FasL shedding is reduced by hypothermia in experimental stroke

Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor-mediated pathways, such as that triggered by Fas/FasL. Male Sprague-Dawley rats subjected to 2 h middle cerebral artery occlusion with 2 h intraischemic mild hypothermia (33°C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by ∼ 50–60% at 6 and 24 h post-insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by ∼ 50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane-bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.     

Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile

Low back pain is a common and debilitating disorder. Current evidence implicates intervertebral disc (IVD) degeneration and herniation as major causes, although the pathogenesis is poorly understood. While several cytokines have been implicated in the process of IVD degeneration and herniation, investigations have predominately focused on Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). However, to date no studies have investigated the expression of these cytokines simultaneously in IVD degeneration or herniation, or determined which may be the predominant cytokine associated with these disease states. Using quantitative real time PCR and immunohistochemistry we investigated gene and protein expression for IL-1beta, TNFalpha and their receptors in non-degenerate, degenerate and herniated human IVDs. IL-1beta gene expression was observed in a greater proportion of IVDs than TNFalpha (79% versus 59%). Degenerate and herniated IVDs displayed higher levels of both cytokines than non-degenerate IVDs, although in degenerate IVDs higher levels of IL-1beta gene expression (1,300 copies/100 ng cDNA) were observed compared to those of TNFalpha (250 copies of TNFalpha/100 ng cDNA). Degenerate IVDs showed ten-fold higher IL-1 receptor gene expression compared to non-degenerate IVDs. In addition, 80% of degenerate IVD cells displayed IL-1 receptor immunopositivity compared to only 30% of cells in non-degenerate IVDs. However, no increase in TNF receptor I gene or protein expression was observed in degenerate or herniated IVDs compared to non-degenerate IVDs. We have demonstrated that although both cytokines are produced by human IVD cells, IL-1beta is expressed at higher levels and in more IVDs, particularly in more degenerate IVDs (grades 4 to 12). Importantly, this study has highlighted an increase in gene and protein production for the IL-1 receptor type I but not the TNF receptor type I in degenerate IVDs. The data thus suggest that although both cytokines may be involved in the pathogenesis of IVD degeneration, IL-1 may have a more significant role than TNFalpha, and thus may be a better target for therapeutic intervention.     

The Fas/Fas Ligand Death Receptor Pathway Contributes to Phenylalanine-Induced Apoptosis in Cortical Neurons

Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.     

X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-α signaling. By contrast, blockade of tumor necrosis factor-α does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.     

Transforming growth factor-beta 1 induces apoptosis through Fas ligand-independent activation of the Fas death pathway in human gastric SNU-620 carcinoma cells

To date, two major apoptotic pathways, the death receptor and the mitochondrial pathway, have been well documented in mammalian cells. However, the involvement of these two apoptotic pathways, particularly the death receptor pathway, in transforming growth factor-beta 1 (TGF-beta 1)-induced apoptosis is not well understood. Herein, we report that apoptosis of human gastric SNU-620 carcinoma cells induced by TGF-beta 1 is caused by the Fas death pathway in a Fas ligand-independent manner, and that the Fas death pathway activated by TGF-beta 1 is linked to the mitochondrial apoptotic pathway via Bid mediation. We showed that TGF-beta 1 induced the expression and activation of Fas and the subsequent caspase-8-mediated Bid cleavage. Interestingly, expression of dominant negative FADD and treatment with caspase-8 inhibitor efficiently prevented TGF-beta 1-induced apoptosis, whereas the treatment with an activating CH11 or a neutralizing ZB4 anti-Fas antibody, recombinant Fas ligand, or Fas-Fc chimera did not affect activation of Fas and the subsequent induction of apoptosis by TGF-beta 1. We further demonstrated that TGF-beta 1 also activates the mitochondrial pathway showing Bid-mediated loss of mitochondrial membrane potential and subsequent cytochrome c release associated with the activations of caspase-9 and the effector caspases. Moreover, all these apoptotic events induced by TGF-beta 1 were found to be effectively inhibited by Smad3 knockdown and also completely abrogated by Smad7 expression, suggesting the involvement of the Smad3 pathway upstream of the Fas death pathway by TGF-beta 1.     

