从黄连根茎中分离出的拓扑异构酶Ⅰ和Ⅱ的抑制剂

DNA 拓扑异构酶可改变 DNA 的拓扑构型,对细胞中的 DNA 复制、转录和重组等起重要作用。有2种类型的 DNA 拓扑异构酶:Ⅰ型和Ⅱ型。除了在 DNA 代谢中的重要作用,拓扑异构酶Ⅰ和Ⅱ在癌症的化疗中引起了广泛的兴趣。在80年代表明拓扑异构酶是临床上应用的抗癌剂的主要目标。如拓扑     

骆驼蓬种子提取物及其β咔保啉生物碱对DNA拓扑异构酶Ⅱ活性的抑制作用

目的探讨去氢骆驼蓬碱、骆驼蓬碱、骆驼蓬总碱及哈尔满碱(止咳药用植物)对DNA拓扑异构酶Ⅱ活性的抑制作用。方法从体外培养的Q3肝癌细胞中,提取分离DNA拓扑异构酶Ⅱ;以阿霉素为阳性对照,用琼脂糖凝胶电泳法检测药物对DNA拓扑异构酶Ⅱ的作用。结果骆驼蓬总碱、去氢骆驼蓬碱、骆驼蓬碱及哈尔满碱对DNA拓扑异构酶Ⅱ活性均有一定的抑制作用。结论对DNA拓扑异构酶Ⅱ活性的抑制作用是骆驼蓬总碱、去氢骆驼蓬碱、骆驼蓬碱等生物碱抗癌作用和细胞毒作用的机制之一。     

恶性胶质瘤DNA拓扑异构酶的表达

目的从细胞分子生物学角度探讨DNA拓扑异构酶(TopoII)在恶性胶质瘤多药耐药中的作用。方法应用流式细胞仪对20例原发与20例复发恶性胶质瘤术后标本中DNA拓扑异构酶进行测定,对照研究。结果20例复发恶性胶质瘤中DNA拓扑异构酶的表达量较20例原发者明显减少(P<0.05)。结论恶性胶质瘤在治疗过程中获得多药耐药性可能是由于DNA拓扑异构酶数量的减少或活性降低造成的。     

拓扑异构酶抑制剂——一种新型的抗肿瘤药物

拓扑异构酶控制、维持和修饰DNA拓扑结构。拓扑异构酶抑制剂通过作用于拓扑异构酶 ,对很多肿瘤发挥抑制活性。论述了拓扑异构酶抑制剂的作用机理、杀伤肿瘤细胞的机制及耐药性问题。     

伊立替康治疗23例晚期结、直肠癌患者的护理体会

伊立替康（喜树碱-11，irinotecan，CPT-11，商品名艾力）为喜树碱水溶液衍生物，是新型的DNA拓扑异构酶Ⅰ（TOPOⅠ）选择性抑制剂，拓扑异构酶Ⅰ是DNA复制延续的关键酶，伊立替康及其活性代谢物SN-38与拓扑异构酶Ⅰ和DNA形成的复合物牢固结合，阻止拓扑异构酶Ⅰ修复DNA缺口，造成DNA不可逆断裂，引起细胞死亡。伊立替康的主要毒性反应包括迟发性腹泻、中性粒细胞减少。其它常见不良反应还包括乙碱胆碱能综合征、胃肠道反应、脱发等。     

结核分枝杆菌中能模拟DNA的氟喹诺酮耐药蛋白

氟喹诺酮类(fluoroquinolones)药物在肺结核治疗中发挥着强大的作用。它作用于菌体DNA促旋酶和DNA拓扑异构酶Ⅳ,通过形成DNA-拓扑异构酶-氟喹诺酮复合物来阻止DNA拓扑异构改变,抑制DNA复制转录的进行,而达到杀菌目的。但是,随着氟喹诺酮在治疗多药耐药结核分枝杆菌中应用日益广     

