Number Of Autophagic Vacuoles Research Articles

P53-TIGAR axis attenuates mitophagy to exacerbate cardiac damage after ischemia

Inhibition of tumor suppressor p53 is cardioprotective against ischemic injury and provides resistance to subsequent cardiac remodeling. We investigated p53-mediated expansion of ischemic damage with a focus on mitochondrial integrity in association with autophagy and apoptosis. p53−/− heart showed that autophagic flux was promoted under ischemia without a change in cardiac tissue ATP content. Electron micrographs revealed that ischemic border zone in p53−/− mice had 5-fold greater numbers of autophagic vacuoles containing mitochondria, indicating the occurrence of mitophagy, with an apparent reduction of abnormal mitochondria compared with those in WT mice. Analysis of autophagic mediators acting downstream of p53 revealed that TIGAR (TP53-induced glycolysis and apoptosis regulator) was exclusively up-regulated in ischemic myocardium. TIGAR−/− mice exhibited the promotion of mitophagy followed by decrease of abnormal mitochondria and resistance to ischemic injury, consistent with the phenotype of p53−/− mice. In p53−/− and TIGAR−/− ischemic myocardium, ROS production was elevated and followed by Bnip3 activation which is an initiator of mitophagy. Furthermore, the activation of Bnip3 and mitophagy due to p53/TIGAR inhibition were reversed with antioxidant N-acetyl-cysteine, indicating that this adaptive response requires ROS signal. Inhibition of mitophagy using chloroquine in p53−/− or TIGAR−/− mice exacerbated accumulation of damaged mitochondria to the level of wild-type mice and attenuated cardioprotective action. These findings indicate that p53/TIGAR-mediated inhibition of myocyte mitophagy is responsible for impairment of mitochondrial integrity and subsequent apoptosis, the process of which is closely involved in p53-mediated ventricular remodeling after myocardial infarction.

Abstract 3789: Induction of autophagy increases the radiation sensitivity of head and neck cancer cells via growth arrest

Abstract Autophagy is frequently activated in cancer cells resistant to various apoptotic stimuli such as ionizing radiation, which supports the premise of enhancing the efficiency of radiotherapy by interventional means of autophagy regulation. In this study, we investigated whether autophagy pathway mediates the radio-sensitizing effect in head and neck cancer cells, and how the mechanism underlying the enhanced sensitivity works in cancer cells. The cellular responses following the ionizing radiation were examined by cell morphologic change, cell proliferation, clonogenic survival, cell cycle distribution, and expression level of several corresponding marker proteins in head and neck cancer HN-9 cells. Induction of autophagy was performed by down-regulating the mTOR signaling pathway using mTOR inhibitors such as rapamycin and PP242. Also, autophagy was inhibited by blocking the formation of autophagic vacuole (3-MA) or by disturbing the autophagosome maturation (bafilomycin A1, pepstatin A/E-46d). The change of autophagic flux was evidenced by the level of autophagy-related proteins and the morphological manifestation of autophagy. Upon exposure to radiation, the number of autophagic vacuoles was increased greatly in radiation dose- and time-dependent manners; however, the increase of apoptotic cells was not observed even in cells exposed to 12 Gy. In the meanwhile, the initial inhibition of cell cycle was maintained at slightly enhanced G1 phase. In some of the cases, it was restored to normal states, which implied that HN-9 cells adopted the mode of autophagy as a survival strategy against radiation. However, unexpectedly, further augmentation of autophagy pathway through the combination of either rapamycin or PP242 and radiation toward HN-9 cells hindered rather than enhanced clonogenic survival and cell proliferation, accompanying prolonged G1 arrest. Taken together, these results suggest that either induction of autophagy could serve as a therapeutic strategy to enhance radio-sensitivity, which is possibly due to irreversible cell cycle arrest following the excessive activation of autophagy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2011-3789

Abstract 5083: Nanoaerosol delivery of beclin1 combined with radiation suppressed tumor growth in the lungs of K-rasLA1 mice

