Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomerase Ⅰ and topoisomerase Ⅱ

OBJECTIVE The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo,as well as the potential mechanisms.METHODS First,the model of C6 glioma in Wistar rats was established and verified by hemotoxylin and eosin staining,immunohistochemical staining and magnetic resonance imaging(MRI).Then different doses of lapachol were gavaged and tumor volumes of the C6 glioma were detected by MRI.The effects of lapachol on C6 cell proliferation,apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)/phen-azinemethosulfate(PMS)assay,Hoechst33358 staining,AnnexinⅤ-FITC/PI staining,and comet assay.Effects of lapachol on topoisomeraseⅠ(TOPⅠ)and topoisomeraseⅡ(TOPⅡ)activities were detected by TOPⅠand TOPⅡmediated supercoiled p BR322 DNA relaxation assay.Molecular docking was used to predict the interaction of lapachol-TOPⅠand lapachol-TOPⅡ.TOP I and TOPⅡexpression levels in C6 cells were determined by Enzymelinked immunosorbent assay kits and real-time polymerase chain reaction(RT-PCR).RESULTS The rat C6 glioma model was successfully established.High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats(P<0.05).MTS/PMS assay,Hoechst 33258 staining,AnnexinⅤ-FITC/PI staining,and comet assay showed that lapachol could inhibit proliferation,induce apoptosis and DNA damage of C6 cells in dose dependent manners.Lapachol could inhibit the activities of both TOPⅠandⅡ.Molecular docking showed that lapachol-TOPⅠshowed relatively stronger interaction than that of lapachol-TOPⅡ.Enzyme-linked immunosorbent assay and RT-PCR showed that lapachol could inhibit TOPⅡexpression levels,but not TOPⅠexpression levels.CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro,which might be related with inhibiting TOPⅠand TOPⅡactivities,as wel as TOPⅡexpression.

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomeraseⅠ and topoisomeraseⅡ

OBJECTIVE Lapachol is a natural naphthoquinone compound that possesses extensive biological activities.The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo,as well as the potential mechanisms.METHODS The antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats.The effects of lapachol on C6 cell proliferation,apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)/phenazinemethosulfate(PMS)assay,hoechst 33358 staining,annexinⅤ-FITC/PI staining,and comet assay.Effects of lapachol on topoisomerase I(TOP I)and topoisomeraseⅡ(TOPⅡ)activities were detected by TOPⅠand TOPⅡmediated supercoiled p BR322DNA relaxation assays and molecular docking.TOPⅠand TOPⅡexpression levels in C6 cells were also determined.RESULTS High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats(P<0.05).It was showed that lapachol could inhibit proliferation,induce apoptosis and DNA damage of C6 cel s in dose dependent manners.Lapachol could inhibit the activities of both TOPⅠ and Ⅱ.Lapachol-TOPⅠshowed relatively stronger interaction than that of lapachol-TOPⅡin molecular docking study.Also,lapachol could inhibit TOPⅡexpression levels,but not TOPⅠexpression levels.CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro,which might be related with inhibiting TOPⅠ and TOPⅡ activities,as wel as TOPⅡ expression.

