD-1 signals displayed robust and sustained activation of AMPK, which mediated a prominent phosphorylation of Ulk1 on the AMPK-specific site Ser555. Furthermore, T cells receiving PD-1 signals displayed robust phosphorylation of Raptor on Ser752, an AMPK-specific site, leading to inactivation of mTORC1. These biochemical events were in complete contrast to those observed in T cells receiving TCR/CD3 and CD28-mediated signals without PD-1, which displayed no AMPK-mediated phosphorylation of Ulk1 or Raptor and had increased mTORC1 activity. Because mTORC1-mediated phosphorylation of Ulk1 disrupts Ulk1-AMPK interaction and prevents Ulk1 activation we examined whether PD-1 induced differential AMPK-Ulk1 interaction and Ulk1 activation. Immunoprecipitation with AMPK-specific mAb revealed a prominent Ulk1-AMPK interaction and Ulk1 activation in T cells stimulated via TCR/CD3, CD28 and PD-1 but not in T cells stimulated via TCR/CD3 and CD28 in the absence of PD-1 signals. To determine the biological consequences of these signaling events we examined whether, via AMPK-Ulk1 activation, PD-1 might induce autophagy in T cells. During autophagy, carboxy-terminal lipid modification of LC3 is a well-characterized phenomenon required for autophagosome formation which can be readily detected by an increased electrophoretic mobility (LC3-II). PD-1 ligation induced a prominent increase of LC3-II, whereas no LC3-II increase was observed during stimulation via TCR/CD3 and CD28. These results uncover an unexpected biochemical mechanism coupling glucose starvation, autophagy and survival downstream of PD-1 and suggest that therapeutic targeting of AMPK might be a novel approach to modulate PD-1-mediated signaling. Disclosures:No relevant conflicts of interest to declare.

Sonic hedgehog promotes autophagy of vascular smooth muscle cells

Sonic hedgehog (Shh) is a morphogen critically involved in development that is reexpressed in atherosclerotic lesions. It also stimulates proliferation of vascular smooth muscle cells (SMCs). Autophagy in vascular SMCs is known to promote SMC survival and increase plaque stability. The aim of this study was to investigate whether Shh induces autophagy of vascular SMCs. Our study showed that both Shh protein and microtubule-associated protein 1 light chain 3 (LC3)-II were increased in SMCs within neointimal lesions of mouse common carotid arteries. In cultured mouse aortic SMCs, recombinant mouse Shh stimulated LC3-II levels. Overexpression of wild-type mouse Shh through the tetracycline-regulated expression-inducible system in human aortic SMCs time-dependently increased the levels of LC3-II and also stimulated protein kinase B (AKT) phosphorylation. Pretreatment with AKT inhibitor IV (AKTI IV) inhibited AKT phosphorylation and the increase in LC3-II. Shh-induced autophagy was further confirmed by the formation of autophagosomes as detected by immunostaining and transmission electron microscopy, which was inhibited by AKTI IV. Shh further increased SMC LC3-II in the presence of bafilomycin A1, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, and pepstatin A or siRNA for the autophagy-related gene 7 (ATG7). In addition, Shh induced SMC proliferation, which was inhibited not only by AKTI IV but also by cyclopamine, an inhibitor of Shh receptor. Inhibition of autophagy with 3-methyladenine (3-MA), bafilomycin A1, or ATG7 siRNA resulted in inhibition of cell proliferation. Treatment with 3-MA, AKTI IV, or cyclopamine inhibited neointima formation in mouse common carotid arteries. Taken together, our results have shown that Shh induces autophagy of vascular SMCs involving AKT activation, suggesting a role of autophagy in Shh-induced cellular responses.

Alleviation of autophagy by knockdown of Beclin-1 enhances susceptibility of hippocampal neurons to proapoptotic signals induced by amino acid starvation

Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.

Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways

Na+/K+-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na+/K+-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G2/M arrest but not apoptosis at IC50 level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.

