Mirabijalone-B的抗肿瘤效应及对DNA拓扑异构酶的影响

目的研究化合物Mirabijalone-B(MB)的抗肿瘤活性及对DNA拓扑异构酶(TOPO)活性的影响。方法以5株人肿瘤细胞株为模型,采用改良MTT法检测化合物MB的体外抗肿瘤活性;以质粒pBR322超螺旋DNA为底物,采用凝胶电泳分别检测MB对TOPOⅠ、Ⅱ介导的pBR322DNA解旋反应的影响。结果化合物MB对5株人肿瘤细胞株K562、HL-60、A549、Bel-7402和SGC-7901生长增殖的半数抑制浓度(IC50)分别为8.73、1.26、4.44、1.93和3.55mg.L-1;MB在80和16mg·mL-1浓度时,分别完全抑制TOPOⅠ、Ⅱ介导的pBR322超螺旋DNA的解旋反应;但在无TO-PO存在的条件下,MB对pBR322超螺旋DNA的解旋反应无直接影响。结论化合物MB体外明显抑制人肿瘤细胞的生长增殖,对DNATOPO尤其是TOPOⅡ活性的抑制可能是其抗肿瘤作用的机制之一。     

Santamarin的抗肿瘤活性及对DNA拓扑异构酶的影响

目的研究Santamarin的体内外抗肿瘤活性及对DNA拓扑异构酶(TOPO)活性的影响。方法以7株人肿瘤细胞株为模型,采用改良MTT法检测Santamarin对肿瘤细胞增殖的影响;以小鼠移植性肉瘤S180和小鼠移植性肝癌H22为模型,检测Santamarin对在体肿瘤生长的影响;以pBR322 DNA为底物,采用琼脂糖凝胶电泳检测Santamarin对TOPO介导的pBR322 DNA解旋反应的影响,以及对pBR322 DNA是否具有直接断裂作用。结果Santamarin对7株人肿瘤细胞株的半数抑制浓度(IC50)在0.99~3.61mg·L-1之间;Santamarin30、90和270mg·kg-1剂量对S180生长的抑制率分别为51.44%、63.60%和56.37%;对H22生长的抑制率分别为40.77%、39.47%和46.73%。Santamarin在2g·L-1时完全抑制TOPOI、Ⅱ的活性,但对DNA无直接断裂作用。结论Santamarin具有较为明显的体内外抗肿瘤活性,对DNA TOPO活性的抑制可能是其作用机制之一。     

氯化两面针碱的抗肝癌活性及对DNA拓扑异构酶的影响

目的研究氯化两面针碱对肝癌HepG2裸小鼠移植瘤的抗肿瘤作用及对DNA拓扑异构酶(TOPO)活性的影响。方法建立人肝癌裸鼠皮下移植瘤模型,观察氯化两面针碱的肿瘤抑制作用。以pBR322DNA为底物,采用琼脂糖凝胶电泳检测氯化两面针碱对TOPO介导的pBR322 DNA解旋反应的影响,以及对pBR322 DNA是否具有直接断裂作用。结果氯化两面针碱2.5、5和10mg·kg-1剂量对肝癌HepG2的抑制率分别为12.06%、35.63%和60.91%,氯化两面针碱在6.25μmol·L-1时可完全抑制TopoI的催化活性,在25μmol·L-1时完全抑制TopoⅡ的催化活性。结论氯化两面针碱具有较为明显的抗肝癌活性,对DNA TOPO活性的抑制可能是其作用机制之一。     

