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三氧化二砷诱导骨髓瘤细胞凋亡机制研究 总被引:27,自引:2,他引:25
目的 了解三氧化二砷(As2O3)诱导多发性骨髓瘤细胞(MM)凋亡的可能机制及其与维甲酸和干扰素的相互作用。方法 联合应用As2O3和巯基还原剂(DTT)、谷胱甘肽耗褐剂(BSO)、全反式维甲酸(ATRA)或干扰素(IFN-α)处理MM细胞系RPM18226和U266细胞;应用台盼蓝拒染法计数细胞活力,经细胞形态学和流式细胞仪等判定细胞凋亡的程度;通过测定细胞内荧光染料Rhodamine123的色强度分析线粒体跨膜电位(△ψm)。结果 BSO可加强As2O3诱导的RPM18226和U266细胞线粒体△ψm下降和凋亡,而DTT则有部分拮抗的作用,ATRA诱导RPMI8226细胞闻过则喜亡,但它和As2O3之间无协同效尖,ATRA并不诱导U266细胞凋亡。此外,INF-α,既不抑制RPMIS226和U266细胞的生长和活力,也不改变As2O3对这些细胞的作用。结论 As2O3诱导多发性骨髓瘤细胞凋亡和巯基有关,但ATRA干扰素和As2O3无协同效应。 相似文献
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As2O3对多发性骨髓瘤细胞的细胞毒作用的机制研究 总被引:1,自引:0,他引:1
背景与目的:多发性骨髓瘤(multiple myeloma,MM)是恶性浆细胞疾病,目前仍难以治愈;已有研究证明三氧化二砷(arsenic trioxide,As2O3)在体外能够抑制骨髓瘤细胞增殖并诱导其凋亡.本研究拟探讨As2O3对多发性骨髓瘤细胞的可能作用机制.方法:采用MTr法检测As2O3对5株骨髓瘤细胞U266、SKO-007、LP-1、HS-Sultan和KM3的抑制作用,求出其IC50,同时研究维生素K3(vitamine K3,VK3)、N-乙酰半胱氨酸(N-acetyl-cysteine,NAC)和还原型谷胱甘肽(glutathione,GSH)对As2O3的协同或拮抗作用;利用光学比色法测定不同浓度As2O3作用后的5株骨髓瘤细胞以及As2O3与VK3、NAC或外源性GSH共同作用后的U266细胞的GSH含量,对细胞GSH含量与IC50进行相关性分析.结果:As2O3对5株骨髓瘤细胞均有增殖抑制作用,但其敏感性不同,细胞内GSH含量与其IC50正相关(r=0.87,P<0.05);氧化剂VK3与As2O3有明显协同作用,抗氧化剂NAC和GSH对As2O3具有拮抗作用.结论:As2O3可能是通过与细胞内的含巯基化合物结合,降低细胞内GSH含量,从而诱导骨髓瘤细胞凋亡. 相似文献
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BCR-ABL mediates arsenic trioxide-induced apoptosis independently of its aberrant kinase activity 总被引:19,自引:0,他引:19
Puccetti E Güller S Orleth A Brüggenolte N Hoelzer D Ottmann OG Ruthardt M 《Cancer research》2000,60(13):3409-3413
In the prechemotherapy era arsenic derivatives were used for treatment of chronic myelogenous leukemia, a myeloproliferative disorder characterized by the t(9;22) translocation, the Philadelphia chromosome (Ph+). In acute promyelocytic leukemia response to arsenic trioxide (As2O3) has been shown to be genetically determined by the acute promyelocytic leukemia-specific t(15;17) translocation product PML/RARalpha. Hence, we reasoned that As2O3 might have a selective inhibitory effect on proliferation of BCR-ABL-expressing cells. Here, we report that: (a) As2O3 induced apoptosis in Ph+ but not in Ph- lymphoblasts; (b) enforced expression of BCR-ABL in U937 cells dramatically increased the sensitivity to As2O3; (c) the effect of As2O3 was independent of BCR-ABL kinase activity; and (d) As2O3 reduced proliferation of chronic myelogenous leukemia blasts but not of peripheral CD34+ progenitors. In summary, these data establish As2O3 as a tumor cell-specific agent, making its clinical application in Ph+ leukemia feasible. 相似文献
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Involvement of CD95-independent caspase 8 activation in arsenic trioxide-induced apoptosis. 总被引:19,自引:0,他引:19
Arsenic trioxide (As2O3)-treatment is effective in acute promyelocytic leukemia (APL) patients with t(15;17). Clinically achievable concentrations of As2O3 induce apoptosis in NB4, an APL cell line, in vitro. Here, to study the mechanism of As2O3-induced apoptosis, we established an As2O3-resistant subline, NB4/As. Growth of NB4/As was inhibited by 50% after 2 day-treatment (IC50) at 1.6 microM As2O3, whereas IC50 of NB4 was 0.3 microM. Degradation of PML-RARalpha and change of the PML-subcellular localization were similarly induced by As2O3 in NB4 and NB4/As, suggesting that their contribution to apoptosis is small. Treatment with 1 microM As2O3 induced the activation of caspase 3 as well as a loss of mitochondrial transmembrane potential (deltapsim) in NB4 but not in NB4/As. Caspase 8 and Bid were also activated by As2O3 in NB4 but not in NB4/As. In NB4, an inhibitor of caspase 8 blocked not only the activation of caspase 3 but also the loss of deltapsim. Neither cell line expressed CD95/Fas, and agonistic anti-Fas antibody (CH-11) failed to cause apoptosis. Neither antagonistic anti-CD95/Fas antibody nor anti-Fas ligand antibodies influenced the As2O3-induced apoptosis. NB4/As had a higher concentration of intracellular glutathione (GSH) than NB4 (96 vs 32 nmol/mg). Reduction of the GSH level by buthionine sulfoxide (BSO) completely restored the sensitivity to As2O3 in NB4/As. Furthermore, caspase activation and the loss of deltapsim were recovered by combination treatment with BSO. These findings suggest that the As2O3 treatment activates caspase 8 in a CD95-independent but GSH concentration-dependent manner. In combination with BSO, As2O3 might be applied to therapy of leukemia/cancers which are insensitive to the clinically achievable concentrations of As2O3. 相似文献