Abstract As incidence and mortality due to lung cancer increase, great effort has been put into developing feasible therapeutics in medical field. Radiation, together with surgery and chemotherapy, is the must in treating lung cancer, however, side effects have to be taken into consideration. To minimize the side effect of radiotherapy, repeated exposures (5 times, 24 hours interval) with low dose (2Gy) of radiation to restricted area (thorax, 2cm) were attempted. After sensitizing lungs of K-rasLA1 mice with radiation, beclin1, known to be a tumor suppressor, involved in autophagy, complexed with nano-sized biodegradable poly(ester amine), was prepared and delivered into K-rasLA1 mice via aerosol 3 times for 4 weeks. Seventeen weeks old K-rasLA1 lung cancer model mice (n=20, 5 mice/group) were sacrificed and collected samples were analysed. K-rasLA1 mice treated with beclin1 and radiation showed significant tumor regression with low progression to adenocarcinoma and increment in number of autophagic vacuoles. Precedent event of beclin1-bcl2 dissociation stimulated synergistic anti-tumor effect via inhibition of protein translation and angiogenesis. Taken together, non-invasive treatment, radiation with PEA-beclin1 aerosol delivery, can be a feasible candidate for developing novel therapeutics in lung cancer treatment. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-2010-0000784). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5083. doi:10.1158/1538-7445.AM2011-5083

Abstract 3775: Autophagy in pancreatic cancer cells is mediated through a non-canonical Beclin-1 independent pathway

Abstract Background & Aims: Autophagy plays a dual role in cancer development. Recent data indicate that autophagy is oncosuppressive and impaired autophagy is emerging as the critical pro-onco “hallmark” of cancer cells. On the other hand, autophagy is a prosurvival mechanism, and its inhibition may increase cancer cell death and thus be beneficial. Beclin-1 plays a key role in mediating autophagy; its interaction with class III PI3K hVPS34 was shown to be necessary for the autophagosome formation in eukaryotic cells and Beclin-1 knockdown promotes tumorigenesis. Here we investigate the role of Beclin-1 in autophagy and apoptosis in pancreatic cancer cells. Methods: Pancreatic cancer (PaCa) cells PANC-1, and CAPAN-2 were subjected to amino-acid (AA) depletion or hypoxia, strong autophagy activators. Measuring autophagic marker LC3-II with western blot or with immunofluorescence assessed the number of autophagic vacuoles. Autophagy induction was measured in cells pretreated with NH4Cl or bafilomycin to block autophagy progression. Fusion of autophagosomes with lysosomes was assessed by co-localization of lysosomal marker LAMP-1 with LC3; the efficiency of autophagic flux by measuring the levels of p62, which is degraded through autophagy; apoptosis by DNA fragmentation, and caspase-3 activity. Results: AA depletion and hypoxia increased LC3-II both in the presence and absence of NH4Cl, indicating autophagy induction. AA depletion and hypoxia decreased p62 level, indicating stimulation of autophagic degradation. In cells treated with or without AA depletion or hypoxia Beclin-1 knockdown had no effect on either autophagic vacuole formation or p62 degradation. Similarly, the inhibition of Beclin-1 partner, hVPS 34 with siRNA or with PI3K inhibitor 3-methyladenine (3MA), decreased neither autophagy induction nor autophagic degradation. Finally, silencing of both Beclin-1 and its downstream mediator, Atg5 did not affect autophagy induction by AA depletion or hypoxia. All these data indicate that autophagy in PaCa cells is mediated through Beclin-1, Atg5, hVPS 34 -independent pathway. Of note, Beclin-1 knockdown increased co-localization of LC3 with LAMP-1 and p62 levels indicating that autophagy progression is delayed in the absence of Beclin-1. AA depletion and hypoxia moderately (∼by 30%) increased apoptosis in PaCa cells. Silencing of Beclin-1, hVPS 34, or Atg5 had no effect on apoptosis, in basal conditions as well as well as in cells subjected to AA depletion or hypoxia. Differently, 3MA stimulated apoptosis through Beclin-1-independent mechanism. Conclusions: In pancreatic cancer cells AA depletion and hypoxia activate autophagy through non-canonical pathway, which does not involve Beclin-1. Silencing of the key mediators of canonical autophagy, Beclin-1, Atg5 and hVPS34 had no effect on autophagy and apoptosis, whereas 3MA stimulated apoptosis through autophagy-independent pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2011-3775

Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson's disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes.

