Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Thiamine attenuates hypoxia-induced cell death in cultured neonatal rat cardiomyocytes

Previous studies have demonstrated that thiamine (vitamin B1) has a cytoprotective effect against ischemic damage to the heart, and that heat shock protein 70 (Hsp70) is capable of protecting cardiac cells from lethal ischemia/hypoxia. We show here that thiamine has a cytoprotective effect on cultured neonatal rat cardiomyocytes under hypoxic insult, and also protects the cardiomyocytes against hypoxia-induced apoptosis; caspase-3 activation, PARP cleavage and DNA fragmentation are all inhibited. Moreover, it increases the level of Hsp70 protein in the cardiomyocytes even under prolonged hypoxic stress and its effects on hypoxia-induced cardiac cell death are antagonized by an Hsp70 inhibitor. These results suggest that the cytoprotective effect of thiamine in cardiomyocytes under hypoxic stress is due to its ability to induce Hsp70.     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid.

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.     

Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.     

Glycosylation of Ceramide Potentiates Cellular Resistance to Tumor Necrosis Factor-α-Induced Apoptosis

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-α (TNF-α)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-α has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC₅₀ for TNF-α, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-α had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-α resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-α.     

Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-alpha has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC(50) for TNF-alpha, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-alpha had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-alpha resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-alpha.     

Hypoxia-induced cell death in human malignant glioma cells: energy deprivation promotes decoupling of mitochondrial cytochrome c release from caspase processing and necrotic cell death

Hypoxia induces apoptosis in primary and transformed cells and in various tumor cell lines in vitro. In contrast, there is little apoptosis and predominant necrosis despite extensive hypoxia in human glioblastomas in vivo. We here characterize ultrastructural and biochemical features of cell death in LN-229, LN-18 and U87MG malignant glioma cells in a paradigm of hypoxia with partial glucose deprivation in vitro. Electron microscopic analysis of hypoxia-challenged glioma cells demonstrated early stages of apoptosis but predominant necrosis. ATP levels declined during hypoxia, but recovered with re-exposure to normoxic conditions unless hypoxia exceeded 8 h. Longer hypoxic exposure resulted in irreversible ATP depletion and delayed cell death. Hypoxia induced mitochondrial release of cytochrome c, but there was no cleavage of caspases 3, 7, 8 or 9, and no DNA fragmentation. Ectopic expression of BCL-XL conferred protection from hypoxia-induced cell death, whereas the overexpression of the antiapoptotic proteins X-linked-inhibitor-of-apoptosis-protein and cytokine response modifier-A had no effect. These findings suggest that glioma cells resist adverse effects of hypoxia until energy stores are depleted and then undergo necrosis rather than apoptosis because of energy deprivation.     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.     

Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages

Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.     

Hypoxia promotes HO-8910PM ovarian cancer cell invasion via Snail-mediated MT1-MMP upregulation

The molecular mechanisms of ovarian cancer cell invasion under hypoxia remain unclear. Here we employed a 3D collagen model and chick chorioallantoic membrane (CAM) invasion assay to explore the influence of hypoxia on ovarian cancer cell invasion. Hypoxia (both 1% O₂ and CoCl₂ 150 and 250 µM) induced HO-8910PM ovarian cancer cell invasion in 3D collagen and collagenolysis determined by hydroxyproline. Pretreatment with a hypoxia inducible factor-1α inhibitor, YC-1, or MMP inhibitor, GM6001, significantly inhibited 3D collagen invasion and degradation and cell proliferation. Hypoxia stimulated both mRNA and protein expressions of membrane-type 1 matrix metalloproteinase (MT1-MMP) and promoted MT1-MMP translocation to the cell surface in an YC-1 sensitive manner. MT1-siRNA transfection inhibited hypoxia-induced invasion, proliferation, and collagen degradation of cells in 3D collagen. Hypoxia stimulated Snail mRNA and protein expression as well as translocation to nucleus in an YC-1 sensitive manner. Overexpression of Snail with a recombinant plasmid in HO-8910PM cells resulted in an enhanced invasion in 3D collagen. Transfection with Snail-specific siRNA significantly decreased MT1-MMP expression and 3D collagen invasion. Hypoxia-treated cells significantly broke the upper CAM surface of 11-day-old chick embryos and infiltrated interstitial tissue, completely blocked in the presence of YC-1 or GM6001, or after MT1-MMP siRNA or Snail siRNA transfection. Together, these data suggest that hypoxia promotes HO-8910PM ovarian cancer cell traffic through 3D matrix via Snail-mediated MT1-MMP upregulation, a possible molecular mechanism of ovarian cancer cell invasion under hypoxia.     

Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.     

Hypoxia-Induced Modulation of Apoptosis and BCL-2 Family Proteins in Different Cancer Cell Types

Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM_EL. BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and –independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.     

Glucosylceramide synthesis inhibition affects cell cycle progression,membrane trafficking,and stage differentiation in Giardia lamblia

Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes.     

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.     

Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.     

Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt^thr308 and ^ser473; these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.     

A defect in cytosolic carboxypeptidase 1 (Nna1) causes autophagy in Purkinje cell degeneration mouse brain

Ceramide is a sphingolipid bioactive molecule that induces apoptosis and other forms of cell death, and triggers macroautophagy (referred to below as autophagy). Like amino acid starvation, ceramide triggers autophagy by interfering with the mTOR-signaling pathway, and by dissociating the Beclin 1:Bcl-2 complex in a c-Jun N-terminal kinase 1 (JNK1)-mediated Bcl-2 phosphorylation-dependent manner. Dissociation of the Beclin 1:Bcl-2 complex, and the subsequent stimulation of autophagy have been observed in various contexts in which the cellular level of long-chain ceramides was increased. It is notable that the conversion of short-chain ceramides (C₂-ceramide and C₆-ceramide) into long-chain ceramide via the activity of ceramide synthase is required to trigger autophagy. The dissociation of the Beclin 1:Bcl-2 complex has also been observed in response to tamoxifen and PDMP (an inhibitor of the enzyme that converts ceramide to glucosylceramide), drugs that increase the intracellular level of long-chain ceramides. However, and in contrast to starvation, overexpression of Bcl-2 does not blunt ceramide-induced autophagy. Whether this autophagy that is unchecked by forced dissociation of the Beclin 1:Bcl-2 complex is related to the ability of ceramide to trigger cell death remains an open question. More generally, the question of whether ceramide-induced autophagy is a dedicated cell death mechanism deserves closer scrutiny.