Role of the mitochondrial pathway in serum deprivation-induced apoptosis of rat endplate cells

The apoptosis of cartilage endplates (CEPs), acting as an initiating factor, plays a vital role in the pathogenesis of intervertebral disc degenerative diseases, the underlying molecular mechanism of the apoptotic process in CEPs is still not clear. The present study aimed to investigate the mechanism of CEP cell apoptosis. We found that low levels of fetal bovine serum (FBS) can induce cell apoptosis. Serum deprivation led to high expression levels of caspase-9, caspase-3, PARP, cytochrome-c and Bax. Flow cytometric analysis showed that inhibition of the intrinsic pathway by a caspase-9 inhibitor (z-LEHD-fmk) significantly suppressed serum deprivation-induced apoptosis. However, a caspase-8 inhibitor (z-IETD-fmk) did not reduce apoptotic cell death. These data suggest that serum deprivation induces apoptosis in rat CEP cells via the activation of the intrinsic apoptotic pathway. The efficacy of a caspase-9 inhibitor in attenuating or preventing apoptosis of serum deprivation-induced disc cell apoptosis suggests that targeting the intrinsic apoptotic pathway may be used as a potential therapy for the treatment of disc degeneration.     

Lansoprazole protects and heals gastric mucosa from non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy by inhibiting mitochondrial as well as Fas-mediated death pathways with concurrent induction of mucosal cell renewal

We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and Bcl(xL) to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.     

Mild thermotolerance induced at 40°C protects HeLa cells against activation of death receptor-mediated apoptosis by hydrogen peroxide

Preexposure to mild temperatures such as 40°C induces thermotolerance, whereby cells resist subsequent exposure to a toxic insult. This study investigates the protective effect of mild thermotolerance (3h, 40°C) against activation of death receptor-mediated apoptosis by H(2)O(2) in HeLa cells. H(2)O(2) (5-50μM) caused rapid activation (1-3h) of the Fas death receptor pathway of apoptosis, which was evident by up-regulation of the death ligand FasL and recruitment of the adaptor protein Fas-associated death domain to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, t-Bid translocation to mitochondria, and caspase-9 activation. These changes were all diminished in thermotolerant cells. Mild thermotolerance also protected cells against cytotoxicity from H(2)O(2) as well as execution-phase events of apoptosis such as caspase-3 activation and chromatin condensation. The antioxidant polyethylene glycol-catalase abolished FasL induction and caspase-8 activation due to H(2)O(2). FasL up-regulation; activation of caspases-8, -2, -9, and -3; and chromatin condensation were decreased by the p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in the activation of death receptor-mediated apoptosis by H(2)O(2). This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against induction of apoptosis by oxidative stress.     

Akt inhibition upregulates FasL, downregulates c-FLIPs and induces caspase-8-dependent cell death in Jurkat T lymphocytes

In T lymphocytes, the role of Akt in regulating Fas/Fas ligand (FasL)-mediated apoptotic signaling and death is not clearly understood. In this study, we observed that inhibition of Akt causes enhanced expression of FasL mRNA and protein and increased death-inducing signaling complex (DISC) formation with Fas-associated death domain (FADD) and procaspase-8 recruitment. Also, caspase-8 was activated at the DISC with accompanying decrease in c-FLIPs expression. FasL neutralizing antibody significantly decreased apoptotic death in the Akt-inhibited T cells. Additionally, Akt inhibition-induced Fas signaling was observed to link to the mitochondrial pathway via Bid cleavage. Further, inhibition of caspase-8 activity effectively blocked the loss of mitochondrial membrane potential and DNA fragmentation, suggesting that DISC formation and subsequent caspase-8 activation are critical initiating events in Akt inhibition-induced apoptotic death in T lymphocytes. These data demonstrate yet another important survival function governed by Akt kinase in T lymphocytes, which involves the regulation of FasL expression and consequent apoptotic signaling.     

