Glutathione-S-transferase pi inhibits As2O3-induced apoptosis in lymphoma cells: involvement of hydrogen peroxide catabolism

Arsenic trioxide (As(2)O(3)) is an effective agent for the treatment of relapsed and refractory acute promyelocytic leukemia by induction of partial differentiation and apoptosis. As(2)O(3), at therapeutic concentrations (1-2 microM), induced apoptosis in Raji lymphoma cells but not in Jurkat lymphoma cells, which inversely correlated with the levels of glutathione-S-transferase pi (GSTP1), but not GSTpi(1) and GSTM(1), expression and activity. GSTP1 mRNA, protein level, and activity were high in Jurkat cells but undetectable in Raji cells. Stable transfection of GSTP1 into Raji cells decreased the amount of As(2)O(3)-induced apoptosis. Apoptosis induced by therapeutic concentrations of As(2)O(3) in Raji cells is related to increasing H(2)O(2) intracellular accumulation but not to JNK activation. Forced expression of GSTP1 by transfection of Raji cells significantly decreased the basal amount of H(2)O(2) and its levels after therapeutic concentration of As(2)O(3) treatment. Added exogenous H(2)O(2) was removed more rapidly, which correlated with a greater decrease in reduced glutathione level in Raji clones expressing GSTP1 than in those clones without GSTP1 expression. Overexpression of GSTP1 in transfected Raji clones was also found to decrease the retention of As(2)O(3). These data suggest that GSTP1 blocks As(2)O(3)-induced apoptosis in lymphoma cells by decreasing intracellular amounts of H(2)O(2) by catabolism and H(2)O(2) production by decreasing the intracellular retention of As(2)O(3).     

Etodolac inhibits EBER expression and induces Bcl-2-regulated apoptosis in Burkitt's lymphoma cells

Cyclooxygenase-2 (COX-2) is reported to be an important cellular target for therapy in malignancies. The growth inhibitory effects of COX-2 inhibitors on malignancies have been demonstrated to be through not only COX-2 dependent, but also independent mechanisms. In this study, we showed that etodolac, COX-2 inhibitor, induced apoptosis via COX-2 independent pathway, and investigated the molecular details of etodolac-induced apoptosis in Burkitt's lymphoma cells. In Daudi and Raji Burkitt's lymphoma cell lines, which expressed no COX-2 enzyme, etodolac more strongly induced apoptosis compared to meloxicam. Moreover, etodolac did not induce apoptosis to normal B-lymphocytes. For the pathway of etodolac-induced apoptosis, reduction of anti-apoptotic bcl-2 mRNA and Bcl-2 protein, activation of Caspase-9 and -3, down-regulation of caspase inhibitors, c-IAP-1 and Survivin were involved. Moreover, EBER-1 and -2 expression in Epstein-Barr virus positive Daudi and Raji cells were reduced to result in down-regulation of Bcl-2 by treatment with etodolac. It has been reported that etodolac has stereoisomers, R- and S-etodolac. We found that racemate of etodolac more strongly induced apoptosis in Daudi and Raji cells compared to R- or S-etodolac. In conclusion, our findings indicated etodolac inhibited EBERs expression and induced apoptosis via a Bcl-2-regulated pathway. Moreover, racemate of etodolac more effectively induced apoptosis than R- and/or S-etodolac. Therefore, these activities of etodolac potentially extend to the treatment of patients with Burkitt's lymphoma resistant to chemotherapy.     

Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway

The activation of the glucagon-like peptide-1 (GLP-1) receptor has been shown to have an important role in the functional activity of islet beta-cells and in the expansion of the islet cell mass. Constant remodeling of islet cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of GLP-1 when cells were challenged by a proapoptotic stimulus. We have shown that activation of the GLP-1 receptor inhibits H(2)O(2)-induced apoptosis in a cultured mouse insulinoma cell line, termed MIN6. GLP-1 reduced DNA fragmentation and improved cell survival. This was mediated by an increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. GLP-1 also prevented the H(2)O(2)-dependent cleavage of poly-(ADP-ribose)-polymerase. Inhibition of the GLP-1-dependent increase of cAMP by Rp-cAMP blocked the antiapoptotic action of GLP-1, as determined by DNA fragmentation and poly-(ADP-ribose)-polymerase assays and by detection of Bcl-2 and Bcl-xL protein levels. Investigation of the role of the protein kinases, PI-3 kinase (PI3K) and MAPK, by use of the inhibitors PD098059 and LY294002 demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H(2)O(2). The present study provides evidence that GLP-1 has an antiapoptotic action mediated by a cAMP- and PI3K-dependent signaling pathway.     

Impairment of death-inducing signalling complex formation in CD95-resistant human primary lymphoma B cells

Multiple mechanisms exist by which tumour cells can escape CD95-mediated apoptosis. Previous studies by our laboratory have shown that primary B cells from non-Hodgkin's Lymphoma (B-NHL) were resistant to CD95-induced cell death. In the current study, we have analysed the mechanisms underlying CD95 resistance in primary human lymphoma B cells. We report that FADD (FAS-associated death domain protein) and caspase-8 were constitutively expressed in lymphoma B cells and that the CD95 pathway was blocked upstream to caspase-8 activation. However, caspase-8 was processed and functional after treatment with staurosporine (STS). We found that the expression levels of FLICE (FADD-like interleukin-1 beta-converting enzyme)-Inhibitory Protein (c-FLIP) and Bcl-2-related proteins were heterogeneous in B-NHL cells and were not related to CD95 resistance. Finally, we report the absence of a CD95-induced signalling complex [death-inducing signalling complex (DISC)] in lymphoma B cells, with no FADD and caspase-8 recruitment to CD95 receptor. In contrast, DISC formation was observed in CD95-resistant non-tumoural (NT) B cells. Therefore, we propose that the absence of DISC formation in primary lymphoma B cells may contribute to protect these cells from CD95-induced apoptosis.     

黄芩苷诱导Burkitt淋巴瘤细胞凋亡的初步研究

目的:探讨黄芩苷(baicalin)对Burkitt淋巴瘤细胞系Daudi细胞凋亡的诱导作用。方法:用3-(4,5-二甲基噻唑)-2,5-二苯基四氮唑溴盐(MTT)法测定细胞生长抑制率;细胞涂片观察细胞形态学改变;流式细胞术检测细胞凋亡变化;Westernblot法检测凋亡相关蛋白天冬氨酸特异性半胱氨酸蛋白酶(caspase-3)、caspase-9。结果:黄芩苷对Daudi细胞作用72h的半数抑制浓度(IC50)为(10.1±0.5)μg/mL,对其生长抑制作用呈剂量依赖性。与对照组相比,黄芩苷组细胞呈现明显的凋亡状态。48h流式检测细胞凋亡率40μg/mL药物组为61.5%±6.3%,20μg/mL药物组为44.2%±4.7%,明显高于对照组(8.2%±0.9%)(P<0.01)。黄芩苷作用Daudi细胞24、36h后,caspase家族的蛋白前体(procaspase)-3、9表达均下降,而激活的活性片段caspase-3则表达上调,且呈时间依赖性。结论:黄芩苷可有效抑制Burkitt淋巴瘤细胞生长,诱导细胞凋亡,上述凋亡蛋白可能参与了黄芩苷诱导Daudi细胞凋亡的过程。     

Defective apoptotic signal transduction pathway downstream of caspase-3 in human B-lymphoma cells: A novel mechanism of nuclear apoptosis resistance.