Autophagy inhibition enhances etoposide-induced cell death in human hepatoma G2 cells

Induction of autophagy usually acts as a survival mechanism of cancer cells in response to chemotherapy. However, the function and molecular mechanism of autophagy in human hepatoma cells under drug treatment is still not clear. To address this issue, we established an experimental model in which HepG2 cells were treated with etoposide, a widely used anticancer agent. We demonstrate the etoposide-induced accumulation of GFP-LC3 dots by fluorescent microscopy, the up-regulation of LC3-II protein expression by Western blotting and the increased number of autophagic vacuoles by electron microscopy, confirming the activation of autophagy by etoposide in HepG2 cells. Inhibition of autophagy by either 3-methyladenine (3MA) or beclin-1 small interfering RNA enhanced etoposide-induced cell death. Furthermore, activation of p53 and AMPK was detected in etoposide-treated cells and inhibition of AMPK triggered apoptosis through suppression of autophagy. On the other hand, inactivation of p53 promoted cell survival through augmentation of autophagy. Collectively, these findings indicate that etoposide-induced autophagy promotes hepatoma cell adaptation and survival, and that autophagy inhibition improves the chemotherapeutic effect of etoposide. Moreover, AMPK activation is clearly associated with etoposide-induced autophagy. We conclude that manipulation of AMPK may be a promising approach of adjuvant chemotherapy for hepatocellular carcinoma.

Insufficient Activation of Autophagy Allows Cellular Damage to Accumulate in Critically Ill Patients

Abstract Context: Responses to critical illness, such as excessive inflammation and hyperglycemia, may trigger detrimental chain reactions that damage cellular proteins and organelles. Such responses to illness contribute to the risk of (nonresolving) multiple organ dysfunction and adverse outcome. Objective: We studied autophagy as a bulk degradation pathway able to remove toxic protein aggregates and damaged organelles and how these are affected by preventing hyperglycemia with insulin during critical illness. Design and Setting: Patients participated in a randomized study, conducted at a university hospital surgical/medical intensive care unit. Patients: We studied adult prolonged critically ill patients vs. controls. Interventions: Tolerating excessive hyperglycemia was compared with intensive insulin therapy targeting normoglycemia. Main Outcome Measures: We quantified (ultra)structural abnormalities and hepatic and skeletal muscle protein levels of key players in autophagy. Results: Morphologically, both liver and muscle revealed an autophagy-deficiency phenotype. Proteins involved in initiation and elongation steps of autophagy were induced 1.3- to 6.5-fold by critical illness (P &amp;lt; 0.01), but mature autophagic vacuole formation was 62% impaired (P = 0.05) and proteins normally degraded by autophagy accumulated up to 97-fold (P &amp;lt; 0.03). Mitophagy markers were unaltered or down-regulated (P = 0.05). Although insulin preserved hepatocytic mitochondrial integrity (P = 0.05), it further reduced the number of autophagic vacuoles by 80% (P = 0.05). Conclusions: Insufficient autophagy in prolonged critical illness may cause inadequate removal of damaged proteins and mitochondria. Such incomplete clearance of cellular damage, inflicted by illness and aggravated by hyperglycemia, could explain lack of recovery from organ failure in prolonged critically ill patients. These data open perspectives for therapies that activate autophagy during critical illness.

