Fate of isolated adult cardiomyocytes undergoing starvation-induced autophagic degeneration

We induced autophagy in isolated adult rat ventricular cardiomyocytes by incubating them in glucose-free medium supplemented with mannitol for up to 4 days. The up-regulation of LC3, and vacuoles containing partially degraded subcellular organelles were readily apparent in glucose-starved cells. Most dead cells in both groups showed features of necrosis, although the survival rate was significantly lower among glucose-starved cells than among the controls. In contrast, the rate of apoptosis was about the same in both groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly reduced the viability of both control and glucose-starved cells in a dose-dependent manner and caused specific morphological alterations: 3-MA reduced the number of autophagic vacuoles, whereas leupeptin greatly increased their number and size. Conversely, rapamycin, an enhancer of autophagy, improved the survival of glucose-starved cells. The reduction in cellular ATP caused by glucose depletion were exacerbated by the inhibitors but mitigated by rapamycin, suggesting that inhibition of autophagy may accelerate energy depletion, leading to necrosis. Our findings suggest that in cardiomyocytes autophagy is a compensatory, prosurvival response to stress and that autophagic death is an unsuccessful outcome brought on by necrosis.

Addendum to: Maruyama R, Goto K, Takemura G, Ono K, Nagao K, Horie T, Tsujimoto A, Kanamori H, Miyata S, Ushikoshi H, Nagashima K, Minatoguchi S, Fujiwara T, Fujiwara H. Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes. Am J Phsyiol Heart Circ Physiol 2008; 295:H1599-607; PMID: 18708438; DOI:10.1152/ajpheart.91449.2007.     

Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.     

No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.     

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.     

Biochemical and Ultrastructural Changes in Tetrahymena pyriformis During Starvation*

SYNOPSIS. Certain of the ultrastructural and biochemical changes occurring during the first 25 hr of starvation in Tetrahymena pyriformis were studied. Ultrastructurally, numerous profiles of degenerating mitochondria were seen in the early stages of starvation. The presence of oxidizable substrate such as glucose and acetate did not prevent this degeneration. Numerous large nucleoli were formed, many of which seemed to be passing into the cytoplasm as forming autophagic vacuoles. There was a transient increase in Oil Red O-positive bodies, presumably lipid (triglycerides). The extent and duration of this increase were pronounced in the presence of acetate. The lipid droplets appeared to arise within the cisternae of the endoplasmic reticulum. Lipid reserves were apparently utilized prior to carbohydrates, as the disappearance of lipid droplets preceded glycogen utilization, both in the presence of acetate and in the absence of exogenous substrate. A considerable loss of cellular protein also occurred. In cells from inorganic medium supplemented with glucose, glycogen occupied much of the cell, leaving only islands of cell organelles. Acid phosphatase was localized, ultrastructurally, mainly in autophagic vacuoles which contained mitochondria and other cell organelles, and in association with small, double-membraned structures which seemed to be sequestering small areas of cytoplasm. Such sequestered areas also appeared within larger autophagic vacuoles. Residual bodies containing concentric whorls of myelin-like membranes surrounding a more solid core accumulated during starvation. Acid phosphatase activity decreased in amount but not in specific activity. The specific activity of cathespin doubled or tripled, but there was little change in total enzyme.     

Autophagy delays apoptotic death in breast cancer cells following DNA damage

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.     

Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture

During Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to programmed cell death (PCD). A rapid death at the end of batch culture is accelerated by nutrient starvation. In this study, type II PCD, autophagy, as well as type I PCD, apoptosis, was found to take place in two antibody-producing CHO cell lines, Ab1 and Ab2, toward the end of batch culture when glucose and glutamine were limiting. The evidence of autophagy was observed from the accumulation of a common autophagic marker, a 16 kDa form of LC3-II during batch culture. Moreover, a significant percentage of the total cells (80% of Ab1 cells and 86% of Ab2 cells) showed autophagic vacuoles containing cytoplasmic material by transmission electron microscopy. An increased level of PARP cleavage and chromosomal DNA fragmentation supported that starvation-induced apoptosis also occurred simultaneously with autophagy.     

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.     

Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.     