Mitochondria play a central role in controlling apoptosis, and activation of the caspase cascade appears to be crucial event during the apoptotic process. Human B lymphoma Raji cells are resistant to nuclear apoptosis induced by various stimuli. Using this cell line, we have asked whether reduction of the mitochondrial transmembrane potential and activation of caspase-3 are sufficient to induce DNA fragmentation during the apoptotic process. After stimulation with cell-permeable C2-ceramide or mitochondrial permeability transition (PT) inducers, not only apoptosis-sensitive cell lines (HL-60, Jurkat, and Daudi cells), but also Raji cells showed reduction of the mitochondrial transmembrane potential (triangle uppsim), activation of caspase-3, and loss of clonogenic potential. However, Raji cells did not show detectable levels of nuclear apoptosis (DNA degradation). In a cell-free system, cell lysates from tetra-butylhydroperoxide (t-BHP)-treated HL-60 cells induced DNA degradation of Raji nuclei, whereas cell lysates from t-BHP-treated Raji cells failed to induce DNA degradation in either apoptosis-sensitive cell lines or apoptosis-resistant Raji cells. Cleavage of DFF-45, which is a downstream target molecule for caspase-3, was observed in Raji cells as well as in apoptosis-sensitive Daudi cells. These results indicate that there is a defective apoptotic pathway in the cytoplasm downstream of caspase-3 in Raji cells.     

Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-x(L)

We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As(2)O(3) (0.5 to 2.0 micromol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli. These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-x(L) (HL-60/Bcl-x(L)), MDR (HL-60/VCR), or MRP (HL-60/AR) protein. The growth-inhibitory IC(50) values for As(2)O(3) treatment for 7 days against all these cell types ranged from 0.8 to 1.5 micromol/L. Exposure to 2 micromol/L As(2)O(3) for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (DeltaPsim) and the increase in reactive oxygen species (ROS). Treatment with As(2)O(3) (2 micromol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase. Significantly, As(2)O(3)-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-x(L), Bax, Apaf-1, Fas, and FasL. Although As(2)O(3 )treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However, in these cells, As(2)O(3 )potently induced hyper-acetylation of the histones H3 and H4. These findings characterize As(2)O(3) as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli.     

白皮杉醇对过氧化氢诱导的血管平滑肌细胞凋亡的影响与机制

目的:研究白皮杉醇(PCT)对过氧化氢(H2O2)诱导的血管平滑肌细胞(VSMCs)凋亡的影响以及所涉及的相关分子机制。方法:组织贴块法培养SD大鼠VSMCs并进行不同分组与处理;MTT法检测各组VSMCs的存活率;LDH、超氧化物歧化酶(SOD)试剂盒测定各组VSMCs中LDH与SOD活力;Annexin V-PI染色检测各组VSMCs凋亡情况,Hoechst 33258染色观察各组VSMCs细胞核形态变化;Western blot检测各组VSMCs中凋亡相关蛋白(Bcl-2、Bax、caspase-3)及PI3K/AKT/eNOS通路蛋白表达水平。结果:不同浓度与时间过氧化氢(H2O2)处理VSMCs后使其存活率降低,而不同浓度PCT预处理可提高细胞存活率,差异有统计学意义(P<0.05、P<0.01或P<0.001)。与对照组相比,H2O2组VSMCs中LDH活力升高而SOD活力下降,细胞凋亡率增加,细胞中Bax、caspase-3蛋白表达升高而Bcl-2蛋白表达下降,p-PI3K、p-Akt、p-eNOS蛋白表达下降,且均呈剂量依赖关系;与H2O2组相比,PCT预处理组VSMCs LDH活力下降而SOD活力升高,细胞凋亡率降低,细胞中Bax、caspase-3下降,Bcl-2蛋白表达升高,p-PI3K、p-Akt、p-eNOS的蛋白表达升高,且均呈剂量依赖关系,差异有统计学意义(P<0.05或P<0.01)。结论:PCT对H2O2引起的VSMCs损伤有保护作用,其机制可能是抑制LDH活力、增强SOD活力以及增强PI3K/AKT/eNOS信号通路进而阻碍VSMCs凋亡的发生。     

