阿糖胞苷,高三尖极酯碱诱导急性非淋巴细胞白血病细胞凋亡的临床研究

运用吖啶橙（ＡＯ）／溴乙锭（ＥＢ）染色法检测阿糖革（Ａｒａ－ｃ）、、高三尖杉酯碱（ＨＨＴ）体外诱导临床初发及复发／难治急性淋巴细胞白血病（ＡＮＬＬ）患的白血病细胞凋亡,观察细胞凋亡指数与临床疗效的关系。结果显示初治ＡＮＬＬ药物诱导的组凋亡率明显高于复发／难治组,初治组完全缓解ＣＢ）凋亡指数明显高于未缓解（ＮＲ）,说明凋亡与耐药,凋亡与临床疗效有一定的关系。     

急性淋巴细胞白血病患者血清乳酸脱氢酶水平与VDLP 方案临床疗效的关系

目的探讨在急性淋巴细胞白血病患者（ALL）中血清乳酸脱氢酶水平与VDLP方案临床疗效的关系。方法选取70例初治儿童急性淋巴细胞白血病患者,并选取同时期40例健康儿童作为对照组。检测治疗前后及复发患者S-LDH水平,并与对照组LDH水平比较,分析LDH水平变化的临床意义。结果ALL患者LDH水平明显高于健康对照组（P〈0．01）,而通过治疗（完全缓解／部分缓解）患者S-LDH明显下降（P〈0．01）,NR患者虽出现下降,但与治疗前比较无统计学差异（P〉0．05）;CR／PR治疗前s-LDH水平亦低于NR治疗前（P〈0．05）;获得CR／PR患者复发后,S-LDH又明显升高,复发患者治疗前S-LDH水平高于未复发患者（治疗前）（P〈0．05）。结论S-LDH可能可以作为成人急性淋巴细胞白血病预后的评估指标。     

健脾益气法辅助化疗治疗急性非淋巴细胞白血病的临床研究

目的探讨健脾益气中药方剂辅助化疗治疗急性非淋巴细胞白血病（ANLL）的临床疗效及不良反应。方法 62例ANLL患者随机分为两组,实验组31例,采用健脾益气中药方剂联合DA化疗方案治疗;对照组31例,单纯用DA方案治疗。观察两组缓解率、临床症状、不良反应等指标。结果实验组CR率为77.42%,总有效率（CR＋PR）为93.55%,均显著高于对照组。实验组各种不良反应发生率明显低于对照组,其中恶心呕吐的发生率显著低于对照组。结论健脾益气中药方剂联合DA化疗方案治疗急性非淋巴细胞白血病可起到减轻化疗的不良反应,提高治疗效果的作用。     

月腺大戟水提物诱导P388白血病细胞的凋亡

目的：研究月腺大戟水提物抗R388白血病及对瘤细胞凋亡的作用。方法：以一级清洁DBA／2型小鼠为实验对象，腹腔接种R388白血病细胞，胃饲10,2．5g·L^-1的月腺大戟溶液0．3mL，连续6d，检测外周血白细胞总数和腹腔瘤细胞总数，分离外周血淋巴细胞，观察瘤细胞及凋亡特征，计算细胞凋亡率，并检测bcl-2和bax凋亡相关基因的表达情况。结果：月腺大戟高剂量组外周血白细胞数量显著低于模型对照组（P〈0．01）。月腺大戟低剂量组外周血白细胞虽低于模型对照组，但差异无显著性（P〉0．05）。两剂量组的细胞凋亡率均明显高于模型对照组，两剂量组间的细胞凋亡率差异显著（P〈0．01）。凋亡细胞中可见核质浓缩、数个圆形凋亡小体位于胞浆，或呈半月形、块状靠近胞膜边缘，还可见核质松散呈网状，细胞膜不完整的退化淋巴细胞，表明高剂量明显诱导细胞凋亡（P〈0．01），低剂量虽能诱导细胞凋亡，但差异无显著性（P〉0．05）。结论：高剂量月腺大戟可通过诱导细胞凋亡明显抑制R388白血病瘤细胞的恶性增殖。     

甲异靛对K562原代细胞凋亡作用研究

目的探讨甲异靛诱导慢性髓系细胞白血病K562细胞株凋亡的作用。方法K562细胞经20μm／L甲异靛处理12h、24h、48h后,采用流式细胞术检测细胞凋亡及细胞周期。结果K562未经甲异靛作用时凋亡率[（1．0±0．36）％]与人体正常细胞系NIH3T3凋亡率[（2．6±0．39）％]相比,差异有统计学意义（P〈0．05）;K562细胞空白组凋亡率为（1．0±0．36）％,12h组为（12．5±0．69）％,24h组为（19．4±1．07）％,48h组为（42．5±3．22）％,与NIH3T3细胞组同时间比较,差异有统计学意义（P〈0．05）;k562组12h、24h、48h与空白组比较,差异有统计学意义（P〈0．05）。甲异靛能使K562细胞阻滞于G2／M期,G2／M期细胞比例增加,G0／G1期和S期细胞减少。结论甲异靛对体外K562细胞有诱导凋亡作用,其作用与时间成正相关关系。甲异靛可使K562细胞阻滞于G2／M期。     

Bcl一2／Bax在溃疡性结肠炎肠黏膜中的表达及与细胞凋亡的关系

目的研究溃疡性结肠炎患者肠黏膜中的凋亡调控蛋白Bcl-2／Bax表达状态及其与细胞凋亡的关系,探讨溃疡性结肠炎的发病机制。方法应用脱氧核糖核酸转移酶介导的缺口末端标记技术和免疫组化方法分析36例溃疡性结肠炎患者和25例正常对照肠黏膜细胞凋亡、凋亡调控蛋白Bcl-2／Bax的表达。结果溃疡性结肠炎组凋亡调控蛋白Bcl-2／Bax：的表达指数显著高于正常对照组（P〈0．05）,凋亡调控蛋白Bcl-2／Bax的表达与凋亡指数呈正相关（r=0．709,P=0．000;r=0．589,P=0．000）。结论溃疡性结肠炎患者肠黏膜凋亡调控蛋白Bel-2／Bax表达增加,诱导肠黏膜细胞凋亡。     

G-CSF对VP16诱导白血病细胞凋亡及Survivin mRNA表达的调控作用

目的：探讨粒细胞集落刺激因子（G—CSF）对足叶乙甙（VP16）诱导白血病细胞凋亡以及Survivin基因表达的影响。方法：用HL-60白血病细胞株进行传代培养，通过DNA凝胶电泳和流式细胞术（FCM）检测细胞凋亡，用RT—PCR方法检测Survivin基凶表达。结果：VP16可诱导HL-60细胞凋亡，下调Survivin表达：G—CSF可上调SurvivinmRNA表达，与VP16联合应用时，细胞凋亡率低于单独应用VP16组（P〈0．01），Survivin mRNA表达水平高于单用VP16组（P〈0．01）。结论：VP16诱导白血病细胞凋亡可能与其抑制Survivin表达有关；G—CSF能抑制VP16的促凋亡作用。     

人参总皂苷对白血病细胞K562凋亡及生存素表达的影响

目的探讨人参总皂苷（TSPG）对人白血病细胞株K562生长和凋亡的影响及其可能机制。方法采用噻唑兰比色法（Mar）观察TSPG对K562细胞生长的影响;流式细胞仪观察TSPG对细胞凋亡的影响;用RT-PCR检测TSPG诱导K562细胞凋亡中生存素（Survivin）基因的表达情况。结果TSPG对K562细胞生长有抑制作用,呈剂量依赖关系（P〈0．05）;10、100、200μg／ml组细胞凋亡率分别为16．67％、23．78％、33．98％,药物组与对照组差异有统计学意义（P〈0．01或P〈0．05）;随着TSPG浓度升高,Survivin基因的表达水平逐渐下调（P〈0．05）。结论TSPG可以抑制人白血病细胞的生长并诱导其凋亡,其机制可能与下调Survivin基因的表达有关。     

Tuftsin促进肝癌细胞凋亡的实验研究

目的 通过研究Ｔｕｆｔｓｉｎ与肝癌细胞凋亡的关系,探讨脾脏及Ｔｕｆｔｓｉｎ因子的抗肿瘤作用及其机制。方法 昆明种小鼠３６只随机分为６组：假手术组（Ａ组）、全脾切除组（Ｂ组）、全脾切除后给予Ｔｕｆｔｓｉｎ组（Ｃ１、Ｃ２组）、１／３脾切除组（Ｄ组）、２／３脾切除组（Ｅ组）。比较这六种小鼠移植性肝癌生长情况;采用流式细胞仪检测各组的腹腔巨噬细胞诱导肝癌细胞凋亡率及Ｔｕｆｔｓｉｎ直接诱导肝癌细胞凋亡（另设     

MA方案治疗成人急性非淋巴细胞白血病的疗效分析

目的评价米托蒽醌、阿糖胞苷（MA）方案作诱导治疗成人急性非淋巴细胞白血病（ANLL）初治患者的疗效。方法采用米托嗯醌（mitoxantrone）和阿糖胞苷（cytarabine）对35例成人ANLL患者进行联合化疗。结果临床和血液学完全缓解（CR）15例（42.9%）,部分缓解（PR）9例（25.7%）,总有效率68.6%。结论在强力支持治疗的基础上,MA方案治疗成人难治性急性非淋巴细胞白血病有较好的疗效,其不良反应可以耐受。     

小剂量化疗药物治疗急性白血病机制的研究

目的　探讨小剂量化疗药物〔高三尖杉酯碱 (HHar)、阿克拉霉素 (ACR)、阿糖胞苷 (Ara- c)〕治疗急性白血病 (AL )的可能机制。方法　以成人 AL原代细胞为研究对象 ,充分利用其遗传性和体内细胞相似的特点 ,模拟人体用药后的最大血药浓度 ,对临床常用的三种小剂量化疗药物进行研究。首先采用光镜观察细胞超微结构变化 ,DNA凝胶电泳、二苯胺法、流式细胞术等一系列国内外检测细胞凋亡的经典方法 ,充分证实它们确可诱导 AL 原代细胞凋亡 ;尔后 ,进一步选取其中较为经济的光镜计数细胞凋亡率、二苯胺法定量测定凋亡细胞的DNA片段化率及台盼蓝染色光镜下计数坏死率三种方法来观察药物对急性淋巴细胞白血病 (AL L)及急性髓细胞白血病 (AML)原代细胞的诱导凋亡和杀伤 ,得出相应的时间—效应关系 ,并进行对比分析。结果　三种药物均可诱导 AL 原代细胞凋亡 ,对比分析显示 ACR的作用最强 ;AML 原代细胞对 HHar诱导凋亡的敏感性高于 AL L原代细胞。结论　诱导 AL 细胞凋亡可能是小剂量化疗药物治疗 AL 的主要机制 ;AL L 白血病细胞对 HHar诱导凋亡的作用不敏感可能是临床上 HHar对 AL L 治疗无效的原因     

Bcl-xL is a dominant antiapoptotic protein that inhibits homoharringtonine-induced apoptosis in leukemia cells

Homoharringtonine (HHT) has been reported to be effective in a portion of patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). To investigate its mechanism of action, cell growth inhibition and cytotoxicity of HHT were investigated in three AML cell lines, HL-60, NB4, and U937, and in three CML cell lines, K562, KU812, and KCL22. AML cells were more sensitive than CML cells to HHT-induced cytotoxicity. Using HL-60 cells, it was revealed that HHT decreased the levels of myeloid cell leukemia 1 (Mcl-1), X-linked inhibitor of apoptosis protein (XIAP), survivin, and B-cell lymphoma 2 (Bcl-2)-homology domain 3 (BH3)-only proteins as well as the mitochondrial membrane potential. The levels of Bcl-2, Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist/killer (Bak) proteins in HL-60 cells were not changed after HHT treatment. U937, K562, KU812, and KCL22 cells expressed B-cell lymphoma-extra large (Bcl-xL) and were less responsive to HHT-induced apoptosis than HL-60 cells. Silencing Mcl-1 or Bcl-xL, but not XIAP or survivin, enhanced HHT-induced apoptosis in U937 cells. The levels of HHT-induced apoptosis in K562, KCL22, and KU812 cells were inversely correlated with the levels of Bcl-xL but not those of Bcl-2 or Mcl-1. K562 cells expressing high levels of Bcl-xL but no Bcl-2 were less responsive to HHT-induced apoptosis than KCL22 cells that expressed lower levels of Bcl-xL and higher levels of Bcl-2 protein. In K562 cells, knockdown of Bcl-xL, but not of Mcl-1, enhanced HHT-induced apoptosis. Transfection of Bcl-xL into KCL22 cells attenuated HHT-induced apoptosis. These data suggest that Bcl-xL plays a more important role than Bcl-2 and Mcl-1 in protecting against HHT-induced apoptosis.     

黄芪对老年急性髓系白血病患者BCL-2家族凋亡因子的相关影响

目的：本研究探讨黄芪（astragalus）对老年急性髓系白血病患者疗效评估,并检测其对患者Bcl-2家族凋亡相关因子的影响,从而探讨黄芪对老年急性髓系白血病患者治疗的作用机制。方法随机分配42例老年急性髓系白血病患者,分为对照组和治疗组,两组均予以化疗,治疗组在此基础上给予黄芪,统计两组病患治疗后完全缓解率,患者治疗后白血病细胞凋亡情况运用流式细胞仪检验,患者治疗前后Bcl-2,Bax、Bak、BCL-xl的mRNA表达状况运用RT-PCR方法进行检测,统计两组患者治疗后疗效情况。结果治疗组患者明显优于对照组,治疗组患者完全缓解率高于对照组（P<0.05）。治疗组患者给予黄芪后,白血病细胞凋亡较对照组明显增高,治疗组治疗后Bcl-2、Bcl-xl、Bax、Bak的表达较治疗前差异有统计学意义（P<0.05）,其中Bcl-2、Bcl-xl较治疗前明显下降,Bax、Bak较治疗前明显升高;对照组治疗后Bcl-2、Bax、Bak、BCL-xl表达水平较对治疗前差异无明显统计学意义（P>0.05）。结论黄芪能促进白血病细胞的凋亡,提高患者疗效,其机制可能是通过减低Bcl-2、BCL-xl的表达,增加Bax、Bak的表达来完成。     

Diethyldithiocarbamate-induced cytotoxicity and apoptosis in leukemia cell lines

Diethyldithiocarbamate (DDTC) has been shown to induce cytotoxicity in several different systems. We examined whether the DDTC-induced cytotoxicity was via apoptosis, or in relation to intracellular glutathione (GSH) in various murine and human leukemia cell lines. The cells most sensitive to DDTC-induced cytotoxicity were P388 lymphoid neoplasma cells and NALM-6, a B cell line of acute lymphocytic leukemia (ALL). The next level of susceptible cells included J774.1, having a macrophage function, HL-60 premyelocytic leukemia cells, MOLT-4, an acute lymphoblastic leukemia cell, and Jurkat, a T-cell leukemia. U937 (expressing many monocyte-like characteristics), K562 erythroleukemia and K562/DXR (a multidrug-resistant clone derived from K562) were almost unaffected by DDTC. P388 was also highly susceptible to H(2)O(2), a most useful exogenous reactive oxygen species generator, and was lower in intracellular total GSH content than other leukemia cells. DDTC-induced cytotoxicity was closely related to intracellular GSH, but the level of cellular GSH did not always correlate with H(2)O(2)-induced cytotoxicity in this experiment. K562 had a higher intracellular total GSH content and showed lower susceptibility to DDTC and H(2)O(2), but with the combination of DDTC and DL-buthionine-(S,R)-sulfoximine (BSO), cytotoxicity increased significantly. The ratio of GSH/GSSG in P388 was reduced by DDTC or H(2)O(2). H(2)O(2)-induced cytotoxicity was completely blocked by catalase (CAT), while it was enhanced by superoxide dismutase (SOD). CAT or SOD did not affect DDTC-induced cytotoxicity. N-Acetylcysteine (NAC: 1 mM), a vanguard substance of GSH, and aurintricarboxylic acid (ATA: 100 microM), an endonuclease inhibitor, ameliorated DDTC-induced cytotoxicity and apoptosis. In conclusion, we suggest that DDTC-induced cytotoxicity was via an oxidative shift in the intracellular redox state, and accompanied the activation of endonuclease through apoptosis in leukemia cell lines.     

Riccardin D, a novel macrocyclic bisbibenzyl, induces apoptosis of human leukemia cells by targeting DNA topoisomerase II

We studied the effect of riccardin D, a macrocyclic bisbibenzyl, which was isolated from the Chinese liverwort plant, on human leukemia cells and the underlying molecular mechanism. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, K562 and its multidrug resistant (MDR) counterpart K562/A02 cells, but showed no effect on the topoisomerase-II-deficient HL-60/MX2 cells, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pBR322 DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase II (topo II). The suppression of topo II activity by riccardin D was stronger than that of etoposide, a known topo II inhibitor. After treatment with riccardin D, nuclear extracts of leukemia K562 and K562/A02 cells left the majority of pBR322 DNA in a supercoiled form. Further examination showed that riccardin D effectively induced HL-60, K562 and K562/A02 apoptosis as evidenced by externalization of phosphatidylserine and formation of DNA ladder fragments. The activation of cytochrome c, caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) was also enhanced, as estimated by Western blot analysis. By contrast, riccardin D was unable to induce apoptosis in the topoisomerase-II-deficient HL-60/MX2 cells, indicating that the induction of apoptosis by riccardin D was due to the inhibition of topo II activity. In addition, riccardin D was able to significantly decrease P-glycoprotein (P-gp) expression in K562/A02 cells. Taken together, our data demonstrate that riccardin D is a novel DNA topo II inhibitor which can induce apoptosis of human leukemia cells and that it has therapeutic potential for both regular and MDR strains of leukemia cells.     

Induction of G2/M phase arrest and apoptosis of human leukemia cells by potent antitumor triazoloacridinone C-1305

In this study, we show that triazoloacridinone derivative C-1305, a potent antitumor compound, in human lymphoblastic (MOLT4) and promyelocytic (HL60) leukemia cells induces G2/M arrest followed by apoptosis. In both type of cells, C-1305 at biological relevant concentrations corresponding to EC(90) value, induced a significant increase in the fraction of G2/M cells. The cell cycle perturbations were accompanied by the appearance of sub-G1 fraction, which can be considered as the apoptotic cells population. In both human leukemia cells apoptosis was additionally proved by appearance of DNA fragmentation, activation of caspase-3, PARP cleavage, externalization of phosphatydilserine as well as decrease of the mitochondrial transmembrane potential DeltaPsi(m) and ATP depletion. Treatment of lymphoblastic MOLT4 cells with the C-1305 at EC(90) concentration, caused massive death by apoptosis. Compared to MOLT4 cells, the capacity of HL60 cells to execute apoptosis after C-1305 treatment at equitoxic dose was significantly weaker, but very effective at high concentration (4x EC(90)). These differences could originate from different sensitivity of both cell types to cytotoxic action of C-1305 (EC(50) value for MOLT4 cells was 8 times lower than for HL60 cells and the EC(90) value was 14 times lower, respectively). Collectively, these results show that C-1305 is a novel and potent compound which induces G2/M arrest and subsequent apoptosis of human leukemia cells. This strong ability to induce apoptosis of tumor cells support the view that C-1305 could be consider as a new potent and promising antitumor agent.     

Molecular basis for the inhibition of anticancer agents-induced apoptosis by thymidine phosphorylase

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. TP was expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. Jurkat cells transfected with TP cDNA (Jurkat/TP) and mutant TP cDNA (Jurkat/TPMu) expressed high levels of TP, while Jurkat/CV cells which were transfected with a control vector did not express TP. A high TP enzyme activity was detected in Jurkat/TP cells, but not in Jurkat/CV and Jurkat/TPMu cells. Sensitivities to cisplatin of these cells were determined by MTT assay. IC50 values for cisplatin of Jurkat/CV, Jurkat/TP, and Jurkat/TPMu cells were 4.50, 14.08, 13.40 microM, respectively. Jurkat/TP and Jurkat/TPMu cells were about three times more resistant to cisplatin than Jurkat/CV cells. TP inhibited activation of caspase 3, 9 and mitochondrial cytochrome c release induced by cisplatin. These findings suggest a mechanism by which TP confers the resistance to cisplatin-induced apoptosis. Moreover, mutant TP that has no enzymatic activity also suppressed the cisplatin-induced apoptosis. These suggest that TP molecules have cytoprotective functions against cytotoxic agents.     

Anti-leukemia activities of Lup-28-al-20(29)-en-3-one,a lupane triterpene

The cytotoxicities of 11 lupane series triterpenes against 3 human leukemias, 2 melanomas, 2 neuroblastomas and normal fibroblast cells were examined. Lupane triterpenes with a carbonyl group at C-17 (7-11) showed inhibitory effects on leukemia, melanoma and neuroblastoma cell growth. Lup-28-al-20(29)-en-3-one (8) markedly inhibited the cell growth of 3 leukemias to a greater extent than the other human cancers and normal lung fibroblast cells. The cytotoxicity profiles of 8 against human cancer cells showed that its cytotoxic effect against 3 lung cancer cell lines was strong and the cytotoxic effects against osteosarcoma, breast cancer and urinary bladder cancer cells were very weak. The morphological observations of leukemia nuclei and the gel electrophoresis analysis of DNA extracted from 8-treated leukemia cells revealed that 8 induced leukemia cell apoptosis. Furthermore, we investigated the cytotoxic effects of 8 on adriamycin (ADM)- and vincristine (VCR)-resistant K562 (K562/ADM and K562/VCR) cells. K562/ADM and K562/VCR cells showed greater resistance toward ADM and VCR when compared to parent K562 cells. However, 8 inhibited the drug-resistant K562 cell growth to the same extent as K562 cells by the induction of apoptosis.     

In vitro susceptibility of CD4+ and CD8+ T cell subsets to fludarabine

Administration of the adenosine analogue fludarabine (FLU) in vivo induces a profound and prolonged T lymphopenia which mainly affects CD4(+) cells. To better understand the mechanistic basis underlying this preferential depletion, we analyzed the in vitro susceptibility of T cell subsets to FLU-induced apoptosis. Contrasting with observations in vivo, our results showed that treatment of peripheral blood mononuclear cells with FLU induced a higher level of apoptosis in CD8(+) than in CD4(+) T lymphocytes. This increased sensitivity of CD8(+) T cells to FLU was observed in samples from both, healthy donors and B cell chronic lymphocytic leukemia patients, and resulted in higher CD4:CD8 ratios in FLU-treated than in untreated cultures (P<0.01). Expression of factors involved in FLU transport and metabolism was then evaluated by quantitative real time-PCR in normal T cell subsets. It was found that mRNA levels of human equilibrative nucleoside transporter-1 nucleoside transporter were higher whereas deoxycytidine kinase and IMP/GMP selective 5'-nucleotidase mRNA levels were lower in CD4(+) cells. However the dCK/cN-II ratio was 2-fold greater in CD8(+) than in CD4(+) T lymphocytes, which could account for the higher apoptosis levels observed in the CD8(+) subset. These results favor the view that decreased CD4:CD8 ratios in FLU-treated patients should be attributed to differences in cell recovery and/or homing between T cell subsets.