Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

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.     

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.     

ER stress induces caspase-8 activation,stimulating cytochrome c release and caspase-9 activation

Excess ER stress induces caspase-12 activation and/or cytochrome c release, causing caspase-9 activation. Little is known about their relationship during ER stress-mediated cell death. Upon ER stress, P19 embryonal carcinoma (EC) cells showed activation of various caspases, including caspase-3, caspase-8, caspase-9, and caspase-12, and extensive DNA fragmentation. We examined the relationship between ER stress-mediated cytochrome c/caspase-9 and caspase-12 activation by using caspase-9- and caspase-8-deficient mouse embryonic fibroblasts and a P19 EC cell clone [P19-36/12 (-) cells] lacking expression of caspase-12. Caspase-9 and caspase-8 deficiency inhibited and delayed the onset of DNA fragmentation but did not inhibit caspase-12 processing induced by ER stress. P19-36/12 (-) cells underwent apoptosis upon ER stress, with cytochrome c release and caspase-8 and caspase-9 activation. The dominant negative form of FADD and z-VAD-fmk inhibited caspase-8, caspase-9, Bid processing, cytochrome c release, and DNA fragmentation induced by ER stress, suggesting that caspase-8 and caspase-9 are the main caspases involved in ER stress-mediated apoptosis of P19-36/12 (-) cells. Caspase-8 deficiency also inhibited the cytochrome c release induced by ER stress. Thus, in parallel with the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in cytochrome c/caspase-9 activation via Bid processing.     

Bid truncation mediated by caspases-3 and -9 in vinorelbine-induced apoptosis

Vinorelbine is a chemotherapeutic vinca alkaloid clinically prescribed for non-small cell lung cancer and breast cancer. Here we studied the mechanism for vinorelbine-induced apoptosis in a human T-cell lymphoma. Although vinorelbine induces DNA fragmentation that is inhibited by specific peptide inhibitors for caspases-9 and -3 in Jurkat cells, caspase-8 deficiency retards vinorelbine-induced apoptosis. Activation of caspase-8 is also observed in vinorelbine-treated cells, and the activity is diminished when the caspase-3 activity is blocked by a specific peptide inhibitor, Ac-DNLC-CHO. Blocking of the Fas receptor with an antagonistic anti-Fas antibody does not affect vinorelbine-induced DNA fragmentation. These results suggest that vinorelbine-induced apoptosis is enhanced by the activation of caspase-8 via caspase-9-mediated activation of caspase-3, but not through a Fas-triggered signal. Western blotting suggests that vinorelbine cleaves caspase-3, -9 and -8 and reduces the amount of mitochondrial cytochrome c. Caspase-8 deficiency suppresses all of these events. A downstream substrate for caspase-8, Bid, is also cleaved in vinorelbine-treated cells, but the Bid truncation is also observed in caspase-8-deficient Jurkat cells. Importantly, recombinant caspases-3 and -9, as well as caspase-8, directly cleaves recombinant Bid in vitro. These results suggest that caspases-3 and -9 participate in Bid truncation, indicating a new mechanism for vinorelbine-induces apoptosis.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Apoptotic cell death by the novel natural compound, cinobufotalin

Cinobufotalin (CB), one of the bufadienolides prepared from toad venom, was investigated for its cytotoxicity, and the underneath mechanism involved. We primarily utilized DNA fragmentation assay and microscopic observation to assess the effect of various doses of CB in human lymphoma U937 cells. Following that, we investigated other parameters involved in cell death mechanism such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptotic proteins activation. HeLa cells were concomitantly used to generalize the data observed. Our results show that CB caused significant DNA fragmentation, decrease of MMP, and an increase in the intracellular Ca(2+) ion and ROS production. In addition, CB induced upregulation of Fas protein, proteolytic activation of cytochrome c, caspase-2, -3, -8 and -9 together with the activation of Bid and Bax. Our findings were further validated using either Fas/FasL antagonist or pan-caspase inhibitor to significantly inhibit CB-induced DNA fragmentation. In our study, we suggest that CB induces caspase dependent cell death in U937 cells, and that Fas plays a role in CB-induced apoptosis. Altogether, our data provides novel insights of the mechanism of action of CB and its potential as a future chemotherapeutic agent.     

