Silymarin causes caspases activation and apoptosis in K562 leukemia cells through inactivation of Akt pathway

Cancer is one of the largest causes of death in both men and women. Akt, overexpressed in a number of human malignancies including leukemia, is an important target in cancer prevention and/or therapy. Silymarin, a flavonoid antioxidant, has high human acceptance being used clinically for the treatment of liver diseases. In this study, Akt activity was inhibited by silymarin without changes in total Akt level associated with a prominent caspases-9 and -3 activation as well as PARP cleavage, accompanied by a strong apoptotic death and growth inhibition of K562 cells. These findings suggest that silymarin could serve as a candidate for anti-leukemia drug.     

锰超氧化物歧化酶模拟化合物通过Fas途径诱导白血病K562细胞凋亡

目的：探讨锰超氧化物歧化酶模拟化合物（mi mics of manganese superoxide dismutase,MnSODm）对人白血病K562细胞的凋亡诱导效应及作用机制。方法：应用MTT比色法、An-nexin V/PI双标记和细胞形态学法观察细胞凋亡;流式细胞术（FCM）测定Fas蛋白表达水平;RT-PCR检测Caspase-3mRNA的表达水平,比色法测定Caspase-3活性变化。结果：MnSODm作用后K562细胞的增殖受到抑制,Annexin V/PI染色显示凋亡细胞明显增多,透射电镜观察呈现典型的凋亡形态改变。同时,Fas蛋白表达水平显著增高,Caspase-3mRNA表达水平明显升高,活性显著增强。结论：MnSODm可能通过Fas途径诱导白血病K562细胞凋亡。     

载As2O3白蛋白纳米粒对人K562细胞的增殖抑制作用

目的采用白蛋白纳米粒包载As2O3,通过肿瘤细胞摄取载药纳米粒来增强As2O3对K562细胞的增殖抑制作用。方法采用去溶剂化法制备白蛋白纳米粒(ALB-NP),以异硫氰酸(FITC)标记ALB-NP,荧光显微镜观察K562细胞对ALB-NP的摄取;以ALB-NP包载As2O3制备载As2O3白蛋白纳米粒(As2O3-ALB-NP),MTT法比较As2O3与As2O3-ALB-NP对K562细胞增殖抑制率的差异。结果 As2O3-ALB-NP在低浓度(<0.8μmol.L-1)即可显著抑制K562细胞增殖,而As2O3在该浓度对其无抑制作用。结论与As2O3相比,利用ALB-NP载As2O3可显著增强其对K562细胞的增殖抑制作用,有望实现对As2O3用药的增效减毒,为其用于抗肿瘤治疗提供了新的给药策略。     

榄香烯诱导人白血病K562细胞凋亡

目的研究榄香烯抗肿瘤的作用机制。方法用流式细胞仪和琼脂糖电泳检测DNA断裂,透射电镜观察超微结构变化。结果260μmol·L-1榄香烯孵育K562细胞6、12、24、48h后流式细胞仪检测到逐渐增强的凋亡峰,电泳发现明显的阶梯状条带,电镜观察到染色体聚集、凋亡小体。结论榄香烯能诱导K562细胞凋亡。     

柔红霉素对K562细胞凋亡及FAKmRNA影响的研究

目的 研究柔红霉素诱导K562细胞增殖和凋亡情况,以及调节细胞周期和FAKmRNA基因,进一步探讨通过调控FAK表达,研究抗白血病的作用机制.方法 应用CCK8细胞增殖法,结合流式细胞仪检测法检测不同浓度柔红霉素在不同时间对K562细胞增殖和凋亡的影响,应用RT-PCR和Western blot技术检测不同浓度柔红霉素作用对K562细胞FAKmRNA以及蛋白表达水平的变化.结果 K562细胞的增殖抑制率随着柔红霉素浓度增加及作用时间延长逐渐升高,同一浓度不同时间组之间,或者同一时间不同浓度组之间比较,差异均有统计学意义(P<0.05);柔红霉素能引起K562细胞凋亡,且随着药物浓度增加,凋亡率也逐渐增加,差异均有统计学意义(P<0.05);柔红霉素能引起K562细胞周期阻滞,多停留在S期;柔红霉素能引起K562细胞FAKmRNA表达和FAK蛋白表达水平的降低.结论 一定浓度的柔红霉素在体外可诱导细胞凋亡增加并抑制分裂期细胞的增殖,对细胞FAK基因和蛋白水平都有显著下调,发生凋亡的机制可能是通过抑制FAK基因表达.     

