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.

Ki-67及TopoisomeraseⅡ在脑胶质母细胞瘤组织中的表达及其生物学意义

目的探讨人胶质母细胞瘤中Ki-67及TopoisomeraseⅡ（TopoⅡ）之间的相关性及其与胶质母细胞瘤病人预后的关系。方法收集O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）低表达的胶质母细胞瘤标本60例,应用免疫组化法检测Ki-67和TopoⅡ的表达,研究其相关性。结果在MGMT低表达的胶质母细胞瘤中,Ki-67与TopoⅡ表达呈正相关（γ=0.83,P〈0.05）。对本组标本来源的60例胶质母细胞瘤病人随访12~23个月,其中死亡38例,肿瘤复发46例。结论 Ki-67和TopoⅡ在胶质母细胞瘤中的表达强度相对一致;其表达能客观反映肿瘤细胞的增殖活性和恶性程度,且可能影响胶质母细胞瘤病人的预后。

免疫组化检测大肠癌GST-π、TopoisomeraseⅡ-α表达的临床意义

目的 探讨GST -π、TopoisomeraseⅡ -α蛋白与大肠癌病理学特征之间的关系。方法 分别用鼠抗人TopoisomeraseⅡ -α单克隆抗体和兔抗人GST -π抗体对 6 0例大肠癌石蜡标本进行免疫组化研究。结果 1.GST -π、TopoisomeraseⅡ -α蛋白的表达和肿瘤大小、部位及类型无关 (P >0 .0 5 ) ;但与肿瘤的分化程度、临床分期、淋巴结转移有统计学差异 (P <0 .0 0 1)。 2 .GST -π在癌灶及癌旁组织中的表达意义不同 ,在肿瘤组织中低分化组高表达而在癌旁组织中则低表达 ,二者有统计学差异 (P <0 .0 0 1) ;TopoisomeraseⅡ -α在高分化组的表达高于低分化组 (P <0 .0 0 1)。结论 1.GST -π在瘤组织中的表达强度及在癌旁组织中的表达均与肿瘤的分化及临床分期有关 ,可作为肿瘤预后的指标。 2 .Topoi someraseⅡ -α在肿瘤组织中的表达和其分化、淋巴结转移有关 。

TopoisomeraseⅡ-α在大肠癌中的表达和临床意义

目的 探讨TopoisomeraseⅡ α蛋白与大肠癌病理学特征之间 ,耐药之间的关系。 方法 分别用鼠抗人TopoisomeraseⅡ α单克隆抗体对 60例大肠癌石蜡标本进行免疫组化研究。 结果 TopoisomeraseⅡ α蛋白及癌旁组织中的表达和性别、肿瘤大小、部位及类型无关 (P >0 .0 5 ) ;但与肿瘤的分化程度、Dukes分期、TMN分期、临床分期、淋巴结转移有统计学差异 (P <0 .0 0 1)。TopoisomeraseⅡ α在高分化组的表达高于低分化组 (P <0 .0 0 1) ,淋巴结转移组表达则低于无淋巴结转移组 (p <0 .0 0 1)。结论 TopoisomeraseⅡ α在肿瘤组织中的表达 ,强度和肠癌生物学特性密切相关 ,与肿瘤的分化及临床分期、淋巴结转移有关 ,可做为衡量恶性程度 ,耐药性及预后的指标之一。

Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

TopoisomeraseⅡαGene as a Marker for Prognostic Prediction of Hepatocellular Carcinoma:A Bioinformatics Analysis

Objective To investigate the expression of topoisomeraseⅡα(TOP2α)in hepatocellular carcinoma(HCC)and its role in predicting prognosis of HCC patients.Methods We used HCC-related datasets in UALCAN,HCCDB,and cBioPortal databases to analyze the expression and mutation of TOP2αand its co-expressed genes in HCC tissues.GO function and KEGG pathway enrichment of TOP2αand its co-expressed genes were identified.The TIMER database was used to analyze infiltration levels of immune cells in HCC.The impacts of TOP2αand its co-expression genes and the infiltrated immune cells on the survival of HCC patients were assayed by Kaplan-Meier plotter analysis.Results TOP2αand its co-expression genes were highly expressed in HCC(P<0.001)and detrimental to overall survival of HCC patients(P<0.001).TOP2αand its co-expression genes were mainly involved in cell mitosis and proliferation,and cell cycle pathway(ID:hsa04110,P=0.001945).TOP2αand its co-expression genes were mutated in HCC and the mutations were significantly detrimental to overall survival(P=0.0247)and disease-free survival(P=0.0265)of HCC patients.High TOP2αexpression was positively correlated with the infiltration of B cell(r=0.459,P<0.01),CD8^(+)T cell(r=0.312,P<0.01),CD4^(+)T cell(r=0.370,P<0.01),macrophage(r=0.459,P<0.01),neutrophil(r=0.405,P<0.01),and dendritic cell(r=0.473,P<0.01)in HCC.The CD8^(+)T cell infiltration significantly prolonged the 3-and 5-year survival of HCC patients(all P<0.05),and CD4^(+)T cell infiltration significantly shortened the 3-,5-,and 10-year survival of HCC patients(all P<0.05).Conclusion TOP2αmay be an oncogene,which was associated with poor prognosis of HCC patients and could be used as a biomarker for the prognostic prediction of HCC.

