Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.
ABSTRACT: Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used ?9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.
Project description:4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.
Project description:Autophagy is of increasing interest as a target for cancer therapy. We find that leucine deprivation causes the caspase-dependent apoptotic death of melanoma cells because it fails to appropriately activate autophagy. Hyperactivation of the RAS-MEK pathway, which is common in melanoma, prevents leucine deprivation from inhibiting mTORC1, the main repressor of autophagy under nutrient-rich conditions. In an in vivo tumor xenograft model, the combination of a leucine-free diet and an autophagy inhibitor synergistically suppresses the growth of human melanoma tumors and triggers widespread apoptosis of the cancer cells. Together, our study represents proof of principle that anticancer effects can be obtained with a combination of autophagy inhibition and strategies to deprive tumors of leucine.
Project description:Glioblastomas (GBMs) are very aggressive tumors with low chemosensitivity. The DNA-alkylating agent temozolomide (TMZ) is currently the most efficient chemotoxic drug for GBM therapy; however, many patients develop resistance to TMZ. Combining TMZ with another agent could present an improved treatment option if it could overcome TMZ resistance and avoid side effects. Sphingosine kinase inhibitors (SKIs) have emerged as anticancer agents. Sphingosine kinases are often overexpressed in tumors where their activity of phosphorylating sphingosine (Sph) contributes to tumor growth and migration. They control the levels of the pro-apoptotic ceramide (Cer) and Sph and of the pro-survival sphingosine-1 phosphate. In the present work, TMZ was combined with a specific SKI, and the cytotoxic effect of each drug alone or in combination was tested on GBM cell lines. The combination of sublethal doses of both agents resulted in the cell death potentiation of GBM cell lines without affecting astrocyte viability. It triggered a caspase-3-dependent cell death that was preceded by accumulation of dihydrosphingosine (dhSph) and dihydroceramide (dhCer), oxidative stress, endoplasmic reticulum stress, and autophagy. Autophagy was identified as the crucial switch that facilitated induction of this cell death potentiation. The sublethal dose of the inhibitor induced these stress events, whereas that of TMZ induced the destructive autophagy switch. Remarkably, neither Cer nor Sph, but rather the Cer intermediates, dhSph and dhCer, was involved in the cytotoxicity from the combination. Cell lines sensitive to the combination expressed low levels of the antioxidant enzyme glutathione peroxidase-1, indicating this enzyme as a potential marker of sensitivity to such treatment. This work shows for the first time a strong interaction between a SKI and TMZ, leading to a tumor cell-specific death induction. It further demonstrates the biological relevance of dihydrosphingolipids in cell death mechanisms and emphasizes the potential of drugs that affect sphingolipid metabolism for cancer therapy.
Project description:There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1(+/-) mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1(+/-) mice to Becn1(+/-) mice. After 1 week on a high fat diet, 4-week-old Pdx1(+/-) Becn1(+/-) mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1(+/-) mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.
Project description:Ceramide has been suggested to be a potent bioactive lipid involved in cell growth, differentiation and apoptosis. Its precursor, dihydroceramide, does not affect these processes. The truncated dihydroceramide analogues N-hexanoyl-[4,5-3H]-d-erythro-sphinganine and N-[1-14C]-hexanoyl-d-erythro-sphinganine were used to study the conversion of dihydroceramide into ceramide by rat hepatocytes. The formation of tritiated water after the addition of the tritiated substrate to intact and permeabilized rat hepatocytes was followed to measure enzyme activity. Desaturation was severely depressed in permeabilized hepatocytes, suggesting loss of cofactors. Of a variety of cofactors tested in the permeabilized cells, NADPH appeared to be stimulatory, pointing to the involvement of a desaturase. In agreement with this, the addition of inhibitors and redox effectors known to affect Delta9-stearoyl-CoA desaturase and Delta1-plasmanyl-ethanolamine desaturase to intact cells resulted in severe inhibition of the desaturation. When added to permeabilized cells fortified with NADPH, these compounds counteracted the NADPH stimulation. The enzyme system was further studied in broken cells. On cell fractionation, the activity was recovered in the microsomal fraction. The results indicate that the conversion of dihydroceramide into ceramide is ctalysed by a desaturase and not by a dehydrogenase or an oxidase as was generally believed.