拓扑异构酶I天然产物抑制剂的研究进展

DNA 拓扑异构酶 I（topoisomerase I）参与 DNA 的复制、转录、重组和修复等所有关键的核过程。近几年,其抗肿瘤小分子抑制剂的研究逐渐成为热点。该文对 21 世纪以来报道的新拓扑异构酶 I 天然产物抑制剂的结构、生物活性及结构改造等进行系统的综述,以期为设计全新结构的拓扑异构酶 I 抑制剂提供指导。 关键词:中图分类号：R914     

原花青素对MNNG致DNA损伤及拓扑异构酶Ⅱ的影响

目的　观察原花青素对DNA损伤的保护作用及对黑色素瘤细胞中拓扑异构酶的影响 ,探讨原花青素癌化学预防的作用机制。方法　单细胞电泳法检测羟自由基对L92 9细胞DNA损伤的尾迹 ,用 [H3]TdR参入细胞DNA中进行标记 ,测定上清液中的放射强度来判定MNNG对DNA损伤 ;从黑色素瘤肿瘤细胞中提取拓扑异构酶 ,用琼脂糖凝胶电泳测定活性。结果　原花青素可以减轻DNA损伤尾迹及MNNG对DNA的损伤。但对黑色素瘤肿瘤细胞中拓扑异构酶无抑制作用。结论　原花青素对羟自由基及致癌剂MNNG引起的细胞DNA损伤有一定的保护作用。但对拓扑异构酶的活性无抑制作用     

鬼臼毒素衍生物GL331的合成及与拓扑异构酶IIα的分子模拟(英文)

本文以鬼臼毒素为起始原料, 三甲基碘硅烷为碘代试剂, 采用一锅法成功合成了鬼臼毒素衍生物GL331。通过分子对接的方法探讨了GL331与DNA拓扑异构酶 IIα的结合位点及作用模式。结果表明,GL331与DNA拓扑异构酶 IIα的ATP结合位点紧密结合, GL331很有可能作为拓扑异构酶 IIα的ATP竞争性抑制剂而发挥抗肿瘤作用。     

思文霉素对胞外DNA拓扑异构酶介导产生的断裂和松驰反应的影响

思文霉素（Siwenmycin）是最先从我国土壤中的链霉菌培养物中提取的一个新型蒽环类抗生素。研究表明该药是DNA拓扑异构酶抑制剂，以ATP依赖性pBR322 DNA断裂松驰反应，观察思文霉素对从哺乳动物细胞中提取的DNA拓扑异构酶Ⅱ活性的抑制作用后发现该药对此酶的最大抑制浓度为25mmol/L。用思文霉素处理Bel 7402细胞后，从中提取的DNA拓扑异构酶Ⅱ所介导的DNA断裂松驰反应活性比对照组增加5倍，研究还发现，思文霉素可抑制胞外DNA拓扑异构酶Ⅰ活性，碱性洗脱实验证明该药可引起DNA单链断裂。     

Catalytic inhibition of DNA topoisomerase II by N-benzyladriamycin (AD 288)

N-Benzyladriamycin (AD 288) is a highly lipophilic, semi-synthetic congener of doxorubicin (DOX). Unlike DOX, which stimulates double-stranded DNA scission by stabilizing topoisomerase II/DNA cleavable complexes, AD 288 is a catalytic inhibitor of topoisomerase II, capable of preventing topoisomerase II activity on DNA. The concentration of AD 288 required to inhibit the topoisomerase II-catalyzed decatenation of linked networks of kinetoplast DNA was comparable to that for DOX. However, AD 288 did not stabilize cleavable complex formation or stimulate topoisomerase II-mediated DNA cleavage. In addition, AD 288 inhibited the formation of cleavable complexes by etoposide in a concentration-dependent manner. Human CCRF-CEM cells and murine J774. 2 cells exhibiting resistance against DOX, teniposide, or 3'-hydroxy-3'-deaminodoxorubicin through reduced topoisomerase II activity remained sensitive to AD 288. These studies suggest that AD 288 inhibits topoisomerase II activity by preventing the initial non-covalent binding of topoisomerase II to DNA. Since AD 288 is a potent DNA intercalator, catalytic inhibition is achieved by prohibiting access of the enzyme to DNA binding sites. These results also demonstrate that specific substitutions on the aminosugar of DOX can alter the mechanism of topoisomerase II inhibition.     