Thiosulfinates from Allium tuberosum L. induce apoptosis via caspase-dependent and -independent pathways in PC-3 human prostate cancer cells

This study was aimed to evaluate the apoptotic effects of thiosulfinates purified from Allium tuberosum L. on PC-3 human prostate cancer cells, and to elucidate detailed apoptosis mechanisms. Thiosulfinates significantly decrease viable cell numbers in dose- and time-dependent manners by apoptotic cell death via DNA fragmentation, chromatin condensation, and an increased sub-G1 phase. Apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8 and -9, and the effector caspase-3. In this study, thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in PC-3 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibit cell proliferation and induce apoptosis in PC-3 cells, which may be mediated via both caspase-dependent and -independent pathways.     

Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells

Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x(L) had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C(2)-ceramide, induced cytochrome c release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x(L) overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells.     

Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.     

Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines

In this study, we investigated the signaling pathways implicated in SSa-induced apoptosis of human colon carcinoma (HCC) cell lines. SSa-induced apoptosis of HCC cells was associated with proteolytic activation of caspase-9, caspase-3, and PARP cleavages and decreased levels of IAP family members, such as XIAP and c-IAP-2, but not of survivin. The fluorescence intensity of DiOC6 was significantly reduced after SSa treatment. CsA significantly inhibited SSa-induced loss of mitochondrial transmembrane potential and moderately inhibited SSa-induced cell death. SSa treatment also enhanced the activities of caspase-2 and caspase-8, Bid cleavage, and the conformational activation of Bax. Additionally, SSa-induced apoptosis was inhibited by both the selective caspase-2 inhibitor z-VDVAD-fmk and the selective caspase-8 inhibitor z-IETD-fmk and also by si-RNAs against caspase-2 and caspase-8. The selective caspase-9 inhibitor, z-LEHD-fmk, also inhibited SSa-induced apoptosis, albeit to a lesser extent compared to z-VDVAD-fmk and z-IETD-fmk, indicating that both mitochondria-dependent and mitochondria-independent pathways are associated with SSa-induced apoptosis. Both z-VDVAD-fmk and z-IETD-fmk significantly attenuated the colony-inhibiting effect of SSa. Moreover, inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk, or by knockdown of protein levels using a si-RNA, suppressed SSa-induced caspase-8 activation, Bid cleavage, and the conformational activation of Bax. Although caspase-8 is an initiator caspase like caspase-2, the inhibition of caspase-8 activation by knockdown using a si-RNA did not suppress SSa-induced caspase-2 activation. Altogether, our results suggest that sequential activation of caspase-2 and caspase-8 is a critical step in SSa-induced apoptosis.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.     

Effector CD4+ T cells generate intermediate caspase activity and cleavage of caspase-8 substrates

Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4(+) T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIP(L), receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4(+) T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling.     

Molecular mechanisms of echinocystic acid-induced apoptosis in HepG2 cells

Echinocystic acid (EA), a natural triterpone enriched in various herbs, has been showed to have cytotoxic activity in some cancer cells, and is used for medicinal purpose in many Asian countries. In the present study, we found that EA could induce apoptosis in human HepG2 cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed at 45 microM for 24 h. Molecular data showed that EA induced the truncation of Bid protein and reduction of Bcl-2 protein. EA also caused the loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to cytosol. Moreover, EA could activate c-Jun NH(2)-terminal kinase (JNK) and p38 kinase, and JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB200235 could block serial molecular events of EA-induced apoptosis such as Bid truncation, Bcl-2 reduction, cytochrome c release, caspase activation, and DNA fragmentation in HepG2 cells. These findings indicate that JNK- and p38 kinase-mediated mitochondrial pathways might be involved in EA-induced apoptosis and enhance our understanding of the anticancer function of EA in herbal medicine.     

