15-Deoxy-Delta(12,14)-prostaglandin J(2): the endogenous electrophile that induces neuronal apoptosis

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J(2)-series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). The observation that the level of 15d-PGJ(2) increased in the tissue cells from patients with sporadic amyotrophic lateral sclerosis suggested that the formation of 15d-PGJ(2) may be closely associated with neuronal cell death during chronic inflammatory processes. In vitro experiments using SH-SY5Y human neuroblastoma cells revealed that 15d-PGJ(2) induced apoptotic cell death. An oligonucleotide microarray analysis demonstrated that, in addition to the heat shock-responsive and redox-responsive genes, the p53-responsive genes, such as gadd45, cyclin G1, and cathepsin D, were significantly up-regulated in the cells treated with 15d-PGJ(2). Indeed, the 15d-PGJ(2) induced accumulation and phosphorylation of p53, which was accompanied by a preferential redistribution of the p53 protein in the nuclei of the cells and by a time-dependent increase in p53 DNA binding activity, suggesting that p53 accumulated in response to the treatment with 15d-PGJ(2) was functional. The 15d-PGJ(2)-induced accumulation of p53 resulted in the activation of a death-inducing caspase cascade mediated by Fas and the Fas ligand.     

15-脱氧-△12,14-前列腺素J2在脑缺血中的神经保护作用

15-脱氧-△12,14-前列腺素J2(15-deoxy-△12,14-prostaglandin J2,15d-PGJ2)是过氧化物酶体增殖物激活受体γ的天然配体之一,在脑缺血中发挥重要的保护作用.文章对15d-PGJ2神经保护机制的研究进展进行了综述.     

Arsenic-interferon-alpha-triggered apoptosis in HTLV-I transformed cells is associated with tax down-regulation and reversal of NF-kappa B activation

Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature activated T cells resistant to conventional chemotherapy. The viral transactivator protein Tax plays a critical role in HTLV-I-induced transformation and apoptosis resistance by inducing I kappa B-alpha degradation, resulting in the activation of the NF-kappa Bpathway. In these HTLV-I-transformed cells, arsenic trioxide (As) and interferon (IFN)-alpha synergize to induce cell cycle arrest and apoptosis. We demonstrate that cell death induction is only partly dependent upon caspase activation and is not associated with modulation of bcl-2, bax, or p53 expression. However, combined As and IFN induce the degradation of Tax, associated with an up-regulation of I kappa B-alpha resulting in a sharp decrease in RelA DNA binding nuclear factor (NF)-kappa B complexes because of the cytoplasmic retention of RelA. Taken the role of Tax in HTLV-I-induced transformation, its down-regulation probably accounts for cell death induction through inactivation of the NF-kappa B pathway. Such specific targeting of the viral oncoprotein by As-IFN treatment, reminiscent of As targeting of promyelocytic leukemia/retinoic acid receptor-alpha in acute promyelocytic leukemia, provides strong rational for combined As-IFN therapy in ATL patients. (Blood. 2000;96:2849-2855)     

过氧化物酶体增殖活化受体γ激动剂对呼吸机相关肺损伤大鼠肺胶原合成的影响及机制

目的探讨过氧化物酶体增殖活化受体(PPAR)γ激动剂[15-去氧-△~(12,14-)前列腺素(PG)J_2 (15d—PGJ_2)]对呼吸机相关肺损伤大鼠肺胶原合成的影响及机制。方法普通级雄性SD大鼠24只,随机分为3组(正常对照组、Ⅰ组、Ⅱ组),每组8只。Ⅰ和Ⅱ组呼吸机条件相同(VT16ml/kg,PEEP为5cmH_2O,吸呼比为2：1,FiO_2为1．0),其中药物干预组(Ⅱ组)通气前1h腹腔注射15d-PGJ_2(1mg/kg),分别通气4h。通气结束后测定肺组织湿/干重(W/D)、肺组织光镜病理、Ⅰ型前胶原肽(PINP)和Ⅲ型前胶原肽(PⅢNP)浓度、Ⅰ型和Ⅲ型前胶原mRNA表达、以及PPARγ和核因子(NF)-kB活性。结果①Ⅰ组肺组织W/ D显著高于正常对照组和Ⅱ组(P＜0．05)。肺组织W/D在正常对照组和Ⅱ组之间无显著差异。②Ⅰ组肺组织Ⅰ型前胶原mRNA表达显著高于正常对照组和Ⅱ组(P均＜0．05)。Ⅱ组与正常对照组肺组织Ⅰ型前胶原mRNA表达无显著差异。③与Ⅰ组比较,正常对照组和Ⅱ组Ⅲ型前胶原mRNA表达均显著降低(P均＜0．05)。与正常组比较,Ⅱ组Ⅲ型前胶原mRNA表达无显著改变。④与正常对照组比较,Ⅰ组和Ⅱ组NF-kB活性均显著增高(P均＜0．05)。Ⅰ组与Ⅱ组NF-kB活性比较,无显著差异。⑤Ⅰ组PPARγ活性显著低于正常对照组(P＜0．05)。与Ⅰ组比较,Ⅱ组PPAγ活性显著增高(P＜0．05)。结论15d- PGJ_2下调呼吸机相关肺损伤大鼠肺组织Ⅰ型和Ⅲ型前胶原mRNA表达,其机制可能与激活PPARγ有关。     