Albaconol的抗肿瘤活性及对DNA拓扑异构酶Ⅱ的影响

目的　研究地花菌提取物Albaconol的体内外抗肿瘤活性及对DNA拓扑异构酶Ⅱ (TOPOⅡ )活性的影响。方法　以人白血病细胞株K5 6 2、人乳腺癌细胞株Bcap 37、人胃腺癌细胞株BGC 82 3和人非小细胞肺癌细胞株A5 4 9为模型 ,采用MTT法测试Albaconol对肿瘤细胞增殖的影响 ;以小鼠移植性肉瘤S180 和小鼠移植性肝癌H2 2 为模型 ,检测Al baconol静脉给药对肿瘤生长的影响 ;以 pBR32 2DNA为底物 ,采用琼脂糖凝胶电泳法测定Albaconol对肿瘤细胞DNATOPOⅡ活性的影响。结果　Albaconol对K5 6 2、Bcap 37、BGC 82 3、A5 4 9的半数抑制浓度 (IC50 )分别为 (2 4 2±1 77)、(1 88± 1 4 1)、(1 0 4± 0 6 4 )、(1 18± 1 10 )mg·L-1;Albaconol0 87、1 73、3 4 6mg·kg-1剂量组对S180 生长的抑制百分率 (抑瘤率 )分别为 2 8 2 %、4 3 2 %、4 7 4 % ;对H2 2 的抑瘤率分别为 15 6 %、2 2 4 %、37 8% ;Albaconol能明显影响DNATOPOⅡ的活性 ,表现为促进其介导的DNA解旋或断裂 ,并抑制其介导的DNA再连接反应。结论　Alba conol具有较强的体内、外抗肿瘤活性 ,DNATOPOⅡ是其抗肿瘤作用的细胞内靶点之一。     

鸦胆子油乳对胶质瘤U251作用的研究

目的该实验拟研究鸦胆子油乳注射液对人脑胶质瘤U251细胞生长抑制及诱导细胞凋亡的作用。方法体外培养人脑胶质瘤细胞U251,利用MTT比色法检测鸦胆子油乳注射液对于U251的增殖抑制影响;用流式细胞术(flowcytometry,FCM)分析药物处理前后U251的细胞周期变化;实时荧光定量PCR检测不同处理组中凋亡基因Bcl-2的表达。结果 MTT显示鸦胆子油乳注射液对U251细胞增殖抑制作用呈剂量依赖性,当浓度为10 g.L-1时,对U251细胞增殖的抑制率达到(75.8±2.3)%(P<0.05);流式细胞仪分析显示,用10 g.L-1鸦胆子油乳注射液培养48 h,细胞周期中G1期所占比例增高,S期占细胞周期的比例降低;实时荧光定量PCR结果显示随着鸦胆子油乳注射液的浓度增加凋亡基因Bcl-2 mRNA的表达逐渐减少。与对照组比较P<0.01。结论中药鸦胆子油乳注射液可显著抑制U251胶质瘤细胞细胞增殖及促进其凋亡。     

本芴醇衍生物LY980503逆转人乳腺癌多药耐药细胞株MCF/DOX对多柔比星的耐药性

目的　探讨本芴醇衍生物LY980 5 0 3对肿瘤多药耐药的逆转作用及其作用机理。方法　采用噻唑蓝 (MTT)法检测细胞毒作用 ;采用流式细胞术测定细胞内多柔比星 (Dox)浓度 ;应用人乳腺癌裸鼠移植瘤模型研究LY980 5 0 3对肿瘤多药耐药的体内逆转作用。结果　LY980 5 0 3在 4 .0 μmol·L- 1(非细胞毒剂量 )能大部逆转人乳腺癌耐Dox细胞株MCF/Dox对Dox的耐药性 ;药物蓄积实验表明 ,LY980 5 0 3能显著增加MCF/Dox细胞内Dox蓄积 ;10 μmol·L- 1LY980 5 0 3作用 96h能明显抑制MCF/Dox细胞mdr 1基因表达水平。将MCF/Dox细胞接种于裸鼠皮下 ,接种后d 4 2 ,合用LY980 5 0 3(2 0 0mg·kg- 1·d- 1×3,ig)的移植瘤体积 (0 .34± 0 .19)cm3较单用Dox的移植瘤体积 (0 .90± 0 .32 )cm3显著缩小。结论LY980 5 0 3在体外及体内均能有效逆转MCF/Dox细胞对Dox的耐药性。     

