Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells

Oxidizedlow-density lipoprotein (oxLDL) is a potent inducer ofapoptosis for vascular cells. In the present study, wedemonstrate that the expression of death mediators, including p53, Fas,and Fas ligand (FasL) was substantially upregulated by oxLDL incultured vascular smooth muscle cells (SMCs). The induction of thesedeath mediators was time dependent and was accompanied by an increase in apoptotic death of SMCs following oxLDL treatment. Twooxysterols, 7-hydroxycholesterol and 25-hydroxycholesterol, werealso effective to induce the expression of death mediators andapoptosis. -Tocopherol and deferoxamine significantlyattenuated the induction of death mediators and cell death induced byoxLDL and oxysterols, suggesting that reactive oxygen species areinvolved in triggering the apoptotic event. Incubation of cellswith FasL-neutralizing antibody inhibited the oxLDL-induced cell deathup to 50%. Furthermore, caspase 8 and caspase 3 activities wereinduced time dependently in SMCs following oxLDL treatment.Collectively, these data suggest that the Fas/FasL death pathway isactivated and responsible for, at least in part, the apoptoticdeath in vascular SMCs upon exposure to oxLDL.

     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.     

Erucylphosphocholine-induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation

Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.     

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.     

Apoptosis-linked gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca(2+)-binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation. The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward. Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fas-mediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein-protein interaction of ALG-2 with Fas was demonstrated using yeast two-hybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process.     

Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.     

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.     

Distinct molecular mechanisms of Fas resistance in murine B lymphoma cells

A panel of murine B lymphoma cell lines, which express different levels of Fas, was extensively studied for sensitivity to Fas-mediated death signals via an anti-Fas mAb and Fas ligand-bearing cell lines. Expression of the Fas receptor on the B lymphoma cell lines did not correlate with their capacity to undergo Fas-mediated apoptosis. Moreover, Fas-associated death domain protein recruitment to the death-inducing signaling complex (DISC) complex occurred in all cell lines expressing Fas, regardless of whether they were sensitive to Fas-mediated death. Interestingly, the protein synthesis inhibitor, cycloheximide, and protein kinase C inhibitors, such as bisindolylmaleimide, rendered one of the resistant cell lines, CH33, sensitive to signals from the Fas receptor, although the levels of Fas were unchanged. This suggests that constitutive PKC activation plays a role in Fas resistance, perhaps by up-regulating NF-kappaB or Bcl-2 family members. Interestingly, CH33 demonstrated caspase 8 activity upon engagement of the Fas receptor in the absence of pharmacological manipulation, suggesting that the block in apoptosis is downstream of the DISC complex. In contrast, the fact that Fas-associated death domain protein was recruited to the DISC complex in other resistant lines, such as WEHI-231, with no caspase 8 activation indicates that these cells may be blocked within the DISC complex. Indeed, Western blot analysis showed that WEHI-231 expressed an isoform of FLICE-like inhibitory protein (cFLIPL), an antiapoptotic protein within the DISC. These studies provide evidence that murine B lymphoma cells utilize different molecular mechanisms along the Fas-signaling cascade to block apoptosis.     

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.     

ExoS of Pseudomonas aeruginosa induces apoptosis through a Fas receptor/caspase 8-independent pathway in HeLa cells

Pseudomonas aeruginosa infection is a serious complication in immunocompromised individuals and in patients with cystic fibrosis. We have previously shown that the type III secreted effector ExoS triggers apoptosis in various cultured cell lines via its ADP-ribosyltransferase (ADPRT) activity. The apoptosis process was further shown to involve intrinsic signalling pathway requiring c-Jun N-terminal kinase (JNK)-initiated mitochondrial pathway. In the present study, we investigated the role of Fas pathway activation in P. aeruginosa-induced apoptosis. P. aeruginosa infection resulted in caspase 8 cleavage in HeLa cells, which was inhibited by overexpression of a dominant negative version of Fas-associated death domain (FADD), suggesting that Fas pathway was activated. In fact, confocal laser scanning microscopy showed that P. aeruginosa induced clustering of FasR. In addition, the ADPRT activity of the ExoS was required for the induction of FasR clustering and caspase 8 cleavage. However, blocking the FasR-FasL interaction by antagonistic antibodies to FasR or to FasL had no effect on P. aeruginosa-induced caspase 8 and caspase 3 activation, neither did the silencing of FasR by small interfering RNA (siRNA), suggesting that caspase 8 activation through the FADD bypasses FasR/FasL-mediated signalling. Thus, FADD-mediated caspase 8 activation involves intracellular ExoS in an ADPRT-dependent manner. Furthermore, silencing of caspase 8 by siRNA did not interfere with P. aeruginosa-induced apoptosis, whereas it rendered HeLa cells markedly increased resistance towards FasL-induced apoptosis. In conclusion, our findings indicate that ExoS of P. aeruginosa induces apoptosis through a mechanism that is independent of Fas receptor/caspase 8 pathway.     

