Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.     

Oxidative stress is not required for the induction of apoptosis upon glutamine starvation of Sp2/0-Ag14 hybridoma cells

L-glutamine (Gln) withdrawal rapidly triggers apoptosis in the murine hybridoma cell line Sp2/0-Ag14 (Sp2/0). In this report, we examined the possibility that Gln deprivation of Sp2/0 cells triggers an oxidative stress which would contribute to the activation of apoptotic pathways. Gln withdrawal triggered an oxidative stress in Sp2/0 cells, as indicated by an increased accumulation of reactive oxygen species (ROS) and an increase in the intracellular content in protein carbonyl groups. Gln starvation also caused a decrease in the intracellular levels of glutathione (GSH). However, a decrease in GSH was not sufficient to induce Sp2/0 cell death since reducing GSH levels with DL-buthionine-[S,R]-sulfoximine did not affect cell viability. The antioxidant N-acetyl-L-cysteine (NAC), while effective in inhibiting ROS accumulation and oxidative stress, did not prevent the loss in cell viability or the processing and activation of caspase-3 triggered by Gln starvation. On the other hand, NAC did reduce the formation of apoptotic bodies in dying cells. Altogether these results indicate that in Sp2/0 cells, Gln deprivation leads to the induction of an oxidative stress which, while involved in the formation of apoptotic bodies, is not essential to the activation of the cell death program.     

p38 MAPK inhibition is critically involved in VEGFR-2-mediated endothelial cell survival

Vascular endothelial growth factor (VEGF) promotes vasculogenesis, arteriogenesis, and angiogenesis by stimulating proliferation, migration, and cell survival of endothelial cells. VEGF mediates its actions through activation of two receptor tyrosine kinases, VEGFR-1 and VEGFR-2. Serum starvation led to apoptosis of human umbilical vein endothelial cells (HUVEC), which was accompanied by activation of p38 MAPK and caspase-3. Stimulation of both VEGF-receptors resulted in a considerable decrease of apoptosis, which was associated with the inhibition of p38 MAPK and caspase-3 activity. Selective stimulation of VEGFR-2 showed similar results, whereas the isolated activation of VEGFR-1 was without effect. Incubation of HUVEC with SB203580, a p38 MAPK inhibitor, resulted in similar effects as VEGF-stimulation: p38 MAPK and caspase-3 enzyme activity were reduced and apoptosis was prevented. These data indicate that activation of VEGFR-2 prevents endothelial cell apoptosis by inhibiting p38 MAPK phosphorylation and thus, reducing caspase-3 activity.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

TRAF2 phosphorylation promotes NF-κB-dependent gene expression and inhibits oxidative stress-induced cell death

Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.     

MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis

Atherosclerosis has been recognized as a chronic inflammatory disease, which can harden the vessel wall and narrow the arteries. MicroRNAs exhibit crucial roles in various diseases including atherosclerosis. However, so far, the role of miR-328 in atherosclerosis remains barely explored. Therefore, our study concentrated on the potential role of miR-328 in vascular endothelial cell injury during atherosclerosis. In our current study, we observed that oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis and inhibited cell viability dose-dependently and time-dependently. In addition, indicated dosage of ox-LDL obviously triggered HUVECs inflammation and oxidative stress process. Then, it was found that miR-328 in HUVECs was reduced by ox-LDL. HUVECs apoptosis was greatly repressed and cell survival was significantly upregulated by overexpression of miR-328. Furthermore, mimics of miR-328 rescued cell inflammation and oxidative stress process induced by ox-LDL. Oppositely, inhibitors of miR-328 strongly promoted ox-LDL-induced endothelial cells injury in HUVECs. By using bioinformatics analysis, high-mobility group box-1 (HMGB1) was predicted as a downstream target of miR-328. HMGB1 has been reported to be involved in atherosclerosis development. The correlation between miR-328 and HMGB1 was validated in our current study. Taken these together, it was implied that miR-328 ameliorated ox-LDL-induced endothelial cells injury through targeting HMGB1 in atherosclerosis.     