中国蓑鲉亚科鱼类DNA条形码及分子系统进化研究

为从分子水平分析我国蓑鲉亚科鱼类分类关系,通过PCR扩增及测序获得了我国8种蓑鲉亚科鱼类COⅠ基因部分序列,结合GenBank下载的6种蓑鲉,共计14种40个个体线粒体COⅠ基因序列,利用MEGA X计算了序列信息和遗传距离,利用基于最大似然法和邻接法构建分子系统进化树。结果显示:除盔蓑鲉(Ebosia bleekeri)和安狭蓑鲉(Pteroidichthys amboinensis)只有一个样本无法测定外,其余12种蓑鲉亚科鱼类的种间平均遗传距离是种内平均遗传距离的69.17倍,显示COⅠ基因可作为蓑鲉亚科鱼类的有效DNA条形码基因。进化树上,蓑鲉亚科部分属并未能聚成属间单系,其中蓑鲉属(Pterois)与短鳍蓑鲉属(Dendrochirus)在进化树上形成多个分支。在蓑鲉属中,翱翔蓑鲉(P.volitans)与斑鳍蓑鲉(P.miles)聚为一支,两者关系较近,但COⅠ遗传距离为0.042,大于Hebert推荐的0.020作为最小区分物种的遗传距离,支持两者为两个独立物种的观点;触须蓑鲉(P.antennata)、辐蓑鲉(P.radiate)和黑颊蓑鲉(P.mombasae)聚为一支,独立于翱翔蓑鲉与斑鳍蓑鲉的分支之外,并与短鳍蓑鲉属(Dendrochirus)的花斑短鳍蓑鲉(D.zebra)以及盔蓑鲉属(Ebosia)的种类聚在一起,与近期有关蓑鲉亚科各种属分子聚类研究结果相似;但这3种蓑鲉是否可以从蓑鲉属中独立出来归为Pteropterus属,还需要后续更多的蓑鲉亚科鱼类样品研究验证。

THE ANTICANCER EFFECT AND ANTI－DNA TOPOISOMERASE II EFFECT OF EXTRACTS OF CAMELLIA PTILOPHYLLA CHANG AND CAMELLIA SINENSIS

The cytotoxic effect of extract of camellia ptilophyllachang(ECPC) and extract of camellia sinensis(ECS) onHeLa cell line, poorly differentiated nasopharyngealcarcinoma cell line(CNE2) and gastric cancer cell line(MGC-803 ) in vitro was studied using MIT assay method.The results showed that ECPC and ECS possessed significantcytotoxic effect on above three cell lines. The anticancer testin mice showed that ECPC had marked inhibitory effectagainst Ehrlich solid carcinoma(ESC) with inhibition ratesof 17. 8 48. 3% and with inhibition rates of 28. 3-54. 5% against reticular cell sarcoma(L2), and that ECShad inhibition rates of 31 . 5 -49. 4 % against ESC and 35. 8- 50% against L2. These two extracts had only marginalinhibitory effect against sarcoma- 180. The unknottingactivity of DNA topoisomerase II was inhibited completelyby ECPC and ECS at the concentration of 50 μg/ mlsuggesting that DNA topoisomerase II might be a targetenzyme of these two extracts.

基于COⅠ基因的5种新疆土著鱼类DNA条形码鉴定及聚类分析

为探讨DNA条形码技术在新疆土著鱼类物种鉴定中的有效性并保护本土渔业重要种质资源,本研究采用DNA条形码技术对裂腹鱼亚科(Schizothoracinae)和高原鳅属(Triplophysa)鱼类进行COⅠ基因序列扩增,并与NCBI数据库进行序列比对,结合遗传距离和系统发育树进行物种鉴定分析。结果表明,33尾个体分属4属5种,其中有13个单倍体;COⅠ基因序列中(G+C) mol%值为46.7%,远小于(A+T) mol%值53.3%,表现出明显的反G偏倚,第三位密码子处的(G+C) mol%值为55.9%,远高于第一和第二密码子处的(G+C) mol%值,R值(转换/颠换)为2.10;种间平均遗传距离(11.55±0.95)%,为种内平均、遗传距离(0.14±0.10)%的43.8倍,远远大于Hebert所提出的“10倍法则”;系统发育树表明,裂腹鱼亚科和高原鳅属鱼类分为两支,其中,裂腹鱼亚科的塔里木裂腹鱼(Schizothorax biddulphi)、宽口裂腹鱼(Schizothorax eurystomus)、扁吻鱼(Aspiorhynchus laticeps)、斑重唇鱼(Diptychus maculatus)的同种不同个体各自独立聚为一支,显示出明显的单系性;高原鳅属中的长身高原鳅(Triplophysa teunis)也独自立为一支,所有物种都被区分开。综上,基于COⅠ基因的DNA条形码技术可以有效鉴定新疆5种土著鱼类。

EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

EFFECT OF ANTITUMOR DRUGS ON THE ACTIVITY OF DNA TOPOISOMERASE I

The activity of DNA topoisomerase Ⅱ prepared from either normal or tumor tissues were compared. It was found that the unknotting activity of the enzyme in malignant tumor cells was higher than that in normal cells. We selected some antitumor drugs including Chinese traditional medicine, and observed their effects on the unknotting activity of topoisomerase Ⅱ. The results showed that inhibition of the unknotting activity of the enzyme required very low concentrations of drugs, but much higher concentrations were required for other tested. Some antitumor drugs had no effect on the enzyme were also proved. It is interesting that carrageenan, an antiviral drug, strongly blocked the unknotting activity although its antitumor activity has not been reported.