Nuclear respiratory factor 1 protects H9C2 cells against hypoxia-induced apoptosis via the death receptor pathway and mitochondrial pathway

Hypoxia-induced cardiomyocyte apoptosis is one of the leading causes of heart failure. Nuclear respiratory factor 1 (NRF-1) was suggested as a protector against cell apoptosis; However, the mechanism is not clear. Therefore, the aim of this study was to elucidate the role of NRF-1 in hypoxia-induced H9C2 cardiomyocyte apoptosis and to explore its effect on regulating the death receptor pathway and mitochondrial pathway. NRF-1 was overexpressed or knocked down in H9C2 cells, which were then exposed to a hypoxia condition for 0, 3, 6, 12, and 24 h. Changes in cell proliferation, cell viability, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) were investigated. The activities of caspase-3, -8, and -9, apoptosis rate, and the gene and protein expression levels of the death receptor pathway and mitochondrial pathway were analyzed. Under hypoxia exposure, NRF-1 overexpression improved the proliferation and viability of H9C2 cells and decreased ROS generation, MMP loss, caspase activities, and the apoptosis rate. However, the NRF-1 knockdown group showed the opposite results. Additionally, NRF-1 upregulated the expression of antiapoptotic molecules involved in the death receptor and mitochondrial pathways, such as CASP8 and FADD-like apoptosis regulator, B-cell lymphoma 2, B-cell lymphoma-extra-large, and cytochrome C. Conversely, the expression of proapoptotic molecules, such as caspase-8, BH3-interacting domain death agonist, Bcl-2-associated X protein, caspase-9, and caspase-3 was downregulated by NRF-1 overexpression in hypoxia-induced H9C2 cells. These results suggest that NRF-1 functions as an antiapoptotic factor in the death receptor and mitochondrial pathways to mitigate hypoxia-induced apoptosis in H9C2 cardiomyocytes.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Resistance to Fas-mediated apoptosis in EBV-infected B cell lymphomas is due to defects in the proximal Fas signaling pathway.

Post-transplant lymphoproliferative disorder is characterized by the outgrowth of EBV-infected B cell lymphomas in immunosuppressed transplant recipients. Using a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from post-transplant lymphoproliferative disorder patients, we assessed the sensitivity of such lymphomas to Fas-mediated cell death. Treatment with either an agonist anti-Fas mAb or Fas ligand-expressing cells identifies two subsets of SLCL based on their sensitivity or resistance to Fas-driven apoptosis. Fas resistance in these cells cannot be attributed to reduced Fas expression or to mutations in the Fas molecule itself. In addition, all SLCL are sensitive to staurosporine-induced cell death, indicating that there is no global defect in apoptosis. Although all SLCL express comparable levels of Fas signaling molecules including Fas-associated death domain protein, caspase 8, and caspase 3, Fas-resistant SLCL exhibit a block in Fas-signaling before caspase 3 activation. In two SLCL, this block results in impaired assembly of the death-inducing signaling complex, resulting in reduced caspase 8 activation. In a third Fas-resistant SLCL, caspase 3 activation is hindered despite intact death-inducing signaling complex formation and caspase 8 activation. Whereas multiple mechanisms exist by which tumor cells can evade Fas-mediated apoptosis, these studies suggest that the proximal Fas-signaling pathway is impeded in Fas-resistant post-transplant lymphoproliferative disorder-associated EBV(+) B cell lymphomas.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.     

