蛋白酶体抑制剂MG132的浓度对人白血病细胞K562凋亡的影响

探讨蛋白酶体抑制剂MG132 在诱导人白血病K562细胞凋亡过程中作用.分别以不同浓度的蛋白酶体抑制剂MG132 处理人白血病细胞K562,通过MTT法检测K562细胞活力,应用Annexin Ⅴ和PI 双染的细胞流式法检测K562细胞凋亡率和细胞内活性氧(ROS) 水平,应用酶标仪法检测K562细胞内Caspase- 3活性变化的情况.结果表明,随着MG132浓度的增加,各个指标与对照组比较差异均有显著性(P<0.05):K562细胞增殖明显受到抑制;细胞凋亡率明显增加,且当MG132浓度为900 nmol/L时,细胞凋亡率达36.5 %;同时,ROS 水平和caspase- 3活性明显升高.因次,蛋白酶体抑制剂MG132可显著抑制人白血病细胞K562增殖并促进其凋亡.     

β—榄香烯吗素抗肿瘤作用的实验研究

β－榄香烯吗素（PIC－BE）是抗癌新药β－榄香烯的水溶性衍生物。本观察了PIC－BE对多药耐药K562／ADM细胞系及其敏感细胞K562的生长抑制和凋亡诱导作用。结果显示,（1）K562／ADM细胞对ADM具有明显的抗性,与K562细胞相比,抗笥倍数约为40倍,而两对PIC－BE的IC50接近,无显差异;（2）PIC－BE（10.0-30.0μg/ml）对K562和K562／ADM细胞不仅具有明显的生长抑制作用,而且显地诱导细胞凋亡,其作用强度在一定的范围内呈相对浓度和时间的依赖性,以上结果提示,PIC－BE是一种有效的抗肿瘤化合物,且已产生MDR的K562／ADM细胞对它不具耐药性。     

蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

JNK信号通路对蜂胶抑制白血病K562细胞增殖的调控作用

目的探讨JNK信号通路对蜂胶抑制K562细胞增殖过程的调控作用。方法体外培养K562细胞,用不同浓度蜂胶、c—Jun氨基末端激酶（c—JanN—terminalkinase,JNK）特异性抑制剂SP600125对白血病K562细胞进行处理,用MTT法检测细胞增殖抑制率,流式细胞术（FCM）检测细胞凋亡率,Western印迹检测JNK下游分子c—Jun以及磷酸化c—Jan（p-c-Jun）的变化。结果蜂胶作用K562细胞后,增殖抑制率、凋亡率显著升高,具有时间和剂量依赖性,并伴随p-c-Jun蛋白水平上调;加入SP600125能下调p-c-Jun的水平,显著提高蜂胶对K562细胞的增殖抑制率和凋亡率。结论JNK信号通路参与了蜂胶抑制K562细胞增殖过程的调控。抑制JNK活性可增强蜂胶对K562细胞的增殖抑制、凋亡诱导作用。     

氧化苦参碱对K562肿瘤细胞增殖的影响

目的:研究氧化苦参碱(OM)对人白血痛细胞系K562生长增殖的影响.方法:运用MTT比色法、活细胞计数法、集落形成法以及透射电镜观察检测OM对人白血病细胞系K562增殖抑制作用.结果:MTT实验、生长曲线及集落形成实验显示OM能明显抑制K562细胞的增殖.随着OM浓度的增加,K562细胞存活细胞显著降低,呈现明显的刺量依赖性,经相关分析,细胞抑制率与OM浓度呈正相关(r=0.9010),其半数抑制浓度(IC50)为0.33 mg/ml.透射电镜下显示在低浓度即有明显的诱导细胞凋亡的作用,出现核固缩、核碎裂、凋亡小体等典型的凋亡形态.结论:OM具有抑制K562白血病细胞增殖诱导肿瘤细胞凋亡的作用.     

青蒿素诱导人白血病细胞K562凋亡的线粒体机制

目的:探讨青蒿素诱导人白血病细胞K562凋亡的线粒体机制.方法:用青蒿素处理K562细胞.通过MTT比色法检别细胞增殖抑制的效果;荧光显微镜观察细胞的凋亡;流式细胞术(flow cytometry,FCM)进行细胞周期分析;Western-blotting测定药物作用前后线粒体、细胞浆细胞色素C的表达.结果:青蒿素抑制K562细胞的增殖,IC5D为1.5× 10-5mol·L-1;Hoechst33342/PI双荧光染色可观察到明显的核浓缩、凝集等细胞凋亡表现;流式细胞仪检测G2期细胞比例增高,S期减少;Western-blotting检测药物处理细胞后线粒体细胞色素C表达水平下调,细胞浆出现明显细胞色素C蛋白条带.结论:青蒿素可能通过线粒体细胞色素C途径诱导K562细胞凋亡.     

