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.

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.

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.

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.

Topoisomerase I Inhibition Radiosensitizing Hepatocellular Carcinoma by RNF144A-mediated DNA-PKcs Ubiquitination and Natural Killer Cell Cytotoxicity

Background and Aims: Topoisomerase I (TOP1) partici-pates the repair of DNA double-strand breaks (DSBs) upon radiation therapy (RT). RNF144A mediates ubiquitination of catalytic subunit of DNA protein kinase (DNA-PKcs), a critical factor in DSB repair. This study aimed to investigate the natural killer (NK) cell-mediated radiosensitization with TOP1 inhibition and the mechanism by DNA-PKcs/RNF144A. Methods: In vitro synergism with TOP1i or cocultured NK cells and RT were evaluated in human hepatocellular car-cinoma (HCC) cell lines (Huh7/PLC5) by clonogenic surviv-als. Orthotopic xenografts were treated with Lipotecan and/or RT. Protein expression was analyzed by western blotting, immunoprecipitation, subcellular fractionation, and confocal microscopy. Results: Lipotecan/RT had a superior synergis-tic effect to RT on HCC cells. Combined RT/Lipotecan reduced the xenograft size by 7-fold than RT (p<0.05). Lipotecan caused more radiation-induced DNA damage and DNA-PKcs signaling. The expression of major histocompatibility com-plex class I-related chain A and B (MICA/B) on tumor cells is associated with the sensitivity to NK cell-mediated lysis. Cocultured NK and HCC cells with Lipotecan radiosensitized HCC cells/tissues with the expression of MICA/B. RNF144A increased more in Huh7 cells with combined RT/TOP1i, and reduced the prosurvival function of DNA-PKcs. The effect was reversed by inhibiting the ubiquitin/proteasome system. In comparison, RNF144A decreased through nuclear transloca-tion with the cumulated DNA-PKcs and radio-resistance of PLC5 cells. Conclusions: TOP1i reinforces NK cell-activated anti-HCC effect of RT through RNF144A mediated DNA-PKcs ubiquitination. RNF144A provides a reason for differentiating radiosensitization effect between HCC cells.

Targeting Tyrosyl-DNA phosphodiesterase I to enhance toxicity of phosphodiester linked DNA-adducts

Our genomic DNA is under constant assault from endogenous and exogenous sources,which needs to be resolved to maintain cellular homeostasis.The eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I(Tdp1)catalyzes the hydrolysis of phosphodiester bonds that covalently link adducts to DNA-ends.Tdp1 utilizes two catalytic histidines to resolve a growing list of DNA-adducts.These DNA-adducts can be divided into two groups:small adducts,including oxidized nucleotides,RNA,and non-canonical nucleoside analogs,and large adducts,such as(drug-stabilized)topoisomerase-DNA covalent complexes or failed Schiff base reactions as occur between PARP1 and DNA.Many Tdp1 substrates are generated by chemotherapeutics linking Tdp1 to cancer drug resistance,making a compelling argument to develop small molecules that target Tdp1 as potential novel therapeutic agents.Tdp1’s unique catalytic cycle,which is centered on the formation of Tdp1-DNA covalent reaction intermediate,allows for two principally different targeting strategies:(1)catalytic inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of DNA-adducts that enhances the effectivity of chemotherapeutics;and(2)poisoning of Tdp1 by stabilization of the Tdp1-DNA covalent reaction intermediate,which would increase the half-life of a potentially toxic DNA-adduct by preventing its resolution,analogous to topoisomerase targeted poisons such as topotecan or etoposide.The catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy best illustrates this concept;however,no small molecules have been reported for this strategy.Herein,we concisely discuss the development of Tdp1 catalytic inhibitors and their results.

