Induction of autophagy by concanavalin A and its application in anti-tumor therapy

Concanavalin A (Con A), a lectin from Jack bean seeds that, once bound to the mannose moiety on the cell membrane glycoprotein, is internalized preferentially to the mitochondria. A BNIP3-mediated mitochondria autophagy is then induced, and causes the tumor cells to undergo autophagic cell death. Con A is also a T cell mitogen that can induce autoimmune hepatitis in mice. Because of the dual properties (autophagic cytotoxicity and immunomodulation) via the specific mannose binding, Con A can exert a potent anti-hepatoma therapeutic effect by inhibiting tumor nodule formation in the liver and prolonging the survival of the tumor-bearing mice. The anti-tumor effect is primarily mediated by activated CD8(+) T cells, and will also establish a tumor antigen-specific immune memory during the hepatic inflammation. This finding provides a novel mechanism in which Con A can be used as an anti-hepatoma agent, and also gives support for the search for natural lectins as anti-cancer compounds.     

Lectin of Concanavalin A as an anti-hepatoma therapeutic agent

Liver cancer is the predominant cause of cancer mortality in males of Southern China and Taiwan. The current therapy is not satisfactory, and more effective treatments are needed. In the search for new therapies for liver tumor, we found that Concanavalin A (Con A), a lectin from Jack bean seeds, can have a potent anti-hepatoma effect. Con A after binding to the mannose moiety on the cell membrane glycoprotein is internalized preferentially to the mitochondria. An autophagy is triggered which leads to cell death. Con A as a T cell mitogen subsequently activates the immune response in the liver and results in the eradication of the tumor in a murine in situ hepatoma model. The liver tumor nodule formation is inhibited by the CD8⁺ T cells, and a tumor antigen-specific immune memory is established during the hepatic inflammation. The dual properties (autophagic cytotoxicity and immunomodulation) via the specific carbohydrate binding let Con A exert a potent anti-hepatoma therapeutic effect. The novel mechanism of the Con A anti-hepatoma effect is discussed. The prototype of Con with an anti-hepatoma activity gives support to the search for other natural lectins as anti-cancer compounds.     

Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.     

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4⁺ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.     

A smARF Way to Die: A Novel Short Isoform of p19ARF is Linked to Autophagic Cell Death

We recently revealed a novel mechanism by which p19ARF can induce cell death. We found that the p19ARF mRNA encodes an additional shorter isoform from the same open reading frame, named smARF. smARF is a short lived protein, which is rapidly degraded by the proteasome, but accumulates after inappropriate proliferative signals generated by oncogenes. Surprisingly, smARF translocates to the mitochondria, impairs the structure of the mitochondria and dissipates the mitochondrial membrane potential in a p53 and Bcl-2 family independent manner, ultimately inducing type II caspase-independent autophagic cell death.

Addendum to:

A Short Mitochondrial form of p19(ARF) Induces Autophagy and Caspase-Independent Cell Death

S. Reef, E. Zalckvar, O. Shifman, S. Bialik, H. Sabanay, M. Oren and A. Kimchi

Mol Cell 2006; 22:463-75     

Distribution of electric charges and concanavalin A binding sites on autophagic vacuoles and lysosomes in mouse hepatocytes

The distributions of electric charges and Concanavalin A binding sites in autophagic vacuoles and lysosomes in mouse hepatocytes were studied by utilizing a frozen ultrathin section labeling method with cationized ferritin (CF) or anionized ferritin and ferritin-conjugated Concanavalin A (Con A-F) as visual probes. Our observations revealed that the inner surface of the autophagic vacuole membrane has more anionic sites (CF binding) than other organelle membranes. This suggests that if the limiting membranes of autophagic vacuoles originate from preexisting membranes, such membranes must undergo structural and compositional alternation during the formation of the autophagic vacuoles. In contrast to CF, Con A-F showed no distinct binding to the membranes of autophagic vacuoles, but the contents of vacuoles displayed varying Con A-F binding, depending on the stage of the autophagic process. Increased binding was seen in more mature autophagic vacuoles. Since lysosomes showed a preferential accumulation of Con A-F particles, molecules with Con A-F binding sites in autophagic vacuoles may be of lysosomal origin. Con A-F distribution varied from lysosome to lysosome in the same cell, indicating heterogeneity of lysosomal contents. These results suggest that ferritin-conjugated lectin labeling methods applied to frozen, ultrathin section are a useful new approach in analyzing the natural history of autophagic vacuoles and the heterogeneity of lysosomes.     