选择性COX-2抑制剂celecoxib对K562细胞的增殖抑制、凋亡诱导作用和分子机制

环氧化酶(cyclooxygenase, COX)家系被显示与恶性肿瘤的增殖和凋亡耐受有关,COX-2可作为恶性肿瘤治疗和预防的重要分子靶标.应用COX-2特异抑制剂——celecoxib,观察了药物对人慢性粒细胞白血病急变细胞株——K562细胞的增殖抑制和凋亡诱导效应.结果证明,celecoxib能够有效地抑制K562细胞增殖(台盼蓝染色,MTT试验及集落形成抑制试验证实),并呈一定的剂量依赖性.Celecoxib抑制K562细胞增殖的IC₅₀为46 μmol/L.通过DNA ladder胶电泳和流式细胞仪检测,凋亡细胞的AO/EB染色等方法证明celecoxib能够诱导K562细胞凋亡,这一效应与Caspase-3蛋白表达上调和裂解激活有关,当阻断Caspase-3的活性,celecoxib诱导的K562细胞凋亡明显受抑.利用RT-PCR分析技术及蛋白质印迹,证明K562细胞存在COX-2 mRNA和COX-2蛋白表达;而且,K562细胞COX-2蛋白表达可被IL-1β诱导性刺激,从而确认K562细胞为COX-2表达阳性细胞;celecoxib在较高浓度(80～160μmol/L)既可抑制K562细胞COX-2 mRNA表达,也可下调COX-2蛋白质表达,提示celecoxib抗K562白血病细胞活性与COX-2的抑制相关,其抗白血病的分子机制部分涉及到COX-2依赖性途径.     

少棘蜈蚣抗菌肽scolopin 2的克隆、表达与抗癌机制研究

旨在研究AMP-scolopin 2的抗菌活性和抗癌机理。利用SUMO融合技术对AMP-scolopin 2进行了体外表达;采用抑菌圈、MTT、流式细胞术和Western blotting等技术分析了其抗菌活性、溶血活性和对肿瘤细胞的促凋亡作用。结果表明,成功表达并纯化了AMP-scolopin 2;AMP-scolopin 2对白血病细胞(K562)和肝细胞癌(Hep G2)具有抑制增殖和促凋亡作用,但对红细胞和正常细胞HEK293的影响很小;AMP-scolopin 2(40μmol/L)可以诱导K562(21.4%)和Hep G2(18.5%)细胞凋亡;此外,AMP-scolopin 2下调了与线粒体相关的凋亡蛋白pro-caspase-3、pro-caspase-9和pro-PARP的表达,且呈浓度依赖性。AMP-scolopin2可以通过线粒体caspase依赖性途径促进肿瘤细胞凋亡。     

Akt/PKB抗凋亡机制与天然活性物质的抗肿瘤作用

Akt/PKB是调控细胞生存与凋亡的重要信号物质之一。它能够影响下游多种效应分子的活化状态,在细胞内发挥着抑制细胞凋亡、促进细胞增殖的作用,并同人类多种肿瘤的发生发展密切相关。天然活性物质能够通过抑制Akt/PKB通路、诱导细胞凋亡来发挥它们的抗肿瘤效应。对Akt/PKB与细胞凋亡关系的研究不但有利于理解细胞凋亡机制,还可以指导开发新型的抗癌活性物质。本文综述了Akt/PKB对细胞凋亡、存活的调节机制及天然活性物质通过PI3K/Akt信号通路抗肿瘤作用的研究进展。     

三氧化二砷对K562细胞凋亡的诱导及生长抑制作用的研究

目的：研究三氧化二砷(As2O3)对人红白血病细胞株K562的生长抑制和凋亡诱导作用。方法：以As2O3作为耐药逆转剂,用台盼兰排染法,噻唑兰(MTT)还原法,Hoechst 33342和PI荧光染色法,流式细胞仪技术和荧光分光光度法,观察了不同浓度的As2O3(0．2—5．0μmol/L)对人红白血病细胞株K562的生长抑制和凋亡诱导作用。结果：As2O3对K562细胞具有明显生长抑制和凋亡诱导作用,其作用强度在一定范围内均具药物浓度和时间依赖性。结论：As2O3主要以诱导肿瘤细胞凋亡而表现其毒性作用。     