Selenium has a protective role in caspase-3-dependent apoptosis induced by H2O2 in primary cultured pig thyrocytes

OBJECTIVE: Hydrogen peroxide (H2O2), necessary for thyroid hormonogenesis, is produced at the apical surface of the thyroid follicular epithelium. Excess H2O2 is potentially cytotoxic and may contribute to the development of hypothyroidism, e.g. in severe selenium deficiency. Yet it is unclear how H2O2 contributes to thyroid cell death. DESIGN AND METHODS: H2O2-induced apoptosis and necrosis were studied in primary cultured pig thyroid cells. Glutathione peroxidase (GPx) activity was altered by culture in low serum with or without selenite substitution. Apoptosis was evaluated by spectrofluorometric measurement of caspase-3-specific substrate cleavage, and by analysis of DNA fragmentation by agarose gel electrophoresis. Necrosis was detected by 51Cr release from prelabeled cells. RESULTS: Exogenous H2O2 dose-dependently (100-400 micromol/l) activated caspase-3 within 3-12 h, and DNA degradation was observed after 24 h. The potency of H2O2 to induce apoptosis was low compared with that of staurosporine, a strong proapoptotic agent. H2O2-treated cells with reduced GPx activity showed increased caspase-3 activation. Incubation of serum-starved cells with selenite (10-100 nmol/l) normalized the GPx activity and reduced the activation of caspase-3 by H2O2. High H2O2 concentrations (400-800 micromol/l) were required to obtain necrosis. The H2O2-induced necrosis was exaggerated by both low GPx activity and catalase inhibition. CONCLUSIONS: Cytotoxic effects of H2O2 on thyroid cells include caspase-3-dependent apoptosis that occurs at H2O2 concentrations insufficient to induce necrosis. Selenium deficiency aggravates the apoptotic response, probably due to impaired capacity of GPx to degrade H2O2.     

Mechanisms which mediate the antiapoptotic effects of angiopoietin-1 on endothelial cells

The main objective of this study was to identify molecular mechanisms through which angiopoietin-1 (Ang-1), a ligand for Tie-2 receptors, influences endothelial cell apoptosis. Human umbilical vein endothelial cells were cultured in a medium enriched with 2% fetal bovine serum (FBS) and growth supplements. Apoptosis was induced over 24 h by reducing FBS to 0.1%. Activation of caspase-9, -8, -7, and -3 and the expression of Bcl-2 family proteins, inhibitors of apoptosis (IAPs), cytochrome c, as well as Smac proteins were evaluated with immunoblotting. Ang-1 clearly attenuated serum deprivation-evoked apoptosis, an effect which required Tie-2 receptor activation. Activation of caspase-9, -7, and -3, but not caspase-8, was inhibited by Ang-1. The inhibitory effects of Ang-1 on apoptosis and caspase activation were reversed by a PI-3 kinase inhibitor (wortmannin). Ang-1 exposure upregulated the expression of Survivin but not XIAP (members of IAPs), reduced the cystosolic levels of Smac, but not that of cytochrome c, and had no effect on the expression of Bcl-2 family proteins. This is the first study to report on the mitochondrial mechanisms through which Ang-1 inhibits apoptosis and to investigate the role of the newly discovered Smac. We conclude that Ang-1 inhibits endothelial cell apoptosis through several pathways, which include PI-3 kinase/AKT activation, inhibition of Smac release from the mitochondria, and upregulation of Survivin protein.     

