A novel role for autophagy in neurodevelopment

We recently showed that Ambra 1, a WD40-containing approximately 130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra 1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra 1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra 1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this article, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.     

The autophagy regulators Ambra1 and Beclin 1 are required for adult neurogenesis in the brain subventricular zone

Autophagy is a conserved proteolytic mechanism required for maintaining cellular homeostasis. The role of this process in vertebrate neural development is related to metabolic needs and stress responses, even though the importance of its progression has been observed in a number of circumstances, both in embryonic and in postnatal differentiating tissues. Here we show that the proautophagic proteins Ambra1 and Beclin 1, involved in the initial steps of autophagosome formation, are highly expressed in the adult subventricular zone (SVZ), whereas their downregulation in adult neural stem cells in vitro leads to a decrease in cell proliferation, an increase in basal apoptosis and an augmented sensitivity to DNA-damage-induced death. Further, Beclin 1 heterozygosis in vivo results in a significant reduction of proliferating cells and immature neurons in the SVZ, accompanied by a marked increase in apoptotic cell death. In sum, we propose that Ambra1- and Beclin 1-mediated autophagy plays a crucial role in adult neurogenesis, by controlling the survival of neural precursor cells.In the adult mammalian brain, neural stem cells are localized in two regions: in the subventricular zone (SVZ), a layer extending along the wall of the lateral ventricle, and in the subgranular zone of the dentate gyrus in the hippocampus.¹ SVZ stem cells are strictly controlled under physiological conditions and are believed to replenish dying cells. In addition to their effect in maintaining brain homeostasis, they are also involved in neuronal replacement in response to injury.² Although several factors are known to affect neurogenesis, understanding of the mechanisms that regulate adult neurogenic niches and their metabolism is still incomplete. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved cellular turnover process in which bulk cytoplasmic materials, long-lived proteins or damaged organelles are sequestered and delivered to lysosomes for degradation.³ A complex crosstalk takes place between apoptosis and autophagy that determines the death or life of cells.⁴ Beclin 1 has a key role in autophagy initiation;⁵ it regulates the autophagy-promoting activity of the Class III PI 3-kinase Vps34,⁶ and is involved in the recruitment of membranes to form the key autophagy vesicles, named autophagosomes. Beclin 1 also interacts with Bcl-2,⁷ and plays an important function in the regulation of cell survival.⁸ Ambra1 (activating molecule in Beclin 1-regulated autophagy) is another modulator of autophagy, which is phosphorylated by the upstream autophagy kinase Ulk1 and acts on Ulk1 stability and function.^{9, 10} Ambra1 also interacts with Beclin 1 upon autophagic stimuli, thereby promoting the binding between Beclin 1 and its target kinase, Vps34. The binding between Ambra1 and mitochondrial Bcl-2 is also important for cell survival.¹¹ Moreover, Ambra1 is crucial for nervous system development and is expressed from early neurulation onwards, with a high specificity for the neural plate.¹²In contrast with studies on the pro-survival impact of autophagy in post-mitotic cells and in disease models, the role of autophagy in the maintenance and function of adult neural stem cells (ANSCs) is poorly understood. Here we have found that expression of upstream autophagy-regulating genes in the adult neurogenic region of SVZ, in physiological conditions, plays a crucial role in the regulation of adult neurogenesis.     

Upregulation of Beclin 1 in the ischemic penumbra

Here we discuss the probable role of autophagy in cerebral ischemia based on our own recent data and the literature. We examined the protein level of Beclin 1 (Bcl-2 interacting protein) and microtubule-associated protein 1 light chain 3 (LC3) which were previously found to promote autophagy. We found a dramatic elevation in Beclin 1 levels and LC3 in the penumbra of rats challenged by cerebral ischemia. We found also that a subpopulation of Beclin 1-upregulating cells is also expressing the active form of caspase-3, and that all Beclin 1 upregulating cells display dense staining of LC3. Neuronal cells that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology, which indicates that not all the Beclin 1-upregulating cells are predestined to die. We conclude that the cell death in the penumbra bears a resemblance not only to necrosis, apoptosis, or a compromise between the two, but exhibits also biochemical and morphological characteristics of autophagic cell death. The question that constantly arises, however, is whether autophagic activity in damaged cells is the cause of death or is actually an attempt to prevent it as a part of an endogenous neuroprotective response.

Addendum to: Rami A et al. Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death. Neurobiol Dis 2007; In press.     

UVRAG: A New Player in Autophagy and Tumor Cell Growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies1 have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, underscoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3- mediated autophagy and tumor cell control.

Addendum to:

Autophagic and Tumor Suppressor Activity of a Novel Beclin 1-Binding Protein UVRAG

Chengyu Liang, Pinghui Feng, Bonsu Ku, Iris Dotan, Dan Canaani, Byung-Ha Oh and Jae U. Jung

Nature Cell Biol 2006; 8:688-99     

Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program.     