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T Kajiguchi K Yamamoto K Hossain A A Akhand I Nakashima T Naoe H Saito N Emi 《Leukemia》2003,17(11):2189-2195
High concentrations (greater than 5 microM) of arsenic trioxide (As(2)O(3)) have been reported to be able to induce apoptosis in several malignant cells. We explored cell lines in which apoptosis was induced with a therapeutic concentration (1-2 microM) of As(2)O(3), and found that 1 microM of As(2)O(3) induced apoptosis in the NKM-1 cell line, which was established from a patient with acute myeloid leukemia (M2). Apoptosis induced by 1 microM of As(2)O(3) in NKM-1 cells was accompanied by an increased cellular content of H(2)O(2), a decreased mitochondrial membrane potential (Deltapsim), and activation of caspase-3. C-Jun-terminal kinase (JNK) was activated only in NKM-1 cells and arsenic-sensitive NB4 cells, but not in arsenic-insensitive HL-60 cells. Activation of JNK in NKM-1 was sustained from 6 to 24 h after As(2)O(3) treatment, and preceded changes in cellular H(2)O(2), Deltapsim, and caspase-3 activation. Moreover, addition of a JNK inhibitor reduced the percentage of apoptotic cells after the As(2)O(3) treatment. Taken together, in the M2 cell line NKM-1, 1 microM of As(2)O(3) induced sustained activation of JNK and apoptosis. This finding may provide a basis to select a subgroup other than acute promyelocytic leukemia, which can benefit from As(2)O(3) treatment. 相似文献
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Anemia of variable severity occurs in more than two-thirds of patients with multiple myeloma (MM). Besides the altered cytokine network, chronic erythropoietin deficiency, blood loss and hemolysis, we have shown that deregulated myeloma cell apoptosis may contribute to progressive destruction of the erythroid matrix by inducing erythroblast cytotoxicity. To exert this effect, highly malignant plasma cells overexpress both Fas-ligand (Fas-L) and TRAIL, which efficiently trigger the death of immature erythroblasts. In view of severe progression of MM in patients with Fas-L/TRAIL-based anemia, overexpression of these apoptogen receptors may characterize a peculiar cytotoxic-apoptogenic phenotype in malignant plasma cells. Early immunophenotyping of myeloma cells could thus help to identify patients with a higher risk of erythropoiesis exhaustion. 相似文献
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Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC. 相似文献
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The potency of arsenic trioxide (As2O3) as chemotherapeutic agent is under investigation in various clinical trials. As2O3 was shown to be a potent inductor of apoptosis, and several publications describe the involvement of caspases, reduction of mitochondrial membrane potential and modulation of intracellular glutathione level. However, little is known about the involvement of membrane bound cell death receptors. We investigated the role of CD95 and CD95L in As2O3 mediated apoptosis in various lympho-haematopoietic cell lines. Basal CD95-expression did not correlate with sensitivity to As2O3 and incubation with As2O3 did not alter CD95-expression. We therefore chose two CD95 positive cell lines (CCRF-CEM and Jurkat) to analyse a potential activation of this pathway. We were able to induce apoptosis in these CD95 positive cell lines with activating anti-CD95 antibodies and could block induction of apoptosis by inhibitory anti-CD95 antibodies. In contrast we were not able to block As2O3-induced apoptosis by inhibitory anti-CD95 antibodies. We could block additive effects of arsenic trioxide and an apoptosis-inducing anti-CD95 antibody against CD95 to levels of arsenic trioxide alone using an inhibitory anti-CD95 antibody. Thus, our data provide no evidence for a role of the CD95L/CD95 pathway in As2O3-induced apoptosis. 相似文献