Hyperglycemia Alters Mitochondrial Fission and Fusion Proteins in Mice Subjected to Cerebral Ischemia and Reperfusion

Preischemic hyperglycemia exacerbates brain damage caused by cerebral ischemia. In the present experiment, we studied the effects of preischemic hyperglycemia on protein markers that are related to mitochondrial fission and fusion, mitochondrial biogenesis, and autophagy in mice subjected to 30-min transient focal ischemia. The fission proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis1), fusion proteins optic atrophy 1 (Opa1) and mitofusin 2 (Mfn2), mitochondrial biogenesis regulators nuclear respiratory factor 1 (NRF1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and autophagy marker beclin 1 and microtubule-associated protein light chain 3 (LC3) were analyzed in control, 30 min middle cerebral artery occlusion (MCAO) plus 6-, 24-, and 72 h of reperfusion in normo- and hyperglycemic conditions. Cerebral ischemia increased the levels of Drp1 and decreased Fis1 after reperfusion. Preischemic hyperglycemia further augmented the increase of Drp1 and induced elevation in Fis1. Ischemia inhibited the levels of Opa1 and Mfn2 and hyperglycemia further decreased the level of Opa1. Further, NRF1 increased after reperfusion in both normo- and hyperglycemic animals. However, such increase was caused by reperfusion rather than glucose level. Finally, ischemia increased beclin 1 level at 6 and 24 h of reperfusion and hyperglycemia further increased the beclin 1 level and caused LC3-II increase as well. Hyperglycemia enhances the ischemia-induced mitochondrial dynamic imbalance towards fission that may favor mitochondrial fragmentation and subsequent damage. Hyperglycemia elevated autophagy markers may represent an adapting reaction to the severe damage incurred in hyperglycemic animals or a third pathway of cell death.

Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration.

Abstract 4654: Docosahexaenoic acid induces autophagy through reactive oxygen species /mTOR pathway in p53 mutant cancer cells

Abstract Our previous studies reported that docosahexaenoic acid (DHA) induces autophagy through p53 inhibition in the wild-type p53 cancer cells. This study attempts to elucidate the molecular mechanism underlying DHA-induced autophagy in PC3 and DU145 prostate cancer cells harboring mutant p53. DHA increased both the level of microtubule-associated protein light-chain 3 (LC3) and the number of autophagic vacuoles. Autophagic flux assay confirmed that DHA-induced increase in LC3-II and autophagic vesicles was an outcome of autophagic process activation, indicating that DHA also induces autophagy in p53 mutant cancer cells. DHA treatment also increased the level of reactive oxygen species (ROS) as measured by dihydroethidine staining, and pretreatment of an antioxidant, N-acetylcysteine (NAC), significantly inhibited the ROS production as well as autophagy induced by DHA, suggesting that ROS regulates the autophagic process triggered by DHA. Further experiments showed that the mechanism of DHA-induced autophagy associated with ROS production was related to a decrease in the activity of mammalian target of rapamycin (mTOR). NAC remarkably restored the decreases in the levels of phospho-mTOR and 4EBP, an mTOR downstream molecule, induced by DHA as analyzed by the Western blot assay. Furthermore, the level of phospho-AMPK, which negatively regulates mTOR was increased, while phospho-Akt was reduced during the DHA-induced autophagy, indicating the involvement of AMPK and Akt signalings. Collectively, our results demonstrate that DHA induces autophagy through the ROS-mediated mTOR inactivation in p53 mutant prostate cancer cells. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0006232]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4654. doi:1538-7445.AM2012-4654

Propofol Prevents Autophagic Cell Death following Oxygen and Glucose Deprivation in PC12 Cells and Cerebral Ischemia-Reperfusion Injury in Rats