Autophagy and Angiogenesis Inhibition

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

Addendum to:

Kringle 5 of Human Plasminogen, an Angiogenesis Inhibitor, Induces Both Autophagy and Apoptotic Death in Endothelial Cells

T.M.B. Nguyen, I.V. Subramanian, A. Kelekar and S. Ramakrishnan

Blood 2007; 109:4793-802     

AMPK Mediates Autophagy during Myocardial Ischemia In Vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.

Addendum to:

Distinct Roles of Autophagy in the Heart During Ischemia and Reperfusion: Roles of AMP-Activated Protein Kinase and Beclin 1 in Mediating Autophagy

Y. Matsui, H. Takagi, X. Qu, M. Abdellatif, H. Sakoda, T. Asano, B. Levine and J. Sadoshima

Circ Res 2007; 100:914-22     

Ambra1 modulates starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in cardiomyocytes is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in cardiomyocytes. To test this hypothesis, we confirmed autophagic activity in serum-starved cardiomyocytes by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes and LC3 protein levels. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in cardiomyocytes. When Ambra1 expression was reduced by siRNA, the cardiomyocytes were more sensitive to staurosporine-induced apoptosis. In addition, co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated cardiomyocytes, suggesting that Ambra1 regulates autophagy in cardiomyocytes by interacting with Beclin1. Finally, we determined that starvation stress-induced activation of Ambra1 contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signaling. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis through AMPK signaling pathway in cardiomyocytes that maintains the balance between autophagy and apoptosis.     

Relationship between Autophagy and Apoptotic Cell Death in Human Neuroblastoma Cells Treated with Paraquat: Could Autophagy be a “Brake” in Paraquat-Induced Apoptotic Death?

Paraquat (PQ) (1, 1’-dimethyl-4, 4’-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson’s disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD.

Addendum to

Inhibition of Paraquat-Induced Autophagy Accelerates the Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

R.A. González-Polo, M. Niso-Santano, M.A. Ortíz-Ortíz, A. Gómez-Martín, J.M. Morán, L. García-Rubio, J. Francisco-Morcillo, C. Zaragoza , G. Soler and J.M. Fuentes

Toxicological Science 2007; In press     

Autophagy Can Be a Killer Even in Apoptosis-Competent Cells

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.

Addendum to:

Role of Phosphoinositide 3-Kinase in the Autophagic Death of Serum-Deprived PC12 Cells.

A. Guillon-Munos, M.X.P. van Bemmelen and P.G.H. Clarke

Apoptosis 2005; 10:1031-41.     

BH3-Only Proteins and BH3 Mimetics Induce Autophagy by Competitively Disrupting the Interaction between Beclin 1 and Bcl-2/Bcl-XL

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X_L) by virtue of its BH3 domain, an amphipathic α-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X_L. The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X_L. This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (ER-phagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X_L can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X_L complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X_L complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X_L at the level of the endoplasmic reticulum.

Addendum to:

Functional and Physical Interaction Between Bcl-X_L and a BH3-Like Domain in Beclin-1

M.C. Maiuri, G. Le Toumelin, A. Criollo, J.-C. Rain, F. Gautier, P. Juin, E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, J.A. Hickman, O. Geneste and G. Kroemer

EMBO J 2007; In press     

Neurodegeneration Induces Upregulation of Beclin 1

Autophagy, a bulk degradation of subcellular constituents, is activated in normal cell growth and development, and represents the major pathway by which the cell maintains a balance between protein synthesis and protein degradation. Autophagy was documented in several neurodegenerative diseases, and under stress conditions the autophagic process can lead to cell death (type II programmed cell death). Beclin 1 is a Bcl-2 interacting protein that was previously found to promote autophagy. We have used Beclin 1 protein as a marker for autophagy following traumatic brain injury in mice. We demonstrated a dramatic elevation in Beclin 1 levels near the injury site. Interestingly Beclin 1 elevation starts at early stages post injury (4 h) in neurons and 3 days later in astrocytes. In both cell types it lasts for at least three weeks. Neuronal cells, but not astrocytes, that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology. These observations may indicate that not all the Beclin 1 overexpressing cells will die. The elevation of Beclin 1 at the site of injury may represent enhanced autophagy as a mechanism to discard injured cells and reduce damage to cells by disposing of injured components.

Addenda to:

Closed Head Injury Induces Upregulation of Beclin 1 at the Cortical Site of Injury

T. Diskin, P. Tal-Or, S. Erlich, L. Mizrachy, A. Alexandrovich, E. Shohami and R. Pinkas-Kramarski

J Neurotrauma 2005; 22:750-62     

Autophagy: An overlooked mechanism of HIV-1 pathogenesis and NeuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4⁺ lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4+ T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.

Addendum to: Zhou D, Spector SA. Human immunodeficiency virus type-1 infection inhibits autophagy. Aids 2008;22:695-9.     