Free cholesterol loading of macrophages is associated with widespread mitochondrial dysfunction and activation of the mitochondrial apoptosis pathway

Macrophage death in advanced atherosclerotic lesions leads to lesional necrosis and possibly plaque rupture and acute vascular occlusion. Among the likely causes of lesional macrophage death is intracellular accumulation of excess free cholesterol (FC), which is known to occur in vivo. We recently showed that FC loading of macrophages causes apoptosis, approximately 50% of which is mediated by activation of cell-surface FasL and triggering of the Fas pathway (Yao, P. M., and Tabas, I. (2000) J. Biol. Chem. 275, 23807-23813). To elucidate other pathways of death in FC-loaded macrophages, we investigated mitochondrial transmembrane potential (DeltaPsi(m)) and the mitochondrial apoptosis pathway in FC-loaded mouse peritoneal macrophages. Starting between 3 and 6 h of FC loading, DeltaPsi(m) was markedly decreased in the majority of macrophages and was independent of the Fas pathway. The decrease in DeltaPsi(m) by FC loading was not prevented by GSH, thus distinguishing it from 7-ketocholesterol-induced mitochondrial dysfunction. Cytochrome c release into the cytosol was noted by 4 h of FC loading, and activation of caspase-9 and effector caspases was observed at 6 h. Finally, we found that both cellular and mitochondrial levels of the pro-apoptotic protein Bax were increased severalfold as early as 4 h after FC loading. Thus, FC loading, perhaps via increased levels of Bax and/or cholesterol overloading of mitochondria, triggers cytochrome c release and activation of caspase-9 and the effector caspases, leading to macrophage apoptosis. These findings and our previous data support a model in which FC loading of macrophages promotes a dual program of caspase-mediated death.     

FADD deficiency sensitises Jurkat T cells to TNF-alpha-dependent necrosis during activation-induced cell death

Activation-induced cell death (AICD) in activated T lymphocytes is largely mediated by Fas/Fas ligand (FasL) interaction. The cytoplasmic adaptor molecule Fas-associated death domain protein (FADD) plays an essential role in the apoptotic signalling of the Fas death pathway. In the present study, we observed that FADD deficient (FADD(-/-)) Jurkat T cells undergo AICD to a similar extent as wild-type cells. AICD in wild-type Jurkat T cells is via apoptosis, whereas it is non-apoptotic in FADD(-/-) cells. The latter took up propidium iodide, exhibit a loss in mitochondrial membrane potential and have no detectable cleavage products of caspase-8 or -3 activation, suggesting that these cells die by necrosis. Wild-type Jurkat T cells undergo apoptosis when incubated with recombinant FasL and Trail but not with TNF-alpha. In contrast, FADD(-/-) Jurkat T cells are resistant to FasL and Trail but die of necrosis when incubated with TNF-alpha. We showed that neutralising anti-TNF-alpha blocked AICD as well as TNF-alpha-induced necrosis in FADD(-/-) Jurkat T cells. Furthermore, down regulating the receptor interacting protein, RIP, with geldanamycin treatment, which is essential for TNF-alpha signalling, markedly inhibited AICD in FADD(-/-) Jurkat T cells. In addition, caspase-8-deficient Jurkat T cells are resistant to Fas- and TNF-alpha-induced cell death. Taken together, our results suggest that a deficiency in FADD and not caspase-8 or the inhibition of the Fas signalling pathway sensitises Jurkat T cells to TNF-alpha-dependent necrosis during AICD.     

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.     

