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。

CD44V6、P-gp、Top-Ⅱ在结直肠癌中的表达与临床病理的关系及其临床意义

目的:探讨CD44拼接变异体V6(CD44variantV6,CD44V6)、P-糖蛋白(P-glycoprotein,P-gp)、拓扑异构酶Ⅱ(TopoisomeraseⅡ,Top-Ⅱ)在结直肠中表达及其临床意义。方法:采用HE方法验证结直肠癌组织和正常大肠黏膜组织。采用免疫组织化学法MaxVision二步法检测80例结直肠癌组织及46例正常大肠黏膜组织中CD44V6、P-gp、Top-Ⅱ蛋白的表达情况,将其与性别,临床Dukes分期,浸润深度,淋巴转移等临床病理学资料进行统计学分析。结果:CD44V6、P-gp、Top-Ⅱ蛋白在结直肠癌组织中的表达显著高于正常大肠黏膜组织。CD44V6在结直肠癌中的表达强度与肿瘤的临床Dukes分期(rs=0.328,P<0.05),浸润深度(rs=0.356,P<0.01),淋巴转移(rs=0.406,P<0.01)成显著正相关性,与性别无相关性;P-gp在结直肠癌中的表达强度与性别,临床Dukes分期,肿瘤的浸润深度,淋巴转移无相关性。Top-Ⅱ在结直肠癌中的表达强度与性别,临床Dukes分期,肿瘤的浸润深度无相关性,但与淋巴转移有相关性(rs=0.245,P<0.05)。结论:P-gp、Top-Ⅱ、CD44V6在结直肠癌中均高表达。CD44V6在结直肠癌中的表达与临床Dukes分期,肿瘤的浸润深度及淋巴转移情况存在正相关性。

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 。

应用组织芯片探讨宫颈癌前病变及宫颈癌中DNA拓扑异构酶Ⅱα的表达及其临床意义

目的：探讨DNA拓扑异构酶Ⅱα（TopoⅡα）蛋白在宫颈癌前病变及宫颈癌中的表达及其在宫颈癌发生发展中的作用。方法选取2007-2008年在山西省肿瘤医院行宫颈病变诊治的患者，其中包括慢性宫颈炎，低级别和高级别宫颈上皮内瘤变（CIN）和宫颈浸润性鳞癌（SCC）的患者分别21、33、30、73例，制作组织芯片，应用免疫组织化学法（SP）检测宫颈组织中TopoⅡα蛋白的表达情况。结果结果TopoⅡα蛋白在慢性宫颈炎上皮组织、CINⅠ、CINⅡ～Ⅲ、SCC中的表达率分别是5％，12％，90％，97％，表达率逐渐增加，高级别CIN和浸润性鳞癌的表达显著高于低级别CIN和正常宫颈组织，差异有统计学意义；而高级别CIN与浸润性癌之间、低级别CIN与正常宫颈上皮之间的表达差异均无统计学意义。结论 TopoⅡα蛋白表达随着宫颈癌前病变的严重程度的加重而增加，因此T o poⅡα蛋白可能参与了宫颈上皮的异常增生和恶性转变。

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。

氟喹诺酮类抗菌药在动物医学上的研究进展

在动物医学上研究和应用的氟喹诺酮类抗菌药有恩诺沙星(Enrofloxacin,乙基环丙沙星),达诺沙星(danofloxacin,甲基环丙沙星),诺氟沙星(norfloxacin,氟哌酸)。氟甲喹(flumequine)等。其共同特点是抗菌谱广,除对革兰氏阴性菌有很强的杀菌作用外,对革兰氏阳性需氧菌、支原体和细胞内病原菌及衣原体也有杀灭作用。杀菌机制为抑制细菌DNA回旋酶亚单位A,该酶为Ⅱ型拓扑异构酶(type—Ⅱtopoisomerase)。

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.

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.

Inhibition of DNA-Topoisomerase I by Acylated Triterpene Saponins from Pittosporum angustifolium Lodd.