The Beclin 1 network regulates autophagy and apoptosis

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, a process of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle. It interacts with several cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, nPIST, VMP1, SLAM, IP(3)R, PINK and survivin) to regulate the lipid kinase Vps-34 protein and promote formation of Beclin 1-Vps34-Vps15 core complexes, thereby inducing autophagy. In contrast, the BH3 domain of Beclin 1 is bound to, and inhibited by Bcl-2 or Bcl-XL. This interaction can be disrupted by phosphorylation of Bcl-2 and Beclin 1, or ubiquitination of Beclin 1. Interestingly, caspase-mediated cleavage of Beclin 1 promotes crosstalk between apoptosis and autophagy. Beclin 1 dysfunction has been implicated in many disorders, including cancer and neurodegeneration. Here, we summarize new findings regarding the organization and function of the Beclin 1 network in cellular homeostasis, focusing on the cross-regulation between apoptosis and autophagy.     

Gtdap-1 and the Role of Autophagy During Planarian Regeneration and Starvation

Planarians have been established as an ideal model organism for stem cell research and regeneration. Planarian regeneration and homeostasis require an exquisite balancing act between cell death and cell proliferation as new tissues are made (epimorphosis) and existing tissues remodeled (morphallaxis). Some of the genes and mechanisms that control cell proliferation and pattern formation are known. However, studies about cell death during remodeling are few and far between. We have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1 together with DAP-kinase has been identified as a positive mediator of programmed cell death induced by gamma-interferon in HeLa cells. We have found that the gene functions at the interface between autophagy and cell death in the remodeling of the organism that occurs during regeneration and starvation in sexual and asexual races of planarians. Our data suggest that autophagy of existing cells may be essential to fuel the continued proliferation and differentiation of stem cells by providing the necessary energy and building blocks to neoblasts.

Addendum to:

Gtdap-1 Promotes Autophagy and is Required for Planarian Remodeling During Regeneration and Starvation

C. González-Estévez, D.A. Felix, A.A. Aboobaker and E. Saló

Proc Natl Acad Sci USA 2007; 104:13373-8     

Beclin 1 bridges autophagy,apoptosis and differentiation

Beclin 1 is a critical component in the class III PI3 kinase complex (PI3KC3) that induces the formation of autophagosomes in mammalian systems. Autophagic triggers upregulate Beclin 1, which in turn binds to PI3KC3 or Bcl-X_L to form complexes of Beclin 1-PI3KC3 or Beclin 1-Bcl-X_L that are physically and functionally independent from each other. Contrary to the previous observations that Beclin 1 binding to Bcl-2 family members is apoptotic and antiautophagic, we found that autophagic trigger-induced Beclin 1-binding to Bcl-X_L is antiapoptotic and has no effect on autophagy, suggesting a convertible role of the Beclin 1-Bcl-X_L complex in response to autophagy stimuli. Both autophagy and differentiation cascades require upregulation of Beclin 1. While the basal Beclin 1 level does not cause autophagy or differentiation, depletion of Beclin 1 cripples both autophagy and differentiation capabilities, but activates apoptosis. These results demonstrate that Beclin 1 is essential for autophagy, differentiation and antiapoptosis, and may play an important role in coordinating inputs for cellular decisions to signaling machinery that mediates different cellular cascades.

Addendum to: Wang J, Lian H, Zhao Y, Kauss MA, Spindel S. Vitamin D3 induces autophagy of human myeloid leukemia cells. J Biol Chem 2008; doi:10.1074/jbc.     

Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy

The Beclin 1-VPS34 complex plays a crucial role in the induction of the autophagic process by generating PtdIns(3)P-rich membranes, which act as platforms for ATG protein recruitment and autophagosome nucleation. Several cofactors, such as Ambra1, ATG14 and UVRAG, are necessary for Beclin 1 complex activity. However, the mechanism by which Beclin 1 complex activity is: stimulated by autophagic stimuli has not yet been fully elucidated. Recently, we reported that autophagosome formation in mammalian cells is primed by Ambra1 release from the dynein motor complex. We found that Ambra1 specifically binds the dynein motor complex under normal conditions through a direct interaction with DLC1. When autophagy is induced, Ambra1-DLC1 are released from the dynein complex in an ULK1-dependent manner, and relocalize to the endoplasmic reticulum, thus enabling autophagosome nucleation. In addition, we found that both DLC1 downregulation and Ambra1 mutations in its DLC1-binding sites strongly enhance autophagosome formation. Ambra1 is therefore not only a cofactor of Beclin 1 in favoring its kinase-associated activity, but also a crucial upstream regulator of autophagy initiation.     

PKCδ and Tissue Transglutaminase are Novel Inhibitors of Autophagy in Pancreatic Cancer Cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKCδ) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKCδ/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKCδ/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.

Addendum to:

Tissue Transglutaminase Inhibits Autophagy in Pancreatic Cancer Cells

U. Akar, B. Ozpolat, K. Mehta, J. Fok, Y. Kondo and G. Lopez-Berestein

Mol Cancer Res 2007; 5:241-9     

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.