Effect of 5-azacytidine on the differentiation of human leukemia K-562 cells

The treatment of K-562 cells with 10(-5) M to 10(-7) M 5-azacytidine induced a marked increase in benzidine-positive cells. Similarly, the exposure of K-562 cells to 2 X 10(-3) M butyric acid or 5 X 10(-7) M 1-beta-arabinofuranosylcytosine or 1 X 10(-3) M hydroxyurea induced an erythroid differentiation of K-562 cells. The activity of DNA-methyltransferase and the level of methylcytosine in newly synthesized DNA were significantly decreased when the cells were treated with 5-azacytidine or butyric acid, while 1-beta-arabinofuranosylcytosine or hydroxyurea had no inhibitory effect on DNA-methylation of K-562 cells. These results suggest that the inhibition of DNA-methylation is not necessarily a specific phenomenon for erythroid differentiation of K-562 cells.     

Improved sensitivity in comparative genomic hybridization analysis of DNA heteroploid cell mixtures after pre-enrichment of subpopulations by fluorescence activated cell sorting.

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K-562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K-562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K-562 samples, a mixture with 32% K-562 cells showed 16 imbalances, and none were detected in mixtures with 13% or 5% K-562 cells. In contrast, 29, 22 and 23 imbalances were detected in K-562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population.     

Detection of surface differences between closely related cell populations by partitioning. Cultured K-562 cell sublines

The K-562 cell line is a culture of human leukemia stem cells originally derived from a patient with chronic myelogenous leukemia in blast crisis. We have applied a sensitive method capable of detecting subtle differences in charge-associated and noncharge-related cell surface properties between closely related cell populations to K-562 cells from different sources and having different histories. The method consists of isotopically labeling aliquots of each of two cell populations to be compared with 51Cr-chromate and mixing the labeled cells with an excess of unlabeled cells with which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a noncharge-sensitive dextran-poly(ethylene glycol) aqueous two-phase system. The distribution curves are analyzed for total cells (in terms of electronic counts) and labeled cells (in terms of cpm). Alterations in relative specific activities through the distribution curves are indicative of differences in surface properties between such cell populations. Using this method we have found surface differences, both charge-associated and noncharge-related, between any two K-562 cell sublines examined. Interestingly, whereas we observed differences among K-562 sublines, we never witnessed a change in surface properties of the respective sublines. The differences among the sublines examined remained unaltered for more than 40 passages in our hands. It thus appears likely that the event(s) leading to an altered K-562 cell surface, detectable by partitioning, does not occur gradually.     

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Erythroglycan biosynthesis in K-562 cells. Inhibition of synthesis by tunicamycin and lack of attachment to the G-protein of vesicular-stomatitis virus.

K-562 cells, which express foetal erythroglycan, are shown to synthesize the lipid-linked oligosaccharide intermediates commonly found in tissues and cultured fibroblasts. The addition of tunicamycin, which blocks the formation of these intermediates and thus of asparagine-linked oligosaccharides, inhibits the synthesis of erythroglycan (Mr 7000-11 000). Vesicular-stomatitis-virus infection of K-562 cells results in the glycosylation of the G-protein with the transferrin-type oligosaccharide (Mr 3000), but not with the larger erythroglycan. These results suggest that, in K-562 cells, the early stages of erythroglycan biosynthesis are the same as those of the transferrin-type oligosaccharides. However, maturation of the oligosaccharide is influenced by protein structure such that erythroglycan is only expressed on specific glycoproteins.     

Activity of human natural killer cells against target cells possessing different sensitivity to interferon]

The cytotoxic activity of peripheral blood natural killers (NK) against target cells (TC) J-96 and L-929 with high sensitivity to interferon (IFN) action, J-41 and MCB resistant to IFN action and line K-562 labelled by H3-uridine was studied in 14 hrs cytotoxic test. It has been shown that human TC J-96 didn't differ from the J-41 in their sensitivity to NK cytotoxicity and they are strongly resistant to NK than TC K-562. The murine TC L-929 as the human TC didn't differ from the MCB in their sensitivity to NK lysis and had also the same sensitivity to NK as the K-562 cells.     

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Natural killers in pregnancy: the problem of heterogeneity and the regulation of activity

Natural killer activity of pregnant women's peripheral blood lymphocytes and of cord blood was investigated. 3H-uridine labeled K-562 and human embryo fibroblasts (HEF) were used as target cells in cytotoxic test. The results of competitive inhibition test led us to a conclusion about the presence of some common K-562 and HEF surface structures recognized by NK cells. It was shown that the decline of NK activity of pregnant women and a low NK activity of cord blood were not associated with the influence of T-lymphocytes or adherent cells.