The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells

In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 microM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells.     

Pyranocoumarin(+/-)-4'-O-acetyl-3'-O-angeloyl-cis-khellactone induces mitochondrial-dependent apoptosis in HL-60 cells

The pyranocoumarin (+)-4'-O-acetyl-3 'O-angeloyl-cis-khellactone (PC) isolated from Radix Peucedani (root of Peucedanum praeruptorum Dunn) showed a dose-dependent effect at 10 -30 pg/mL on causing apoptotic DNA and nuclear fragmentations in HL-60 cells. After 24 h of PC treatment there were losses of mitochondrial membrane potential and cytochrome c. PC also increased total cellular and mitochondrial Bax protein, stimulated an increase in caspase-dependent Bcl-2 cleavage but showed no effect on Bcl-Xv. These observations strongly suggest activation of the mitochondria apoptotic pathway. The pan-specific caspase inhibitor, ZVAD-fmk, abolished the PC-induced apoptosis,whereas the caspase-8 inhibitor IETD-fmk showed no effect, implying the involvement of the caspase 9 pathway. PC caused a 2 to 12 hour transient increase in phospho-ERK, and a 72 h-long activation of JNK. Pre-treatment with the MEK inhibitor PD98059, which suppresses ERK activation, paradoxically promoted PC-induced mitochondrial cytochrome c release, procaspase-3 and -8 cleavage, and enhanced apoptosis. Our results show that PC triggers mitochondria-mediated apoptosis in HL-60 cells, and the involvement of ERK and JNK signal pathways in the process.     

Heterogeneous role of caspase-8 in fenretinide-induced apoptosis in epithelial ovarian carcinoma cell lines

The mechanism of action of fenretinide, a synthetic retinoid currently undergoing testing as a chemopreventive and chemotherapeutic agent, is incompletely understood. In the present study, fenretinide caused apoptotic changes, including DNA fragmentation and cleavage of caspase substrates, in six low-passage ovarian cancer cell lines. However, the caspase activation pathway used by this agent varied. Transient transfection of cDNA-encoding cytokine response modifier A (CrmA), a caspase-8 inhibitor, diminished fenretinide-induced death in OV177 cells. Likewise, IETD(OMe)-fluoromethylketone (fmk) inhibited fenretinide-induced apoptosis by >80% in OV177 or OV266 cells and by approximately 50% in OV17, OV167, or OV207 cells. Further analysis demonstrated that inhibition of Fas ligand, tumor necrosis factor-alpha, or TRAIL signaling with blocking reagents did not affect fenretinide-induced apoptosis, raising the possibility that fenretinide activates caspase-8 in a death receptor-independent manner. In contrast, CrmA transfection or IETD(OMe)-fmk treatment did not inhibit fenretinide-induced apoptosis in OV202 cells. These divergent behaviors did not correlate with increased levels of procaspase-10, which is relatively resistant to CrmA and IETD(OMe)-fmk, nor with the expression of procaspase-8 and -9, apoptotic protease activating factor-1, or cellular FLICE-like inhibitory protein. Similarly, fenretinide treatment increased ceramide levels equally in cells that do (OV177) and do not (OV202) rely on caspase-8 to initiate apoptosis. These results indicate that synthetic retinoids can use caspase-8 as an initiating caspase, but they also indicate unexpected heterogeneity in caspase activation pathways among closely related cell lines.     

Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: any single lysine or lysine pair of the small subunit is sufficient for ubiquitination

Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.     

Differential modulation of apoptosis-associated proteins by ethanol in rat cerebral cortex and cerebellum

Chronic ethanol treatment caused a differential modulation of apoptosis-associated proteins, cytochrome c release, concomitant with procaspase-9 and procaspase-3 activation leading to oligonucleosomal DNA fragmentation in rat cerebral cortex and cerebellum. Caspase-3 proform (32 kDa) showed decreased immunoreactivity in cortex and cerebellum, while the cleaved active fragment (17 kDa) increased significantly in cerebellum after ethanol treatment. Further, chronic ethanol treatment increased caspase-3 activity in cortex and to a higher extent in cerebellum, which was further confirmed by blocking experiments with caspase-3 specific inhibitor, N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). We tested whether activated caspase-3 cleaves downstream substrates such as poly (ADP-ribose) polymerase-1 and protein kinase C-delta (PKC-δ). Western blots showed poly (ADP-ribose) polymerase-1 cleavage to its signature fragment of 85 kDa and decreased levels of PKC-δ in cerebral cortex and cerebellum after ethanol treatment, suggestive of caspase-3 activation. Elevated caspase-3 activity in cerebellum than cortex correlating with cytochrome c, caspase-9, active caspase-3 (p17), poly (ADP-ribose) polymerase-1 and PKC-δ data, suggests a mechanism by which ethanol might be exerting pro-apoptotic events in brain and how selective brain regions such as cerebellum are vulnerable to ethanol neurotoxicity in terms of cell death.     

