Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42345 (mTOR)
 26,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.
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PMID:HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death. 1904 21

Safingol, the synthetic L-threo-stereoisomer of endogenous (D-erythro-) sphinganine, is an inhibitor of protein kinase C and sphingosine kinase in vitro, and in some cell types has been implicated in ceramide generation and induction of apoptosis. Utilizing electron microscopy, acridine orange staining, and immunoblot and fluorescent localization studies of the myosin light chain-associated protein (LC3), we determined that safingol induces cell death of an exclusively autophagic character and lacking any of the hallmarks of apoptosis. Safingol inhibited PKCbeta-I, PKC delta and PKC epsilon, and inhibited phosphorylation of critical components of the PI3k/Akt/mTOR pathway (Akt, p70S6k and rS6) and the MAPk pathway (ERK). Inhibition of PI3k with LY294002 or suppression of PKC delta and PKC epsilon with siRNA in HCT-116 cells induced autophagy, though not to the extent caused by safingol. Conversely, activation of PKCs with phorbol 12,13-dibutyrate (PDBu) or transient transfection of a constitutively active form of Akt each reduced safingol's autophagic induction, but not completely, indicating that Akt- and PKC-dependent pathways both contribute partially and independently to safingol-induced autophagy. Accordingly, combining siRNA depletion of PKC epsilon with LY294002 inhibition of PI3k induced autophagy to a degree comparable to safingol. Liquid chromatography, electrospray tandem mass spectrometry analysis indicated that safingol did not elevate levels of any endogenous sphingolipids previously shown to induce autophagy (ceramide, sphingosine-1-phosphate and dihydroceramide); therefore, these effects may be due to safingol per se or another metabolite. Thus, our studies establish that safingol induces autophagy through inhibition of PKCs and PI3k by safingol directly rather than via changes in endogenous sphingolipids.
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PMID:Safingol (L-threo-sphinganine) induces autophagy in solid tumor cells through inhibition of PKC and the PI3-kinase pathway. 1909 47

Glycosylated antitumor ether lipids (GAELs) have superior anticancer properties relative to the alkyllysophospholipid class, but there have been no studies of the mechanisms of these compounds. The prototype GAEL, 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-beta-D-glucopyranosyl)-sn-glycerol (Gln), effectively killed mouse embryonic fibroblasts (MEFs) lacking key molecules involved in caspase-dependent apoptosis, and cell death was not prevented by caspase inhibitors. Gln did not cause a loss of mitochondrial membrane potential, even in rounded-up dying cells. Gln stimulated the appearance and accumulation of LC3-II, a protein marker for autophagy, in a variety of cells, including wild-type MEFs, but not in MEFs lacking ATG5, a key protein required for autophagy. Gln induced LC3 puncta formation in Chinese hamster ovary cells stably expressing a LC3-green fluorescent protein fusion protein. Thus, Gln appears to induce autophagy. Autophagy was mTOR-independent and was not inhibited by 3-methyladenine or wortmannin. Although Gln is toxic, cellular ability to undergo autophagy was not essential for its toxicity. Furthermore, the GAEL analog 2-deoxy-C-Glc induced LC3 puncta formation but did not kill the cells. Gln, but not 2-deoxy-C-Glc, caused the accumulation of cytoplasmic acidic vacuoles in the cells. Our data suggest that GAELs may activate autophagy; however, GAELs do not kill cells by apoptosis or autophagy but rather by a paraptosis-like cell death mechanism.
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PMID:A glycosylated antitumor ether lipid kills cells via paraptosis-like cell death. 1937 58

Dopamine at 100-500 microM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1-2h after treatment we found an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1alpha and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12h of 500muM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6h of treatment with both 100 and 500 microM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.
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PMID:Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model. 1941 Jun 1

The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age-related neurodegenerative disorders, including Parkinson's disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ-1 have been reported to be associated with autosomal recessive, early-onset parkinsonism. In this paper we established a model system to study the involvement of the DJ-1 protein in paraquat-induced autophagy. When human neuroblastoma SH-SY5Y cells were transfected with DJ-1-specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat-induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3-methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ-1 in the autophagic response produced by paraquat, providing evidence for the role of PD-related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.
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PMID:Silencing DJ-1 reveals its contribution in paraquat-induced autophagy. 1942 77

Sphingosine 1-phosphate (S1P) is a platelet- and endothelial cell-released lysophospholipid that regulates various cellular functions through activating a specific family of G protein-coupled receptors. Both platelet activation and angiogenesis play important roles in cancer development, implying that cancer cells might encounter a large amount of S1P during these processes. Cancer cells, in the meantime, may experience nutrient deprivation and rely on autophagy for early development. Whether extracellular S1P regulates autophagy remains to be tested. In the present work, we investigated whether autophagy is regulated by S1P in PC-3 cells. Through monitoring the modification patterns of LC3 by Western blotting, we demonstrated that autophagy was induced by exogenously applied S1P in PC-3 cells. This observation was further confirmed by fluorescence microscopy using PC-3 cells stably expressing enhanced green fluorescent protein-LC3. By applying small interfering RNA and dihydro-S1P, S1P(5) activation was found to be involved in this process. Besides, mammalian target of rapamycin signaling was inhibited upon S1P treatment. Taken together, our results suggest that, under serum-starved conditions, S1P further upregulates autophagic activity through S1P(5)-dependent pathways in PC-3 cells.
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PMID:S1P(5) is required for sphingosine 1-phosphate-induced autophagy in human prostate cancer PC-3 cells. 1947 91