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Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines 总被引:6,自引:0,他引:6
Although c-Jun NH(2)-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27(Kip1), without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-kappaB activation in a dose-dependent fashion, associated with phosphorylation of IkappaB kinase alpha (IKKalpha) and degradation of IkappaBalpha. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-kappaB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-kappaB in MM cells. 相似文献
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目的了解三氧化二砷(As2O3)诱导慢粒细胞系K562细胞株凋亡是否与线粒体跨膜电位(mitochondrial transmembrane potentials,△ψm)改变有关及其可能机制。方法联合应用As2O3、DTT和BSO处理K562细胞株,通过细胞内荧光染料PI/Rh123的色强度分析(△ψm),测定细胞活力、亚G1期细胞含量及形态学观察等鉴定细胞凋亡。结果As2O3可明显诱导K562细胞线粒体△ψm下降。BSO可加强As2O3诱导的K562细胞凋亡和线粒体△ψm下降,而DTT则有部分拮抗作用。在2×10-6mol/L As2O3处理72h的K562细胞,PI-Rh123-细胞达(26.0±2.5)%,当1×10-3mol/LBSO同时处理时,PI-Rh123-细胞和凋亡细胞数上升达(37.2±5.7)%和(39.1±4.5)%;而当2×10-4mol/L DTT同时处理时,PI-Rh123-细胞和凋亡细胞数分别降至(11.5±1.3)%和(15.4±3.5)%。结论线粒体跨膜电位下降是As2O3诱导K562细胞凋亡的关键环节,其机制可能与巯基氧化有关,巯基可能是As2O3凋亡细胞的重要靶分子。 相似文献
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Objective The effect of arsenic trioxide on apoptosis gene expression of Raji cell was explored when Raji cells were incubated with 0.5μmol/L of arsenic trioxide for 6h。Methods Cell culture,extraction and isolation of mRNA,preparation of probes labeled with fluorescence,hybridization technique of DNA chip(each chip containing 200 apoptosis genes,Chinese Shanghai Biostar,In.)were used.Results Arsenic trioxide induced significant changes in 10%(20/200 genes)of the apoptosis genes:18 genes were downregulated,only two upregulated.In particular,inhibitors of apoptosis protein,such as X-linked inhibitor of apoptosis protein,were significantly downregulated.P53 and the other apoptosis genes were also downregulatec.Of the upregulated genes,high expression of heat-shock protein could promote apoptosis of Raji cells.Conclusion The inhibitors of apoptosis protein play an important role in the process of arsenic trioxide-induced apoptosis of Raji cells. 相似文献
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AS2O3对多发性骨髓瘤细胞凋亡及其对自分泌VEGF的影响 总被引:3,自引:1,他引:3
目的研究三氧化二砷(AS2O3)对多发性骨髓瘤(MM)细胞株KM3凋亡及其对自分泌VEGF的影响。方法不同浓度AS2O3分别作用于KM3细胞24、48及72小时,用锥虫蓝拒染法检测细胞生长抑制率,经形态学(光镜,透射电镜)和DNA电泳观察KM3细胞凋亡;用酶联免疫吸附试验(ELISA)检测培养液上清血管内皮生长因子(VEGF)的含量。结果AS2O3可抑制KM3细胞的生长,诱导KM3细胞凋亡;AS2O3对KM3细胞白分泌VEGF的抑制作用具有代偿机制,且与时间浓度有关。24小时时,随着AS2O3浓度的增加,VEGF分泌逐渐增强;48和72小时时,随着AS2O3浓度的增加,VEGF分泌则先上升后下降。结论AS2O3可诱导MM细胞凋亡,同时可抑制MM细胞自分泌VEGF。 相似文献
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Ogawa M Nishiura T Oritani K Yoshida H Yoshimura M Okajima Y Ishikawa J Hashimoto K Matsumura I Tomiyama Y Matsuzawa Y 《Cancer research》2000,60(15):4262-4269
A new human myeloma cell line, OPM-6, was established from the peripheral blood of a patient with advanced IgG-kappa plasma cell leukemia. Cytogenetic and phenotypic analysis confirmed that the cells were derived from the patient's leukemic cells. Insulin-like growth factor-1 (IGF-1) acts as an autocrine growth factor in these cells. In addition, OPM-6 cells were particularly sensitive to dexamethasone (DEX), when endogenous IGF-1 was blocked. Under these conditions, >95% of the DEX-treated cells died within 36 h. Therefore, OPM-6 represents a potentially powerful tool for the analysis of the