Induction of apoptosis by tumor suppressor FHIT via death receptor signaling pathway in human lung cancer cells

FHIT is a novel tumor suppressor gene located at human chromosome 3p14.2. Restoration of wild-type FHIT in 3p14.2-deficient human lung cancer cells inhibits cell growth and induces apoptosis. In this study, we analyzed potential upstream/downstream molecular targets of the FHIT protein and found that FHIT specifically targeted and regulated death receptor (DR) genes in human non-small-cell lung cancer (NSCLC) cells. Exogenous expression of FHIT by a recombinant adenoviral vector (Ad)-mediated gene transfer upregulated expression of DR genes. Treatment with a recombinant TRAIL protein, a DR-specific ligand, in Ad-FHIT-transduced NSCLC cells considerably enhanced FHIT-induced apoptosis, further demonstrating the involvement of DRs in FHIT-induced apoptosis. Moreover, we also found that FHIT targeted downstream of the DR-mediated signaling pathway. FHIT overexpression disrupted mitochondrial membrane integrity and activated multiple pro-apoptotic proteins in NSCLC cell. These results suggest that FHIT induces apoptosis through a sequential activation of DR-mediated pro-apoptotic signaling pathways in human NSCLC cells.     

Fas/Fas ligand pathway, apoptosis, and clonal anergy involved in systemic acetylcholine receptor T cell epitope tolerance

The cellular mechanisms of high dose systemic acetylcholine receptor (AChR) T cell epitope, alpha 146--162 peptide-induced tolerance in experimental myasthenia gravis were examined. CD4 cells are the prime target for alpha 146--162 peptide-induced tolerance. The expression of CD69, Fas, and B7.2 molecules on AChR-immune lymphocytes was enhanced within 4--12 h after tolerance induction. A high dose of alpha 146--162 peptide in IFA failed to suppress T cell proliferation and/or clinical myasthenia gravis in lpr and gld mice deficient in Fas and Fas ligand, respectively. A high dose of alpha 146--162 peptide in IFA in AChR-immunized mice induced apoptosis of BV6 cells. Further, reconstitution of IL-2 in vitro-recovered alpha 146--162 peptide tolerized T cell proliferation, IFN-gamma, and IL-10 production. The findings implicate the possible role of Fas-/Fas ligand-mediated apoptosis and the resulting clonal anergy as the mechanisms of high dose AChR alpha 146--162 peptide-induced tolerance on CD4 cells.     

Rapid and transient intracellular oxidative stress due to novel macrosphelides trigger apoptosis via Fas/caspase-8-dependent pathway in human lymphoma U937 cells

The ability of the derivatives of macrosphelides (MS) core (simplified 16-membered core structure of natural MS) to induce apoptosis in human lymphoma U937 cells was investigated. Of the five compounds examined, MS core with ketones at 8 and 14 positions (MS5) showed the highest potency to induce apoptosis, while another, MS3 with one ketone, was minimal potent. MS5 was found to induce apoptosis in the U937 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis. MS5 treated cells showed increase in intracellular reactive oxygen species (ROS), glutathione depletion, Bid activation and lipid peroxidation. Pretreatment of cells with pancaspase inhibitor resulted in the complete inhibition of MS5-induced apoptosis. N-Acetyl-l-cysteine (NAC) pretreatment resulted in the increase in glutathione concentration, reduction of intracellular ROS, complete inhibition of DNA fragmentation, mitochondrial membrane potential (MMP) collapse, Fas externalization and caspase-8 activation. Furthermore, MS5-induced oxidative stress also triggered transient increase in intracellular calcium ion ([Ca2+]i) concentration which was completely inhibited by NAC. Pretreatment with an intracellular Ca2+ chelator, BAPTA-AM reduced MS5-induced DNA fragmentation and caspase-8 activation while it has marginal effects on MMP collapse. Taken together our present data showed that a rapid increase in intracellular ROS by MS5 triggers apoptosis via the Fas/caspase-8-mediated mitochondrial pathway suggesting that the presence of diketone makes the compound more potent to induce apoptosis. These characteristics of MS5 will make it useful for therapeutic applications of targeted apoptosis.     

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling

15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a naturally occurring cyclopentenone metabolite of PGD(2) that possesses both peroxisome proliferator-activated receptor gamma (PPAR-gamma)-dependent and PPAR-gamma-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ(2) as well as synthetic PPAR-gamma agonists inhibit lymphocyte proliferation. However, only 15d-PGJ(2), but not the specific PPAR-gamma activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain(-/-) or caspase-8(-/-) Jurkat T cells has no effect on apoptosis induction by 15d-PGJ(2). Conversely, overexpression of Bcl-2 or Bcl-x(L) completely inhibits the initiation of apoptosis, indicating that 15d-PGJ(2)-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ(2) induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Deltapsi(m)) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ(2). We also demonstrate that 15d-PGJ(2) potently stimulates reactive oxygen species production in Jurkat T cells, and Deltapsi(m) loss induced by 15d-PGJ(2) is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ(2) may modulate immune responses even independent of PPAR-gamma by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.