p38 mitogen-activated protein kinase mediates bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide

p38 mitogen-activated protein kinase is activated and involved in cleavage of caspase-3 during apoptosis induced by a number of stimuli. However, the signaling events triggered by p38 that result in caspase-3 activation are still unknown. In human leukemia cells, two reactive oxygen species, singlet oxygen and hydrogen peroxide (H(2)O(2)), selectively stimulated the phosphorylation of p38. Preincubation of cells with SB203580, a specific inhibitor of p38, dose dependently inhibited DNA fragmentation induced by singlet oxygen but not by H(2)O(2). Protection from apoptosis by SB203580 correlated with inhibition of caspase-3, and several events that are associated with caspase-3 activation, including Bid cleavage, decrease in mitochondrial transmembrane potential and release of cytochrome c from mitochondria, whereas caspase-8 cleavage was not affected by this inhibitor. In contrast, blockade of caspase-8 with Ile-Glu-Thr-Asp-fluoromethyl ketone is sufficient to prevent formation of DNA fragments and to inhibit all the above signaling events, with exception of p38 phosphorylation, in both singlet oxygen- and H(2)O(2)-treated cells. These data suggest that caspase-3 activation is regulated through redundant signaling pathways that involve p38 and caspase-8 acting upstream of Bid during singlet oxygen-induced apoptosis, whereas the activation of caspase-3 by H(2)O(2) is only governed by a caspase-8-mediated apoptotic pathway.     

A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages

A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells. As an NF-kappaB target gene, A20 is also induced in many other cell types by a wide range of stimuli. Expression of A20 has been shown to protect from TNF-induced apoptosis and also functions via a negative-feedback loop to block NF-kappaB activation induced by TNF and other stimuli. To date, there are no reports on whether A20 can protect OxLDL-induced apoptosis in macrophages. For the first time we report that A20 expression blocks OxLDL-mediated cell toxicity and apoptosis. OxLDL induced the expression of Fas and FasL, and the subsequent caspase-8 cleavage and treatment with a neutralizing ZB4 anti-Fas antibody blocked apoptosis induced by OxLDL. Expression of dominant negative FADD efficiently prevented OxLDL-induced apoptosis and caspase-8 activation. A20 expression significantly attenuated the increased expression of Fas and FasL, and Fas-mediated apoptosis. These findings suggest that A20-mediated protection from OxLDL may occur at the level of Fas/FADD-caspase-8 and be FasL dependent. Treatment of RAW264.7 cells with OxLDL induces a series of time-dependent events, including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol, activation of caspase-9, -6, -2, and -3, which are blocked by A20 expression. No cleaved form of Bid was detected, even treatment with OxLDL for 48 h. Expression of dominant negative FADD also efficiently prevented OxLDL-induced the above apoptotic events. The release of cyto c, Smac and Omi from mitochondria to cytosol, activated by OxLDL treatment, and the activation of caspase-9 may not be a downstream event of caspase-8-mediated Bid cleavage. Therefore, the protective effect of A20 on mitochondrial apoptotic pathway activated by OxLDL may be dependent on FADD. A20 expression reversed OxLDL-mediated G(0)/G(1) stage arrest by maintaining the expression of cyclin B1, cyclin D1, and cyclin E, and p21 and p73. Thus, A20 expression blocks OxLDL-mediated apoptosis in murine RAW264.7 macrophages through disrupting Fas/FasL-dependent activation of caspase-8 and the mitochondria pathway.     

Cytochrome c release into cytosol with subsequent caspase activation during warm ischemia in rat liver

Apoptosis plays an important role in liver ischemia and reperfusion (I/R) injury. However, the molecular basis of apoptosis in I/R injury is poorly understood. The aims of this study were to ascertain when and how apoptotic signal transduction occurs in I/R injury. The apoptotic pathway in rats undergoing 90 min of warm ischemia with reperfusion was compared with that of rats undergoing prolonged ischemia alone. During ischemia, mitochondrial cytochrome c was released into the cytosol in a time-dependent manner in hepatocytes and sinusoidal endothelial cells, and caspase-3 and an inhibitor of caspase-activated DNase were cleaved. However, apoptotic manifestation and DNA fragmentation were not observed. After reperfusion, nuclear condensation, cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, and DNA fragmentation were observed and caspase-8 and Bid cleavage occurred. In contrast, prolonged ischemia alone induced necrosis rather than apoptosis. In summary, our results show that release of mitochondrial cytochrome c and caspase activation proceed during ischemia, although apoptosis is manifested after reperfusion.