The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571

Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor kappa B (NF-kappa B) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies.     

Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells

Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl- cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned.     

PGC-1α对高糖诱导的血管内皮细胞内ROS生成的影响

目的探讨过氧化物酶体增殖物激活受体辅激活因子1α（PGC-1α）对高糖诱导的血管内皮细胞内活性氧簇（ROS）生成的影响。方法将人脐带静脉内皮细胞（HUVECs）分成对照组、空病毒Ad组、d-PGC-1α感染组、PGC-1αsiRNA转染组,分别将四组细胞置于正常糖及高糖环境中进行培养。用活性氧检测试剂盒处理标本,以酶标仪检测细胞内荧光强度,显示细胞内ROS浓度。结果高糖组HUVECs内ROS浓度明显高于正常糖组（P〈0.01）;与对照组及空病毒Ad组比较,d-PGC-1α感染组ROS浓度明显降低,PGC-1αsiRNA转染组明显升高（P均〈0.01）。结论与正常糖组比较,高糖组HUVECs内ROS浓度明显升高;过度表达PGC-1α蛋白后,HUVECs内ROS浓度明显降低;抑制细胞内PGC-1α蛋白表达,则HUVECs内ROS浓度明显升高。提示PGC-1α可作为防治糖尿病血管并发症的新靶点之一。     

Fas-ligand (CD178) and TRAIL synergistically induce apoptosis of CD40-activated chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells become sensitive to Fas (CD95)-mediated apoptosis 3 to 5 days after CD40 ligation. However, CD4+ cytotoxic T lymphocytes (CTLs) can kill CLL B cells via a Fas-ligand (CD178)-dependent process within 24 hours after CD40 cross-linking, when ligation of CD95 alone is insufficient to induce apoptosis. In addition to CD95, CD40-activated CLL cells also express DR5, a receptor for tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that is expressed by CD4+ CTL. In addition, CD40 ligation in vitro and in vivo induces CLL cells to express the proapoptotic protein, BH3 interacting domain death agonist (Bid), which can facilitate crosstalk between mitochondrial-dependent, apoptosis-inducing pathways and death receptors, such as death receptor 5 (DR5). To evaluate whether ligation of CD95 and/or DR5 can induce apoptosis of CD40-activated CLL cells, we generated artificial cytotoxic effector cells that express both human TRAIL and CD178 (Chinese hamster ovary [CHO]-CD178/TRAIL) or only TRAIL (CHO-TRAIL) or CD178 (CHO-CD178). CHO-CD178/TRAIL cells were significantly more effective in killing CD40-activated CLL cells than either CHO-TRAIL or CHO-CD178 and, unlike the latter, could kill CLL cells 24 hours after CD40 ligation. We conclude that CD40 ligation induces CLL cells to express the proapoptotic molecule Bid and the death receptors CD95 and DR5, the latter of which can act synergistically to induce caspase-dependent apoptosis of CD40-activated CLL B cells.     

Generation of reactive oxygen species is not involved in idarubicin-induced apoptosis in human leukaemic cells

The anthracycline antibiotic idarubicin (IDA) induces double-stranded DNA breaks, the generation of reactive oxygen species (ROS) and apoptosis in human leukaemic cells. It is unclear whether the generation of ROS is associated with the apoptotic process. Using the T-lymphoblastic leukaemic CEM cell line, we found that IDA-induced DNA breaks were correlated with final cell death. The reduction in mitochondrial membrane potential (Deltapsim) and the generation of ROS occurred simultaneously with IDA-induced activation of caspase-9 and caspase-3. Inhibition of caspases by a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) completely blocked IDA-induced reduction of Deltapsim, apoptosis and final cell death. Interestingly, ROS generation was significantly enhanced by Z-VAD-fmk. ROS generation was neither caspase dependent nor part of the apoptotic process. IDA-mediated reduction in Deltapsim is caspase dependent and is not a consequence of the generation of ROS. These results indicate that IDA-induced generation of ROS and apoptosis are separate events. Inhibition of caspases facilitates IDA-mediated generation of ROS.     

C-peptide reduces high-glucose-induced apoptosis of endothelial cells and decreases NAD(P)H-oxidase reactive oxygen species generation in human aortic endothelial cells

Aims/hypothesis  

Reactive oxygen species (ROS) generated during hyperglycaemia are implicated in the development of diabetic vascular complications. High glucose increases oxidative stress in endothelial cells and induces apoptosis. A major source of ROS in endothelial cells exposed to glucose is the NAD(P)H oxidase enzyme. Several studies demonstrated that C-peptide, the product of proinsulin cleavage within the pancreatic beta cells, displays anti-inflammatory effects in certain models of vascular dysfunction. However, the molecular mechanism underlying this effect is unclear. We hypothesised that C-peptide reduces glucose-induced ROS generation by decreasing NAD(P)H oxidase activation and prevents apoptosis     

Triptolide cooperates with chemotherapy to induce apoptosis in acute myeloid leukemia cells

OBJECTIVE: Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. MATERIALS AND METHODS: The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry. RESULTS: A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis. CONCLUSION: These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.     