Project description:Sex-dependent differences in adaptation to famine have long been appreciated, thought to hinge on female versus male preferences for fat versus protein sources, respectively. However, whether these differences can be reduced to neurons, independent of typical nutrient depots, such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A vital adaptation to starvation is autophagy, a mechanism for recycling amino acids from organelles and proteins. Here we show that segregated neurons from males in culture are more vulnerable to starvation than neurons from females. Nutrient deprivation decreased mitochondrial respiration, increased autophagosome formation, and produced cell death more profoundly in neurons from males versus females. Starvation-induced neuronal death was attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7 knockdown using small interfering RNA; or L-carnitine, essential for transport of fatty acids into mitochondria, all more effective in neurons from males versus females. Relative tolerance to nutrient deprivation in neurons from females was associated with a marked increase in triglyceride and free fatty acid content and a cytosolic phospholipase A2-dependent increase in formation of lipid droplets. Similar sex differences in sensitivity to nutrient deprivation were seen in fibroblasts. However, although inhibition of autophagy using Atg7 small interfering RNA inhibited cell death during starvation in neurons, it increased cell death in fibroblasts, implying that the role of autophagy during starvation is both sex- and tissue-dependent. Thus, during starvation, neurons from males more readily undergo autophagy and die, whereas neurons from females mobilize fatty acids, accumulate triglycerides, form lipid droplets, and survive longer.
Project description:We applied a metabolic approach to investigate the role of sphingolipids in cell density-induced growth arrest in neuroblastoma cells. Our data revealed that sphingolipid metabolism in neuroblastoma cells significantly differs depending on the cells' population context. At high cell density, cells exhibited G0/G1 cell-cycle arrest and reduced ceramide, monohexosylceramide, and sphingomyelin, whereas dihydroceramide was significantly increased. In addition, our metabolic-labeling experiments showed that neuroblastoma cells at high cell density preferentially synthesized very long chain (VLC) sphingolipids and dramatically decreased synthesis of sphingosine-1-phosphate (S1P). Moreover, densely populated neuroblastoma cells showed increased message levels of both anabolic and catabolic enzymes of the sphingolipid pathway. Notably, our metabolic-labeling experiments indicated reduced dihydroceramide desaturase activity at confluence, which was confirmed by direct measurement of dihydroceramide desaturase activity in situ and in vitro. Importantly, we could reduce dihydroceramide desaturase activity in low-density cells by applying conditional media from high-density cells, as well as by adding reducing agents, such as DTT and L-cysteine to the media. In conclusion, our data suggest a role of the sphingolipid pathway, dihydroceramides desaturase in particular, in confluence-induced growth arrest in neuroblastoma cells.
Project description:Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.
Project description:Dihydroceramide desaturases are evolutionarily conserved enzymes that convert dihydroceramide (dhCer) to ceramide (Cer). While elevated Cer levels cause neurodegenerative diseases, the neuronal activity of its direct precursor, dhCer, remains unclear. We show that knockout of the fly dhCer desaturase gene, infertile crescent (ifc), results in larval lethality with increased dhCer and decreased Cer levels. Light stimulation leads to ROS increase and apoptotic cell death in ifc-KO photoreceptors, resulting in activity-dependent neurodegeneration. Lipid-containing Atg8/LC3-positive puncta accumulate in ifc-KO photoreceptors, suggesting lipophagy activation. Further enhancing lipophagy reduces lipid droplet accumulation and rescues ifc-KO defects, indicating that lipophagy plays a protective role. Reducing dhCer synthesis prevents photoreceptor degeneration and rescues ifc-KO lethality, while supplementing downstream sphingolipids does not. These results pinpoint that dhCer accumulation is responsible for ifc-KO defects. Human dhCer desaturase rescues ifc-KO larval lethality, and rapamycin reverses defects caused by dhCer accumulation in human neuroblastoma cells, suggesting evolutionarily conserved functions. This study demonstrates a novel requirement for dhCer desaturase in neuronal maintenance in vivo and shows that lipophagy activation prevents activity-dependent degeneration caused by dhCer accumulation.
Project description:We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (? = 0.74-0.81, P ? 0.04) and C24:0-dihydroceramide (? = 0.84-0.90, P ? 0.004), but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ? 0.001) and cytotoxicity (P ? 0.001). These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer approach.