斑腿蝗科Catantopidae七种蝗虫线粒体COⅠ基因的DNA条形码研究

以我国常见的斑腿蝗科Catantopidae7种蝗虫为对象测定了COⅠ基因序列,探讨COⅠ基因作为DNA条形码在识别蝗虫物种方面的可行性。结果表明,斑腿蝗科3属7种的DNA分类和形态学分类基本一致,该基因可以探讨蝗虫属、种分类单元的系统发育问题,为将线粒体基因组的COⅠ基因作为蝗虫DNA条形码进行分类鉴定手段的可行性提供一定的参考。

以重组人DNA拓扑异构酶Ⅰ为靶位从天然产物及其衍生物中筛选抗肿瘤化合物

为了在体外以人DNA拓扑异构酶Ⅰ(hTopoⅠ)为靶位进行抗肿瘤化合物的快速筛选,首次使用毕赤酵母表达了hTopoⅠ。在微量反应体系中,测定了化合物抑制hTopoⅠ松驰活性的能力,并用MTT实验验证筛选到的hTopoⅠ抑制剂的抗肿瘤活性。从74个结构新颖的天然产物及人工合成的黄酮类衍生物中筛到6个hTopoⅠ抑制剂,有4个化合物抑制肿瘤细胞生长的能力较强。

马兜铃酸Ⅰ致肾小管上皮细胞DNA损伤的实验研究

目的:研究马兜铃酸Ⅰ(AAⅠ)致肾小管上皮细胞(LLC-PK1细胞)DNA损伤和对细胞周期的影响,并探讨其与P53途径的关系。 方法:不同浓度的AAⅠ纯品(80、160、320、640和1280ng/ml)体外刺激LLC-PK1细胞24h,采用单细胞凝胶电泳和流式细胞仪观察AAⅠ对LLC-PK1细胞DNA损伤和细胞周期的影响。用免疫荧光染色和流式细胞仪检测小管上皮细胞P53蛋白的表达。 结果:单细胞凝胶电泳实验发现 AAⅠ浓度为160,320,640和1280ng/ml时能导致LLC-PK1细胞产生彗星拖尾现象,且剂量越大,拖尾越明显,尾长和尾部荧光值与对照组比较有明显差异(P<0.05或P<0.01)。此剂量的AAI使LLC-PK1细胞在G2/M期的比例明显增高,且剂量越大,增高越明显,与对照组比较有明显差异(P<0.05或P<0.01)。各组小管上皮细胞P53蛋白的表达无明显差异(P>0.05)。 结论:AAⅠ可导致LLC-PK1细胞DNA损伤,使细胞周期阻滞在G2/M期,这可能是马兜铃酸肾毒性损伤后肾小管上皮细胞再生修复能力差和形成泌尿道肿瘤的机制之一。AAⅠ对LLC-PK1细胞DNA损伤和细胞周期阻滞作用是非P53依赖的,具体机制有待进一步研究。

基于COⅠ基因的厦门海域鱼类DNA条形码鉴定

为研究DNA条形码技术在厦门海域鱼类分类鉴定中的可行性,随机选取了厦门海域23种鱼类样品进行COⅠ基因扩增,结果共获取23条序列,平均长度为656 bp,序列中T、C、G、A碱基的平均含量分别为:29.40%、28.10%、18.40%、24.10%.样品种内、种间、科内和目内的遗传距离(K2P)分别为:0.21%,20.50%、24.47%和25.62%,遗传距离随着分类阶元的提高而增大,种间遗传距离明显大于种内遗传距离.所选厦门海域鱼类样品仅蓝圆鯵鉴定到圆鯵属,未能鉴定到种,其余22种全部鉴定到种,表明COⅠ基因序列可以作为鱼类分类鉴定的有效DNA条形码.