Inducible loss of NF-kappaB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 induces NF-kappaB activity by targeting IkappaBalpha. To understand the role of NF-kappaB activation in EBV-related oncogenesis, we have subcloned mutated IkappaBalpha(32/36A) cDNA into a pHEBo vector containing doxycycline regulatory sequences and stably transfected this construct into a lymphoblastoid cell line. Two tightly regulated clones were obtained in which IkappaBalpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IkappaBalpha(32/36A) peaked at day 2. Inhibition of NF-kappaB activity was closely correlated with levels of inducible IkappaBalpha(32/36A). Levels of 3 well-known NF-kappaB-dependent genes, CD54, p105, and endogenous IkappaBalpha, were decreased when IkappaBalpha(32/36A) was induced, and the growth of IkappaBalpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappaB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IkappaBalpha(32/36A)-overexpressing cells. Together these results show that it is possible to control IkappaBalpha(32/36A) levels, ie, NF-kappaB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappaB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IkappaBalpha(32/36A) may be useful in the identification of genuine NF-kappaB target genes in EBV-infected B cells. (Blood. 2000;95:2068-2075)     

二硫代氨基甲酸吡咯烷对苦参碱诱导肝癌细胞凋亡的影响

目的 观察二硫代氨基甲酸吡咯烷(PDTC)抑制核因子-κB(NF-κB)活化后对苦参碱诱导肝癌细胞HepG2凋亡的影响.方法 MTT法观察苦参碱(分0.8、1.0、1.5、2.0、2.5 g/L组)及PDTC联合苦参碱对HepG2细胞增殖的抑制作用.将HepG2细胞随机分为细胞对照组、PDTC组(20μmol/L)、苦参碱组(1.5 g/L)和PDTC+苦参碱联合组,流式细胞仪和末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测细胞凋亡;电泳迁移率改变实验检测细胞核内NF-κB的活化水平.结果 PDTC增强了苦参碱对细胞增殖的抑制作用(F=183.92,P＜0.01).苦参碱同时具有诱导HepG2细胞凋亡和NF-κ B活化的作用;PDTC能显著增加苦参碱诱导的HepG2细胞凋亡和抑制苦参碱诱导的HepG2的NF-κB活化,细胞凋亡率由6.11%±0.81%增加至12.95%±0.02%(χ2=9.67,P＜0.05),NF-κB活化的灰度值由38.82±0.17降至32.01±0.69(χ2=10.38,P＜0.05).结论 苦参碱诱导HepG2细胞凋亡的同时激活NF-κB;PDTC可通过抑制NF-κB活化,增强苦参碱诱导HepG2细胞凋亡的作用.     

Molecular sequelae of histone deacetylase inhibition in human malignant B cells

Histone acetylation modulates gene expression, cellular differentiation, and survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of suberoylanilide hydroxamic acid (SAHA), the prototype of a series of hydroxamic acid-based HDAC inhibitors, in cell lines and patient cells from B-cell malignancies, including multiple myeloma (MM) and related disorders. SAHA induced apoptosis in all tumor cells tested, with increased p21 and p53 protein levels and dephosphorylation of Rb. We also detected cleavage of Bid, suggesting a role for Bcl-2 family members in regulation of SAHA-induced cell death. Transfection of Bcl-2 cDNA into MM.1S cells completely abrogated SAHA-induced apoptosis, confirming its protective role. SAHA did not induce cleavage of caspase-8, -9, or -3 in MM.1S cells during the early phase of apoptosis, and the pan-caspase inhibitor ZVAD-FMK did not protect against SAHA. Conversely, poly(ADP)ribose polymerase (PARP) was cleaved in a pattern indicative of calpain activation, and the calpain inhibitor calpeptin abrogated SAHA-induced cell death. Importantly, SAHA sensitized MM.1S cells to death receptor-mediated apoptosis and inhibited the secretion of interleukin 6 (IL-6) induced in bone marrow stromal cells (BMSCs) by binding of MM cells, suggesting that it can overcome cell adhesion-mediated drug resistance. Our studies delineate the mechanisms whereby HDAC inhibitors mediate anti-MM activity and overcome drug resistance in the BM milieu and provide the framework for clinical evaluation of SAHA, which is bioavailable, well tolerated, and bioactive after oral administration, to improve patient outcome.     

NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells.

A sensitive DNA-protein crosslinking approach has been used to characterize four inducible T-cell proteins (50 kDa, 55 kDa, 75 kDa, and 85 kDa) that specifically bind to kappa B enhancer elements. Partial proteolytic mapping revealed a distinct cleavage pattern for three of these proteins. These polypeptides are sequestered as inactive precursors in the cytosol of unstimulated T cells but can be converted into active forms in vivo by phorbol ester stimulation or in vitro by detergent treatment. The induction of these proteins by phorbol ester results in a strikingly biphasic pattern of nuclear expression with the 55-kDa and 75-kDa species appearing within minutes, whereas the 50-kDa and 85-kDa species appear only several hours after cellular stimulation. These data suggest that NF-kappa B-binding activity may not correspond to a single polypeptide but rather a family of at least four inducible and differentially regulated DNA-binding proteins that are expressed with distinct kinetics in human T lymphocytes.     