Effect of Inhibitor K-23 on O_2-evolution and Hill Activity of PSⅡ Membrane Fragments of Spinacia oleracea

研究了新的抑制剂K_2 3对波菜 (SpinaciaoleraceaMill.)PSⅡ放氧活性和 2 ,6_dichlorophenolindophenol (DCIP)光还原活性的影响。研究发现 :抑制剂K_2 3在低浓度时对PSⅡ放氧活性有明显促进作用 ,而对DCIP光还原活性的促进作用不太明显。在高浓度时抑制PSⅡ放氧活性和DCIP光还原。对K_2 3的抑制部位进行了初步探讨。

A Copper(Ⅱ)Complex Based on N-(4-hydroxybenzyl)-L-serine:Synthesis,Crystal Structure and Inhibitory Activity on PTP1B and TCPTP

A novel copper（II） complex with the reduced Schiff base, [Cu（L）2]·H2O （I, HL = N-（4-hydroxybenzyl）-L-serine）, was prepared in aqueous solution and characterized by elemental analysis, FT-IR, electrospray ionization mass spectrometry and single-crystal X-ray diffraction. Complex I crystallizes in the orthorhombic system, space group P212121, with a = 8.9040（18）, b = 9.1530（18）, c = 24.891（5）A^°, V = 2028.6（7） A^°3, Z = 4, C20H26CuN2O9, Mr = 501.97, Dc = 1.644g·cm^3, μ = 1.135 mm^-1, F（000） = 1044, GOOF = 1.194, the final R = 0.0484 and wR = 0.1420 for 6186 observed reflections （I 〉 2σ（I））. In I, two L^- anions are coordinated to the copper ion in tridentate and bidentate chelating modes, respectively, resulting in the coordinated geometry of copper ion to be a distorted square pyramid. The intermolecular hydrogen bonds between the complexes, complexes and lattice water molecules lead to a 2D supramolecular network. The bioactivity of the complex as a potential PTPs inhibitory agent in vitro was investigated, displaying potent inhibition against PTP1B （IC50, 0.27 μM） and TCPTP （IC50, 0.57 μM） with a moderate selectivity.

Current Role of Platelet Glycoprotein Ⅱ b/Ⅲ a Receptor Inhibitors in Clinical Trials of Cardiovascular Diseases

Thrombosis formation on disrupted atherosclerotic plaque is the most common acuse of cardiovascular diseases, in the pathophysiology, increased platelet reactivity is a descriptor of the risk of cardiovascular events in healthy persons and in patients with overt coronary artery disease. Regardless of the stimulus for activation platelet-platelet interation and thrombus formation is ultimately regulated through the GP Ⅱ b/Ⅲ a receptor

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。

TOPⅡ表达与乳腺癌新辅助化疗疗效的关系分析

目的探讨乳腺癌新辅助化疗方案疗效及与化疗前后TOPⅡ表达的关系。方法采用免疫组化方法检测乳腺癌患者(73例)新辅助化疗前后乳腺癌组织中TOPⅡ的表达,了解其与化疗方案及疗效的关系。结果 TOPⅡ的表达与疗效相关(P=0.015),TOPⅡ阳性表达患者对蒽环类药物敏感(P=0.005),多西他赛类药物疗效与TOPⅡ表达无相关性(P=0.495)。化疗前后TOPⅡ表达差异具有统计学意义(P=0.027),阳转阴者中获得pCR为35.3%,阴转阳或无变化者获得pCR为9.1%,两者比较差异具有统计学意义(P=0.017)。结论 TOPⅡ阳性表达者对蒽环类化疗方案敏感,TOPⅡ可能成为预测疗效的指标,化疗后TOPⅡ表达由阳转阴者获得较好的病理缓解,TOPⅡ可能成为指导术后化疗方案选择的指标之一。