椭圆食粉螨线粒体DNA COⅠ基因片段序列分析

采用苯酚-氯仿-异戊醇抽提法提取了采自江西南昌和广州潮州2个地理种群的椭圆食粉螨Aleuro-glyphus ovatus Troupeau,1878基因组DNA。以相应引物对椭圆食粉螨线粒体DNA细胞色素氧化酶I亚基基因(mtDNA COI)进行PCR扩增,直接测序,得到379bp的碱基片断(国际基因库索引号EF527825),其碱基序列A、C、G、T含量分别为71bp(18.73%)、53bp(13.98%)、89bp(23.48%)、166bp(43.80%);江西南昌和广州潮州2个地理种群间的椭圆食粉螨mtDNACOI基因片段完全一致,未发现地理差异。

基于线粒体CO Ⅰ基因15个鸡种的DNA编码研究

【目的】探明地方鸡种与引进鸡种遗传多态性特点,探讨COⅠ这一特定基因的特定区段作为DNA条形码在识别鸡种方面的可行性和有效性。【方法】以13个中国地方鸡种和2个国外引进品种为研究对象,利用DNA测序技术测定了线粒体细胞色素C氧化酶亚基Ⅰ(cytochrome c oxidaseⅠ,COⅠ)基因部分序列。【结果】选择的这段COⅠ基因序列有38个突变位点,15个鸡种单倍型多样度平均为0.963,核苷酸多样度平均为0.00518,其中引进鸡种明显低于地方鸡种(除藏鸡外);15个鸡种品种间Kimura双参数遗传距离为0.056%—0.917%,种内遗传距离为0—0.346%,15个鸡种的DNA分类和形态学分类基本一致,引进鸡种与地方鸡种分歧较远。【结论】利用线粒体细胞色素C氧化酶Ⅰ(COⅠ)这一特定基因的特定区段来做DNA条形编码的基础,进行不同鸡品种鉴定,具有可行性和有效性。

我国耐力运动员线粒体DNA高变区Ⅰ序列多态性分析

为了探讨人类运动能力相关的基因标记与分子机制 ,对mtDNA高变区I作了序列多态性分析。研究选取汉族耐力运动员 95人 ,相应汉人对照 92人 ,对其mtDNA高变区I特异性片段进行扩增、测序 ,分析其序列多态性改变。结果显示 :中国汉族耐力运动员的多态位点有 83个 ,其中 ,碱基替换位点 6 8,缺失位点 5个 ,插入位点 10个 ,位点 16 2 2 8碱基缺失及 16 113- 16 114和 16 335 -16 336碱基插入为运动员独有 ;中国汉族运动员mtDNA高变区I同质性多态主要表现为碱基转换、碱基颠换、缺失及插入四种类型 ,其中 ,碱基转换发生率最高 ,碱基颠换发生率次之 ,碱基缺失及插入频率最低。运动员碱基颠换频率明显高于常人 (P <0 0 5 ) ,而碱基缺失及插入频率则显著低于常人 (P <0 0 5 )。运动员T -C转换频率显著高于常人 (P <0 0 5 ) ,运动员A -G转换频率则显著低于常人 (P <0 0 1)。研究结果还提示 ,mtDNA扩增产物直接测序分析作为一种简便快捷的mtD NA序列多态性和单核苷酸多态性研究方法 ,为运动能力遗传标记的筛选与研究提供了有效方法 。