Previous phytochemical investigation of the leaves and seeds of Pittosporum angustifolium Lodd.led to the isolation and structural elucidation of polyphenols and triterpene saponins.Evaluation for cytotoxicity of isolated saponins revealed that the predominant structural feature for a cytotoxic activity are acyl substituents at the oleanane aglycon backbone.The present work reports the results of a screening of 10 selected acylated saponins for their potential to inhibit the human DNA-topoisomerase I,giving rise to IC50 values in a range of 2.8-46.5 lM.To clarify the mode of observed cytotoxic action and,moreover,to distinguish from a pure surfactant effect which is commonly accompanied with saponins,these results indicate an involvement of the topoisomerase I and its role as a possible target structure for a cytotoxic activity.In addition,computational predictions of the fitting of saponins to the topoisomerase I-DNA complex,indicate a similar binding mode to that of clinically used topoisomerase I inhibitors.Graphical Abstract Ten acylated triterpene saponins from Pittosporum angustifolium were investigated for their potential to inhibit the human DNA-topoisomerase I and computational predictions of the fitting of saponins to the topoisomerase I-DNA complex were carried out.

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.

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.

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.

Therapy-related myeloid neoplasms - what have we learned so far?

Therapy-related myeloid neoplasms are neoplastic processes arising as a result of chemotherapy, radiation therapy, or a combination of these modalities given for a primary condition. The disease biology varies based on the etiology and treatment modalities patients receive for their primary condition. Topoisomerase II inhibitor therapy results in balanced translocations. Alkylating agents, characteristically, give rise to more complex karyotypes and mutations in p53. Other etiologies include radiation therapy, high-dose chemotherapy with autologous stem cell transplantation and telomere dysfunction. Poor-risk cytogenetic abnormalities are more prevalent than they are in de novo leukemias and the prognosis of these patients is uniformly dismal. Outcome varies according to cytogenetic risk group. Treatment recommendations should be based on performance status and karyotype. An in-depth understanding of risk factors that lead to the development of therapy-related myeloid neoplasms would help developing risk-adapted treatment protocols and monitoring patients after treatment for the primary condition, translating into reduced incidence, early detection and timely treatment.

Cu(II) Propionyl-Thiazole Thiosemicarbazone Complexes: Crystal Structure, Inhibition of Human Topoisomerase IIα, and Activity against Breast Cancer Cells

Two new thiosemicarbazone ligands, 2-propionylthiazole ethylthiosemicarbazone (PTZ-ETSC), and 2-propionylthiazole tert-butylthiosemicarbazone (PTZ-tBTSC), along with their two copper(II) complexes, [Cu(PTZ-ETSC)Cl] and [Cu(PTZ-tBTSC)Cl], are reported here for the first time. Once characterized by NMR and MS, these mono-anionic tridentate ligands were reacted with Cu2+ to form the square planar metal complexes [Cu(PTZ-ETSC)Cl] and [Cu(PTZ-tBTSC)Cl]. The x-ray crystal structure of the [Cu(PTZ-tBTSC)Cl] complex shows that the complex adopts a square planar arrangement around the copper(II) ion, but forms a sulfur-bridged dimer in the solid state. Both of the copper complexes displayed strong inhibition of human topoisomerase IIα at activities between 2-4 μM for [Cu(PTZ-ETSC)Cl], and between 8-10 μM for the [Cu(PTZ-tBTSC)Cl] complex. The EC50 values for the MDA-MB-231 breast cancer cell line were 82.6 μM for (PTZ-ETSC), 17.9 μM for [Cu(PTZ- ETSC)Cl], 97.8 μM for (PTZ-tBTSC), and 1.41 μM for [Cu(PTZ-tBTSC)Cl]. The EC50 values for the MCF7 breast cancer cell lines were 9.36 μM for (PTZ-ETSC), 0.13 μM for [Cu(PTZ-ETSC)Cl], 0.333 μM for (PTZ-tBTSC), and 0.093 μM for [Cu(PTZ-tBTSC)Cl].

Associations with Organ Involvement and Autoantibodies in Systemic Sclerosis: Results from the Canadian Scleroderma Research Group (CSRG)