Melatonin prevents hydrogen peroxide-induced Bax expression in cultured rat astrocytes

During oxidative stress, cell apoptosis is promoted through the mitochondrial death pathway. Increased reactive oxygen species (ROS) are linked to excess cell loss and mediate the induction of apoptosis in various cell types. However, the role of ROS in the apoptotic pathway has not been clearly established. The aims of this study were to investigate the biochemical and morphological responses of rat astrocytes to hydrogen peroxide-mediated cell death and to define the role that melatonin might play in the apoptotic cascade. Hydrogen peroxide (H2O2; 0.1-1.0 mM) significantly reduced cell viability. Astrocyte death was associated with enhanced ROS production in a dose-dependent manner, as measured by 2',7'-dichloro-fluorescein fluorescence. H2O2-induced cell death was found to be mediated through an apoptotic pathway as treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H2O2 also triggered caspase-3 activation and Bax expression. The ability of different antioxidants to prevent H2O2-induced apoptosis was examined by pre-incubating rat astrocytes with N-acetylcysteine (10 mM), glutathione (0.5 mM) or melatonin (0.1 mM and 10 nM). Results showed that N-acetylcysteine and glutathion can protect astrocytes against ROS accumulation and caspase-3 activation, whereas 0.1 mM melatonin can inhibit H2O2-induced apoptosis by regulating Bax expression and by inhibiting caspase-3 activation. Antiapoptotic effect of 10 nM melatonin associated to inhibition of Bax expression, give rise to new therapeutic approaches.     

Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.     

Nip21 gene expression reduces coxsackievirus B3 replication by promoting apoptotic cell death via a mitochondria-dependent pathway

Our previous studies, using differential mRNA display, suggested that the mouse Nip21 gene may be involved in myocarditis development in the coxsackievirus B3 (CVB3)-infected mouse heart. Sequence comparison indicated that the mouse Nip21 gene shares high sequence homology to human Nip2. This human protein is known to interact with both the apoptosis inhibitor Bcl-2 and a homologous protein, the adenovirus E1B 19-kDa protein. Such interactions implicate Nip21 gene in cell death pathways. To study the function of this gene, we have cloned Nip21 from mouse hearts and established a Tet-On doxycycline-inducible HeLa cell line and a cardiomyocyte H9c2 cell line expressing Nip21 to characterize gene function in relation to apoptosis. We demonstrated that Nip21 expression could induce apoptosis via caspase-depended mitochondria activation. To further determine the function of Nip21 in CVB3-induced apoptosis, the Tet-On/Nip21 HeLa cell line was induced by doxycycline followed by CVB3 infection. We found that activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase occurred 2 hours earlier than in vector-transfected control cells, suggesting that Nip21 expression enhances CVB3-induced apoptosis. We also demonstrated a significant decrease in HeLa cell and H9c2 cell viability. Particularly, as illustrated by viral plaque assay, CVB3 replication was dramatically reduced in Tet-On HeLa cells, due at least in part to the earlier killing of the host cells by Nip21 overexpression.     

Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway

Previous studies have shown that mitofusin 2 (Mfn-2) (or hyperplasia suppressor gene [HSG]) inhibits vascular smooth muscle cell (VSMC) proliferation. Here, we demonstrate that Mfn-2 is a primary determinant of VSMC apoptosis. First, oxidative stress with H2O2, inhibition of protein kinase C with staurosporine, activation of protein kinase A with forskolin, and serum deprivation concurrently elevate Mfn-2 expression and induce VSMC apoptosis. Second, overexpression of Mfn-2 also triggers apoptosis of VSMCs in culture and in balloon-injured rat carotid arteries, thus contributing to Mfn-2-mediated prevention of neointima formation after angioplasty. Third, Mfn-2 silencing protects VSMCs against H2O2 or Mfn-2 overexpression-induced apoptosis, indicating that upregulation of Mfn-2 is necessary and sufficient for oxidative stress-mediated VSMC apoptosis. The Mfn-2 proapoptotic effect is independent of its role in mitochondrial fusion but mainly mediated by inhibition of Akt signaling and the resultant activation of the mitochondrial apoptotic pathway, as manifested by decreased Akt phosphorylation, increased mitochondrial Bax/Bcl-2 ratio, cytochrome c release, and activation of caspases-9 and caspase-3. Furthermore, Mfn-2-induced apoptosis was blocked by overexpression of an active phosphoinositide 3-kinase mutant or Bcl-xL or inhibition of caspase-9 but not caspases-8. Thus, in addition to its antiproliferative effects, Mfn-2 constitutes a primary determinant of VSMC apoptosis.     

