c-myc induces autophagy in rat 3Y1 fibroblast cells

The proto-oncogene c-myc is a multifunctional gene that regulates cell division, cell growth, and apoptosis. Here we report a new function of c-myc: induction of autophagy. Autophagy is a bulk degradation system for intracellular proteins. Autophagy proceeds with characteristic morphologies, which begins with the formation of a double-membrane structure called the autophagosome surrounding a portion of the cytoplasm, after which its outer membrane then fuses with the lysosomal membrane to become an autolysosome. Autophagosomes and autolysosomes are generally called autophagic vacuoles. When c-Myc protein was overexpressed in rat 3Y1 fibroblasts or when the chimeric protein c-MycER was activated by estrogen, the number of autophagic vacuoles in cells increased significantly. The formation of autophagic vacuoles induced by c-Myc was completely blocked by a specific inhibitor of autophagosome formation, 3-methyladenine. A c-Myc mutant lacking Myc Box II induced neither apoptosis nor oncogenic transformation, but still stimulated autophagy. An inhibitor of caspases suppressed apoptosis but not autophagy. These results suggest that the autophagy caused by c-myc is not due to the apoptosis or tumorigenesis induced by c-myc. Taken together, our results suggest that the induction of autophagy is a novel function of c-myc.     

Inhibition of autophagic vacuole formation and protein degradation by amino acids in isolated hepatocytes

An amino acid mixture, specifically developed to suppress endogenous protein degradation in isolated hepatocytes, inhibited lysosornal (propylamine-sensitive) protein degradation by 70–75% and reduced the cytoplasmic volume fraction of the autophagic/lysosomal compartment to a similar extent. Incubation with the amino acid mixture for 1 h reduced the subcompartment of early autophagic vacuoles by 95%. These results support the hypothesis that autophagy is the major route of delivery of endogenous proteins to the lysosomes, and that amino acids exert their regulatory function on protein degradation by controlling the sequestration step of autophagy.     

Analysis of autophagy in Aspergillus oryzae by disruption of Aoatg13, Aoatg4, and Aoatg15 genes

Autophagy is a degradation system in which cellular components are digested via vacuoles/lysosomes, and involved in differentiation in addition to helping cells to survive starvation. The autophagic process is composed of several steps: induction of autophagy, formation of autophagosomes, transportation to vacuoles, and degradation of autophagic bodies. To further understand autophagy in the filamentous fungus Aspergillus oryzae, we first constructed A. oryzae mutants defective for the Aoatg13, Aoatg4, and Aoatg15 genes and examined the resulting phenotypes. The ΔAoatg13 mutant developed conidiophores and conidia, although the number of conidia was decreased compared with the wild-type strain, while conidiation in the ΔAoatg4 and ΔAoatg15 mutants was not detected. The ΔAoatg15 mutants displayed a marked reduction of development of aerial hyphae. Moreover, autophagy in these mutants was examined by observation of the behavior of enhanced green fluorescent protein (EGFP)-AoAtg8. In the ΔAoatg13 mutant, the slight accumulation of EGFP-AoAtg8 in vacuoles, preautophagosomal structures (PAS), and autophagosomes was observed, whereas only PAS-like structures were detected in the ΔAoatg4 mutant. In the ΔAoatg15 mutant, autophagic bodies accumulated in vacuoles, suggesting that the uptake process proceeded. We therefore propose that the level of autophagy is closely correlated with the degree of differentiation in A. oryzae.     

Activation of autophagy of aggregation-prone ubiquitinated proteins by timosaponin A-III

Chemical modulators of autophagy provide useful pharmacological tools for examination of autophagic processes, and also may lead to new therapeutic agents for diseases in which control of cellular sequestration and degradation capacity are beneficial. We have identified that timosaponin A-III (TAIII), a medicinal saponin reported to exhibit anticancer properties and improve brain function, is a pronounced activator of autophagy. In this work, the salient features and functional role of TAIII-induced autophagy were investigated. In TAIII-treated cells, autophagic flux with increased formation of autophagosomes and conversion into autolysosomes is induced in association with inhibition of mammalian target of rapamycin activity and elevation of cytosolic free calcium. The TAIII-induced autophagy is distinct from conventional induction by rapamycin, exhibiting large autophagic vacuoles that appear to contain significant contents of endosomal membranes and multivesicular bodies. Furthermore, TAIII stimulates biosynthesis of cholesterol, which is incorporated to the autophagic vacuole membranes. The TAIII-induced autophagic vacuoles capture ubiquitinated proteins, and in proteasome-inhibited cells TAIII promotes autophagy of aggregation-prone ubiquitinated proteins. Our studies demonstrate that TAIII induced a distinct form of autophagy, and one of its pharmacological actions is likely to enhance the cellular quality control capacity via autophagic clearance of otherwise accumulated ubiquitinated protein aggregates.     

Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi.     

The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells

The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 (Atg7) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood.     

Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components

alpha-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti-alpha-amylase antibody showed that alpha-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

GFP‐LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP‐LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP‐LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP‐Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin‐positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP‐LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP‐LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP‐LC3, a fusion protein widely used as a marker for autophagic vesicles and pre‐autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley‐Liss, Inc.     