Cellular irinotecan resistance in colorectal cancer and overcoming irinotecan refractoriness through various combination trials including DNA methyltransferase inhibitors: a review

Treatment with pharmacological drugs for colorectal cancer(CRC)remains unsatisfactory.A major cause of failure in pharmacotherapy is the resistance of colon cancer cells to the drugs,creating an urgent issue.In this review,we summarize previous studies on the resistance of CRC cells to irinotecan and discuss possible reasons for refractoriness.Our review presents the following five major causes of irinotecan resistance in human CRC:(1)cellular irinotecan resistance is induced mainly through the increased expression of the drug efflux transporter,ABCG2;(2)cellular irinotecan resistance is also induced in association with a nuclear receptor,pregnane/steroid X receptor(PXR/SXR),which is enriched in the CYP3A4 gene enhancer region in CRC cells by exposing the cells to SN-38;(3)irinotecan-resistant cells possess either reduced DNA topoisomerase I(Top1)expression at both the mRNA and protein levels or Top1 missense mutations;(4)alterations in the tumor microenvironment lead to drug resistance through intercellular vesicle-mediated transmission of miRNAs;and(5)CRC stem cells are the most difficult targets to successfully treat CRC.In the clinical setting,CRC gradually develops resistance to initially effective irinotecan-based therapy.To solve this problem,several clinical trials,such as irinotecan plus cetuximab vs.cetuximab monotherapy,have been conducted.Another clinical trial on irinotecan plus guadecitabine,a DNA-methyltransferase inhibitor,has also been conducted.

Entrapment of DNA topoisomerase-DNA complexes by nucleotide/nucleoside analogs

Topoisomerases are well-validated targets for cancer chemotherapy and DNA topoisomerase 1(Top1)is the sole target of the camptothecin(CPT)class of anticancer drugs.Over the last 20 years,multiple studies have shown Top1 activity is modulated by non-native DNA structures and this can lead to trapping of Top1 cleavage complexes(Top1cc)and conversion to DNA double strand breaks.Among the perturbations to DNA structure that generate Top1cc are nucleoside analogs that are incorporated into genomic DNA during replication including cytarabine,gemcitabine,and 5-fluoro-2’-deoxyuridine(FdU).We review the literature summarizing the role of Top1cc in mediating the DNA damaging and cytotoxic activities of nucleoside analogs.We also summarize studies demonstrating distinct differences between Top1cc induced by nucleoside analogs and CPTs,particularly with regard to DNA repair.Collectively,these studies demonstrate that,while Top1 is a common target for both Top1 poisons such as CPT and nucleoside analogs such as FdU,these agents are not redundant.In recent years,studies have shown that Top1 poisons and nucleoside analogs together with other anti-cancer drugs such as cisplatin cause replication stress and the DNA repair pathways that modulate the cytotoxic activities of these compounds are being elucidated.We present an overview of this evolving literature,which has implications for how targeting of Top1 with nucleoside analogs can be used more effectively for cancer treatment.

The interplay between DNA topoisomerase 2a posttranslational modifications and drug resistance

The type 2 DNA topoisomerases(Top2)are conserved enzymes and biomarkers for cell proliferation.The catalytic activities of the human isoform Top2a are essential for the regulation of DNA topology during DNA replication,transcription,and chromosome segregation.Top2a is a prominent target for anti-cancer drugs and is highly regulated by post-translational modifications(PTM).Despite an increasing number of proteomic studies,the extent of PTM in cancer cells and its importance in drug response remains largely uncharacterized.In this review,we highlight the different modifications affecting the human Top2a in healthy and cancer cells,taking advantage of the structure-function information accumulated in the past decades.We also overview the regulation of Top2a by PTM,the level of PTM in cancer cells,and the resistance to therapeutic compounds targeting the Top2 enzyme.Altogether,this review underlines the importance of future studies addressing more systematically the interplay between PTM and Top2 drug resistance.

Effects of DNA topoisomerase IIα splice variants on acquired drug resistance

DNA topoisomerase IIα(170 kDa,TOP2α/170)induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation,replication,and segregation.Therefore,TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance.Although many resistance mechanisms have been defined,acquired resistance of human cancer cell lines to TOP2αinterfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels.Recent studies by our laboratory,in conjunction with earlier findings by other investigators,support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing.Specifically,several TOP2αmRNA splice variants have been reported which retain introns and are translated into truncated TOP2αisoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition.In addition,intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine(Tyr805)necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs.Ultimately,these truncated TOP2αisoforms result in decreased drug activity against TOP2αin the nucleus and manifest drug resistance.Therefore,the complete characterization of the mechanism(s)regulating the alternative RNA processing of TOP2αpre-mRNA may result in new strategies to circumvent acquired drug resistance.Additionally,novel TOP2αsplice variants and truncated TOP2αisoforms may be useful as biomarkers for drug resistance,prognosis,and/or direct future TOP2α-targeted therapies.

