NGF augments the autophosphorylation of Ret via inhibition of ubiquitin-dependent degradation

Nerve growth factor (NGF) is required for the trophic maintenance of postnatal sympathetic neurons. A significant portion of the growth-promoting activity of NGF is from NGF-dependent phosphorylation of the heterologous receptor tyrosine kinase, Ret. We found that NGF applied selectively to distal axons of sympathetic neurons maintained in compartmentalized cultures activated Ret located in these distal axons. Inhibition of either proteasomal or lysosomal degradation pathways mimicked the effect of NGF on Ret activation. Likewise, NGF inhibited the degradation of Ret induced by glial cell line-derived neurotrophic factor-dependent activation, a process that requires ubiquitination and proteasomal degradation. NGF induced the accumulation of autophosphorylated Ret predominantly in the plasma membrane, in contrast to GDNF, which promoted the internalization of activated Ret. An accretion of monoubiquitinated, but not polyubiquitinated, Ret occurred in NGF-treated neurons, in contrast to glial cell line-derived neurotrophic factor that promoted the robust polyubiquitination of Ret. Thus, NGF stimulates Ret activity in mature sympathetic neurons by inhibiting the ongoing ubiquitin-mediated degradation of Ret before its internalization and polyubiquitination.     

Pathway choice between proteasomal and autophagic degradation

Efficient degradation of abnormal or aggregated proteins is crucial to protect the cell against proteotoxic stress. Selective targeting and disposal of such proteins usually occurs in a ubiquitin-dependent manner by proteasomes and macroautophagy/autophagy. Whereas proteasomes are efficient in degrading abnormal soluble proteins, protein aggregates are typically targeted for degradation by autophagic vesicles. Both processes require ubiquitin-binding receptors, which are targeted to proteasomes via ubiquitin-like domains or to phagophores (the precursors to autophagosomes) via Atg8/LC3 binding motifs, respectively. The use of substrate modification by ubiquitin in both pathways raised the question of how degradative pathway choice is achieved. In contrast to previous models, proposing different types of ubiquitin linkages for substrate targeting, we find that pathway choice is a late event largely determined by the oligomeric state of the receptors. Monomeric proteasome receptors bind soluble substrates more efficiently due to their higher affinity for ubiquitin. Upon substrate aggregation, autophagy receptors with lower ubiquitin binding affinity gain the upper hand due to higher avidity achieved by receptor bundling. Thus, our work suggests that ubiquitination is a shared signal of an adaptive protein quality control system, which targets substrates for the optimal proteolytic pathway.     

白藜芦醇通过诱导细胞自噬性死亡抑制宫颈癌的研究

白藜芦醇作为一种广泛存在于药食同源植物中的非黄酮类多酚化合物,其抗肿瘤效果受到广泛关注,但在抑制宫颈癌方面仍缺乏体内效应的实验依据.本研究通过体内实验发现白藜芦醇具有明显的抗肿瘤生长作用,组织水平LC3B、P62和Beclin-1表达改变,推测白藜芦醇可能通过促进癌细胞的过度自噬抑制宫颈癌的进展;进一步通过体外细胞实验...     

Tetrandrine blocks autophagic flux and induces apoptosis via energetic impairment in cancer cells

Lysosomes are acidic organelles that have a crucial role in degrading intracellular macromolecules and organelles during the final stage of autophagy. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, was reported as an autophagy activator. Here, in contrast with previous studies, we show that Tet is a potent lysosomal deacidification agent and is able to block autophagic flux in the degradation stage. Single-agent Tet induces significant apoptosis both in vitro and in xenograft models. In the presence of Tet, apoptosis was preceded by a robust accumulation of autophagosomes and an increased level of microtubule-associated protein 1 light chain 3, type II (LC3-II). However, Tet increased the level of sequestosome 1 and decreased the turnover of LC3, indicating the blockade of autophagic flux in the degradation stage. As blockade of autophagic flux decreases the recycling of cellular fuels, Tet reduces the uptake of glucose in cancer cells. These effects lead to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. Blunting autophagosome formation using 3-methyladenine or genetic knockdown of Beclin-1 failed to rescue cells upon Tet treatment. By contrast, addition of methyl pyruvate to supplement TCA substrates protected Tet-treated tumor cells. These results demonstrate that energetic impairment is required in Tet-induced apoptosis. Tet, as a potent lysosomal inhibitor, is translatable to the treatment of malignant tumor patients.     

Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demonstrate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endosomes/lysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autophagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus (MLV) assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by nonspecific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possibility that similar regulations can occur in other biological contexts.     

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

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

Induction of apoptotic but not autophagic cell death by Cinnamomum cassia extracts on human oral cancer cells

Cinnamomum cassia has been widely studied in different fields to reveal its antidiabetic, antidepressive, antiviral, anti-inflammatory, antiosteoporotic, and anticancer effects. Its antimalignant activities have been explored in lung cancer, breast cancer, colorectal cancer, and even oral cancer, but the detailed signaling mechanism and effects of this plant on animal models need to be clarified. In the current study, C. cassia extract (CCE) was used to investigate the antitumorigenesis mechanism in vitro and in vivo. The major constituents of CCE used in this study were coumarin, cinnamic acid, and cinnamic aldehyde. CCE reduced the viability, number, and colony formation of human oral cancer cells, and induced their apoptosis. Caspase-3 activation, Bcl-2 reduction, and phosphatidylserine inversion were involved in CCE-stimulated apoptosis. CCE also enhanced the expression of autophagic markers, including acidic vesicular organelle, microtubule-associated protein 1 light chain 3-I, autophagy-related protein 14, rubicon, and p62. The combined treatment of CCE and caspase inhibitor significantly restored mitochondrial membrane potential (Δ ψ _m) and cell viability. However, the combined treatment of CCE and autophagy inhibitor further reduced the cell viability indicating that autophagy might be a survival pathway of CCE-treated SASVO3 cells. In contrast, CCE treatment for 12 days did not adversely affect SASVO3 tumor-bearing nude mice. CCE also elicited dose-dependent effects on the decrease in tumor volume, tumor weight, and Ki-67 expression. These results suggested that CCE showed the potential for the complementary treatment of oral caner.     

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death.     

PRKAA/AMPK restricts HBV replication through promotion of autophagic degradation

Adenosine monophosphate-activated protein kinase (AMPK) is a crucial energy sensor that maintains cellular energy homeostasis. AMPK plays a critical role in macroautophagy/autophagy, and autophagy facilitates hepatitis B virus (HBV) replication. To date, the intrinsic link among AMPK, autophagy and HBV production remains to be elucidated. Here, we demonstrate that PRKAA (a catalytic subunit of AMPK) is activated in response to HBV-induced oxidative stress, which in turn decreases the production of HBV. Mechanistic studies reveal that the autophagy machinery is associated with the inhibitory effect of PRKAA/AMPK on HBV production. Activation of PRKAA/AMPK promotes autolysosome-dependent degradation through stimulation of cellular ATP levels, which then leads to the depletion of autophagic vacuoles. Taken together, our data suggest that the activation of AMPK might be a stress response of host cells to restrict virus production through promotion of autophagic degradation. These findings therefore indicate that AMPK could provide a potential therapeutic target for HBV infection.     

DNA ploidy and autophagic protein degradation as determinants of hepatocellular growth and survival