Activation of the death receptor pathway of apoptosis by the aldehyde acrolein

Reactive alpha,beta-unsaturated aldehydes such as acrolein are major components of common environmental pollutants. As a toxic by-product of lipid peroxidation, acrolein has been implicated as a possible mediator of oxidative damage to cells and tissues in a wide variety of disease states, including atherosclerosis and neurodegenerative and pulmonary diseases. Although acrolein can induce apoptotic cell death in various cell types, the biochemical mechanisms are not understood. This study investigates the implication of the death receptor pathway in acrolein-induced apoptosis. Exposure of Chinese hamster ovary cells to acrolein caused translocation of adaptor protein Fas associated with death domain to the cytoplasmic membrane and caspase-8 activation. Kp7-6, an antagonist of Fas receptor activation, blocked apoptotic events downstream of caspase-8, such as caspase-7 activation and nuclear chromatin condensation. Acrolein activated the cross-talk pathway between the death receptor and mitochondrial pathways. Bid was cleaved to truncated-Bid, which was translocated to mitochondria. Activation of the mitochondrial pathway by acrolein was confirmed by caspase-9 activation. Inhibition of activation of either the Fas receptor or caspase-8 partially decreased acrolein-induced caspase-9 activation. These findings indicate that acrolein activates the Fas receptor pathway, which occurs upstream of the mitochondrial pathway. Caspase-9 activation still occurred despite inhibition of the Fas receptor pathway, suggesting that acrolein could also trigger the mitochondrial pathway independent of the receptor pathway. These findings improve our understanding of mechanisms of toxicity of the reactive aldehyde acrolein, which has widespread implications in multiple disease states which seem to be mediated by oxidative stress and lipid peroxidation.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Inhibition of drug-induced Fas ligand transcription and apoptosis by Bcl-XL.

Fas/Fas ligand system triggers apoptosis in many cell types. Bcl-XL overexpresion antagonizes Fas/Fas ligand-mediated cell death. The mechanism by which Bcl-XL influences Fas-mediated cell death is unclear. We have found that microtubule-damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL-dependent manner. Inhibition of Fas/FasL pathway by anti-FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel-induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase-8 and caspase-3. Overexpression of Bcl-XL leads to inhibition of paclitaxel-induced FasL expression and apoptosis. Bcl-XL prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl-XL can suppress the anti-apoptotic function of Bcl-XL and may be a target for regulatory post-translational modifications. Upon phosphorylation, Bcl-XL loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl-XL-mediated resistance to these agents is related to failure to induce FasL expression.     

Fas/FasL-dependent and -independent activation of caspase-8 in doxorubicin-treated human breast cancer MCF-7 cells: ADAM10 down-regulation activates Fas/FasL signaling pathway

The contribution of Fas-mediated death pathway to doxorubicin-induced death of MCF-7 cells is not unambiguously elucidated. Thus, this study was conducted to explore doxorubicin-induced Fas/FasL signaling pathway activation in MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Dox) cells. Doxorubicin-induced caspase-8 activation was found to be mediated through Akt/ERK inactivation and FasL-independent Fas pathway in MCF-7 cells, while caspase-8 activation in MCF-7/Dox cells depended exclusively on FasL-stimulated Fas pathway. Suppression of caspase-8 activation restored the viability of doxorubicin-treated MCF-7 cells and MCF-7/Dox cells. Contrary to FasL surface expression exclusively detected in MCF-7/Dox cells, intracellular FasL expression was noted with MCF-7 cells. Promotion of FasL translocation to the cell surface by lysophosphatidic acid evoked a FasL-activated Fas death pathway in MCF-7 cells. Doxorubixin-evoked β-TrCP up-regulation promoted Sp1 degradation, which subsequently suppressed ADAM10 expression in MCF-7 and MCF-7/Dox cells. Doxorubicin-induced down-regulation of ADAM10 reduced FasL shedding, leading to Fas pathway activation in MCF-7/Dox cells. Knock-down of ADAM10 induced death in MCF-7/Dox cells, but marginally reduced the viability of MCF-7 cells. Taken together, our data indicate that Akt/ERK-mediated caspase-8 activation and Fas/FasL-mediated caspase-8 activation mostly elucidate doxorubicin-induced death in MCF-7 cells and MCF-7/Dox cells, respectively. These observations suggest a promising therapeutic modality for overcoming doxorubicin-resistant breast cancer by targeting ADAM10 sheddase activity.     

Synthetic glycosidated phospholipids induce apoptosis through activation of FADD,caspase-8 and the mitochondrial death pathway

Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD−/− and caspase-8−/− cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas−/− Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.