FTY720 demonstrates promising preclinical activity for chronic lymphocytic leukemia and lymphoblastic leukemia/lymphoma

FTY720 is an immunosuppressant developed to prevent organ transplant rejection. Recent studies indicate an additional role for FTY720 in inducing cell apoptosis. We demonstrate here that FTY720 mediates toxic effects in cell lines representing different B-cell malignancies and primary B cells from patients with chronic lymphocytic leukemia (CLL). In contrast to previous reports in T-cell lines, FTY720-induced toxicity in the Raji cell line and primary CLL B cells is independent of activation of caspases or poly(ADP-ribose) polymerase processing. Further, pancaspase inhibitor Z-VAD-fmk failed to rescue these cells from apoptosis mediated by FTY720. FTY720 induced down-regulation of Mcl-1 but not Bcl-2 in CLL B cells. Overexpression of Bcl-2 failed to protect transformed B cells from FTY720-induced apoptosis, suggesting a Bcl-2-independent mechanism. Interestingly, FTY720 induced protein phosphatase 2a (PP2a) activation and downstream dephosphorylation of ERK1/2, whereas okadaic acid at concentrations that inhibited the FTY720-induced PP2a activation also resulted in inhibition of FTY720-mediated apoptosis and restoration of baseline ERK1/2 phosphorylation in primary CLL cells, indicating a role for PP2a activation in FTY720-induced cytotoxicity. Further, FTY720 treatment resulted in significant prolonged survival in a xenograft severe combined immunodeficiency (SCID) mouse model of disseminated B-cell lymphoma/leukemia. These results provide the first evidence for the potential use of FTY720 as a therapeutic agent in a variety of B-cell malignancies, including CLL.     

The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3'-kinase

Recent studies have shown that chronic beta-adrenergic receptor (beta-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective beta(1)- and beta(2)-AR subtype stimulation on apoptosis induced by hypoxia or H(2)O(2) in rat neonatal cardiac myocytes. Although neither beta(1)- nor beta(2)-AR stimulation had any significant effect on the basal level of apoptosis, selective beta(2)-AR stimulation protected myocytes from apoptosis. beta(2)-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3'-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. beta(1)-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked beta(2)-AR-mediated protection from apoptosis as well as the beta(2)-AR-stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked beta(2)-AR-mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on beta(2)-AR-mediated protection. These findings demonstrate that beta(2)-ARs activate a PI-3K-dependent, pertussis toxin-sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.     

Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease inhibitor 9

Immune surveillance against malignant cells is mediated by cytotoxic T-lymphocytes and NK-cells (CTL/NK) that induce apoptosis through the granzyme-B-dependent pathway. The serine protease inhibitor serpinB9/protease inhibitor-9 (PI-9) is a known inhibitor of granzyme B. Ectopic expression of PI-9 in tumour cells has been reported. However, the impact of PI-9 on granzyme-B-induced apoptosis in tumour cells remains unclear. The aim of this study was to investigate the influence of constitutive PI-9 expression in leukaemia cell lines on the activity of granzyme B and apoptosis induction. PI-9 negative (lymphoblastic Jurkat cells; myeloblastic U937 cells) and PI-9-expressing cell lines (myeloblastic K562 cells, EBV-transformed LCL-1 and LCL-2 B-cells, lymphoblastic Daudi cells, AML-R cells f leukaemia and the U937 subclone U937PI-9⁺). For accurate granzyme B activity determination a quantitative substrate (Ac-IEPD-pNA) cleavage assay was established and caspase-3 activation measured for apoptosis assessment. Cells were treated with a cytotoxic granule isolate that has previously been shown to induce apoptosis through granzyme B signalling. We found a robust correlation between constitutive PI-9 expression levels and the suppression of granzyme B activity. Further, inhibition of granzyme B translated into reduced caspase-3 activation. We conclude, suppression of granzyme B initiated apoptosis in PI-9-expressing cells could contribute to immune evasion and the measurement of granzyme B activity with our assay might be a useful predictive marker in immune-therapeutic approaches against cancer.     