Topoisomerase IB:a relaxing enzyme for stressed DNA

DNA topoisomerase I enzymes relieve the torsional strain in DNA;they are essential for fundamental molecular processes such as DNA replication,transcription,recombination,and chromosome condensation;and act by cleaving and then religating DNA strands.Over the past few decades,scientists have focused on the DNA topoisomerases biological functions and established a unique role of Type I DNA topoisomerases in regulating gene expression and DNA chromosome condensation.Moreover,the human enzyme is being investigated as a target for cancer chemotherapy.The active site tyrosine is responsible for initiating two transesterification reactions to cleave and then religate the DNA backbone,allowing the release of superhelical tension.The different steps of the catalytic mechanism are affected by various inhibitors;some of them prevent the interaction between the enzyme and the DNA while others act as poisons,leading to TopI-D NA lesions,breakage of DNA,and eventually cellular death.In this review,our goal is to provide an overview of mechanism of human topoisomerase IB action together with the different types of inhibitors and their effect on the enzyme functionality.

Phylogenetic positions of several amitochondriate protozoa——Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, En-tamoeba and Microsporida, were once widely considered to be the most primitive extant eu-karyotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and pos-sessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then com-bining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.

Study of isolation of fluoroquinolone-resistant Ureaplasma urealyticum and identification of mutant sites

OBJECTIVE: To study the resistance mechanism of clinical isolates of Ureaplasma urealyticum resistant to fluoroquinolones. METHODS: Thirteen isolates of Ureaplasma urealyticum resistant to six fluoroquinolones were selected out of 184 clinical isolates and their QRDRs (quinolone resistance-determining region) gyrA, gyrB, parC and parE were amplified by PCR. Sequencing results were compared to those susceptible reference strains and a comparison of deduced amino acid sequences were performed. RESULTS: Sequence comparison revealed a C to A change at 87nt of gyrA QRDR leading to the substitution of Asp95 with glutamic acid and a C to T change at 50nt of parC QRDR leading to the substitution of Ser80 with leucine. CONCLUSION: These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with fluoroquinolone resistance of Ureaplasma urealyticum.

A homeopathic nosode, Hepatitis C 30 demonstrates anticancer effect against liver cancer cells in vitro by modulating telomerase and topoisomerase Ⅱ activities as also by promoting apoptosis via intrinsic mitochondrial pathway

OBJECTIVE： Homeopathic nosodes have seldom been scientifically validated for their anticancer effects. This study was conducted to examine if a recently developed hepatitis C nosode has demonstrable anticancer potential in cancer cells in vitro.METHODS： Anticancer effects of Hepatitis C 30C（Hep C 30）, if any, were initially tested on three cancer cell lines, HepG2（liver cancer）, MCF-7（breast cancer） and A549（lung cancer） and one normal liver cell line WRL-68 cells and subsequently a more thorough study using further scientific protocols was undertaken on HepG2 cells（against WRL-68 cells as the normal control） as HepG2 cells showed better anticancer response than the other two. Three doses, one at 50% lethal dose（LD50） and the other two below LD50, were used on HepG2 cells subsequently. Protocols like apoptosis induction and its possible signaling mechanism were deployed using immunoblots of relevant signal proteins and confocal microscopy, with particular reference to telomerase and topoisomerase Ⅱ（Top Ⅱ） activities, two strong cancer biomarkers for their direct relationship with divisional activities of cells and DNAs. RESULTS： Hep C 30 induced apoptosis, caused distorted cell morphology typical of apoptotic cells, increased reactive oxygen species generation and produced increased DNA nicks. Further it enhanced pro-apototic signal proteins like Bax, cytochrome c and inhibited anti-apoptotic signal proteins, Bcl-2, cytochrome c and caspase-3, changed mitochondrial membrane potential and caused externalization of phosphatidylserine. The drug also decreased expression of two cancer biomarkers, Top Ⅱ and telomerase, consistent with its anticancer effect. CONCLUSION： Hep C 30 has demonstrable anticancer effects against liver cancer cells in vitro.