Antibody-induced nonapoptotic cell death in human lymphoma and leukemia cells is mediated through a novel reactive oxygen species-dependent pathway

Monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell malignancies. Although Fc-dependent mechanisms of mAb-mediated tumor clearance have been extensively studied, the ability of mAbs to directly evoke programmed cell death (PCD) in the target cell and the underlying mechanisms involved remain under-investigated. We recently demonstrated that certain mAbs (type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD through an actin-dependent, lysosome-mediated process. Here, we reveal that the induction of PCD by these mAbs, including the type II anti-CD20 mAb GA101 (obinutuzumab), directly correlates with their ability to produce reactive oxygen species (ROS) in human B-lymphoma cell lines and primary B-cell chronic lymphocytic leukemia cells. ROS scavengers abrogated mAb-induced PCD indicating that ROS are required for the execution of cell death. ROS were generated downstream of mAb-induced actin cytoskeletal reorganization and lysosome membrane permeabilization. ROS production was independent of mitochondria and unaffected by BCL-2 overexpression. Instead, ROS generation was mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These findings provide further insights into a previously unrecognized role for NADPH oxidase-derived ROS in mediating nonapoptotic PCD evoked by mAbs in B-cell malignancies. This newly characterized cell death pathway may potentially be exploited to eliminate malignant cells, which are refractory to conventional chemotherapy and immunotherapy.     

糖化终末产物对MIN6细胞活力及活性氧(ROS)水平的影响

探讨糖化终末产物(AGE)对小鼠胰岛细胞株MIN6细胞活力及活性氧(ROS)水平的影响.制备BSA-AGE,用不同浓度AGE(100、200、400 mg/L)干预MIN6细胞不同时间后,MTF比色法检测细胞活力变化.以活性氧捕获剂双氢-乙酰乙酸二氯荧光黄(DCFH-DA)孵育细胞,通过流式细胞仪检测细胞内二氯荧光黄(DCF)的荧光强度而测得细胞内活性氧水平,并测定胰岛素分泌的变化.随着AGE浓度的升高和作用时间的延长,细胞活力明显下降(P＜0.05).经DCFH-DA孵育后流式细胞仪检测显示,AGE处理组细胞内DCF平均荧光强度较对照组明显升高(P＜0.05).胰岛素分泌量随着AGE浓度的增高和时间的延长,呈下降趋势(P＞0.05).提示BSA-AGE抑制MIN6活力,使细胞内活性氧生成增加,诱导MIN6细胞氧化应激.

Abstract：

To explore the effect of advanced glycation end-products(AGEs)on cell viability and level of reactive oxygen species(ROS)in MIN6 cells. After intervention of various concentrations(100,200, and 400 mg/L)of AGEs for some time, cell viability was detected by MTT assay. 2', 7'-dichlorofluorescein diacetate(DCFH-DA)was used as a reactive oxygen species capture agent. The fluorescent intensity of 2', 7'-dichlorofluorescein(DCF), which was the product of cellular oxidation of DCFH-DA, was detected by flow cytometry. The level of ROS and insulin secretion was thus measured. Viability of MIN6 cells was inhibited by AGEs in a dose and time dependent manner(P＜0.05).Intracellular fluorescent intensity of DCF was markedly elevated in the AGEs groups as compared with that in the control group(P＜0.05).Insulin secretion was decreased in the AGEs groups than that in the control group(P＞0.05). The results suggest that AGEs inhibit the viability and induce oxidative stress in MIN6 cells by overproduction of ROS.     

Aging might augment reactive oxygen species (ROS) formation and affect reactive nitrogen species (RNS) level after myocardial ischemia/reperfusion in both humans and rats

Previous studies indicate aging results in significantly decreased cardiac function and increased myocardial apoptosis after myocardial ischemia/reperfusion (MI/R) in humans or rats. The underlying mechanisms of aging-exacerbated effects remain unknown. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to play vital roles in aging-related MI/R injury. Heretofore, the effects of aging upon ROS and RNS formation were not investigated in humans, which is the focus of the current study. Due to experimental limitations with clinical trials, an additional animal experiment was performed. All enrolled acute myocardial infarction (AMI) patients received percutaneous coronary intervention (PCI) therapy. AMI patients were assigned into two groups: adult (age <65, n = 34) and elderly (age ≥65, n = 45) AMI patients. Blood samples were obtained from all study participants at 24 h and 3 days post-PCI. Plasma/white blood cell (WBC) ROS and RNS markers (malondialdehyde (MDA), myeloperoxidase (MPO), reduced glutathione (GSH), inducible nitric oxide synthase (iNOS) activity, NOx, and nitrotyrosine) were determined. The same markers were determined in rat cardiac tissue after 24 h MI/R. Compared to the adult group, elderly patients manifested increased plasma MDA and MPO and decreased plasma GSH concentrations. No significant differences in plasma NOx or nitrotyrosine concentration existed between adult and elderly patients. Furthermore, WBC iNOS activity in elderly patients was significantly decreased compared to the adult group. The measurement of ROS markers in the rat experiments was consistent and supported human study data. Surprisingly, RNS markers (NOx and nitrotyrosine) in blood and heart tissue increased from young to middle-aged rats but decreased from middle age to old age. Aging augments ROS, which might exacerbate MI/R injury. Additionally, our data support aging-induced changes of RNS levels in humans and rats in vivo.     