TRAIL-induced apoptosis in human vascular endothelium is regulated by phosphatidylinositol 3-kinase/Akt through the short form of cellular FLIP and Bcl-2

BACKGROUND: Apoptosis of vascular endothelial cells plays a central role in angiogenesis and atherosclerosis. This study investigates the molecular mechanisms of endothelial apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following inhibition of phosphatidylinositol 3-kinase (PI3K). It examines downstream regulation and activation of the extrinsic and intrinsic pathways. METHODS AND RESULTS: By flow cytometry, TRAIL receptors 2 and 3 were present to a greater extent than receptors 1 and 4. TRAIL reduced cell numbers in combination with the PI3K inhibitor LY 294002. TRAIL (100 ng/ml) with LY 294002 (20 micromol/l) activated the extrinsic pathway, causing progressive cleavage of caspase-8 and caspase-3. Activation of the intrinsic pathway proceeded by release of mitochondrial factors Smac/DIABLO and cytochrome c, and caspase-9 cleavage. LY 294002 reduced phosphorylated Akt (p-Akt), with early loss of the short form of cellular FLIP (c-FLIP(S)) and concurrent reduction of Bcl-2. Treatment with small interfering RNA against PI3K also reduced c-FLIP(S) and Bcl-2, and cotreatment with TRAIL triggered caspase-3 cleavage. CONCLUSIONS: This study details the molecular regulation of TRAIL-induced apoptosis in vascular endothelium. Inhibition of PI3K reduces p-Akt, with concurrent reductions in c-FLIP(S) and Bcl-2, and so renders endothelium sensitive to TRAIL-induced apoptosis through the extrinsic and intrinsic pathways.     

Erythropoietin attenuates hydrogen peroxide-induced damage of hepatocytes.

Background/aims: High levels of hydrogen peroxide (H2O2) are observed during inflammatory and ischemic states of the liver and usually lead to cellular dysfunction and cytotoxicity. Recently, it has been reported that erythropoietin and mitochondrial K (ATP) channel openers have a protective effect via a pharmacological preconditioning action during ischemia reperfusion injury of the liver and heart. However, it remains unclear as to whether K (ATP) channel blockers can reduce the protective effect of erythropoietin in the H2O2-induced injury of hepatocytes. Methods: To determine whether erythropoietin treatment decreases H2O2-induced toxicity, we used human hepatocyte cell line Hep3B for assays. Cells were pretreated with different dosages of erythropoietin (0.1-1-10-50 IU/ml) 2 h before H2O2 application. For determination of effects of blockage of mitochondrial K (ATP) channels during erythropoietin treatment, glibenclamide treatment was applied to the medium 2 h before H2O2 toxicity. Cell number, lactate dehydrogenase and caspase- 3 levels were measured in erythropoietin, glibenclamide and/or H2O2-treated groups. Results: Erythropoietin treatment significantly increased cell number at the 24th and 48th h compared to the control group. H2O2 application induced apoptosis and lactate dehydrogenase release from Hep3B cells and decreased cell number. Erythropoietin prevents H2O2 toxicity in hepatocytes. The K channel inhibitor glibenclamide decreased the cytoproliferative and cytoprotective effect of erythropoietin during H2O2 toxicity of Hep3B cells. Conclusions: Erythropoietin treatment may be considered as a therapeutic agent during oxidative injuries of hepatocytes and its cytoprotective effect is abolished by glibenclamide.