斑蝥素诱导人胰腺癌细胞凋亡的实验研究

目的 探讨斑蝥素（Cantharidin）对人胰腺癌细胞凋亡的影响及其作用机制。方法采用MTT法观察斑蝥素对人胰腺癌细胞株SW1990细胞增殖的抑制作用。采用Hoechst33258染色、TUNNEL染色、流式细胞术检测细胞凋亡改变，并以RT—PCR和Westernblot检测凋亡调节基因p53和Bcl-2、Bax的表达。结果 5mol／L斑蝥素能明显抑制人胰腺癌SW1990细胞的生长，呈现凋亡特征。RT—PCR和Westernblot检测可见Bax、p53基因表达显著增加，而Bcl—2基因表达减少。结论 斑蝥素能诱导人胰腺癌细胞凋亡，其作用可能与上调p53、Bax基因和下调Bcl-2基因有关。     

斑蝥素诱导人胰腺癌细胞凋亡的实验研究

目的 探讨斑蝥素(Cantharidin)对人胰腺癌细胞凋亡的影响及其作用机制.方法 采用MTT法观察斑蝥素对人胰腺癌细胞株SW1990细胞增殖的抑制作用.采用Hoechst 33258染色、TUNNEL染色、流式细胞术检测细胞凋亡改变,并以RT-PCR和Western blot检测凋亡调节基因p53和Bcl-2、Bax的表达.结果 5μmol/L斑蝥素能明显抑制人胰腺癌SW1990细胞的生长,呈现凋亡特征.RT-PCR和Western blot检测可见Bax、p53基因表达显著增加,而Bc1-2基因表达减少.结论 斑蝥素能诱导人胰腺癌细胞凋亡,其作用可能与上调p53、Bax基因和下调Bcl-2基因有关.     

Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells

Whether resveratrol, a component of red grapes, berries, and peanuts, could suppress the proliferation of multiple myeloma (MM) cells by interfering with NF-kappaB and STAT3 pathways, was investigated. Resveratrol inhibited the proliferation of human multiple myeloma cell lines regardless of whether they were sensitive or resistant to the conventional chemotherapy agents. This stilbene also potentiated the apoptotic effects of bortezomib and thalidomide. Resveratrol induced apoptosis as indicated by accumulation of sub-G(1) population, increase in Bax release, and activation of caspase-3. This correlated with down-regulation of various proliferative and antiapoptotic gene products, including cyclin D1, cIAP-2, XIAP, survivin, Bcl-2, Bcl-xL, Bfl-1/A1, and TRAF2. In addition, resveratrol down-regulated the constitutive activation of AKT. These effects of resveratrol are mediated through suppression of constitutively active NF-kappaB through inhibition of IkappaBalpha kinase and the phosphorylation of IkappaBalpha and of p65. Resveratrol inhibited both the constitutive and the interleukin 6-induced activation of STAT3. When we examined CD138(+) plasma cells from patients with MM, resveratrol inhibited constitutive activation of both NF-kappaB and STAT3, leading to down-regulation of cell proliferation and potentiation of apoptosis induced by bortezomib and thalidomide. These mechanistic findings suggest that resveratrol may have a potential in the treatment of multiple myeloma.     

Tamoxifen induces suppression of cell viability and apoptosis in the human hepatoblastoma cell line HepG2 via down-regulation of telomerase activity.

BACKGROUND/AIMS: Antiproliferative action of tamoxifen in the estrogen receptor-alpha-negative human hepatoblastoma cell line HepG2 was investigated. METHODS: HepG2 cells, seeded at different densities (4000-36 000 cells/cm(2)), were incubated with tamoxifen (1, 10, or 20 microM) or the telomerase inhibitor 3'-azido-3'-deoxythymidine (AZT) (0.6-3.0 mM) up to 72 h. Cell viability was assessed (MTT-test), flow cytometric analysis was performed, and telomerase activity was measured (telomeric repeat amplification protocol assay). RESULTS: Ten or 20 microM tamoxifen induced a reduction of cell viability. Basically reduction of viability was related to an increase in the fraction of G0/1-phase. When tamoxifen was present at higher concentration (20 microM) or at low cell density (4000/cm(2)) an additional increase of the rate of apoptotic cells occurred with a delay, aggravating the effect of tamoxifen on cell viability substantially. When apoptosis was induced a significant suppression of telomerase activity preceded regularly. Direct inhibition of telomerase activity with AZT resulted in a decrease of cell viability and apoptosis. CONCLUSION: The tamoxifen-induced reduction of cell viability in HepG2 cells depends on drug concentration and cell density and is due to cytostatic and cytocide effects. The latter may be mediated by a down-regulation of telomerase activity.