Objective: Serum from SSc patients was analyzed centrally to determine ANA patterns and extractable nuclear antigens (ENAs) between lcSSc and dcSSc and associations with organ involvement. Methods: 1145 SSc patients had ANA and ENA analyzed by indirect immunofluorescence on HEp-2 substrate at a screening serum dilution of 1/160. Most ENA antibodies [Sm. U1-RNP, Ro52, SS-A/Ro60, topoisomeraseI (Topo1), SS-B/La, chromatin, ribosomal P and Jo1] were measured by laser bead immunoassay;and RNA polymerase III (RNAP) by ELISA. Results: ANA was positive in 95% (same in lcSSc, and dcSSc). Centromere pattern was present in 34%, speckled 22%, nucleolar 18%, homogeneous and speckled (H&S) 16%, multiple nuclear dots 6%. Anti-centromere Ab (ACA) occurred in 46% of lcSSc and 11% of dcSSc (P = 0.0001). ENAs that differed between lcSSc and dcSSc subsets were Topo1 (OR 2.4, P = 0.0001) and RNAP (OR 5.6, P 0.0001) more common in dcSSc. Overall, 15% had positive Topo1;usually with a H&S pattern (67%);Topo1 was associated with ILD on CXR (OR 2.3;95% CI 1.5 - 3.5) and HRCT (OR 3.8;95% CI 1.8 - 8.2). RNAP occurred in 18.5% (35.4% in dcSSc vs. 8.9% in lcSSc). Scleroderma renal crisis (SRC) was 13 times more likely if RNAP positive;P = 0.0001. ACA was only weakly associated with sPAP > 50 mmHg (OR 1.8;95%CI 1.1 - 3.0). Conclusion: ANA homogeneous pattern alone is rare in SSc;ACA was significantly more common in lcSSc. Many ENAs are equal in lcSSc and dcSSc except RNAP and Topo1. RNAP has the highest OR of SRC. Topo1 is less strongly associated with ILD. Abstract word count: 249, Body word count 1246, Figures 2, Tables 2. Key Messages: 1) 95% of SSc has a positive ANA and ANA patterns in SSc include centromere, nucleolar, and homogeneous and speckled together;2) Most ENAs are equal in both dcSSc and lcSSc except anti RNA polymerase III and topoisomerase I;3) RNA polymerase III has the highest association (odds ratio) with scleroderma renal crisis, topoisomerase I is associated with interstitial lung disease;whereas anticentromere was not associated with elevated pulmonary arterial pressures on echocardiogram.

Cu(II) Benzoylpyridine Thiosemicarbazone Complexes: Inhibition of Human Topoisomerase IIα and Activity against Breast Cancer Cells

The focus of this research is on the study of a series of copper (II) benzoylpyridine thiosemicarbazone complexes. Of the six benzoylpyridine thiosemicarbazone ligands used in this study, two are reported for the first time;2-benzoylpyridine tert-butyl thiosemicarbazone (BZP-tBTSC), and 2-benzoylpyridine benzyl thiosemicarbazone (BZP-BzTSC). Once characterized by NMR, melting point, and MS, these mono-anionic tridentate ligands were then reacted with Cu2+ to form the new square planar metal complexes [Cu(BZP-tBTSC)Cl] and [Cu(BZP-BzTSC)Cl]. All of the copper complexes display marked inhibition of human topoisomerase IIα. The [Cu(BZP-tBTSC)Cl] complex shows marked activity against human breast cancer cell lines.

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.

Fitness profiling links topoisomeraseⅡregulation of centromeric integrity to doxorubicin resistance in fission yeast

OBJECTIVE To address the molecular implication of Top2 in the context of its interaction with doxorubicin resistance(DXR)genes.METHODS To perform epistasis analyses of top2 with 63genes representing doxorubicin resistance(DXR)genes in fission yeast.Fission yeast cells with single and double mutants were serial diluted and spotted to plates containing 15-75μg·mL-1 doxorubicin.Plates were scanned after 3and 7d.Cell growth was measured and compared between single mutants and double mutants.Nucleus morphology was performed by staining the cells with 4′,6-diamidino-2-phenylindole(DAPI)to observe chromosome segregation.Reverse transcriptase PCR(RT-PCR)was employed to visualize the changes in transcription level and evaluate the stability of chromatin structure.RESULTS Our findings revealed a subset that synergistically collaborate with Top2 to confer DXR and showed that the chromatin-regulating RSC and SAGA complexes act with Top2 in a cluster that is functionally distinct from the Ino80 complex.In various DXR mutants,doxorubicin hypersensitivity was unexpectedly suppressed by a concomitant top2 mutation.Several DXR proteins showed centromeric localization,and their disruption resulted in centromeric defects and chromosome missegregation.An additional top2 mutation could restore centromeric chromatin integrity,suggesting a counterbalance between Top2 and these DXR factors in conferring doxorubicin resistance.CONCLUSION The findings reported here show a functional interaction between Top2 and factors that confer genomic stability at centromeric chromatin under doxorubicin condition.Overall,this molecular basis for mitotic catastrophe associated with doxorubicin treatment will help to facilitate drug combinatorial usage in Doxorubicin-related chemotherapeutic regimens.