A novel role for connexin hemichannel in oxidative stress and smoking-induced cell injury

Oxidative stress is linked to many pathological conditions, including ischemia, atherosclerosis and neurodegenerative disorders. The molecular mechanisms of oxidative stress induced pathophysiology and cell death are currently poorly understood. Our present work demonstrates that oxidative stress induced by reactive oxygen species and cigarette smoke extract depolarize the cell membrane and open connexin hemichannels. Under oxidative stress, connexin expression and connexin silencing resulted in increased and reduced cell deaths, respectively. Morphological and live/dead assays indicate that cell death is likely through apoptosis. Our studies provide new insights into the mechanistic role of hemichannels in oxidative stress induced cell injury.     

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.     

Effect of resveratrol on high glucose-induced stress in human leukemia K562 cells

Hyperglycemia, a symptom of diabetes mellitus, induces hyperosmotic responses, including apoptosis, in vascular endothelial cells and leukocytes. Hyperosmotic shock elicits a stress response in mammalian cells, often leading to apoptotic cell death. In a previous report, we showed that hyperosmotic shock induced apoptosis in various mammalian cells. Importantly, apoptotic biochemical changes (i.e., caspase-3 activation and DNA fragmentation) were blocked by antioxidant pretreatment during hyperosmotic shock-induced cell death. In the present study, we report that resveratrol, a phytoalexin present in grapes with known antioxidant and anti-inflammatory properties, attenuates high glucose-induced apoptotic changes, including c-Jun N-terminal kinase (JNK) activation and caspase-3 activation in human leukemia K562 cells. Experiments with the cell permeable dye, 2',7'-dichlorofluorescein diacetate (DCF-DA), an indicator of reactive oxygen species (ROS) generation, revealed that high glucose treatment directly increased intracellular oxidative stress, which was attenuated by resveratrol. In addition, high glucose-treated K562 cells displayed a lower degree of attachment to collagen, the major component of vessel wall subendothelium. In contrast, cells pretreated with resveratrol followed by high glucose exhibited higher affinity for collagen. The results of this report collectively imply the involvement of oxidative stress in high glucose-induced apoptosis and alterations in attachment ability. Moreover, resveratrol blocks these events by virtue of its antioxidant property.     

Enhanced 15-HPETE production during oxidant stress induces apoptosis of endothelial cells

Oxidant stress plays an important role in the etiology of vascular diseases by increasing rates of endothelial cell apoptosis, but few data exist on the mechanisms involved. Using a unique model of oxidative stress based on selenium deficiency (-Se), the effects of altered eicosanoid production on bovine aortic endothelial cells (BAEC) apoptosis was evaluated. Oxidant stress significantly increased the immediate oxygenation product of arachidonic acid metabolized by the 15-lipoxygenase pathway, 15-hydroxyperoxyeicosatetraenoic acid (15-HPETE). Treatment of -Se BAEC with TNFalpha/cyclohexamide (CHX) exhibited elevated levels of apoptosis, which was significantly reduced by the addition of a specific 15-lipoxygenase inhibitor PD146176. Furthermore, the addition of 15-HPETE to PD146176-treated BAEC, partially restored TNF/CHX-induced apoptosis. Increased exposure to 15-HPETE induced apoptosis, as determined by internucleosomal DNA fragmentation, chromatin condensation, caspase-3 activation, and caspase-9 activation, which suggests mitochondrial dysfunction. The expression of Bcl-2 protein also was decreased in -Se BAEC. Addition of a caspase-9 inhibitor (LEHD-fmk) completely blocked 15-HPETE-induced chromatin condensation in -Se BAEC, suggesting that 15-HPETE-induced apoptosis is caspase-9 dependent. Increased apoptosis of BAEC as a result of oxidant stress and subsequent production of 15-HPETE may play a critical role in a variety of inflammatory based diseases.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway

Even though retinoids are widely used as adjuvant in chemotherapeutic interventions to improve cancer cell death, their mechanism(s) of action involves multiple overlapping pathways that remain unclear. We have previously shown that vitamin A, the natural precursor of the retinoids, induces oxidative-dependent cytochrome c release from isolated mitochondria, suggesting a putative mechanism for apoptosis activation. Using Sertoli cells in culture, we show that retinol causes mitochondrial-dependent apoptosis, involving oxidative stress. Apoptosis was evaluated by nuclear morphology, DNA fragmentation, and caspase-3/7 activity. Retinol induced oxidant- and time-dependent imbalance of several mitochondrial parameters, cytochrome c release and caspase-3/7 activation, leading cells to commit apoptosis. All parameters tested were attenuated or blocked by trolox co-administration, suggesting that retinol induces apoptosis through oxidative damage, which mitochondria plays a pivotal role.     