Intracellular protein degradation and autophagy in isolated pancreatic acini of the rat

Simultaneous investigation of protein degradation and autophagy of isolated exocrine pancreatic cells is carried out here for the first time in a systematic way by a complex biochemical, morphological and morphometrical approach. Protein degradation proceeds with a decreasing rate of 4-1.5 per cent per h over a 4-h period indicating a comparatively low degradation capacity. Cells in freshly isolated acini do not contain autophagic vacuoles but the latter appear within an hour in vitro and their quantity remains close to a steady state during the subsequent 3 h. Both traditional inhibitors of the autophagic-lysosomal pathway, e.g. vinblastine, leupeptin, and lysosomotropic amines together with the recently introduced 3-methyladenine, inhibit degradation to a similar maximal extent, offering the possibility of the estimation of the ratio of lysosomal/non-lysosomal degradation. In pancreatic acinar cells autophagic sequestration is unaffected and protein degradation is inhibited inside secondary lysosomes by leupeptin and lysosomotropic amines, while 3-methyladenine prevents the formation of autophagosomes. Vinblastine seems to act by inhibiting the fusion of autophagosomes with lysosomes and there is no evidence for the stimulation of autophagic sequestration by vinblastine in the present system. The effect of inhibitors of protein breakdown on protein synthesis is variable and does not correlate with their influence on degradation. Amino acids strongly stimulate protein synthesis, but in contrast to what is found in liver cells, they do not seem to affect protein degradation or autophagy significantly, thus indicating major regulatory differences of these processes between pancreatic acinar cells and hepatocytes.     

The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins.

ts85, a cell line that harbors a mutant thermolabile ubiquitin-activating enzyme, E1, fails to degrade short lived proteins at the restrictive temperature (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, the involvement of the ubiquitin system in the degradation of long lived proteins (most cellular proteins fall in this category) has not been addressed. In the present study we show that upon shifting the mutant cells to the restrictive temperature, there is no change in the rate of degradation of long lived proteins. In contrast, shifting the wild-type cells (FM3A) to the high temperature is accompanied by a 2-fold increase in the rate of proteolysis of this group of proteins. This heat-induced accelerated degradation can be inhibited completely by NH4Cl and chloroquine. Similarly, exposure of the cells to starvation, a stimulus that activates the autophagic-lysosomal pathway, has no effect on the degradation of long lived proteins in the mutant cells after inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20 (Kulka, R. G., Raboy, B., Schuster, R., Parag, H. A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem. 263, 15726-15731)). Cycloheximide and 3-methyladenine, known inhibitors of formation of autophagic vacuoles, inhibit the heat-induced accelerated degradation of long lived proteins in wild-type cells. Taken together, the results suggest that 1) heat stress induces enhanced degradation of intracellular proteins; 2) the process occurs most probably in autophagic vacuoles; and 3) activation of ubiquitin is required for the formation of these vacuoles. As there is no change in the basal rate of degradation of intracellular proteins in the mutant cells at the restrictive temperature, it appears that the ubiquitin system is not involved in their breakdown.     

Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.     

In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5′-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.     

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial- lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.     

Effect of different protein synthesis inhibitors on the exocrine pancreatocytic autophagocytosis in vivo.

The translational inhibitor cycloheximide is also used as an inhibitor of cellular autophagy and intracellular degradation of endogenous cellular proteins. Some evidence for a similar effect of other inhibitors of protein biosynthesis is also available (largely from in vitro systems). In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the fact that when the formation of autophagosomes is inhibited, a regression of the autophagolysosomal compartment can be measured, provided intralysosomal degradation in the pre-existing autophagic vacuoles continues at an unchanged rate. To make the measurements easier, autophagolysosomal compartment of the cells was enlarged by administering vinblastine (10 mg/kg b.wt.) for 2 h when the inhibitors were given for an additional 30 min. During this time cycloheximide (0.2 mg/g b.wt.), emetine (0.12 mg/g b.wt.) and puromycin (0.2 mg/g b.wt.), respectively caused 35, 25 and 52% regression of the pancreatocytic autophagolysosomal compartment. Since all the above translational inhibitors inhibited autophagocytosis as well, the possibility of a coupling between the regulation of synthesis and inhibition of proteins arises.     

Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

Lysosomal Turnover,but Not a Cellular Level,of Endogenous LC3 is a Marker for Autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D, and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors

Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.     

A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors

Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.     

Role of Ca2+ for protein turnover in isolated rat hepatocytes.

Experiments with bivalent-cation chelators (EGTA and EDTA), a Ca2+ ionophore (A23187) and a Ca2+-channel blocker (verapamil) indicate that Ca2+ is required for the lysosomal degradation of endogenous protein in hepatocytes. A distinction is made between lysosomal and non-lysosomal degradation by using the lysosomotropic agent methylamine. As Ca2+ does not appear to be required for the lysosomal degradation of endocytosed asialo-fetuin, the Ca2+-dependence for the degradation of endogenous protein is probably connected with the formation of autophagic vacuoles or the fusion of autophagic vacuoles with lysosomes. EGTA and EDTA had a slight inhibitory effect on the non-lysosomal degradation. This effect could be due to the activity of non-lysosomal Ca2+-dependent thiol proteinases. Together with previous experiments with thiol-proteinase inhibitors, the present experiments indicate that these proteinases have a very limited impact on the bulk protein degradation in the isolated hepatocytes.