Enterovirus 71 (EV71) is an important pathogen causing death in children under 5 years old worldwide. However, the underlying pathogenesis remains unclear. This study reveals that EV71 infection in rhabdomyosarcoma (RD) and neuroblastoma (SK-N-SH) cells stimulated the autophagic process, which was demonstrated by an increase of punctate GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3), the level of autophagosome-bound LC3-II protein and double-membrane autophagosome formation. EV71-induced autophagy benefited EV71 replication, which was confirmed by the autophagic inducer rapamycin and the inhibitor 3-methyladenine. Signaling pathway investigation revealed that the decreased expression of phosphorylated mTOR and phosphorylated p70S6K is involved in EV71-induced autophagy in a cell-specific manner. The expression of phosphorylated extracellular signal-regulated kinase (Erk) was suppressed consistently in EV71-infected cells. However it did not participate in the autophagic response of the cell. Other signaling pathway molecules, such as Erk, PI3K/Akt, Bcl-2, BNIP3, and Beclin-1 were not affected by infection with EV71. Electron microscopy showed co-localization of autophagosome-like vesicles with either EV71-VP1 or LC3 protein in neurons of the cervical spinal cord in ICR mice infected with EV71. In conclusion, EV71 infection triggered autophagic flux and induced autophagosome formation both in vitro and in vivo. Autophagy induced by EV71 is beneficial for viral replication. Understanding the role of autophagy induced by EV71 in vitro and the formation of autophagosome-like vesicle in vivo provide new insights into the pathogenesis of EV71 infection.
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PMID:Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication. 1947 21

Continuous macroautophagic activity is critical for the maintenance of neuronal homeostasis; however, unchecked or dysregulated autophagy can lead to cell death. Cultured Purkinje neurons die by an autophagy-associated cell death mechanism when deprived of trophic support. Here, we report that insulin-like growth factor-I (IGF-I) completely blocked the autophagy-associated cell death of Purkinje neurons. To examine the mechanism by which IGF-I influences autophagy, neurons were infected with adeno-RFP-LC3 and subjected to trophic factor withdrawal, and the size and number of autophagosomes were analyzed by live-cell fluorescence imaging. In control neurons, autophagy occurred at a constitutive low level with most autophagosomes measuring less than 0.75 microm. Trophic factor withdrawal increased the number and size of autophagosomes with most autophagosomes ranging between 0.75 and 1.5 microm and some reaching 1.5-2.25 microm. IGF-I added at the time of trophic factor withdrawal prevented the accumulation of the larger autophagosomes; however, it had no effect on the conversion of LC3, an indicator of autophagy induction. Instead, the rate of autophagosome-to-lysosome fusion measured by colocalization of RFP-LC3 and LysoSensor Green was accelerated by IGF-I. Treating the neurons with bafilomycin A(1) in the presence of IGF-I led to the accumulation of autophagosomes even larger than those induced by trophic factor withdrawal alone, indicating that IGF-I regulates autophagic vesicle turnover. Finally, the effect of IGF-I on autophagy was mediated by an Akt/mTOR-de pend ent and an ERK-independent pathway. These data suggest a novel role for IGF-I in protecting Purkinje neurons from autophagy-associated cell death by increasing autophagy efficiency downstream of autophagy induction.
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PMID:Insulin-like growth factor-I prevents the accumulation of autophagic vesicles and cell death in Purkinje neurons by increasing the rate of autophagosome-to-lysosome fusion and degradation. 1950 89

Autophagy is a membrane process that results in the transporting of cellular contents to lysosomes for degradation. Autophagic cell death is another way of programed cell death called type II PCD, which has complicated connection with apoptosis, both of these two types of cell death play an important role in tumor development. In this study, we investigated chemotherapeutic agent induced cell death pathway in wild type (WT), Bax(-/-) and PUMA(-/-) HCT116 cells. Bax or PUMA deficient cells had similar chemosensitivity to WT cells but were defective in undergoing apoptosis. The results of electron microscopy and GFP-LC3 localization assay showed that autophagy was induced in Bax or PUMA deficient cells but not in WT cells. mTOR activity was decreased in Bax or PUMA deficient cells which further indicated the up-regulation of autophagy. Inhibition of autophagy by 3-Methyladenine (3-MA) decreased the cell death in Bax or PUMA deficient cells. Taken together, these results suggest that autophagic cell death can be used as an alternative cell death pathway in apoptosis defective cells and may bring a new target for cancer therapy.
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PMID:Autophagic cell death induced by 5-FU in Bax or PUMA deficient human colon cancer cell. 1966 Aug 60

Recent evidence suggests that autophagy plays a role in oxidative injury-induced cell death. Here we examined whether glutamate-mediated oxidative toxicity induces autophagy in murine hippocampal HT22 cells and if autophagy induction affects the molecular events associated with cell death. Markers for autophagy induction including LC3 conversion, suppression of mTOR pathway, and GFP-LC3 dot formation were enhanced by glutamate treatment. By contrast, autophagy inhibition blocked glutamate-induced LC3 conversion and consequently reduced cell death. Activation of ERK1/2, a hallmark of glutamate-induced cytotoxicity, was also decreased by autophagy inhibition. Interestingly, autophagy inhibition also affected the expression of chaperones including Hsp60 and Hsp70, which are differentially regulated during HT22 cell death. Conversely, knock-down of Hsp60 greatly decreased LC3 conversion. Together these results suggest that glutamate-induced cytotoxicity involves autophagic cell death and chaperones may play a role in this process.
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PMID:Activation of autophagy during glutamate-induced HT22 cell death. 1966 9


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