Insufficient Activation of Autophagy Allows Cellular Damage to Accumulate in Critically Ill Patients

Responses to critical illness, such as excessive inflammation and hyperglycemia, may trigger detrimental chain reactions that damage cellular proteins and organelles. Such responses to illness contribute to the risk of (nonresolving) multiple organ dysfunction and adverse outcome. We studied autophagy as a bulk degradation pathway able to remove toxic protein aggregates and damaged organelles and how these are affected by preventing hyperglycemia with insulin during critical illness. Patients participated in a randomized study, conducted at a university hospital surgical/medical intensive care unit. We studied adult prolonged critically ill patients vs. controls. Tolerating excessive hyperglycemia was compared with intensive insulin therapy targeting normoglycemia. We quantified (ultra)structural abnormalities and hepatic and skeletal muscle protein levels of key players in autophagy. Morphologically, both liver and muscle revealed an autophagy-deficiency phenotype. Proteins involved in initiation and elongation steps of autophagy were induced 1.3- to 6.5-fold by critical illness (P ≤ 0.01), but mature autophagic vacuole formation was 62% impaired (P = 0.05) and proteins normally degraded by autophagy accumulated up to 97-fold (P ≤ 0.03). Mitophagy markers were unaltered or down-regulated (P = 0.05). Although insulin preserved hepatocytic mitochondrial integrity (P = 0.05), it further reduced the number of autophagic vacuoles by 80% (P = 0.05). Insufficient autophagy in prolonged critical illness may cause inadequate removal of damaged proteins and mitochondria. Such incomplete clearance of cellular damage, inflicted by illness and aggravated by hyperglycemia, could explain lack of recovery from organ failure in prolonged critically ill patients. These data open perspectives for therapies that activate autophagy during critical illness.

Autophagy Is Constitutively Active in Normal Mouse Sino-Atrial Nodal Cells

This study was designed to examine the autophagy in sino-atrial (SA) nodal cells from the normal adult mouse heart. Autophagy is the cellular process responsible for the degradation and recycling of long-lived and/or damaged cytoplasmic components by lysosomal digestion. In the heart, autophagy is known to occur at a low level under physiological conditions, but to become upregulated when cells are exposed to certain stresses, such as ischemia. We examined whether the basal level of autophagy in SA nodal cells was different from that in ventricular or atrial myocytes. An ultrastructural analysis revealed that the SA nodal cells contained a number of autophagic vacuoles (autophagosomes) with various stages of degradation by lysosomal digestion, whereas the number of those in ventricular or atrial myocytes was either negligible or very small. The immunostaining of autophagosome marker microtubule-associated protein 1 light chain 3 (LC3) and lysosome marker lysosome-associated membrane protein 1 (LAMP1) indicated that the content of both autophagosomes and lysosomes were much greater in SA nodal cells than in ordinary cardiomyocytes. Our results provide evidence that the autophagy is active in normal SA nodal cells, which is not a stress-activated process but a constitutive event in the mouse heart.

Morphological analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most common cause of autosomal recessive Parkinson’s disease. The PINK1 protein has a mitochondrial localization and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. In particular, PINK1 is able to recruit parkin to the surface of dysfunctional mitochondria, to promote the ubiquitination of several mitochondrial proteins and the subsequent activation of the mitophagy cascade. Aim of this study was to use a dopaminergic cell model and transmission electron microscopy to characterize whether the modulation of PINK1 expression: (i) modifies the number and morphology of mitochondria and of autophagy organelles (autophagosomes); (ii) alters the recruitment of beclin1, parkin and ubiquitin to the mitochondria; (iii) affects cell survival. We used PC12 cells transfected either with the empty vector (pcDNA), or vectors expressing wild type PINK1 (PINK1wt), a pathogenic mutant (PINK1 W437X ), shRNA against rat PINK1 (shPINK1) or scramble (shSCR). Samples were analyzed both in baseline conditions and following methamphetamine (METH) treatment to provide a neurotoxic, autophagy-dependent stimulation. We showed that, especially upon METH exposure, the modulation of PINK1 levels dramatically affected the morphology and clearance of mitochondria. In fact, the number of abnormal mitochondria was reduced in PINK1wt, while it was significantly increased upon shPINK1 and also, to a lesser extent, in PINK1 W437X cells. In keeping with this, mitochondrial ubiquitin clusters and mitochondrial levels of parkin and beclin1 were increased in PINK1wt cells while they were reduced both in PINK1 silenced and PINK1 W437X cells. Interestingly, the number of autophagic vacuoles was unaffected by PINK1 modulation in baseline conditions, and was significantly reduced only in cells lacking functional PINK1 and upon METH exposure. All these effects were significantly associated with a modulation of apoptotic cell death. Our data provide robust sub-cellular evidence that PINK1 counteracts neurodegeneration by simultaneously recruiting beclin1, parkin and ubiquitin and thus enhancing the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observed a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase in apoptotic cell death.