Magnesium tanshinoate B protects endothelial cells against oxidized lipoprotein-induced apoptosis

The activation of c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in the induction of cell apoptosis. We previously reported that magnesium tanshinoate B (MTB), a compound purified from a Chinese herb danshen (Salvia miltiorrhiza), could inhibit ischemia/reperfusion-induced myocyte apoptosis in the heart. The objective of the present study was to investigate whether MTB can prevent oxidized lipoprotein-induced apoptosis in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated with copper-oxidized very low density lipoprotein (Cu-OxVLDL) or copper-oxidized low density lipoprotein (Cu-OxLDL). Treatment of cells with Cu-OxVLDL or Cu-OxLDL resulted in a 3-fold increase in the JNK activity. The amount of cytochrome c released and the activity of caspase-3 in cells treated with Cu-OxVLDL or Cu-OxLDL were significantly elevated, indicating the occurrence of apoptosis. The presence of MTB was able to abolish the JNK activation, cytochrome c release, and caspase-3 activation induced by Cu-OxVLDL or Cu-OxLDL, resulting in a marked reduction in apoptosis in endothelial cells. The data from this study indicate that oxidized lipoproteins induce apoptosis in endothelial cells. We postulate that the inhibition of the JNK signaling pathway by MTB is a key mechanism that protects these cells from oxidized lipoprotein-induced apoptosis.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.     

Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.     

Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7β-hydroxycholesterol (7βOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24 h, which was preceded by early lipid accumulation (6 h) and lysosomal membrane permeabilization (6−12 h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7βOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7βOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.     

Comparative proteomic analysis of indioside D-triggered cell death in HeLa cells

Medicinal plants represent a rich source of cancer drug leads. Indioside D, a furostanol glycoside isolated from Solanum mammosum, was found to possess antiproliferative activity toward a panel of human cancer cell lines. Proteomic analysis of indioside D-treated HeLa cells revealed profound protein changes related to energy production and oxidative stress, suggesting that mitochondria dysfunction plays a role in indioside D-induced apoptosis. Indioside D caused a rapid dissipation of mitochondrial transmembrane potential (DeltaPsim) and the generation of reactive oxygen species (ROS), leading to the activation of caspase-dependent apoptotic cell death. The Fas death receptor pathway was also activated following indioside D treatment, and triggered the activation of caspase-8 and cleavage of Bid, which also acted through the mitochondrial apoptosis pathway. These results suggest that indioside D induced apoptosis in HeLa cells via both intrinsic and extrinsic cell death pathways.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Protective Effects of Spatholobi Caulis Extract on Neuronal Damage and Focal Ischemic Stroke/Reperfusion Injury

Neuronal apoptotic cell death plays an important role in many neurological disorders, including Alzheimer’s disease, Parkinson’s disease, and ischemic stroke. Spatholobi Caulis (SC) has been widely used in traditional herbal medicine for the treatment of cancer, inflammation, viral infection, and anemia. However, the protective effects of SC extract (SCE) against apoptotic cell death in the brain have not been reported. We investigated the protective effects of SCE against neuronal injury etoposide-induced neurotoxicity and in rats subjected to focal transient ischemic stroke middle cerebral artery occlusion (MCAO) for 45 min, followed by 7 days of reperfusion. The in vitro study demonstrated that SCE protected cells against etoposide-induced cell viability loss in SH-SY5Y cells. Apoptotic phenotypes, such as cleaved PARP and caspase-3, and oxidative stress in etoposide-treated cells were ameliorated by SCE treatment. In MCAO-reperfusion injury, SCE promoted neuronal survival and level of brain-derived neurotrophic factor (BDNF) by reducing glial activation, oxidative stress, and apoptosis in the ipsilateral cortex. These results indicated that SCE exerted protective effects under etoposide treatment and in a MCAO-reperfusion model by reducing JNK and p38 MAPK activation. This study presents the first evidence that SCE has therapeutic potential for the treatment of ischemic stroke or neurological disorder-related cell death.