毛叶茶和龙井茶提取物的抗瘤作用以及对DNA拓扑异构酶Ⅱ的抑制作用

作者用噻唑蓝(MTT)法研究了毛叶茶提取物(毛茶C)和龙井茶提取物(龙井A)对人宫颈癌HeLa细胞、鼻咽低分化鳞癌CNE_2细胞和胃低分化粘液腺癌MGC—803细胞的细胞毒作用。毛茶C对上述3种细胞株的半数抑制浓度(IC_(50))分别为495.6μg/ml、243.4μg/ml、268.6μg/ml;而龙茶A分别为421.1μg/ml、311.4和274.6μg/ml。体内抑瘤试验表明,毛茶C在2.5—10mg//kg剂量范围内,对小鼠艾氏腹水癌实体型(ESC)有明显抑制作用,抑瘤率为17.8％—48.3％;对小鼠网织细胞肉瘤(L_2)的抑瘤率为28.3％—54.5％;龙茶A在2.5—10mg/kg剂量范围内对艾氏实体瘤ESC的抑制率为31.5％—49.4％;对L_2的抑瘤率为35.8％—50％。而二种茶叶提取物对小鼠肉瘤S—180仅有边缘的抗瘤活性。用琼脂糖凝胶电泳方法则得毛茶C和龙茶A在1—50μg/ml浓度范围内,对DNA拓扑异构酶Ⅱ的抑制率分别为75—100％,抑酶作用有明显的量效关系。在50μg/ml浓度均可抑制全部酶活性,推测DNA拓扑异构酶Ⅱ可能是这两种茶叶提取物的靶酶。

铜绿假单胞菌环丙沙星耐药株Ⅱ类拓扑异构酶基因突变的研究

用PCR方法 ,扩增 2 6株临床分离铜绿假单胞菌环丙沙星耐药株、4株环丙沙星敏感菌株和PA0 1菌株的Ⅱ类拓扑异构酶 gyrA、gyrB、parC和 parE基因的喹诺酮类药物耐药决定区 (QRDR)基因片断 ;并对铜绿假单胞菌II类拓扑异构酶基因突变与耐药性关系进行研究。结果表明 90 %以上的铜绿假单胞菌耐药株表现出Ⅱ类拓扑异构酶基因突变 (2 6株中有 2 4株 )。突变主要发生在 gyrA基因 (2 2株 )和 parC基因 (14株 )上 ,其中 gyrA基因的第 83位氨基酸密码子表现出高频突变 (2 2株中有 2 1株 ,ACC→ATC ,占 90 % ) ,parC基因第 80位氨基酸密码子也表现出较高的突变率 (14株中有 12株 ,TCG→TTG ,占 60 % ) ,4株耐药株的 gyrB和 3株耐药株的 parE基因发生单点突变 ,2株耐药株II类拓扑异构酶基因未检测到突变。用二倍稀释法 ,测定部分喹诺酮和 β 内酰胺类药物对耐药突变株的体外抗菌活性。表明铜绿假单胞菌Ⅱ类拓扑异构酶基因突变株对诺氟沙星、左氧氟沙星、哌拉西林、头孢他啶和亚胺培南表现出不同的敏感性。试验所用 2 4株突变株对诺氟沙星呈现 10 0 %的耐药 ;对左氧氟沙星 65 %的耐药 ;40 %的菌株对哌拉西林表现出耐药 ;3 0 %的菌株对头孢他啶耐药 ;75

乳腺癌原发灶及淋巴结转移灶中P-gp、GST-π、TopoⅡ的表达及临床意义

目的探讨乳腺癌原发灶和淋巴结转移灶中P-gp、GST-π、TopoⅡ的表达及相关性。方法选择2005年1～12月所有在我科行手术治疗的乳腺癌患者70例,其中有腋下淋巴结转移的36例,无腋下淋巴结转移的34例,用免疫组化SP法检测原发灶和淋巴结转移灶P-gp、GST-π、TopoⅡ的表达。结果(1)有腋下淋巴结转移的乳腺癌中P-gp、GST-π的表达高于无腋下淋巴结转移的乳腺癌(P=0.043、P=0.012),TopoⅡ的表达两者间差异无统计学意义。(2)乳腺癌原发灶和淋巴结转移灶的GST-π、TopoⅡ表达有相关性(P=0.001、P=0.01),P-gp的表达无相关性。结论(1)P-gp、GST-π高表达与腋下淋巴结转移显著相关。(2)乳腺癌原发灶与淋巴结转移灶的耐药性不尽一致。