Deficiency in the incorporation of labeled thymidine and inhibition in the biosynthesis of interleukin-2 in lymphocytes obtained from Histoplasma capsulatum infected mice

The incorporation of (³H) thymidine and the biosynthesis of interleukin-2(IL-2) were investigated in Concanavalin A (ConA) and histoplasmin stimulated lymphocytes from spleen of infected Balb/c mice with the yeast phases of Histoplasma capsulatum. The ability to incorporate (³H) thymidine of Con A stimulated lymphocytes in culture from spleen of Histoplasma capsulatum infected mice, as well as the IL-2 content present in the supernatants of that cultures, were depressed along the first three weeks of the experiments, but starting week five, normal values were restored or even discretly increased. Incorporation of (³H) thymidine in histoplasmin stimulated lymphocytes remained inhibited along the seven weeks the experiment lasted. Results showed that inoculation of H. capsulatum yeast in mice provoked a temporary immunosuppression on cell mediated immunity, that can be explained by means of the inability of T cells to produce enough IL-2 necessary for the proliferation of T cells in culture.     

A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice

BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.     

Differential and cell-type specific microheterogeneity of high mannose-type Asn-linked oligosaccharides of human transferrin receptor.

The structural analysis of high mannose-type Asn-linked (N-linked) oligosaccharides of the human transferrin receptor (hTR) from D-[2-3H]mannose metabolic-radiolabeled human cells--A431, K562, BeWo, and HL60--was investigated. The radiolabeled hTR glycopeptides were prepared and fractionated by a lectin chromatography of Concanavalin A-Sepharose. The composition analysis of hTR glycopeptides revealed that Con A-I contains both mannose and fucose, whereas Con A-III has mannose exclusively. The Con A-III glycopeptides were treated with Endo H. The released oligosaccharides were charge-fractionated by QAE-Sephadex. The neutral oligosaccharides were further size-fractionated by an amine absorption high performance liquid chromatography (HPLC). Our results demonstrate that the high mannose-type oligosaccharides of hTR ranged in size from Man5-R to Man9-R with cell-type specific patterns. A relative amount of each component was found to be differentially heterogeneous among the four different human cell lines.     

Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner

Abstract

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.     

Autophagy,Mitochondria and Cell Death in Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) are debilitating genetic conditions that frequently manifest as neurodegenerative disorders. They severely affect eye, motor and cognitive functions and, in most cases, abbreviate the lifespan. Postmitotic cells such as neurons and mononuclear phagocytes rich in lysosomes are most often affected by the accumulation of undegraded material. Cell death is well documented in parts of the brain and in other cells of LSD patients and animal models, although little is known about mechanisms by which death pathways are activated in these diseases, and not all cells exhibiting increased storage material are affected by cell death. Lysosomes are essential for maturation and completion of autophagy-initiated protein and organelle degradation. Moreover, accumulation of effete mitochondria has been documented in postmitotic cells whose lysosomal function is suppressed or in aging cells with lipofuscin accumulation. Based upon observations in the literature and our own data showing similar mitochondrial abnormalities in several LSDs, we propose a new model of cell death in LSDs. We suggest that the lysosomal deficiencies in LSDs inhibit autophagic maturation, leading to a condition of autophagic stress. The resulting accumulation of dysfunctional mitochondria showing impaired Ca2+ buffering increases the vulnerability of the cells to pro-apoptotic signals.