A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 microM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent.     

Role of c-Jun N-terminal kinase/p38 stress signaling in 1-beta-D-arabinofuranosylcytosine-induced apoptosis

1-beta-D-Arabinofuranosylcytosine (ara-C) induced apoptosis in HL-60 cells, which was preceded by the activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK). 2'-Amino-3'-methoxyflavone (PD098059) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) were used to inhibit the activity of ERK and p38, respectively. SEK-AL, a dominant-negative mutant of SEK1, was transfected into HL-60 cells (HL-60/SEK-AL) to assess the role of JNK/SAPK activity in apoptosis. PD098059 (25 microM) inhibited ara-C-induced caspase-3-like activity but was ineffective in altering ara-C-mediated apoptotic DNA fragmentation and clonogenicity. On the other hand, SB203580 (20 microM) inhibited ara-C-induced caspase-3-like activity, apoptotic DNA fragmentation, and clonogenicity. The inhibition of JNK1 activation in HL-60/SEK-AL cells did not block ara-C-induced apoptotic DNA fragmentation. These results suggest that ara-C-induced apoptotic DNA fragmentation and loss of clonogenicity occur through a p38-dependent pathway.     

A dual role for poly(ADP-ribose) polymerase-1 during caspase-dependent apoptosis

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway.     

Cadmium induces apoptosis partly via caspase-9 activation in HL-60 cells.

Cadmium (Cd), a potent immunotoxic metal, induces apoptosis both in vitro and in vivo. However, the mode of action remains unclear. We previously reported that Cd-induced apoptosis was partly dependent on mitochondria. In the present study, we investigated the involvement of caspase-9, which is the apex caspase in the mitochondoria-dependent apoptosis pathway, in Cd-induced apoptosis in human promyelocytic leukemia HL-60 cells. A specific inhibitor of caspase-9, Z-LEHD-FMK, partly inhibited DNA fragmentation induced by Cd treatment in HL-60 cells. Moreover, treatment of HL-60 cells with Cd resulted in the appearance of Cytochrome c (Cyt c), a potent activator of caspase-9, in the cytosol at 3 h, which closely paralleled the activation of caspase-9. Caspase-9 is an initiator caspase that is a potent activator of downstream effector caspases such as caspase-3. Caspase-3 activation was subsequent to the Cyt c release at 6 h. DNA fragmentation, an index of induction of apoptosis, also appeared 6 h after Cd treatment. The effects were more pronounced at 9 h after Cd addition. A broad-specificity inhibitor of caspases, Z-Asp-CH(2)-DCB, inhibited caspase-3 activation and DNA fragmentation induced by Cd in a dose-dependent fashion. The results suggest that Cd-induced apoptosis is partly caused by caspase-9 activation triggered by Cyt c.     

Reduction of Cys–Cys and Cys–Cys disulfide bonds in recombinant human granulocyte colony stimulating factor

The Cys³⁶–Cys⁴² and Cys⁶⁴–Cys⁷⁴ disulfide bonds in recombinant methionyl human granulocyte colony-stimulating factor were reduced to sulfhydryls with dithiothreitol or mercury. Both reduction reactions are dependent on the pH. The reduction reaction with dithiothreitol increased in rate with increasing pH; between pH 7–9 and above pH 10.5 this increase was less than in other regions. These observations are explained by repulsive forces between dithiothreitol and regions in granulocyte colony-stimulating factor which intensify in these pH-regions. The hydroxyl catalysis causes the overall increase in k_obs in the pH-region studied. The reduction of the disulfides with mercury is, as could be expected from the Nernst equation for disulfide reduction, also pH dependent: the half-wave potential decreases with increasing pH as predicted by theory.     