乳腺浸润性导管癌中拓扑异构酶Ⅱ等相关因素表达与腋窝淋巴结转移的关系研究

目的分析拓扑异构酶Ⅱ(TopoisomeraseⅡ)、nm23基因、雌激素受体(ER)、孕激素受体(PR)和C-erbB-2基因等在乳腺浸润性导管癌中的表达与腋窝淋巴结转移的关系,并探讨其临床意义,为进一步治疗、准确判断患者预后提供数据支持。方法采用免疫组织化学-SP法检测101例乳腺浸润性导管癌患者病理组织中的Topoi-someraseⅡ、nm23基因、ER、PR和C-erbB-2基因等,分析其与腋窝淋巴结转移的关系。结果 C-erbB-2(-)、(+)、(++)、(+++)4组的腋窝淋巴结转移发生率依次升高,分别为18.2%(2/11)、69.7%(23/33)、70.0%(21/30)、81.5%(22/27),差异有统计学意义(χ2=14.718,P=0.002)。TopoisomeraseⅡ(-)组腋窝淋巴结转移发生率(45.2%)低于TopoisomeraseⅡ(+)组(77.1%),差异有统计学意义(χ2=9.990,P=0.002)。而nm23(-)组腋窝淋巴结转移发生率(85.0%)高于nm23(+)组(55.7%),差异有统计学意义(χ2=9.404,P=0.002)。Logistic回归分析结果显示,TopoisomeraseⅡ(OR=8.870,P=0.001)、C-erbB-2(OR=1.848,P=0.041)是腋窝淋巴结转移的危险因素;而nm23(OR=0.151,P=0.008)是保护因素。结论 C-erbB-2基因和TopoisomeraseⅡ的阳性表达与腋窝淋巴结转移有关,nm23基因低表达患者腋窝淋巴结转移的危险较高。

拓扑异构酶Ⅱ在宫颈癌组织中的表达及其临床意义

目的 探讨DNA拓扑异构酶Ⅱ (TopoisomeraseⅡ ,TopoⅡ )在正常宫颈组织、宫颈上皮内瘤变(cervicalintraepithelialneoplasm ,CIN)和宫颈癌中的表达。 方法 采用免疫组化的方法对 1 8例正常宫颈组织 ,2 8例CIN及 5 8例浸润性宫颈癌进行TopoⅡ的检测。 结果 TopoⅡ在正常宫颈组织、CIN及宫颈癌中均有表达 ,但在CINⅢ和宫颈癌中表达水平高于正常组织和CINⅠ组。其表达与浸润性宫颈癌组织学分级无关。结论 TopoⅡ是检测肿瘤细胞增殖的一种免疫标记物 ,其在宫颈癌中明显高表达 。

肝癌及肝硬化组织中ICAM-1和TopoⅡ的表达

目的 探讨细胞间黏附分子 1(ICAM 1)和拓扑异构酶Ⅱ (TopoⅡ )作为判断肝硬化及肝癌发展程度及预后指标的可能性。方法 应用免疫组化S P法对 32例肝细胞癌和癌旁组织、2 6例肝硬化和 10例正常肝组织标本进行ICAM 1和TopoⅡ标记。结果 ICAM 1和TopoⅡ表达率分别为肝癌 84 3%、6 2 5 % (2 7/32 ,2 0 /32 ) ;癌旁组织为 6 8 7%、4 6 8% (2 2 /32 ,15 /32 ) ;肝硬化为 6 5 3% ,38 4 % (17/2 6 ,10 /2 6 )。 10例正常肝组织无表达及弱表达 ,阳性率与组织学分类相关 ,肝癌组织中I CAM 1和TopoⅡ含量明显高于癌旁及肝硬化组织 (P <0 0 1) ,而癌旁及肝硬化组织中表达差异无显著性 (P >0 0 5 )。 结论肝组织中ICAM 1和TopoⅡ的过度表达在一定程度上可以反应肝癌、肝硬化发展及细胞增殖活性 ,有可能作为判断预后的指标之一。