Addendum to:

Mitochondrial Aberrations in Mucolipidosis Type IV

J.J. Jennings Jr., J.H. Zhu, Y. Rbaibi, X. Luo, C.T. Chu and K. Kiselyov

J Biol Chem 2006; 281:39041-50     

Nervous system myelin in the electric ray, Torpedo marmorata: Morphological characterization of the membrane and biochemical analysis of its protein components

In Torpedo, PNS as well as CNS myelines are characterized by clearly separated double intraperiod lines. CNS myelin of Torpedo contains two glycosylated hydrophobic proteins labelled T1 (25,800 Da1) and T2 (29,700 Da1), and two basic proteins BP1 and BP2, migrating like mammalian large basic protein (BP2) and pre-small basic protein (BP1) (Barbarese et al., 1977). PNS myelin of Torpedo carries only BP1 and is characterized by a closely spaced doublet of the glycosylated hydrophobic proteins Con A+ (29,700 Da1) and Con A? (31,000 Da1); the latter does not bind Concanavalin A. These glycosylated proteins (T1, T2, Con A+, Con A?) contain mannose, N-acetylglucosamine and galactose, but lack fucose and sialic acids. They have isoleucine at their amino terminus. They bind anti-rat PNS myelin P₀ antibodies but do not react with anti-rat CNS myelin PLP antibodies. Limited proteolyses of isolated proteins suggest sequence homologies between T1 and T2, and possibly between Con A+ and Con A?. The two basic proteins BP1 and BP2 bind antibodies directed against human myelin basic protein. All Torpedo myelin proteins electrofocus in pH regions characteristic of their mammalian counterparts.     

Autophagy Induction and Autophagic Cell Death in Effector T Cells

The population size of the T cells is tightly regulated. The T cell number drastically increases in response to their specific antigens. Upon antigen clearance, the T cell number decreases over time. Apoptosis, also called type I programmed cell death, plays an important role in eliminating T cells. The role of autophagic cell death, also called type II programmed cell death, is unclear in T cells. Our recent work demonstrated that autophagy is induced in both Th1 and Th2 cells. Both TCR signaling and IL-2 increase autophagy in T cells, and JNK MAP kinases are required for the induction of autophagy in T cells, whereas caspases and mTOR inhibit autophagy in T cells. Autophagy is required for mediating growth factor withdrawal-dependent cell death in T cells. Here, we hypothesize that autophagic cell death plays an important role in T cell homeostasis.

Addendum to:

Autophagy is Induced in CD4⁺ T Cells and Important for the Growth Factor-Withdrawal Cell Death

C. Li, E. Capan, Y. Zhao, J. Zhao, D. Stolz, S.C. Watkins, S. Jin and B. Lu

J Immunol 2006; 177:5163-8     

Growth Stimulation of Tumor-Specific Cytotoxic T Lymphocytes on Concanavalin A-Immobilized Carrier Beads

Human tumor-specific CD4⁺ cytotoxic T lymphocytes (CTL) were generated against duodenum papilloma cell line TGBC18TKB from HLA type-matched peripheral blood mononuclear cells. Concanavalin A (Con A) immobilized on carrier beads stimulated growth of the CTL in a long-term culture without repeated antigen stimulation, while soluble Con A induced death of the CTL. The CTL exhibited the target-specific cytotoxicity in a more potent manner than those before the long-term culture in the presence of the immobilized Con A. Enhanced expression of the adhesion molecule, CD11b, was observed on the CTL. These results suggest that immobilized Con A will be useful for continuous growth stimulation and large scale expansion of CTL without tumor antigen.     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Suppression of in vitro lymphocyte stimulation in mice bearing primary Moloney sarcoma virus-induced tumors