苦参碱对U251胶质瘤细胞的增殖抑制和原癌基因表达的影响

目的　探讨苦参碱对胶质瘤细胞株 (U2 5 1细胞株 )的抑制作用及其作用机制。方法　用MTT比色法检测不同浓度苦参碱对U2 5 1细胞的增殖抑制 ,流式细胞仪观察苦参碱对U2 5 1细胞周期的影响 ,RT PCR观察原癌基因C myc表达的变化。结果　苦参碱对U2 5 1细胞增殖抑制作用成剂量依赖性 ,当浓度为 0 10g·L-1时 ,对U2 5 1细胞增殖的抑制率达到 5 3 7%± 6 0 %。流式细胞仪分析显示 ,用 0 10g·L-1苦参碱培养 3d ,细胞周期中G0 /G1期所占比例增高 ,S期占细胞周期的比例减低。RT PCR结果显示 ,随着苦参碱作用剂量的增加 ,C myc基因的表达被明显抑制。结论　苦参碱对人胶质瘤细胞系U2 5 1的增殖具有明显的抑制作用 ,并对原癌基因C myc的表达具有抑制作用     

以DNA拓扑异构酶Ⅱ为靶点的抗癌药物

DNA拓扑异构酶Ⅱ（Topoisomerase Ⅱ,TOPO Ⅱ)是一种真核生物生存所必需的泛酶，在几乎所有DNA代谢过程中发挥重要作用。TOPOⅡ使一条完整的DNA双链穿过一个移过性的双链断口，从而导致DNA解结或解旋。因为TOPOⅡ具有重要的生理功能，它已成为抗癌药物的重要作用靶点。以TOPOⅡ为靶点的药物按作用方式可分为2类：一类通过稳定TOPOⅡ介导的可切割复合物而杀死肿瘤细胞，称为TOPOⅡ毒剂（TOPOⅡ poison)；另一类通过抑制TOPOⅡ的催化活性而达到抑制肿瘤的作用，称为TOPOⅡ催化抑制剂（TOPOⅡ inhibi-tor)。近年来，对TOPOⅡ催化机制和药物作用方式的研究取得了很大进展，这些发现有助于进一步了解TOPOⅡ的生理功能，进而研究出更有效的治疗方案和新的抗癌药。本文介绍了以TOPOⅡ为靶点的抗癌药物的作用机制及其发展现状。     

鸦胆子油乳对小鼠黑色素瘤高转移细胞黏附、侵袭及转移能力的影响

目的研究鸦胆子油乳对B16-BL6小鼠高转移细胞黏附、侵袭及转移能力的影响.方法采用细胞黏附实验、重组基底膜侵袭实验以及小鼠黑色素瘤自发性转移模型,观察鸦胆子油乳对B16-BL6细胞黏附、侵袭及转移能力的影响.结果在3和6μL·mL-1浓度时,鸦胆子油乳与B16-BL6细胞作用24h,可抑制其与纤粘连蛋白的黏附,抑制率分别为24.5%和63.2%;也抑制其与层粘连蛋白的黏附,抑制率分别为17.0%和34.5%;对B16-BL6细胞侵袭重组基底膜的抑制率为26.7%和41.3%.在小鼠黑色素瘤自发性转移模型中,鸦胆子油乳2.5,5.0,10.0mL·kg-1连续注射给药1个月,小鼠肺转移灶与对照组相比明显减少.结论鸦胆子油乳具有抑制B16-BL6细胞黏附、侵袭及转移的作用.     

黄芩苷对肺腺癌细胞多药耐药性的影响及机制

目的探讨黄芩苷对人肺腺癌细胞株A549多药耐药性(MDR)的影响及作用机制。方法体外培养人肺腺癌细胞株A549后给予0、0.5、1、2、4、8mg/L黄芩苷对细胞进行处理,MTT法检测不同浓度黄芩苷对A549细胞生长的影响情况;逆转录-聚合酶链反应法(RT-PCR)检测各组细胞多药耐药基因1(MDR-1)、多药耐药相关蛋白-1(MRP1)、谷胱苷肽转移酶-π(GST-π)、拓扑异构酶(TOPO)ⅡαmRNA的表达情况。结果与对照组相比,黄芩苷组细胞生长明显受到抑制(P<0.05或<0.01);黄芩苷处理后肿瘤细胞MDR-1、MRP1、GST-π、TOPOⅡαmRNA表达水平降低,与对照组比较差异均有统计学意义(P<0.01)。结论 baicalin可以调节多种MDR基因的表达,具有明显的逆转肺腺癌细胞MDR的作用。     