Propofol protects the autophagic cell death induced by the ischemia/reperfusion injury in rats.

Autophagy has been implicated in cardiac cell death during ischemia/reperfusion (I/R). In this study we investigated how propofol, an antioxidant widely used for anesthesia, affects the autophagic cell death induced by the myocardial I/R injury. The infarction size in the myocardium was dramatically reduced in rats treated with propofol during I/R compared with untreated rats. A large number of autophagic vacuoles were observed in the cardiomyocytes of I/R-injured rats but rarely in I/R-injured rats treated with propofol. While LC3-II formation, an autophagy marker, was up-regulated in the I/R-injured myocardium, it was significantly down-regulated in the myocardial tissues of I/R-injured and propofol-treated rats. Moreover, propofol inhibited the I/R-induced expression of Beclin-1, and it accelerated phosphorylation of mTOR during I/R and Beclin-1/Bcl-2 interaction in cells, which indicates that it facilitates the inhibitory pathway of autophagy. These data suggest that propofol protects the autophagic cell death induced by the myocardial I/R injury.

Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in one in 2000 Americans. The Z mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes and chronic liver injury. Autophagy is one of several proteolytic mechanisms activated to cope with this hepatocellular protein burden, and is likely important in disposal of the unique polymerized conformation of the a1AT mutant Z protein, which is thought to be especially injurious to the cell. Recent data indicate that rapamycin may more efficiently upregulate autophagy when given in weekly dose pulses, as compared with a daily regimen. Therefore, we evaluated the effect of rapamycin on PiZ mice, a well-characterized model which recapitulates human a1AT liver disease. Daily dosing had no effect on autophagy, on accumulation of a1AT mutant Z protein or on liver injury. Weekly dosing of rapamycin did increase autophagic activity, as shown by increased numbers of autophagic vacuoles. This was associated with reduction in the intrahepatic accumulation of a1AT mutant Z protein in the polymerized conformation. Markers of hepatocellular injury, including cleavage of caspase 12 and hepatic fibrosis, were also decreased. In conclusion, this is the first report of a successful in vivo method for reduction of intrahepatic a1AT mutant Z polymerized protein. Application of this finding may be therapeutic in patients with a1AT deficiency by reducing the intracellular burden of the polymerized, mutant Z protein and by reducing the progression of liver injury.

Enhancement of Autophagy during Lytic Replication by the Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Activator

Autophagy is one of two major degradation systems in eukaryotic cells. The degradation mechanism of autophagy is required to maintain the balance between the biosynthetic and catabolic processes and also contributes to defense against invading pathogens. Recent studies suggest that a number of viruses can evade or subvert the host cell autophagic pathway to enhance their own replication. Here, we investigated the effect of autophagy on the KSHV (Kaposi's sarcoma-associated herpesvirus) life cycle. We found that the inhibition of autophagy reduces KSHV lytic reactivation from latency, and an enhancement of autophagy can be detected during KSHV lytic replication. In addition, RTA (replication and transcription activator), an essential viral protein for KSHV lytic reactivation, is able to enhance the autophagic process, leading to an increase in the number of autophagic vacuoles, an increase in the level of the lipidated LC3 protein, and the formation of autolysosomes. Moreover, the inhibition of autophagy affects RTA-mediated lytic gene expression and viral DNA replication. These results suggest that RTA increases autophagy activation to facilitate KSHV lytic replication. This is the first report demonstrating that autophagy is involved in the lytic reactivation of KSHV.