A marked depression of phytohemagglutinin (PHA) reactivity was observed in spleen cell cultures of C57B1/6N mice bearing primary Moloney sarcoma virus (MSV)-induced tumors. This defect was most pronounced 14 days after virus inoculation (MSV 14) and was reversed after regression of the tumor. Spleen cells from mice with primary methylcholanthrene-induced sarcomas were similarly deficient while no such effect was observed during the first weeks after inoculation of Moloney leukemia virus. The responses of MSV 14 spleen cells to Concanavalin A (Con A) were as consistently depressed as those to PHA, but reactivity to bacterial lipopolysaccharide was affected to a lesser degree. Stimulation by pokeweed mitogen (PWM) was not significantly lower in tumor-bearing mice than in control animals. Passage of MSV 14 spleen cells over rayon adherence columns which removed about 75% of the initial cell population led to an almost complete restoration of their PHA and Con A responses on a per cell basis. This may indicate that within MSV 14 spleens, T lymphocytes reactive to PHA and Con A are diluted out by a majority of unreactive cells. However, the possibility also exists that column passage removes a suppressor cell that actively inhibits these responses.     

Mitoptosis: Different Pathways for Mitochondrial Execution

Mitoptosis was described as a sort of mitochondrial death program. It could be associated with both necrosis and apoptosis, although degenerating mitochondria are also found in autophagic vacuoles. It was demonstrated that several molecules might contribute to the remodeling and rearrangement of mitochondrial membranes, leading to mitochondria rupture and disruption. Here, we hypothesize that, at least in T cells, two main pathways of mitoptosis can occur: an inner membrane mitoptosis (IMM), in which only the internal matrix and cristae are lost while the external mitochondrial envelope remains unaltered, and an outer membrane mitoptosis (OMM) where only swollen internal cristae are detected as remnants. We suggest that the study of these processes could provide useful insights not only to the field of cell death but also to the study of the pathogenic mechanisms of mitochondria-associated human diseases.

Addendum to:

Death Receptor Ligation Triggers Membrane Scrambling Between Golgi and Mitochondria

S. Ouasti, P. Matarrese, R. Paddon, R. Khosravi-Far, M. Sorice, A. Tinari, W. Malorni, M. Degli Esposti

Cell Death Differ 2006; Epub ahead of print     

Roles of the Akt/mTOR/p70S6K and ERK1/2 Signaling Pathways in Curcumin-Induced Autophagy

Curcumin has a potent anticancer effect and is a promising new therapeutic strategy. We previously demonstrated that curcumin induced non-apoptotic autophagic cell death in malignant glioma cells in vitro and in vivo. This compound inhibited the Akt/mammalian target of rapamycin/p70 ribosomal protein S6 kinase pathway and activated the extracellular signal-regulated kinases 1/2 thereby inducing autophagy. Interestingly, activation of the first pathway inhibited curcumin-induced autophagy and cytotoxicity, whereas inhibition of the latter pathway inhibited curcumin-induced autophagy and induced apoptosis, thus augmenting the cytotoxicity of curcumin. These results imply that these two autophagic pathways have opposite effects on curcumin’s cytotoxicity. However, inhibition of nuclear factor κB, which is the main target of curcumin for its anticancer effect, was not observed in malignant glioma cells. These results suggest that autophagy but not nuclear factor κB plays a central role in curcumin anticancer therapy and warrant further investigation toward application in patients with malignant gliomas. Here, we discuss the therapeutic role of two autophagic pathways influenced by curcumin.

Addendum to:

Evidence That Curcumin Suppresses the Growth of Malignant Gliomas in Vitro and in Vivo through Induction of Autophagy: Role of Akt and Extracellular Signal-Regulated Kinase Signaling Pathways

H. Aoki, Y. Takada, S. Kondo, R. Sawaya, B. B. Aggarwal and Y. Kondo

Mol Pharmacol 2007; 72:29-39