吡格列酮对Aβ_(25-35)引起的皮层神经元损伤保护作用部分机制的研究

目的研究吡格列酮对抗淀粉样β蛋白片段25-35(Amyloid-β,Aβ25-35)所致培养皮层神经元损伤作用的机制。方法取培养7d大鼠乳鼠大脑皮层神经元,Aβ组加入Aβ25-35(20μmol.L-1)作用24h;吡格列酮组和各种阻断剂组,先加入吡格列酮(0.1、1、10μmol.L-1)或各种阻断剂作用1h,然后加入Aβ25-35(20μmol.L-1)作用24h;正常对照组加入等量培养基。MTT法测定细胞存活率;免疫荧光染色法测定活性的caspase-3细胞内定位;Westernblot检测活性的caspase-3表达水平;Griess法测定培养细胞上清液中一氧化氮(NO)含量。结果神经元经NSE和NF200免疫荧光鉴定,其阳性率可达90%以上。Aβ25-35(20μmol.L-1)可使神经元细胞存活率下降、caspase-3表达明显增加,同时神经元培养液中的NO含量也明显增加。吡格列酮可明显抑制Aβ25-35诱导的神经元细胞存活率下降、抑制caspase-3表达的增加,吡格列酮还可明显抑制Aβ25-35诱导的神经元培养液中NO含量增加,且呈浓度依赖性。GW9662(10μmol.L-1)能明显对抗吡格列酮对Aβ25-35诱导的神经元细胞存活率下降、活性的caspase-3表达增加、NO增加的抑制作用。SP600125(5μmol.L-1)、SB203580(20μmol.L-1)和SMT(1mmol.L-1)可明显对抗Aβ25-35诱导的神经元细胞存活率下降及培养液中NO含量增加。结论吡格列酮能够明显的抑制Aβ25-35引起的皮层神经元损伤作用,这种作用可能与激活PPARγ受体、抑制JNK信号传导通路和p38MAPK信号传导通路有关。     

Procaspase-3小分子激活剂的研究进展

Caspase-3作为细胞凋亡通路的下游关键执行蛋白,在细胞凋亡信号传导中扮演着重要的角色。开发直接通过激活高表达状态的procaspase-3成为caspase-3,进而重启肿瘤细胞凋亡的小分子药物,已成为克服中/上游凋亡蛋白突变的有效策略。目前,以procaspase-3为靶点已经报道了一些特异性小分子激活剂,至今还没有上市药物,开发直接激活procaspase-3的新型药物仍然是一项重要和艰难的挑战。本文介绍了procaspase-3与肿瘤之间的关系及小分子procaspase-3激活剂的研究进展。     

DNA引物酶抑制剂碘化-3,3′-二乙基-9-甲基-硫杂羰花青诱导人白血病HL-60细胞凋亡

目的研究DNA引物酶抑制剂碘化-3,3′-二乙基-9-甲基-硫杂羰花青(DMTCCI)诱导人粒细胞性白血病HL-60细胞凋亡并探索其机制。方法分别采用不同浓度的DMTCCI处理培养于RPMI-1640培养基的HL-60细胞。采用MTT法检测DMTCCI对HL-60细胞的生长抑制作用。采用流式细胞仪和DNA琼脂糖凝胶电泳方法检测细胞凋亡。采用蛋白免疫印迹(Western blotting)法观察凋亡相关蛋白survivin， Bcl-xL， Bad， Bax， Bcl-2， caspase-9， caspase-3， caspase-6， PARP， DFF45和lamin B的表达。采用ApoAlert Caspase-3分析试剂盒检测caspase-3的活性。结果DMTCCI具有抑制人白血病HL-60细胞增殖的作用，其IC₅₀值为0.24 μmol·L^-1。流式细胞仪和DNA琼脂糖凝胶电泳结果显示，DMTCCI可诱导HL-60细胞凋亡。在经DMTCCI处理的HL-60细胞中，survivin和Bcl-xL蛋白的表达水平下调，Bad和Bax蛋白的表达水平上调，Bcl-2蛋白的表达水平无变化，caspase-9，caspase-3，caspase-6，PARP，DFF45和lamin B被分别裂解，产生相应裂解产物。在HL-60细胞中，caspase-3的活性在1 μmol·L^-1 DMTCCI处理3 h时明显升高，在处理12 h时达到最高峰。结论DMTCCI可抑制人白血病HL-60细胞的增殖并诱导其发生细胞凋亡。Bcl-2家族蛋白、survivin和caspases家族蛋白可能参与了上述诱导HL-60细胞凋亡的过程。     