Effect of active fraction of Eriocaulon sieboldianum on human leukemia K562 cells via proliferation inhibition,cell cycle arrest and apoptosis induction

Eriocaulon sieboldianum (Sieb. & Zucc. ex Steud.), a genus of Eriocaulon in the Eriocaulaceae family, is an edible and medicinal plant used in traditional Chinese medicine. It was processed into healthcare beverages for expelling wind-heat, protecting eyes, and reducing blood fat. Also, it has been used with other herbs as Traditional Chinese herbal compound to treat cancer as adjuvants in tumor therapy in China. However, the active fractions and precise cellular mechanisms of E. sieboldianum extract remain to be illustrated. The goal of this study was to investigate the effects of the active fraction of E. sieboldianum on the growth of K562 cells and understand the possible mechanisms of its action. Our findings suggested that the fraction E3 of E. sieboldianum could effectively inhibit the activity of Aurora kinase and induce apoptosis via blocking cell cycle, up-regulating the expression of proapoptotic proteins including p53 and Bax and reducing the expression of Bcl-2. The levels of Cytochrome C, cleaved caspase-9, cleaved caspase-3 and cleaved PARP were also found to be increased after treatment with fraction E3 of E. sieboldianum.This study could improve the development of E. sieboldianum and raise its application value in cancer adjuvant therapy. Considering it is both a dietary supplement and a traditional Chinese herbal medicine which exhibits anticancer activities, it can be developed into functional food.     

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。     

DuP-697对K562白血病细胞凋亡诱导作用观察及机制研究

目的：观察选择性COX-2抑制剂DuP697对慢粒白血病K562细胞的凋亡诱导效应,并探讨其作用机制。方法：细胞培养加入DuP-697作用后,透射电镜观察细胞凋亡的形态,流式细胞仪检测细胞周期和凋亡率,Western印迹检测K562细胞Caspase-8蛋白表达;用Z—IETD—FMK阻断Caspase8活性,以证实Caspase-8蛋白表达和细胞凋亡的关系。结果：DuP-697能诱导K562细胞凋亡,其作用呈浓度依赖性,这-效应与Caspase-8蛋白表达上调和裂解激活有关;用Z-IETD—FMK阻断Caspase-8的活性,细胞凋亡明显受抑。结论：DuP-697能诱导K562细胞凋亡,其机制涉及Caspase-8活化的信号转导途径。     

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。     

分枝石蕊多糖诱导人白血病K562细胞凋亡

目的：研究分枝石蕊多糖（CFP-1）是否能诱导K562细胞凋亡。方法：抑制细胞增殖的测定采用MTT法;用荧光显微镜和透射电镜观察细胞的形态学变化;采用琼脂糖凝胶电泳法观测DNA碎片;用流式细胞仪检测凋亡细胞数。结果：CFP-1（50-800mg/L）明显抑制K562细胞增殖,并且呈浓度依赖性,K562细胞与CFP-1300mg/L共同培养5d后,观察到典型的凋亡形态变化,电泳呈现梯形条带。结论：CFP-1诱导人白血病K562细胞凋亡。     

辛伐他汀对K562细胞Ras-MAPK信号通路的体内外研究

目的通过体内、外实验研究辛伐他汀对K562细胞Ras-MAPK信号通路的影响,探讨辛伐他汀诱导K562细胞凋亡的可能机制。方法体外培养CML细胞株K562细胞,做以下实验:①用流式细胞术(FCM)检测辛伐他汀处理K562细胞后其凋亡率的变化,②18只Balb/c-nu/nu裸小鼠皮下接种K562细胞,构建裸鼠的K562细胞移植瘤模型,③TUNEL法检测辛伐他汀诱导裸鼠体内K562细胞早期凋亡的变化,④RT-PCR检测裸鼠体内外K562细胞N-ras、c-Raf-1、ERK1mRNA水平的变化。结果辛伐他汀在体外能明显诱导K562细胞凋亡,Ras-MAPK通路大多数基因出现差异表达。不同剂量的辛伐他汀能够诱导裸鼠体内K562细胞凋亡,并随剂量的增加凋亡率逐渐增高(P<0.01);辛伐他汀能够引起N-ras、c-Raf-1、ERK1mRNA水平的明显差异表达(P<0.01)。结论辛伐他汀在体内外均能够引起参与K562细胞凋亡的Ras分子和Ras分子下游分子mRNA水平的变化,从一定角度说明辛伐他汀能依赖Ras-MAPK信号通路诱导K562细胞凋亡。     