BackgroundPropofol exerts protective effects on neuronal cells, in part through the inhibition of programmed cell death. Autophagic cell death is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. We therefore studied whether propofol could attenuate the formation of autophagosomes, and if so, whether the inhibition of autophagic cell death mediates the neuroprotective effects observed with propofol.Methodology/Principal FindingsThe cell model was established by depriving the cells of oxygen and glucose (OGD) for 6 hours, and the rat model of ischemia was introduced by a transient two-vessel occlusion for 10 minutes. Transmission electron microscopy (TEM) revealed that the formation of autophagosomes and autolysosomes in both neuronal PC12 cells and pyramidal rat hippocampal neurons after respective OGD and ischemia/reperfusion (I/R) insults. A western blot analysis revealed that the autophagy-related proteins, such as microtubule-associated protein 1 light chain 3 (LC3-II), Beclin-1 and class III PI3K, were also increased accordingly, but cytoprotective Bcl-2 protein was decreased. The negative effects of OGD and I/R, including the formation of autophagosomes and autolysosomes, the increase in LC3-II, Beclin-1 and class III PI3K expression and the decline in Bcl-2 production were all inhibited by propofol and specific inhibitors of autophagy, such as 3-methyladenine (3-MA), LY294002 and Bafilomycin A1 (Baf),. Furthermore, in vitro OGD cultures and in vivo I/R rats showed an increase in cell survival following the administration of propofol, as assessed by an MTT assay or histochemical analyses.Conclusions/SignificanceOur data suggest that propofol can markedly attenuate autophagic processes via the decreased expression of autophagy-related proteins in vitro and in vivo. This inhibition improves cell survival, which provides a novel explanation for the pleiotropic effects of propofol that benefit the nervous system.

Tumor suceptibility gene 101 modulates autophagy through regulation of LC3‐II and ATG3 level

Autophagy and endosomal vesicular trafficking cooperate to regulate the turnover of intracellular macromolecule, defective organelles and signaling receptor. Tumor Susceptibility Gene 101(TSG101) is a member of endosomal sorting complex required for transport‐I, ESCRT‐I, which regulates membrane receptor recycling and vesicular trafficking. Previous reports indicated that TSG101 is required for autophagy because its deprivation led to the accumulation of autophagosome. Here, we observed the increase of TSG101 and autophagic marker proteins LC3‐II and ATG3 upon nutrient starvation. Knockdown of TSG101 in HeLa cell resulted in the elevation of LC3‐II, ATG3 and ubiquitinated protein aggregates marker protein p62. However, in neuroblastoma SH‐SY5Y cell, transfection of siRNA led to the decrease of LC3‐II and ubiquitinated protein level. These results indicated that TSG101 might be essential for autophagy and the maintenance of steady‐state level of cellular ubiquitinated proteins known to be linked with the pathologic condition of neuronal cell type. Ectopic expression of HA‐TSG101 led to the increase of LC3‐II in both cell types and the elevation of ATG3 level in HeLa cell, indicating ectopic HA‐TSG101 might facilitate the execution of autophagic flow.

Polyamine supplementation enhances autophagy and reverses age‐related arterial stiffening

Aging leads to stiffening of large elastic arteries as a result of changes in structural proteins and increased macromolecular damage. Polyamines (PA) play a causal role in survival/longevity and have been reported to induce autophagy, the cellular process of recycling damaged biomolecules. However, the potential for PA to reduce age‐related arterial stiffness is unknown. We studied young (Y: 5 mo, n=7) and old (O: 28–29 mo, n=7) male C57/BL6 mice, and old mice treated with the PA spermidine (OPA: 29 mo, n=7). Aortic pulse wave velocity (aPWV), an index of arterial stiffness, was higher in O vs. Y mice (465 ± 25 vs. 383 ± 21 cm/s, p&lt;0.05). This was associated with ~50% greater aortic levels of both protein carbonyls, an index of oxidative damage, and advanced glycation end products (AGEs), a marker of protein cross‐linking (dot and western blotting, both p&lt;0.05, O vs. Y). PA supplementation (3 mM drinking water × 4w) ameliorated aortic stiffening in old mice (OPA aPWV pre: 482 ± 17 vs. OPA post: 343 ± 14 cm/s, P&lt;0.05) and reversed age‐related increases in protein carbonyls and AGEs. The effects of PA treatment were associated with enhanced autophagy as indicated by an ~20% reduction in acetylation (inhibition) of histone H3, an ~25% increase in expression of the transcription factors AMPK and FOXO3a, and a corresponding increase in LC3‐II and decrease in p62, markers of autophagy and autophagic protein aggregation/damage, respectively (western blotting, all p&lt;0.05, O vs. OPA). These results suggest that PA supplementation may be a novel strategy for reversing age‐related arterial stiffening, perhaps by enhancing autophagic clearance of damaged/aggregated proteins.NIH AG013038, AG039210, HL007822