基于COⅠ的中国西施舌DNA条形码

以我国大连(DL)、日照(RZ)、启东(QD)、长乐(CL)、漳州(ZZ)和北海(BH)6个野生群体西施舌为研究对象,用引物LCO1490和HCO2198扩增线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因片段序列,PCR产物双向测序,用ClustalX进行DNA序列比对,用MEGA4.1和PhyML软件构建NJ树和ML树,用Arlequin3.11软件进行分子方差分析,研究COⅠ这一特定基因区段作为DNA条形码在识别西施舌地理群体的可行性和有效性。结果获得658bp的COⅠ核苷酸片段,其T、C、A、G含量分别为42.4%、14.8%、21.8%、21.0%,A+T(64.2%)含量明显高于G+C含量。变异位点占15.3%(101/658),其中简约信息位点占14.1%。群体内遗传距离为0.0020～0.0043,群体间遗传距离为0.0024～0.0883。群体间氨基酸序列无变异。长乐、漳州群体(DH组)与其他群体(HB组)间有显著的遗传分化(FST=0.956～0.970)。NJ树和ML树显示长乐、漳州群体聚为一支,北方的大连、日照、启东群体和南方的北海群体聚为置信度较高的另一支(BPN=99),2支间的遗传距离为0.0837,是2支内群体间遗传距离的17.8～23.5倍。本研究结果表明,COⅠ基因片段序列作为鉴定福建西施舌条形码是可行的,但不能将其余群体区别开来。

紫草素对DNA拓扑异构酶Ⅰ活性的抑制作用和诱导人白血病K562细胞的凋亡(英文)

目的 :研究紫草素对DNA拓扑异构酶I催化活性和K5 6 2白血病细胞凋亡的影响。方法 :从K5 6 2白血病细胞提取拓扑异构酶I,通过DNA解螺旋试验评价该药对拓扑异构酶I催化活性的抑制作用。用MTT法测定了该药对K5 6 2白血病细胞增殖的抑制作用。应用流式细胞术和琼脂糖凝胶电泳观察了该药对细胞凋亡的诱导作用。结果 :紫草素可显著抑制拓扑异构酶I的解螺旋活性 (IC50 =75 0 μmol/L)。该药能显著抑制K5 6 2的细胞增殖 ,并随剂量增大而抑制作用增强。紫草素 0 3～ 3μmol/L对K5 6 2细胞作用 2 4h后 ,其凋亡率为 2 0 %～ 70 %。琼脂糖凝胶电泳出现典型的DNA“lad der”。结论 :紫草素显著抑制拓扑异构酶I的催化活性 ,并能诱导K5 6

两株高DNA拓扑异构酶Ⅰ抑制活性的药用植物内生真菌筛选

从7种中国南方药用植物中分离了278株内生真菌,分析了其发酵液甲醇提取物对人DNA拓扑异构酶Ⅰ(hTo-poI)松驰活性的抑制能力。对2株具有高hTopoI抑制力的内生真菌(LF4-7L和LF6-1)进行了深入研究。通过发酵物对hTo-poI抑制能力的时间变化曲线,表明2株内生真菌的hTopoI抑制物质在其生长停滞阶段才大量产生和积累。经分子鉴定,LF4-7L可能是阴炭团菌(Hypoxylon stygium),而与LF6-1L最相近的是毛竹基腐病菌(Arthrinium phaeospermum)。

基于线粒体COⅠ的DNA条形码技术在贻贝科种类鉴定中的应用

DNA条形码（DNA barcoding）技术作为近年来发展起来的物种鉴定的一门新兴技术,已引起越来越多的关注。其原理是利用一段短DNA标准序列（对动物而言一般为线粒体细胞色素c氧化酶亚单位Ⅰ,mitochondrial cytochrome c oxidase subunitⅠ,COⅠ）来实现物种鉴定。它的两大主要目的是鉴定已知种和发现新种。

基于COⅠ基因的西南大西洋部分经济鱼类DNA条形码鉴定

目前,国际上广泛使用FishBase(http://fishbase.org)等鱼类分类检索系统进行鱼类形态学鉴定,但由于部分地区鱼类开发和研究水平不一,因此单一的形态学鉴定无法完全满足物种鉴定的需求。DNA序列辅助分类作为一个理想的辅助形态学分类手段应运而生,迄今已有30多年历史。2003年,Hebert等[1,2]明确提出了DNA条形码(DNA barcoding)的概念,即通过使用短的、标准化的基因片段来进行物种鉴定,以提高真核生物识别的效率。