Modulation of topoisomerase II catalytic activity by DNA minor groove binding agents distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole

The effects of distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole (DAPI) on the catalytic activity of topoisomerase II from L1210 cells were determined. These compounds were used as model agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range alterations to the DNA structure. Two types of reactions catalyzed by topoisomerase II were examined, relaxation of supercoiled DNA and decatenation of highly catenated DNA. Distamycin at low concentrations (0.2-2 microM) substantially stimulated relaxation of supercoiled pBR322 DNA. Higher drug levels (25-50 microM) resulted in a potent inhibition of relaxation. At the stimulatory concentrations of distamycin, only completely relaxed reaction products were observed, as in the absence of the drug. The onset of inhibition (caused by 5-10 microM distamycin) was accompanied by the appearance of partially relaxed intermediates. Similar inhibition of relaxation was observed for Hoechst 33258 and DAPI but, unlike distamycin, these agents produced only marginal stimulation of relaxation when added in low noninhibitory concentrations. Another reaction of topoisomerase II, decatenation of catenated kinetoplast DNA, was also inhibited by distamycin, Hoechst 33258, and DAPI at concentrations similar to those inhibiting the relaxation reaction. This study demonstrates that agents binding to the minor groove of DNA represent a new class of drugs interfering with topoisomerase II and provides possibilities for modulation of this important enzyme.     

Biochemical and proteomics approaches to characterize topoisomerase IIalpha cysteines and DNA as targets responsible for cisplatin-induced inhibition of topoisomerase IIalpha

Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase IIalpha. This inhibition was not accompanied by stabilization of a covalent topoisomerase IIalpha-DNA intermediate. Pretreatment of kinetoplast plasmid DNA (kDNA) or pBR322 DNA with submicromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase IIalpha catalytic assay. Cisplatin nearly equally inhibited growth of a parental K562 and an etoposide-resistant K/VP.5 cell line that contained decreased topoisomerase IIalpha levels, a result consistent with isolated enzyme experiments demonstrating that cisplatin was not a topoisomerase IIalpha poison. Because cisplatin is known to react with protein sulfhydryl groups, the 13 cysteine groups in the topoisomerase IIalpha monomer were evaluated by mass spectrometry to determine which cysteines were free and disulfide-bonded to identify possible sites of cisplatin adduction. High-pressure liquid chromatography-matrix-assisted laser desorption ionization mass spectrometry showed that topoisomerase IIalpha contained at least five free cysteines (170, 216, 300, 392, and 405) and two disulfide-bonded cysteine pairs (427-455 and 997-1008). Cysteine 733 was also disulfide-bonded, but its partner cysteine could not be identified. Cisplatin antagonized the formation of a fluorescence adduct between topoisomerase IIalpha and the sulfhydryl-reactive maleimide reagent 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic acid methyl ester (ThioGlo-1). Dithiothreitol, which was shown by spectrophotometry to react rapidly with cisplatin (6-min half-time), diminished the capacity of cisplatin to interfere with ThioGlo-1 binding to topoisomerase IIalpha. The results of this study suggest that cisplatin may exert some of its cell growth inhibitory and antitumor activity by inhibition of topoisomerase IIalpha through reaction with critical enzyme sulfhydryl groups and/or by forming DNA adducts that render the DNA substrate refractory to topoisomerase IIalpha.     

海南粗榧新碱衍生物HH07A对肿瘤细胞内DNA拓扑异构酶Ⅱ和蛋白激酶C活性的影响

HH07A 5.5 μmol·L^-1作用L1210细胞24 h后, 可使细胞的DNA拓扑异构酶Ⅱ活性下降; 在无细胞系统, HH07A 0.55 mmol·L^-1也能直接促进DNA拓扑异构酶Ⅱ引起的DNA链断裂. 经HH07A (L1210细胞5.5 μmol·L^-1, HL-60细胞8.25 μmol·L^-1)作用24 h后, L1210及HL-60细胞的胞浆蛋白激酶C(PKC)活性升高, 胞膜PKC活性下降, 而全细胞PKC活性变化不大. 在无细胞系统中, HH07A 1.1 mmol·L^-1能明显抑制PKC的活性.     