RNA干扰靶向cyclin D1基因对K562细胞增殖、细胞周期及凋亡的影响

目的 利用RNA干扰（RNAi）技术体外观测其对K562细胞cyclin D1基因的沉默效应及对细胞增殖、细胞周期及凋亡的影响。方法 通过壳聚糖介导转染K562细胞、Westernblot分析检测转染前后CyclinD1蛋白表达变化；集落形成实验检测细胞增殖能力；流式细胞仪检测细胞周期分布及凋亡率的影响。结果 构建靶向cyclin D1基因的shRNA表达质粒（pshRNA-419和pshtNA-575）经壳聚糖转染后．能显著抑制cyclin D1基因表达；抑制K562细胞增殖，影响其集落形成能力；影响K562细胞周期分布，诱导细胞凋亡。而设计一碱基突变的序列所构建的质粒并无上述生物学效应。结论 cyclin D1基因表达下调可抑制K562细胞生长，并诱导细胞凋亡。提示cyclin D1基因可能是白血病治疗的一个有效靶点的。     

Mitochondrial dysfunction as an early event in the process of apoptosis induced by woodfordin I in human leukemia K562 cells

Tannins are a group of widely distributed plant polyphenols, some of which are beneficial to health because of their chemopreventive activities. In the present study, we investigated the effects and action mechanisms of woodfordin I, a macrocyclic ellagitannin dimer, on human chronic myelogenous leukemia (CML) K562 cells. The results showed that woodfordin I was able to suppress the proliferation and induce apoptosis in K562 cells. Apoptosis was evaluated by cytomorphology, internucleosomal DNA fragmentation, and externalization of phosphatidylserine. Woodfordin I treatment caused a rapid and sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS), transient elevation of intracellular Ca2+ concentration, and cytosolic accumulation of cytochrome c. The activation of caspase-9 and 3, but not caspase-8, was also demonstrated, indicating that the apoptotic signaling triggered by woodfordin I was mediated through the intrinsic mitochondria-dependent pathway. Western blot and immunofluorescence analysis revealed that the anti-apoptotic Bcl-2 and Bcl-xL levels were downregulated, together with the pro-apoptotic Bax protein. Significantly, woodfordin I-induced apoptosis was associated with a decline in the levels of c-Abl, Bcr-Abl, and cellular protein tyrosine phosphorylation. Considering the consequence of all the events in the process of woodfordin I-induced apoptosis, the mitochondrial dysfunction is directly responsible for the pro-apoptotic effects on K562 cells. Furthermore, because CML is a malignancy of pleuripotent hematopoietic cells caused by the dysregulated tyrosine kinase activity of Bcr-Abl, these findings suggest that woodfordin I may be a potential lead compound against CML.     

二烯丙基二硫诱导K562细胞凋亡及与G2 / M期阻滞关系研究

目的探讨二烯丙基二硫（DADS）对体外培养的人白血病细胞系K562细胞增殖、凋亡和细胞周期的影响。方法采用细胞计数法、形态学观察、MTT分析法、AO／EB染色、流式细胞术探讨DADS对K562细胞增殖、凋亡和细胞周期的影响。结果1．DADS在10-80mg-L。范围内,对K562细胞的抑制作用呈剂量一时间依赖效应。2．DADS浓度在10-80mg．L-1。时作用K562细胞24h后,凋亡率逐渐升高,差异有统计学意义（p〈0．05或P〈0．01）。3．不同浓度DADS作用于K562细胞24h后,K562细胞阻滞于G2／M期。结论DADS有抑制K562细胞增殖和促进K562细胞凋亡的作用,其作用的可能机制与k562细胞阻滞于G2／M期有关。     

Gambogic acid induces G0/G1 arrest and apoptosis involving inhibition of SRC-3 and inactivation of Akt pathway in K562 leukemia cells

Gambogic acid (GA), a major active component of gamboge, exhibits potent anticancer activity in many kinds of cancer cells. However, the anticancer mechanism of GA is not clearly understood. Here we showed that GA could cause growth inhibition, induce the G0/G1 phase cell cycle arrest and apoptosis in human chronic myelogenous leukemia cell line K562 cells. Since steroid receptor coactivator-3 (SRC-3), overexpressed in many human malignancies including leukemia, is a central target for cancer therapy, we also explored the effects of GA on SRC-3 and SRC-3-regulated gene products in K562. GA treatment downregulated the expression of SRC-3 and then inhibited the activity of Akt kinase and its downstream targets p70 S6 kinase 1 (S6K1) and glycogen synthase kinase 3β (GSK3β) without changes in total protein levels of these three proteins, which thus influenced the expression of the apoptosis related gene Bcl-2 in K562 cells. These results suggest that GA might exhibit its strong antitumor effects via the interruption of SRC-3.     