molecular mechanisms of DEX-induced apoptosis, because it is possible to easily analyze the direct effects of DEX using this system. Using this culture system of OPM-6, we demonstrated that the treatment with DEX plus a monoclonal antibody to the human IGF-1 receptor (alphaIGF-1R) leads to the down-regulation of the gene expression of Bcl-xL, an antiapoptotic gene, and the activation of CPP32 during this apoptotic process. IFN-alpha as well as IL-6 prevented DEX plus alphaIGF-1R-induced apoptosis, and this prevention was blocked by the mitogen-activated protein kinase kinase inhibitor, PD098059, or the phosphatidylinositol 3-kinase inhibitor, wortmannin. Therefore, both IL-6 and IFN-alpha blocked DEX plus alphaIGF-1R-induced apoptosis through activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. 相似文献
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Phosphorylation of PML by mitogen-activated protein kinases plays a key role in arsenic trioxide-mediated apoptosis 总被引:6,自引:0,他引:6
The promyelocytic leukemia (PML) protein is a potent growth suppressor and proapototic factor, whereas aberrant fusions of PML and retinoic acid receptor (RAR)-alpha are causal agents in human acute promyelocytic leukemia. Arsenic trioxide (As(2)O(3)) treatment induces apoptosis in acute promyelocytic leukemia cells through an incompletely understood mechanism. We report here that As(2)O(3) treatment induces phosphorylation of the PML protein through a mitogen-activated protein (MAP) kinase pathway. Increased PML phosphorylation is associated with increased sumoylation of PML and increased PML-mediated apoptosis. Conversely, MAP kinase cascade inhibitors, or the introduction of phosphorylation or sumoylation-defective mutations of PML, impair As(2)O(3)-mediated apoptosis by PML. We conclude that phosphorylation by MAP kinase cascades potentiates the antiproliferative functions of PML and helps mediate the proapoptotic effects of As(2)O(3). 相似文献
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Dib A Gabrea A Glebov OK Bergsagel PL Kuehl WM 《Journal of the National Cancer Institute. Monographs》2008,2008(39):25-31
Translocations involving an MYC gene (c > N >L) are very late tumor progression events and provide a paradigm for secondary translocations in multiple myeloma. Using a combination of fluorescent in situ hybridization and comparative genomic hybridization arrays (aCGH), we have identified rearrangements of an MYC gene in 40 of 43 independent myeloma cell lines. A majority of MYC translocations involve an Ig locus (IgH > Iglambda > Igkappa), but the breakpoints only infrequently occur near or within switch regions or V(D)J sequences. Surprisingly, about 40% of MYC translocations do not involve an Ig locus. The MYC translocations mostly are nonreciprocal translocations or insertions, often with the involvement of three chromosomes and sometimes with associated duplication, amplification, inversion, and other associated chromosomal abnormalities. High-density aCGH analyses should facilitate the cloning of MYC breakpoints, enabling the determination of their structures and perhaps elucidating how rearrangements not involving an Ig gene cause dysregulation of an MYC gene. 相似文献
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砷诱导肝母细胞瘤HepG2细胞凋亡机理研究 总被引:6,自引:3,他引:3
目的 研究三氧化二砷(As203)诱导肝母细胞瘤细胞系HePG2细胞凋亡的机理及其对细胞核基质相关蛋白——前髓白血病蛋白(PML)表达的影响。方法 Hep2细胞培养于MEM培养基中,用不同浓度As2O3分别处理24或96h。运用原位末端脱氧核苷转换酶(TdT)标记法(TUNEL)和DNA阶梯法检测细胞凋亡。以激光共振聚交显微镜及Western杂交观察PML的表达。结果 TUNEL阳性的凋亡细胞及DNA阶梯片段可见于As203处理组。用2μmol/L As203处理的Hep2细胞核基质蛋白内PML表达减弱。激光共振聚交图像显示,经2μmol/L As203处理的Hep2细胞核中,PML蛋白的表达明显减弱;用5μmol/L As203处理后,PML在Hep2细胞核中的微小点状表达几乎消失。结论 As203在体外可明显抑制Hep2细胞生长,其诱导HePG2细胞凋亡的作用依时间及剂量不同而异。As203可降解HepG2细胞核中PML蛋白,且PML的表达降低与细胞凋亡密切相关。核基质相关蛋白PML可能是As203作用的靶蛋白。 相似文献
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A critical role for the NFkB pathway in multiple myeloma 总被引:1,自引:0,他引:1