Topoisomerase I poisons and suppressors as anticancer drugs

Inhibitors of topoisomerase I constitute a novel family of antitumor agents. The camptothecin derivatives topotecan and irinotecan represent new weapons in our arsenal for battling human cancer. These two drugs act specifically at the level of the topoisomerase I-DNA complex and stimulate DNA cleavage. This mechanism of action is not restricted to the camptothecins. Numerous topoisomerase I poisons including DNA minor groove binders such as Hoechst 33258 and DNA intercalators such as benzophenanthridine alkaloids and indolocarbazole derivatives have been discovered and developed. Another important group of topoisomerase I inhibitors contains drugs which prevent or reverse topoisomerase I-DNA complex formation. Many of these topoisomerase I suppressors are natural products (beta-lapachone, diospyrin, topostatin, topostin, flavonoids) which are believed to interact directly with the enzyme. This review is concerned with the different families of topoisomerase I poisons and suppressors. Their origin, chemical nature and mechanism of action are presented. The relationships between drug binding to DNA and topoisomerase I inhibition are discussed.     

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II

Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258–DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.     

DNA topoisomerases as anticancer drug targets: from the laboratory to the clinic

DNA topoisomerases play important roles in basic cellular biology. Recently they have been identified as the molecular targets of a variety of pharmaceutical agents. Some of the drugs that target the topoisomerases are anticancer drugs. These anticancer drugs work by a novel mechanism of action. They inhibit the topoisomerase molecule from religating DNA strands after cleavage. This leaves a cell with DNA breaks, which if not repaired, become lethal. In other words, these drugs convert the topoisomerase molecule into a DNA damaging agent. This is a stoichiometric relationship. Each anticancer drug molecule has the potential of interacting with one topoisomerase molecule to cause one DNA lesion. The clinical implication of this mechanism of drug action is that sensitivity to topoisomerase targeting drugs should be dependent on high topoisomerase levels. This is clearly true in laboratory systems. With new developments in in situ immunohistochemistry, topoisomerase expression can now be easily estimated in human cancers. From this information, it may be possible to predict the sensitivity or resistance of human cancers to topoisomerase targeting anticancer drugs.     

Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are both sensitive to camptothecin, an inhibitor of DNA topoisomerase I. An S. cerevisiae DNA repair mutant, rad52, is hypersensitive to the drug. In both species, topoisomerase I mutants totally lacking the enzyme are completely resistant to the drug. A strain with a mutation leading to a temperature-sensitive topoisomerase I exhibits temperature dependence in its in vivo response to camptothecin. A strain carrying a plasmid that overproduces topoisomerase I is hypersensitive to the drug. The rad52 mutant is killed by overproduction of the enzyme, even in the absence of the drug. The response of several of these strains to camptothecin analogs, to DNA topoisomerase II inhibitors, and to other drugs is reported. The cytotoxic effects of camptothecin are discussed in terms of the drug extending the lifetime of a topoisomerase I-DNA covalent intermediate, which is recognized as DNA damage by a DNA repair system.     

Etoposide, topoisomerase II and cancer

Etoposide is an important chemotherapeutic agent that is used to treat a wide spectrum of human cancers. It has been in clinical use for more than two decades and remains one of the most highly prescribed anticancer drugs in the world. The primary cytotoxic target for etoposide is topoisomerase II. This ubiquitous enzyme regulates DNA under- and overwinding, and removes knots and tangles from the genome by generating transient double-stranded breaks in the double helix. Etoposide kills cells by stabilizing a covalent enzyme-cleaved DNA complex (known as the cleavage complex) that is a transient intermediate in the catalytic cycle of topoisomerase II. The accumulation of cleavage complexes in treated cells leads to the generation of permanent DNA strand breaks, which trigger recombination/repair pathways, mutagenesis, and chromosomal translocations. If these breaks overwhelm the cell, they can initiate death pathways. Thus, etoposide converts topoisomerase II from an essential enzyme to a potent cellular toxin that fragments the genome. Although the topoisomerase II-DNA cleavage complex is an important target for cancer chemotherapy, there also is evidence that topoisomerase II-mediated DNA strand breaks induced by etoposide and other agents can trigger chromosomal translocations that lead to specific types of leukemia. Given the central role of topoisomerase II in both the cure and initiation of human cancers, it is imperative to further understand the mechanism by which the enzyme cleaves and rejoins the double helix and the process by which etoposide and other anticancer drugs alter topoisomerase II function.     