Efficacy of combined therapy with paclitaxel and low-level ultrasound in human chronic myelogenous leukemia cell line K562

Abstract

Background: Sonochemotherapy, which applies ultrasound in cancer chemotherapy, has been proven to be a promising therapeutic modality by some previous researches.

Purpose: To investigate the interaction between an antineoplastic agent – paclitaxel (PTX) and low-level ultrasound in human chronic myelogenous leukemia cell line K562, their combined efficacy, and the potential mechanisms in sonochemotherapy.

Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis and Guava Viacount assay were adopted to examine cell viability. Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D staining. Changes in plasma membrane permeability were monitored by FD500-uptake and lactate dehydrogenase (LDH) release assays. Additionally, the ultrastructure changes were evaluated under scanning electron microscope (SEM).

Results: When 5?ng/ml PTX was combined with sonication at Load Power (LP)?=?2?W, the expected synergistic effects on cell viability loss (p?<?0.05) and apoptosis enhancement could be significantly detected, the plasma membrane permeability showed the best response, and relatively serious cell damages were observed under SEM.

Conclusion: The interaction between these two therapies depended on specific parameter settings, such as PTX dose and ultrasound intensity. Under synergistic conditions, ultrasound significantly potentiated the efficacies of PTX, including cytotoxicity, apoptosis and cell damage induction, which may be due to the enhanced membrane permeability and the increased intracellular PTX level.     

Toxic-dose warfarin-induced apoptosis and its enhancement by gamma ionizing radiation in leukemia K562 and HL-60 cells is not mediated by induction of oxidative stress

The purpose of this study was to test the hypothesis that warfarin may enhance free radical production and oxidative damage on cancer cells. We examined the possible concentration-dependent effect of warfarin on cytotoxicity with respect to oxidative stress on leukemia cell lines (K562 and HL-60) and normal human peripheral blood mononuclear cells (PBMC). Gamma radiation was used as a positive control agent for oxidative stress. At all concentrations of warfarin (5–200 μM), 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol)- and bis-N-methylacridinium nitrate (lucigenin)-amplified chemiluminescence responses and lipid peroxidation and protein oxidation were stable after 72 h incubation at 37°C. However, The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) oxidation was increased when cells were incubated with high concentrations (50–200 μM) of warfarin. In these concentration ranges, warfarin reduced cell growth in a dose-dependent manner, producing apoptosis. Our results also revealed that at concentrations above 5 μM, warfarin had a potentiating effect on radiation-mediated growth inhibition and apoptosis. Furthermore, marked effects were observed on leukemic cells compared with PBMC. We report here that the increase of DCFH oxidation might be due to the increase in the release of cytochrome C caused by warfarin, as cytosolic cytochrome C content was significantly elevated in the warfarin-treated cells compared with control cells, and because cotreatment with antioxidants N- acetylcysteine or 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) was unable to prevent cytochrome C release and DCFH oxidation induced by the drug. Taken together, these results suggest that high warfarin concentrations may be toxic to leukemic cells in vitro through apoptosis, although at the pharmacological concentrations (<50 μM), warfarin has no prooxidant or cytotoxic effect on PBMC, K562, and HL-60 cells. In addition, when the treatment of leukemic cells with warfarin at concentrations above 5 μM is combined with radiation, we observed an increase in radiation-induced cytotoxicity. The mechanism by which warfarin potentiates this cytotoxicity is unclear, but it may not be directly due to toxic damage induced by warfarin-generated free radicals.     

Elemene induces apoptosis and regulates expression of bcl 2 protein in human leukemia K562 cells 1

目的：研究榄香烯的抗肿瘤作用机制．方法：抑制细胞增殖采用ＭＴＴ法;用荧光显微镜观察细胞的形态学变化;ＤＮＡ电泳、流式细胞仪技术检测ＤＮＡ断裂;用流式细胞仪检测ｂｃｌ２蛋白的表达．结果：榄香烯６５－５２０μｍｏｌ·Ｌ－１明显抑制Ｋ５６２细胞增殖,ＩＣ５０（９５％可信区间）为２２０（１５２－３１９）μｍｏｌ·Ｌ－１,电泳可见ＤＮＡ断裂形成的阶梯状条带,形态学上表现为染色体聚集,核固缩、断裂,而ｂｃｌ２蛋白的水平明显下降．结论：榄香烯诱导人白血病Ｋ５６２细胞凋亡,这与ｂｃｌ２蛋白表达的下降有关     

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.     

黄芪总黄酮对K562细胞凋亡及细胞周期的影响

<正>白血病是严重危害人类健康的恶性肿瘤,儿童尤其多见。黄芪总黄酮(total flavonoids of Astragalus,TFA)是从蒙古黄芪中分离的抗氧化、清除自由基的主要活性成分[1,2],