Hepatocytes have the ability to go through specialized cell cycles, which, during normal developmental liver growth, result in the formation of binuclear and polyploid cells. In the adult rat liver, the majority of the hepatocytes (about 70%) are tetraploid, 15-20% are octoploid, and only 10-15% are diploid (about 50% in humans). One-third of the hepatocytes in either rats or humans are binuclear (with two diploid or two tetraploid nuclei). Among cultured rat hepatocytes stimulated with growth factors (EGF and insulin), one-half of the mitoses are of the binucleating type (suggesting a "quantal" mechanism), causing one-third of the postmitotic cells to become binuclear. In contrast, regenerative liver growth, induced by partial hepatectomy, is predominantly nonbinucleating. During rat liver carcinogenesis, the early populations of phenotypically altered cells (foci) are predominantly diploid, as are the later neoplastic nodules and carcinomas, which can be shown to have a regeneration-like, largely nonbinucleating growth pattern. A negative correlation between growth capacity and ploidy can be demonstrated in cultured hepatocytes, regenerating livers, neoplastic nodules, and hepatocellular carcinomas, suggesting that suppression of binucleation and polyploidization may carry a growth advantage, in addition to helping to maintain a large population of diploid, potential stem cells. Since a diploid genome is less protected against mutagenic change than a polyploid genome, diploid tumor cells may, furthermore, be more prone than polyploid cells to undergo mutation-based progression toward increasing malignancy. The ability of liver tumor promoters like 2-acetylaminofluorene, cyproterone acetate, -hexachlorocyclohexane and methylclofenapate to induce nonbinucleating hepatocyte growth may, therefore, cooperate with the selective growth stimulation of cancer cells and cancer cell precursors to promote liver carcinogenesis.Autophagy, a mechanism for the bulk degradation of cytoplasm, contributes to intracellular protein turnover and serves to restrict cellular growth. Rat liver carcinogenesis is accompanied by a progressive reduction of autophagic capacity, preneoplastic livers having 50% and hepatocellular carcinoma cells only 20% as much autophagy as normal hepatocytes. The ascites hepatoma cell line AH-130 has virtually no autophagy during logarithmic growth, but some autophagy is turned on when the cells become growth-arrested at high cell density. Ascitic fluid from AH-130 cells is able to completely inhibit autophagy in normal hepatocytes, suggesting that the cancer cells may improve their growth ability through an autocrine, autophagy-suppressive mechanism. Hepatocytes from preneoplastic livers similarly maintain a low autophagic activity under restrictive culture conditions, thereby surviving much better than normal hepatocytes, which switch on their autophagy. In the presence of an autophagy inhibitor (3-methyladenine), normal and preneoplastic hepatocytes survive equally well, testifying to the importance of autophagy as a determinant of cell survival and growth.     

Xanthine dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation

Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown. Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis. Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells. Similar results were obtained in XDH-knockout cells. Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells. Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH. Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo. In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: The cellular mechanism

Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti‐tumor properties. To better understand berberine‐induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97‐L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine‐induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3‐methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97‐L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97‐L cells through activation of Beclin‐1 and inhibition of the mTOR‐signaling pathway by suppressing the activity of Akt and up‐regulating P38 MAPK signaling. This is the first study to describe the role of Beclin‐1 activation and mTOR inhibition in berberine‐induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms. J. Cell. Biochem. 111: 1426–1436, 2010. © 2010 Wiley‐Liss, Inc.     

Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy

Autophagy is a catabolic process in which cell components are degraded to maintain cellular homeostasis by nutrient limitations. Defects of autophagy are involved in numerous diseases, including cancer. Here, we demonstrate a new role of phospholipase D (PLD) as a regulator of autophagy. PLD inhibition enhances autophagic flux via ATG1 (ULK1), ATG5 and ATG7, which are essential autophagy gene products critical for autophagosome formation. Moreover, PLD suppresses autophagy by differentially modulating phosphorylation of ULK1 mediated by mTOR and adenosine monophosphate-activated protein kinase (AMPK), and by suppressing the interaction of Beclin 1 with vacuolar-sorting protein 34 (Vps34), indicating that PLD coordinates major players of the autophagic pathway, AMPK-mTOR-ULK1 and Vps34/Beclin 1. Ultimately, PLD inhibition significantly sensitized in vitro and in vivo cancer regression via genetic and pharmacological inhibition of autophagy, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PLD inhibition. Collectively, we show a novel role for PLD in the molecular machinery regulating autophagy.