NADPH氧化酶在瘦素诱导肝星状细胞株HSC-T6产生活性氧中的作用及其机制

目的 研究NADPH氧化酶(NOX)是否参与瘦素诱导肝星状细胞(HSC)产生活性氧(ROS),并探讨其机制. 方法取对数生长期的HSC-T6细胞,随机分为9组:瘦素组、瘦素+ROS生成系统抑制剂[包括氯化二亚苯基碘嗡(DPI)、鱼藤酮、甲双吡丙酮、别嘌呤醇和吲哚美辛]组成的干预组、瘦素+JAK抑制剂AG490组成的干预组、正常对照组和阴性对照组.HSC-T6细胞经药物作用1、12、24 h后,荧光显微镜和(或)流式细胞术观察细胞内二氯荧光素强度来反应ROS水平;分光光度计检测NADPH的吸光度,以NADPH的消耗量表示NOX活性;逆转录聚合酶链反应检测Rac1和p22Phox的mRNA相对表达量.数据分析用单因素方差分析、SNK-q检验或秩和检验,P＜0.05为差异有统计学意义.结果 HSC-T6细胞加入瘦素(100 ng/ml)作用1 h后,荧光强度较正常对照组明显升高(92.91±4.19比27.56±6.27,P＜0.01);DPI(20 μmol/L)和AG490(50 μ mol/L)能抑制ROS的产生,荧光强度值分别为37.35±4.66和53.12±6.63,均低于瘦素组的92.91±4.19(q值分别为31.78和22.76,P值均＜0.01).瘦素作用HSC-T6细胞1 h后,NADPH的消耗量较正常对照组明显升高[(1.90±0.22)pmol·min-1·mg1比(0.76±0.06)pmol·min-1·mg-1,P＜0.05)],而作用12、24 h后的消耗量明显高于1 h时的消耗量(x2值均为7.54,P值均＜0.05);DPI及AG490干预能抑制瘦素诱导NOX活性的上调(q值分别为16.58和16.23,P值均＜0.05).瘦素作用12 h后,HSC-T6细胞内Racl和p22Phox的mRNA相对表达量明显高于正常对照组(分别为0.41±0.13比0.14±0.08和0.45±0.12比0.20±0.08,P值均＜0.05).结论 瘦素诱导HSC产生的ROS主要来源于NOX,其机制可能是瘦素通过JAK信号通路直接激活NOX,并通过上调NOX的亚基表达使NOX活性持续增高而产生大量ROS.     

Endothelin-1 inhibits apoptosis through a sphingosine kinase 1-dependent mechanism in uterine leiomyoma ELT3 cells

Uterine leiomyomas, or fibroids, are the most common tumors of the myometrium. The ELT3 cell line, derived from Eker rat leiomyoma, has been successfully used as a model for the study of leiomyomas. We have demonstrated previously the potent mitogenic properties of the peptidic hormone endothelin (ET)-1 in this cell line. Here we investigated the antiapoptotic effect of ET-1 in ELT3 cells. We found that 1) serum starvation of ELT3 cells induced an apoptotic process characterized by cytochrome c release from mitochondria, caspase-3/7 activation, nuclei condensation and DNA fragmentation; 2) ET-1 prevented the apoptotic process; and 3) this effect of ET-1 was fully reproduced by ETB agonists. In contrast, no antiapoptotic effect of ET-1 was observed in normal myometrial cells. A pharmacological approach showed that the effect of ET-1 on caspase-3/7 activation in ELT3 cells was not dependent on phosphatidylinositol 3-kinase, ERK1/2, or phospholipase D activities. However, inhibitors of sphingosine kinase-1 (SphK1), dimethylsphingosine and threo-dihydrosphingosine, reduced the effect of ET-1 by about 50%. Identical results were obtained when SphK1 expression was down-regulated in ELT3 cells transfected with SphK1 small interfering RNA. Furthermore, serum starvation induced a decrease in SphK1 activity that was prevented by ET-1 without affecting the level of SphK1 protein expression. Finally, sphingosine 1-phosphate, the product of SphK activity, was as efficient as ET-1 in inhibiting serum starvation-induced caspase-3/7 activation. Together, these results demonstrate that ET-1 possesses a potent antiapoptotic effect in ELT3 cells that involves sphingolipid metabolism through the activation of SphK1.