Protection by ascorbic acid from denaturation and release of cytochrome c, alteration of mitochondrial membrane potential and activation of multiple caspases induced by H(2)O(2), in human leukemia cells

We investigated peroxide and superoxide accumulation, cytochrome c nature and release from mitochondria, as well as caspase activation during exposure of HL-60 cells to H(2)O(2) and the protective effect of ascorbic acid. Exposure of the cells to 100 microM H(2)O(2) resulted in intracellular accumulation of peroxides, denaturation of cytochrome c that was identified in the mitochondria and cytosol, release of native cytochrome c to the cytosol and fall in mitochondrial membrane potential (DeltaPsi(m)). Loading of cells with ascorbic acid before exposure to H(2)O(2) resulted in a dose-dependent protective effect against: intracellular accumulation of peroxides, DeltaPsi(m) alteration, cytochrome c denaturation and release. The accumulation of peroxides induced processings and activations of procaspase-8, -9 and -3. Double staining with antiserum which recognizes the p18 subunit of cleaved caspase-3 and with Hoechst had shown that a high percentage of cells exposed to 100 microM H(2)O(2) stained positively with the antibody and showed features of apoptosis. Ascorbic acid loading prevented caspase activation induced by H(2)O(2). We conclude that ascorbic acid protects against activation of apoptotic cascades in HL-60 cells exposed to H(2)O(2) and against apoptosis.     

Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56 in human Jurkat T cells

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of caspase-12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by overexpression of Bcl-xL. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132-induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, Δψm loss, and cleavage of procaspase-9 (47 kDa) to active form (35 kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G₁ peak, Bak activation, and Δψm loss were markedly reduced by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and mitochondria-dependent activation of caspase cascade were more significant in p56^lck-stable transfectant JCaM1.6/lck than in p56^lck-deficient JCaM1.6/vector. The cytotoxicity of MG132 toward p56^lck-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56^lck enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.     

The cytotoxicity of eutigosides fromEurya emarginata against HL-60 promyelocytic leukemia cells

Two phenolic glucosides, eutigoside B and eutigoside C were isolated from the fresh leaves of Eurya emarginata. These two phenolic glucosides exerted a significant inhibitory effect on the growth of HL-60 promyelocytic leukemia cells. Furthermore, when the HL-60 cells were treated with eutigoside C, several apoptotic characteristics such as DNA fragmentation, morphologic changes, and increase of the population of sub-G1 hypodiploid cells were observed. In order to understand the mechanism of apoptosis induction by eutigoside C, we examined the changes of Bcl-2 and Bax expression levels. The eutigoside C reduced Bcl-2 protein and mRNA levels, but slightly increased Bax protein and mRNA levels in a time-dependent manner. When we examined the activation of caspase-3, an effector of apoptosis, the eutigoside C increased the expression of active form (19-kDa) of caspase-3 and the increase of their activities was demonstrated by the cleavage of poly (ADP-ribose) polymerase, a substrate of caspase-3, to 85-kDa. The results suggest that the inhibitory effect of eutigoside C from E. emarginata on the growth of HL-60 appears to arise from the induction of apoptosis via the down-regulation of Bcl-2 and the activation of caspase.     

Differential protection with inhibitors of caspase-8 and caspase-3 in murine models of tumor necrosis factor and Fas receptor-mediated hepatocellular apoptosis