Glucose-starved Cells Do Not Engage in Prosurvival Autophagy

In response to nutrient shortage or organelle damage, cells undergo macroautophagy. Starvation of glucose, an essential nutrient, is thought to promote autophagy in mammalian cells. We thus aimed to determine the role of autophagy in cell death induced by glucose deprivation. Glucose withdrawal induces cell death that can occur by apoptosis (in Bax, Bak-deficient mouse embryonic fibroblasts or HeLa cells) or by necrosis (in Rh4 rhabdomyosarcoma cells). Inhibition of autophagy by chemical or genetic means by using 3-methyladenine, chloroquine, a dominant negative form of ATG4B or silencing Beclin-1, Atg7, or p62 indicated that macroautophagy does not protect cells undergoing necrosis or apoptosis upon glucose deprivation. Moreover, glucose deprivation did not induce autophagic flux in any of the four cell lines analyzed, even though mTOR was inhibited. Indeed, glucose deprivation inhibited basal autophagic flux. In contrast, the glycolytic inhibitor 2-deoxyglucose induced prosurvival autophagy. Further analyses indicated that in the absence of glucose, autophagic flux induced by other stimuli is inhibited. These data suggest that the role of autophagy in response to nutrient starvation should be reconsidered.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.     

Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death

The term "autophagic cell death" was coined to describe a form of cell death associated with the massive formation of autophagic vacuoles without signs of apoptosis. However, questions about the actual role of autophagy and its molecular basis in cell death remain to be elucidated. We recently reported that adult hippocampal neural stem (HCN) cells undergo autophagic cell death following insulin withdrawal. Insulin-deprived HCN cells exhibit morphological and biochemical markers of autophagy, including accumulation of Beclin 1 and the type II form of microtubule-associated protein 1 light chain 3 (LC3) without evidence of apoptosis. Suppression of autophagy by knockdown of Atg7 reduces cell death, whereas promotion of autophagy with rapamycin augments cell death in insulin-deficient HCN cells. These data reveal a causative role of autophagy in insulin withdrawal-induced HCN cell death. HCN cells have intact apoptotic capability despite the lack of apoptosis following insulin withdrawal. Our study demonstrates that autophagy is the default cell death mechanism in insulin-deficient HCN cells, and provides a genuine model of autophagic cell death in apoptosis-intact cells. Novel insight into molecular mechanisms of this underappreciated form of programmed cell death should facilitate the development of therapeutic methods to cope with human diseases caused by dysregulated cell death.     

Glucose starvation reduces IGF-I mRNA in tumor cells: evidence for an effect on mRNA stability

The purpose of this study was to characterize the mechanisms by which glucose regulates IGF-I gene expression in rat C6 glioma cells and in rat GH3 pituitary adenoma cells. Glucose starvation for periods of 12 to 48 h decreased IGF-I mRNA levels. In contrast, there was no stimulation of IGF-I mRNA by medium glucose between 1 and 25 mM over a 24-h period. Studies with hexoses and glycolytic metabolites suggested that glucose metabolism was required to maintain IGF-I mRNA. Glucose starvation lowered IGF-I mRNA half-life in both C6 and GH3 cells. Protein synthesis inhibition lowered IGF-I mRNA by about 20% in glucose-fed C6 and GH3 cells, while potently increasing IGF-I mRNA in glucose-starved C6 cells and not altering IGF-I mRNA in glucose-starved GH3 cells. Our results suggest that in these tumor cells, IGF-I mRNA stability is reduced by glucose starvation, secondary to a deficiency in intracellular glucose metabolism. Ongoing protein synthesis is not required for this mRNA de-stabilizing effect in GH3 cells. Rather, in glucose-starved C6 cells, decreased IGF-I mRNA stability may result from the action of a labile protein.     

Biogenesis of multilamellar bodies via autophagy

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.     

A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase‐dependent manner by serum‐starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho‐associated, coiled‐coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.     

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.     

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.     

A life-span extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for life-span extension, it is controversial which forms or aspects of autophagy are responsible for this effect. We addressed this topic by analyzing the life span of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macroautophagy and several forms of microautophagy were dispensable for life-span extension. The only autophagy protein that is critical for life-span extension was Atg15, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for life-span extension, which was indicated by the shortened life span of mutants missing proteins (ypt7Delta, nyv1Delta, vac8Delta) or lipids (erg6Delta) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Delta accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macroautophagy is not blocked by erg6Delta, we propose that a new form of autophagy transports Atg15 via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in life-span extension.     

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 μM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1 fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells. This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent.