二烯丙基二硫活化NADPH氧化酶诱导人白血病K562细胞凋亡

目的研究DADS诱导人白血病K562细胞凋亡的分子机制。方法应用MTT法检测细胞的活性;流式细胞术检测细胞内的活性氧(reactive oxygen species,ROS)水平以及凋亡细胞百分率;Real-time PCR检测NADPH氧化酶各亚基mRNA水平;免疫共沉淀检测蛋白Rac2与蛋白p67phox的结合;Western blot检测Rac2蛋白的表达。结果 DADS能明显抑制K562细胞的增殖,呈时间和剂量依赖性;6 mg·L-1DADS作用人白血病K562细胞6 h后,NADPH氧化酶复合物的6个亚基mRNA水平都明显上调;5.0、10.0 mg·L-1DADS作用人白血病K562细胞24 h后蛋白Rac2的表达水平明显上调;免疫共沉淀结果显示,DADS诱导的K562细胞凋亡过程中有Rac2与p67phox结合;流式细胞术检测凋亡细胞百分率结果显示,PMA能明显提高DADS诱导K562细胞凋亡的作用,而DPI能抑制DADS诱导K562细胞凋亡。PMA能提高DADS诱导K562细胞活性氧的水平,而DPI明显抑制了活性氧的产生。结论 NADPH氧化酶的活化是DADS诱导K562细胞凋亡过程中活性氧的主要来源,DADS通过活化NADPH氧化酶诱导K562细胞凋亡。     

二烯丙基二硫下调DJ-1抑制人白血病HL-60细胞增殖及诱导分化

目的研究二烯丙基二硫(DADS)通过下调DJ-1对人白血病细胞系HL-60增殖抑制及诱导分化的影响。方法通过细胞形态法观察细胞分化;Western blot及免疫细胞化学检测DADS对HL-60细胞DJ-1表达的影响;针对DJ-1mRNA序列设计合成siRNA转染人白血病细胞HL-60,Western blot检测基因干扰效果;利用MTT法及间接免疫荧光检测DADS及DJ-1沉默对HL-60细胞的生长及细胞分化的影响。结果 DADS可以诱导HL-60细胞向成熟粒细胞系样分化。DADS呈时间依赖性抑制DJ-1的表达(P<0.05),DJ-1干扰组细胞生长减慢(P<0.05),与DADS共同作用后可诱导HL-60细胞分化。检测细胞生长情况发现干扰组细胞生长减慢(P<0.05)。结论 DADS通过下调DJ-1抑制细胞增殖并诱导HL-60细胞分化;干扰DJ-1基因可以增强DADS对HL-60细胞的增殖抑制诱导分化作用。     

Rac2在二烯丙基二硫诱导人白血病K562细胞凋亡中的作用

目的研究二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病K562细胞凋亡的分子机制。方法 Real-time PCR检测Rac2基因mRNA水平;Western blot检测Rac2、JNK、p38等蛋白的表达;基因沉默Rac2蛋白的表达;流式细胞术检测凋亡细胞百分率以及细胞内的活性氧(reactive oxygen species,ROS)水平;琼脂糖凝胶电泳检测晚期凋亡。结果随着DADS质量浓度的增加,Rac2基因mRNA水平明显上调(P<0.05)。与未干扰组相比,siRac2RNA组凋亡率明显降低(P<0.05)。Rac2siRNA干扰的DADS诱导的K562细胞并未出现梯状条带。与未干扰组相比,siRac2RNA组在5.0,10.0和15.0mg·L-1 DADS作用于K562细胞8h后,荧光强度显著降低(P<0.05)。Western blot分析结果显示:与对照组相比,10.0和15.0mg·L-1 DADS作用于K562细胞24h后,蛋白Rac2的表达水平明显上调(P<0.05);用siRNA抑制Rac2蛋白的表达后,JNK1的表达显著降低,p38和磷酸化的p38的表达在Rac2干扰前后没有明显变化。结论 Rac2与DADS诱导K562细胞凋亡、活性氧的产生密切相关。活化的Rac2选择性地激活JNK信号通路而不是p38信号通路导致细胞凋亡。     

菊苣酸诱导人白血病细胞HL-60凋亡作用及机制研究

目的观察菊苣酸诱导人急性髓系白血病细胞株HL-60凋亡的作用并初步探讨其机制。方法培养HL-60细胞,分别给予终浓度为10¹00μmol·L-1的菊苣酸48 h,检测细胞活性和凋亡、Caspase-3活性以及Bcl-2蛋白表达水平。结果终浓度为10100μmol·L-1的菊苣酸48 h,检测细胞活性和凋亡、Caspase-3活性以及Bcl-2蛋白表达水平。结果终浓度为10¹00μmol·L-1的菊苣酸能呈浓度依赖性降低HL-60细胞增殖活性和增加凋亡,增强Caspase-3活性和下调Bcl-2蛋白表达。结论菊苣酸具有抑制白血病细胞株HL-60增殖活性和诱导其凋亡作用,其机制与降低抗凋亡蛋白Bcl-2表达及增强Caspase-3活性有关。     

二烯丙基二硫启动人白血病HL-60细胞凋亡模型的建立

目的寻找二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病HL-60细胞凋亡的启动点,建立DADS启动HL-60细胞凋亡模型。方法实验设未处理组、处理组和撤药组,分别采用细胞计数、流式细胞术、DNA凝胶电泳、Western blot等方法,绘制生长曲线,检测凋亡率、活化的caspase-3表达率以及DNALadder、凋亡相关蛋白的检测。结果3.6mg.L-1DADS作用HL-60细胞1d,与对照组相比,细胞数没有明显变化,而作用2～6d,细胞数分别减少24.1%、36.5%、44.2%、52%、53.6%。DADS作用1、2d,凋亡率分别为3.1%、4.3%,与未处理组(3.0%)差异无显著性,而作用3～5d的凋亡率分别达到8.5%,15.2%,27.4%,均高于未处理组(P<0.05)。DADS作用2～5d撤药后再培养至d6,凋亡率分别为7.9%,12.4%,16.5%,18.8%,高于未处理组的3.3%(P<0.05)。处理2～5d后,活化的caspase-3的表达率分别为10.0%、10.4%、14.9%、17.3%,均高于未处理组5.4%(P<0.05)。处理组中DADS处理4d后DNA凝胶电泳出现梯状条带,而DADS作用2～5d撤药后再培养至d6均出现明显梯状条带。Westernblot结果表明,从作用2d起,caspase-3表达开始上调,而Bcl-2表达开始下调。结论3.6mg.L-1DADS诱导人白血病HL-60细胞凋亡的启动点为处理2d。     

二烯丙基二硫通过活性氧介导的JNK信号通路诱导人白血病K562细胞凋亡

目的研究二烯丙基二硫(DADS)诱导人白血病K562细胞凋亡的分子机制。方法应用MTT法检测细胞的活性;用流式细胞术检测细胞凋亡以及细胞内的活性氧(reac-tive oxygen species,ROS)水平;Western blot检测JNK以及磷酸化JNK的活化。结果 DADS能明显抑制K562细胞的增殖,呈时间和剂量依赖性;5.0 mg.L-1DADS处理K562细胞,细胞内ROS水平在1 h后明显增加,8 h达到高峰,随后又开始下降。随着DADS剂量的增加,JNK的活性明显增强,在DADS处理K562细胞8 h后,磷酸化的JNK达到最高值,而在随后的4 h又明显降低。Sp600125和NAC能明显减少磷酸化JNK的表达和抑制DADS诱导的细胞凋亡。结论 ROS是DADS诱导K562细胞凋亡过程中JNK活化的有效调节剂,DADS通过ROS介导的JNK活化诱导人白血病K562细胞凋亡。     

二烯丙基二硫诱导人白血病细胞系HL-60细胞分化的实验研究

目的　探讨二烯丙基二硫 (DADS)对人急性髓性白血病细胞系HL 6 0细胞增殖抑制及诱导分化作用的影响。方法　采用MTT法检测对细胞增殖的影响 ,通过观察细胞形态、硝基四氮唑蓝 (NBT)还原反应、流式细胞仪测定鉴定细胞分化。结果　HL 6 0细胞经DADS处理后 ,细胞增殖受抑 ,呈剂量效应关系。 0 6 2 5～ 1 2 5mg·L-1时NBT还原反应增强 (P <0 0 1或 <0 0 5 ) ,流式细胞术显示可诱导表面分化抗原CD11b表达升高及细胞周期阻滞于G1期。结论　小剂量DADS长时间作用对HL 6 0细胞具有诱导分化作用 ,其作用相当于全反式维甲酸 ,对于白血病的治疗具有潜在的应用价值     

中华眼镜蛇毒诱导HL-60细胞凋亡及其机制探讨

目的　观察中华眼镜蛇毒体外对白血病细胞凋亡的影响 ,并探讨其可能的作用机制。方法　本研究采用血清药理学方法 ,以人白血病细胞HL 60细胞为靶细胞 ,通过流式细胞仪分析、DNA片段凝胶电泳、免疫组织化学 (S P法 )观察中华眼镜蛇毒兔血清体外对细胞凋亡的诱导作用及癌基因表达的影响。结果　经DNA电泳、流式细胞仪分析显示 :中华眼镜蛇毒兔血清与HL 60细胞共同培养 48h可诱导其细胞凋亡增加 ,bcl 2、c myc基因蛋白表达下调。结论　中华眼镜蛇毒兔血清体外抑制HL 60细胞增殖可能与其诱导细胞凋亡及使细胞bcl 2、c myc基因蛋白表达下调密切相关     

二烯丙基二硫诱导人白血病HL-60细胞分化差异表达cDNA文库的构建

目的　应用抑制性消减杂交技术构建二烯丙基二硫(diallyldisulfide ,DADS)诱导人白血病细胞分化的消减杂交cDNA文库 ,以期克隆DADS诱导人白血病HL 6 0细胞分化的相关基因。方法　用DADS诱导人白血病HL 6 0细胞分化 ,提取polyA+ RNA ,反转录合成cDNA ,消化成短片段后分成两组 ,分别与两种不同的接头连接 ,再与未处理的白血病细胞cDNA进行两次消减杂交及两次抑制性PCR扩增 ,将PCR产物与pGEM T线性载体连接 ,转化大肠杆菌进行文库扩增 ,随机挑取克隆进行酶切鉴定。结果　成功地构建了具有高消减效率的DADS诱导白血病细胞分化的cDNA文库 ,随机挑取消 2 0 0个克隆制备质粒并酶切分析 ,其中 84 5 %的克隆均具有 1 0 0～ 6 0 0bp左右的插入片段 ,说明每一克隆中均含有特异性的目的片段 ,从而为大批量筛选、克隆DADS诱导人白血病细胞分化的相关未知新基因奠定了基础。结论　DADS能诱导人白血病HL 6 0细胞分化 ,并引起相关的基因发生改变 ,而抑制性消减杂交技术能有效地分离差异表达的基因。     

DADS抑制JAK1/STAT3信号通路诱导人白血病HL-60细胞分化

目的探讨JAKs/STATs信号转导通路在二烯丙基二硫(DADS)诱导人白血病HL60细胞分化中的变化及其调控机制。方法将HL60细胞与DADS或JAKs/STATs信号通路的激酶抑制剂AG490在体外共同培养,观察细胞形态变化,检测药物作用前后细胞NBT还原能力及细胞表面分化抗原CD11b的改变;用Westernblot检测JAKs,STATs各家族成员在DADS诱导HL60细胞分化中的改变;并用免疫细胞化学法检测核转录基因STATs,cmyc,cfos,cjun的表达变化。结果DADS和AG490均可诱导HL60细胞向成熟粒系分化,且DADS在1.25mg·L-1时诱导分化作用达峰值;Westernblot检测JAK1,STAT3的酪氨酸激酶发生了磷酸化改变;免疫细胞化学示STAT3与cmyc基因蛋白核内表达下降,cjun,cfos基因蛋白核内表达上升。结论JAK1,STAT3酪氨酸激酶的磷酸化抑制参与了DADS诱导HL60细胞分化的调控,其机制可能通过调控与HL60细胞增殖分化相关的基因表达,抑制细胞DNA合成,从而抑制细胞增殖,诱导分化。DADS的作用相当于JAK1/STAT3信号通路的阻断剂。     

二烯丙基二硫对人白血病HL-60细胞株survivin表达的影响

目的:观察二烯丙基二硫(diallyl disulfide,DADS)作用于人白血病HL-60细胞株后,细胞增殖及survivin蛋白表达的变化,探讨DADS对HL-60细胞增殖的影响及其机制。方法:DADS作用HL-60后,MTT法检测细胞增殖情况,SP-免疫组化法检测细胞survivin蛋白表达的变化。结果:DADS可以呈时间-浓度依赖性抑制HL-60细胞增殖并下调survivin蛋白的表达。结论:DADS能通过下调survivin蛋白的表达而抑制HL-60细胞的增殖。     

二烯丙基二硫对人白血病HL-60细胞体外抑制机制的初步研究

大蒜及其烯丙基硫化物抗肿瘤活性的研究是抗癌药物研究的一个新领域.二烯丙基二硫(diallyl disulfide,DADS,C6H10S2)是用来研究大蒜抗癌疗效最适当的一种油溶性烯丙基硫化物[1].目前,有关DADS抑制人肿瘤细胞的报道尚少,本研究拟在探讨DADS对体外培养的人白血病HL-60细胞的抑制作用及其机制,为进一步探讨DADS的抗肿瘤作用提供理论基础.     

Apoptosis induced by α-anordrin in human promyelocytic leukemia HL-60 cells

Induction of apoptosis in human promyelocytic leukemia HL-60 cells by α-anordrin was estimated with the use of morphological and biochemical methods. α-Anordrin 2.5-50.0 μmol·L^-1 inhibited the growth of HL-60 cells in a dose-dependent manner and arrested them at G₁ phase of cell cycle. The cells exhibited marked morphological changes such as cell shrinkage, nuclear fragmentation, chromatin condensation. Agarose gel electrophoresis of DNA extracted from α-anordrin-treated cells revealed a “ladder” pattern. Nearly 63% of cells underwent apoptosis at α-anordrin concentration of 50 μmol·L^-1 as determined by flow cytometry. The data demonstrate that α-anordrin induces HL-60 cell apoptosis, which might be related to its antitumor action.     

Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells.

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.     

Sphinganine potentiation of cellular differentiation induced by various anti-leukemia drugs in human leukemia cell line HL-60

A slight induction of cellular differentiation (myelocytes and granulocytes) of HL-60 cells occurred after treatment with anti-tumor agents etoposide (VP-16), mitoxantrone (MXT), mitomycin C (MMC), actinomycin D (Act-D) or novobiocin (NOVO). Addition of sphinganine (SP), an inhibitor of protein kinase C (PKC) enhanced (2–3fold) the VP-16, MXT, MMC or Act-D-induced differentiation but not the NOVO-induced differentiation. No induction of differentiation was observed with 5-fluorouracil (5-FU) in the absence or presence of SP. The addition of SP in the fresh medium after the removal of VP-16, MXT, or MMC (0.5 h treatment) enhanced the induction of differentiation. In contrast, SP post-treatment did not have any effect on enhancing the differentiation which was induced by Act-D short exposure (0.5 h).In an attempt to characterize the biochemical requirements for potentiation of VP-16-induced differentiation, we examined the effects of calcium depletion using calcium chelator ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA) or calcium channel blocker verapamil. Potentiation of VP-16-induced differentiation by SP was not observed in EGTA- or verapamiltreated cells. Calcium supplementation to the cells during the treatment with EGTA restored the SP-potentiation of VP-16-induced differentiation. Our results also showed that the induction of differentiation was accompanied by a decrease in PKC activity (70% of the control). PKC activity decreased to a greater extent (50% of control) in SP potentiation of differentiation induction.Our results suggested that calcium-dependent biological action of antitumor agents and the inhibition of PKC activity are required for SP-potentiation of differentiation induction.This work was supported by Chang Gung Research Grant CMRP 352 and National Science Council (R.O.C.) Grants NSC 81-0412-B-182-41 and NSC 82-0412-B-182-006     

Inhibition of ERK and activation of p38 are involved in diallyl disulfide induced apoptosis of leukemia HL-60 cells

We investigated the effects of diallyl disulfide (DADS) on the induction of apoptosis in human Leukemia cell line HL-60 and explored the roles of mitogen-activated protein kinase (ERK and p38 MAPK) pathways in the growth inhibition and apoptosis induced by DADS. MTT assay was used to determine the DADS induced cell growth inhibition in HL-60 cells. Flow cytometry and DNA fragmentation were used to examine the roles of apoptosis in DADS-mediated cell death. Western blot analysis of the expression of phospho-MAPKs (ERK and p38) was employed to elucidate the possible mechanisms of DADS induced apoptosis. We found that growth inhibition of HL-60 cells treated with DADS exhibited a dose-dependent response (P<0.05) and DADS induced significant apoptosis. DADS at the concentration of 10 mg/L persistently activated p38 and simultaneously reduced ERK activity. PD98059, an inhibitor of ERK upstream activators MAPK kinase MKK1 and MKK2, promoted cytotoxicity and apoptosis in HL-60 cells treated with DADS. In contrast, SB203580, an inhibitor of p38, decreased cytotoxicity and apoptosis induced by DADS. Therefore, DADS can effectively inhibit the proliferation and induce apoptosis of human leukemia cell line HL-60. Inhibition of ERK signaling pathways and activation of p38 signaling pathways are likely involved in DADS induced apoptosis in HL-60 cells.