Characterization of the topoisomerase II-induced cleavage sites in the c-myc proto-oncogene. In vitro stimulation by the antitumoral intercalating drug mAMSA

In an attempt to get an insight into the activity of mAMSA (a DNA topoisomerase II-mediated drug) on the human proto-oncogene c-myc, an in vitro system consisting of purified calf thymus DNA topoisomerase II and a c-myc DNA inserted in lambda phage was utilized. The occurrence of discrete bands, detected by hybridization of Southern blots with appropriate c-myc probes, indicated the presence of cleavage sites in the sole presence of DNA topoisomerase II. The band intensity increased in the presence of mAMSA, while no significant difference occurred in the cleavage pattern. The location of the cleavage sites along the c-myc locus revealed a striking correspondence with that of some DNase hypersensitive sites. These results indicate that DNA topoisomerase II is most certainly implicated in the mAMSA activity and that the drug stimulates the topoisomerase II cleaving activity at specific sites, which may be involved in the biological activity of the drug.     

The cardioprotective and DNA topoisomerase II inhibitory agent dexrazoxane (ICRF-187) antagonizes camptothecin-mediated growth inhibition of Chinese hamster ovary cells by inhibition of DNA synthesis

Dexrazoxane (ICRF-187), which is clinically used to reduce doxorubicin-induced cardiotoxicity, has cell growth inhibitory properties through its ability to inhibit the catalytic activity of DNA topoisomerase II. A study was undertaken to investigate whether preincubating Chinese hamster ovary cells (CHO) with dexrazoxane prior to camptothecin treatment resulted in potentiation. Camptothecin is a DNA topoisomerase I poison. It was found that pretreating CHO cells with concentrations of dexrazoxane sufficient to strongly inhibit topoisomerase II for periods from 18 to 96 h resulted in significant antagonism of camptothecin-mediated growth inhibition. Lower concentrations that were sufficient to cause partial inhibition of topoisomerase II and partial dexrazoxane-mediated cell growth inhibition had little effect on camptothecin-mediated growth inhibition. Neither topoisomerase I protein levels nor camptothecin-induced topoisomerase I-DNA covalent complexes were affected by dexrazoxane concentrations that were sufficient to cause antagonism of camptothecin-induced growth inhibition. However, under these experimental conditions, dexrazoxane caused a decrease in DNA synthesis. Therefore, results presented here confirm the importance of the DNA synthesis-dependent replication fork interaction with topoisomerase I-DNA covalent complexes for the expression of camptothecin activity. It is concluded that dexrazoxane and camptothecin analogs should be used with caution in combination chemotherapy.     

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.     

Stimulation of topoisomerase II-mediated DNA cleavage by an indazole analogue of lucanthone.

Lucanthone is an antitumour drug used as an adjuvant in radiation therapy. The drug intercalates into DNA and inhibits topoisomerase II. An indazole analogue of lucanthone (IA-5) was examined for its ability to modulate topoisomerase II-DNA cleavable complex formation in vitro. The drug contains a methylbenzothiopyranoindazole chromophore instead of the methyl-thioxanthenone nucleus of lucanthone. Using a radiolabelled linear plasmid DNA as a substrate, both lucanthone and the indazole analogue were shown to promote the cleavage of DNA by human topoisomerase II. Sequencing experiments with different restriction fragments indicated that the indazole drug promoted DNA cleavage primarily at sites having a C on the 3' side of the cleaved bond (-1 position). By contrast, in the same sequencing methodology lucanthone exerted a much weaker effect on topoisomerase II. The sequence selectivity of IA-5 is reminiscent of that of the anticancer drug mitoxantrone and its anthrapyrazole analogue losoxantrone, which is structurally close to IA-5. Binding to DNA and topoisomerase II inhibition are two distinct processes contributing separately to the cytotoxic activity of the indazole drug.