Excessive apoptosis has been implicated in a number of acute and chronic human diseases. The activation of caspases has been shown to be critical for the apoptotic process. The objective of this investigation was to evaluate the beneficial effects and mechanism of action of the caspase-8 inhibitor IETD-CHO and the caspase-3 inhibitor DEVD-CHO against tumor necrosis factor (TNF)-induced hepatocellular apoptosis in vivo and compare these results to effects of the same inhibitors against Fas-induced apoptosis. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine/100 microg/kg endotoxin induced parenchymal apoptosis (indicated by caspase-3 activation and morphology) and severe liver injury (indicated by the increase in plasma alanine aminotransferase activities and histology) at 7 h. Treatment with IETD-CHO or DEVD-CHO (10 mg/kg at 3, 4.5, and 5.5 h) significantly attenuated caspase-3 activation and liver injury. Western analysis showed that DEVD-CHO had no effect while IETD-CHO substantially reduced procaspase-3 and procaspase-9 processing. On the other hand, caspase-3 activation and liver injury by the anti-Fas antibody Jo-2 was completely prevented by a single dose of DEVD-CHO and, as previously shown, by IETD-CHO at 90 min. Both inhibitors prevented procaspase-3 and procaspase-9 processing. Thus, there are fundamental differences in the efficacy of caspase inhibitors in these two models. We conclude that Fas may rely exclusively on caspase-8 activation and mitochondria to activate caspase-3, which can process more procaspase-8 and thus propagate the amplification of the apoptotic signal. TNF can activate a similar signaling pathway. However, alternative signaling mechanisms seem to exist, which can compensate if the main pathway is blocked.     

Activation of p38 mitogen-activated protein kinase during ent-11alpha-hydroxy-16-kauren-15-one-induced apoptosis in human leukemia HL-60 cells

Kaurene-type diterpenes possess various biological activities including antitumor and anti-inflammatory effects. Indeed, we have found that an ent-kaurene diterpene, ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis via caspase-8 activation in human promyelocytic leukemia HL-60 cells. However, the mechanism of caspase-8 activation by KD is not clear. In this study, we investigated the involvement of p38 mitogen-activated protein kinase (p38 (MAPK)) in KD-induced apoptosis. p38 (MAPK) was activated by treatment with KD parallel to DNA ladder formation. Pretreatment with SB203580, a specific inhibitor of p38 (MAPK), attenuated induction of apoptosis by KD and inhibited activation of caspase-8. Cleavage of Bid, a typical substrate of caspase-8, was also inhibited by treatment with SB203580, suggesting that activation of p38 (MAPK) occurs upstream of caspase-8 during KD-induced apoptosis.     

Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56lck in human Jurkat T cells

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of caspase-12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by overexpression of Bcl-xL. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132-induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, Δψm loss, and cleavage of procaspase-9 (47kDa) to active form (35kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G(1) peak, Bak activation, and Δψm loss were markedly reduced by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and mitochondria-dependent activation of caspase cascade were more significant in p56(lck)-stable transfectant JCaM1.6/lck than in p56(lck)-deficient JCaM1.6/vector. The cytotoxicity of MG132 toward p56(lck)-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56(lck) enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.     

Trimidox induces apoptosis via cytochrome c release in NALM-6 human B cell leukaemia cells

Trimidox (3,4,5-trihydroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase accompanied by growth inhibition and the differentiation of mammalian cells. Here we examine the induction of apoptosis by trimidox in several human leukaemia cell lines, focusing on the release of cytochrome c and the activation of caspase proteases in the human B cell line NALM-6. Induction of apoptosis by trimidox (300 microM) was detected in NALM-6, HL-60 (premyelocytic leukaemia cells), MOLT-4 (an acute lymphoblastic leukaemia cells), Jurkat (a T-cell leukaemia cells), U937 (expressing many monocyte-like characteristics), and K562 (erythroleukaemia). NALM-6 was most affected by trimidox among leukaemia cells; therefore, we employed NALM-6 cells in the subsequent experiments. The cells showed a time-dependent increase in DNA damage after trimidox (250 microM) treatment. A significant increase in the amount of cytochrome c release was detected after treatment with trimidox. Bcl-2 and Bax protein expressions were not changed by trimidox. Caspase-3 and -9 were activated by incubation with trimidox, whereas caspase-8 was not. Furthermore, trimidox-induced apoptosis was prevented by a broad-spectrum caspase inhibitor, a caspase-3, and a caspase-9 inhibitor, but not by a caspase-8 inhibitor. Inhibition of c-Jun NH2-terminal kinase (JNK) by SP600125 appreciably protected cells from trimidox-induced apoptosis, but no effect inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580. In contrast, extracellular signal-regulated kinase (ERK) inhibitors U0126 and PD98059 strongly potentiated the apoptotic effect of trimidox. This report shows that the induction of apoptosis by trimidox occurs through a cytochrome c-dependent pathway, which sequentially activates caspase-3 and caspase-9.