Reduced DNA topoisomerase II activity and drug-stimulated DNA cleavage in 9-hydroxyellipticine resistant cells

We have isolated a Chinese hamster lung cell line resistant to 9-hydroxyellipticine (DC-3F/9-OH-E) which is also cross-resistant to topoisomerase II inhibitors such as amsacrine and etoposide. In this work we have studied quantitatively both DNA topoisomerase II activity by decatenation of kinetoplast DNA and drug-stimulated DNA cleavage of pBR 322. DNA topoisomerase II activity of DC-3F/9-OH-E nuclear extract was reduced by 3.5-fold as compared to that from DC-3F (sensitive parent) nuclear extract. We also found that DC-3F/9-OH-E nuclear extracts have a reduced capacity to induce in vitro topoisomerase II-mediated DNA cleavage upon stimulation by etoposide and amsacrine (7- and 10-fold respectively). Besides, mixing nuclear extracts from both sensitive and resistant cells indicates that either the enzyme in resistant cells is modified or a modulating factor is associated to it. Our results suggest that the resistance of the DC-3F/9-OH-E cell line to topoisomerase II inhibitors might be due to both a reduced amount of the enzyme and its reduced ability to form the cleavable complex in the presence of drugs.     

DNA拓扑异构酶Ⅱ是salvicine作用于酿酒酵母的主要靶点

目的：确定DNA拓扑异构酶Ⅱ（TopoⅡ）是否为salvicine在酿酒酵母细胞内的主要作用靶点及其作用方式。方法：用TopoⅡ介导的超螺旋pBR322解旋反应检测salvicine在无细胞体系中对TopoⅡ催化活力的影响；用克隆形成实验检测salvicine对四种酵母细胞系生长的作用。结果：salvicine能明显抑制无细胞体系中TopoⅡ对超螺旋pBR322的解旋作用。salvicine对JN394母系细胞及TOP1缺失的JN394topl^-细胞毒作用相似，验证了TopoⅠ不是其作用靶点，在25℃时，salvicine对温度敏感型JN394t2-1细胞具有良好的细胞毒作用；但在30℃时，TopoⅡ的活力大为降低，在有意义的浓度范围内salvicine对此类细胞的生长无明显抑制作用，另外，TopoⅡ发生突变的JN394t2-5细胞显示出对salvicine和etoposide(VP16)高度的耐受性，结论：TopoⅡ是salvicine细胞内主要作用靶点；salvicine通过捕获TopoⅡ-DNA断裂复合物而杀死细胞。此外，salvicine与VP16在TopoⅡ上有相似的作用位点。     

Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes

Salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, is a novel anticancer drug candidate. The compound has significant in vitro and in vivo activity against malignant tumor cells and xenografts, especially some human solid tumor models. This anticancer activity of salvicine is associated with its ability to induce tumor cell apoptosis. Salvicine was also found to have a profound cytotoxic effect on multidrug-resistant (MDR) cell lines by down-regulating the expression of MDR-1 mRNA of MDR cells. Salvicine acted as a topoisomerase II (Topo II) poison through its marked enhancement effect on Topo II-mediated DNA double-strand breaks as observed in the DNA cleavage assay. Strong inhibitory activity of salvicine against Topo II was observed in a kDNA decatenation assay, with an approximate IC(50) value of 3 microM. A similar result was obtained by a Topo II-mediated supercoiled DNA relaxation assay. In contrast, no inhibitory activity was observed against the catalytic activity of Topo I. When the effects of salvicine on individual steps of the catalytic cycle of Topo II were dissected, it was found that the mechanism by which salvicine inactivates Topo II is different from that by other anti-Topo II agents. Salvicine greatly promoted Topo II-DNA binding and inhibited pre- and post-strand Topo II-mediated DNA religation without interference with the forward cleavage steps. In addition, salvicine was not a DNA intercalative agent, as demonstrated by DNA unwinding assays. The results of this study indicate that the inhibitory activity of salvicine against Topo II was derived from its ability to stabilize DNA strand breaks through interactions with the enzyme alone or with the DNA-enzyme complex. It is therefore postulated that salvicine acts on Topo by trapping the DNA-Topo II complex, which in turn produces anticancer effects.