Transcription Factor GATA4 Inhibits Doxorubicin-induced Autophagy and Cardiomyocyte Death

Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure. The transcription factor GATA4 antagonizes DOX-induced cardiotoxicity. However, the protective mechanism remains obscure. Autophagy is the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles, and its activation could be either protective or detrimental depending on specific pathophysiological conditions. Here we investigated the ability of GATA4 to inhibit autophagy as a potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat cardiomyocytes. DOX markedly increased autophagic flux in cardiomyocytes as indicated by the difference in protein levels of LC3-II (microtubule-associated protein light chain 3 form 2) or numbers of autophagic vacuoles in the absence and presence of the lysosomal inhibitor bafilomycin A1. DOX-induced cardiomyocyte death determined by multiple assays was aggravated by a drug or genetic approach that activates autophagy, but it was attenuated by manipulations that inhibit autophagy, suggesting that autophagy contributes to DOX cardiotoxicity. DOX treatment depleted GATA4 protein levels, which predisposed cardiomyocytes to DOX toxicity. Indeed, GATA4 gene silencing triggered autophagy that rendered DOX more toxic, whereas GATA4 overexpression inhibited DOX-induced autophagy, reducing cardiomyocyte death. Mechanistically, GATA4 up-regulated gene expression of the survival factor Bcl2 and suppressed DOX-induced activation of autophagy-related genes, which may likely be responsible for the anti-apoptotic and anti-autophagic effects of GATA4. Together, these findings suggest that activation of autophagy mediates DOX cardiotoxicity, and preservation of GATA4 attenuates DOX cardiotoxicity by inhibiting autophagy through modulation of the expression of Bcl2 and autophagy-related genes.

Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy

To study the antitumor effect of matrine in human hepatoma G2 (HepG2) cells and its molecular mechanism involved in antineoplastic activities. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect viability of HepG2 cells. The effect of matrine on cell cycle was detected by fl ow cytometry. Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under an inverted phase contrast microscope. Transmission electron microscopy was performed to further examine ultrastructural structure of the cells treated with matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy. Whether autophagy is blocked by 3-methyladenine (3-MA), an autophagy inhibitor, was evaluated. Expression levels of Bax and Beclin 1 in HepG2 cells were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Matrine significantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner, and induced G1-phase cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner. The total apoptosis rate was 0.14% for HepG2 cells not treated with matrine. In contrast, the apoptosis rate was 28.91%, 34.36% and 38.80%, respectively, for HepG2 cells treated with matrine at the concentration of 0.5, 1.0 and 2.0 mg/mL. The remarkable morphological changes were observed under an inverted phase contrast microscope. Abundant cytoplasmic vacuoles with varying sizes were observed in HepG2 cells treated with matrine. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in HepG2 cells after matrine treatment. When the specific autophagic inhibitor, 3-MA, was applied, the number of autophagic vacuoles greatly decreased. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in HepG2 cells was greater in matrine treatment group than in control group. Fewer autophagic vacuoles were observed in the combined 3-MA and matrine treatment group when 3-MA was added before matrine treatment, indicating that both autophagy and apoptosis are activated when matrine-induced death of hepatoma G2 cells occurs. Real-time quantitative RT-PCR revealed that the expression levels of Bax gene, an apoptosis-related molecule, and Beclin 1 gene which plays a key role in autophagy were higher in matrine treatment group than in control group, indicating that Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanism of matrine may be related to its upregulation of Bax expression. Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma.

Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P < 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy

Liver pathology induced by chemotherapy (steatosis or vascular injury) is known to increase the liver's sensitivity to ischemia/ reperfusion (I/R) injury, thereby increasing morbidity and mortality after liver resection. Our aim was to assess whether ischemic preconditioning (IP) reduces I/R injury to livers with chemotherapy-induced pathology. We analyzed a series of livers from patients treated with chemotherapy for colorectal cancer who underwent IP (n=30) or not (n=31) before hepatectomy. All but one of the livers exhibited chemotherapy-induced steatosis and/ or peliosis before the I/R insult. Necrosis was less frequent (p=0.038) in livers with IP than in the others. IP had no influence on apoptosis as assessed by terminal transferase uridyl nick-end labeling (TUNEL) assay or caspase-3, -8 and -9 expression. IP induced a twofold increase in B-cell leukemia/ lymphoma 2 (Bcl-2; p<0.05), which was localized to hepatocytes of centrolobular and peliotic areas and colocalized with the autophagy protein beclin-1 in livers with IP, suggesting their coordinated role in autophagy. Increased expression of the phosphorylated Bcl-2 was observed in preconditioned livers and was associated with a decreased immunoprecipitation of beclin-1 and the increased expression of light chain 3 type II (LC3-II). The increased number of autophagic vacuoles seen by electron microscopy confirmed an association of autophagy in chemotherapy-injured livers following IP. However, the differences in protein expression were not reflected in postresection liver-injury tests or measure of patient morbidity. IP is associated with a reduction in necrosis of hepatocytes already damaged by chemotherapy and an activation of autophagy. Bcl-2 and beclin-1 could be major targets in the regulation of cell death during I/R injury.

Adult triclabendazole-resistantFasciola hepatica: morphological changes in the tegument and gut followingin vivotreatment with artemether in the rat model

A study has been carried out to determine the morphological changes to the adult liver fluke, Fasciola hepatica after treatment in vivo with artemether. Rats were infected with the triclabendazole-resistant Sligo isolate of F. hepatica, dosed orally with artemether at a concentration of 200 mg/kg and flukes recovered at 24, 48 and 72 h post-treatment (p.t.). Surface changes were monitored by scanning electron microscopy and fine structural changes to the tegument and gut by transmission electron microscopy. Twenty-four hours p.t., the external surface showed minor disruption, in the form of mild swelling of the tegument. The tegumental syncytium and sub-tegumental tissues appeared relatively normal. Forty-eight and seventy-two hours p.t., disruption to the tegumental system increased, with isolated patches of surface blebbing and reduced production of secretory bodies by the tegumental cells being the main changes seen. The gastrodermal cells showed a relatively normal morphology 24 h p.t. By 48 h, large numbers of autophagic vacuoles and lipid droplets were present. Autophagy increased in magnitude by 72 h p.t. and substantial disruption to the granular endoplasmic reticulum was observed. Results from this study show that flukes treated in vivo with artemether display progressive and time-dependent alterations to the tegument and gut. Disruption to the gut was consistently and substantially more severe than that to the tegument, suggesting that an oral route of uptake for this compound predominates. This is the first study providing ultrastructural information on the effect of an artemisinin compound against liver fluke.

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35, Aβ1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.

Linking of Autophagy to Ubiquitin-Proteasome System Is Important for the Regulation of Endoplasmic Reticulum Stress and Cell Viability

Two major protein degradation systems exist in cells, the ubiquitin proteasome system and the autophagy machinery. Here, we investigated the functional relationship of the two systems and the underlying mechanisms. Proteasome inhibition activated autophagy, suggesting that the two are functionally coupled. Autophagy played a compensatory role as suppression of autophagy promoted the accumulation of polyubiquitinated protein aggregates. Autophagy was likely activated in response to endoplasmic reticulum stress caused by misfolded proteins during proteasome inhibition. Suppression of a major unfolded protein response pathway mediated by IRE1 by either gene deletion or RNA interference dramatically suppressed the activation of autophagy by proteasome inhibitors. Interestingly, c-Jun NH(2)-terminal kinase (JNK) but not XBP-1, both of which are the known downstream targets of IRE1, seemed to participate in autophagy induction by proteasome inhibitors. Finally, proteasome inhibitor-induced autophagy was important for controlling endoplasmic reticulum stress and reducing cell death in cancer cells. Our studies thus provide a mechanistic view and elucidate the functional significance of the link between the two protein degradation systems.