HER2 and topoisomerase Ⅱα： possible predictors of response to neoadjuvant chemotherapy for breast cancer patients

Background Surrogate markers may be used to assess the response to neoadjuvant treatment. The association between HER2 overexpression and favorable response to specific therapy in breast cancer is controversial, and the mechanism unclear. The purpose of the study was to evaluate HER2 and topoisomerase Ⅱα （Topo Ⅱα） as candidates for predicting the response to neoadjuvant chemotherapy in breast cancer patients.Methods Between 1999 and 2006, seventy-six breast cancer patients who had received neoadjuvant chemotherapy were studied. Regimens including either CEF （cyclophosphamide, epirubicin, 5-fluorouracil） or CMF （cyclophosphamide, methotrexate, 5-fluorouracil） were given in more than three cycles to this group of patients. Protein expression of HER2 and Topo Ⅱα were determined by immunohistochemistry. The primary endpoint was pathological and clinical response.Results Of 76 primary breast cancer samples, 27 （35.5%） showed overexpression of either HER2 （25%） or Topo Ⅱα protein （10.5%）, whereas in 7 tumors （9.2%） both proteins were found to be overexpressed. Ten patients （13.2%） had a clinical complete response and 21 （27.6%） had a clinical partial response. Five women （6.6%） had a pathological complete response, 5 （6.6%） had microscopic residual disease, and 46 （60.5%） had macroscopic residual disease. HER2 and Topo Ⅱα overexpression was significantly associated with a favorable response （P 〈0.001 and P=0.005 respectively）.Conclusion Our study suggests that HER2 and Topo Ⅱα overexpression could be predictors of the response to neoadjuvant chemothrapy in both the CEF and CMF arms.

Novel berberine derivatives:Design,synthesis,antimicrobial effects,and molecular docking studies

A series of berberine derivatives were synthesized by introducing substituted benzyl groups at C-9.All these synthesized compounds（4a–4m）were screened for their in vitro antibacterial activity against four Gram-positive bacteria and four Gram-negative bacteria and evaluated for their antifungal activity against three pathogenic fungal strains.All these compounds displayed good antibacterial and antifungal activities,compared to reference drugs including Ciprofloxacin and Fluconazole;Compounds 4f,4g,and 4l showed the highest antibacterial and antifungal activities.Moreover,all the synthesized compounds were docked into topoisomerase Ⅱ-DNA complex,which is a crucial drug target for the treatment of microbial infections.Docking results showed that H-bond,π-πstacked,π-cationic,andπ-anionic interactions were responsible for the strong binding of the compounds with the target protein-DNA complex.

Experiment alstudies on topoisomerase inhibitor camptothecin as an antipsoriatic agent

Objectives To elucidate the therapeutic mechanism of topical camptothecin (CPT) in treating psoriasis and to detect the effects of CPT on keratinocyte proliferation, differentiation and apoptosis Methods Mitotic numbers in mouse vaginal epithelium at estrus and numbers of scale with granular layer per 100 scales in mouse tail epidermis were determined in vivo Experiments on cultured normal human keratinocytes were performed using the methods of crystal violet staining, absorbance cell number converting, cell counting and quantitation of morphologic changes during differentiation, transglutaminase assay and nucleosomal enrichment assay Results Inhibition of cell proliferation and promotion of cell differentiation by camptothecin were showed in animal models and were reconfirmed in cultured keratinocytes Apoptosis was induced by camptothecin and was showed by activation of “tissue” transglutaminase and increase in nucleosomes The endonuclease activity was reduced by an endonuclease inhibitor aurintricarboxylic acid Conclusion The therapeutic effects of camptothecin on psoriasis can at least partly be explained by its multiple effects on DNA as a topoisomerase inhibitor

Topoisomerase I in Human Disease Pathogenesis and Treatments

Mammalian topoisomerase 1（TOP1） is an essential enzyme for normal development.TOP1 relaxes supercoiled DNA to remove helical constraints that can otherwise hinder DNA replication and transcription and thus block cell growth.Unfortunately,this exact activity can covalently trap TOP1 on the DNA that could lead to cell death or mutagenesis,a precursor for tumorigenesis.It is therefore important for cells to find a proper balance between the utilization of the TOP1 catalytic activity to maintain DNA topology and the risk of accumulating the toxic DNA damages due to TOP1 trapping that prevents normal cell growth.In an apparent contradiction to the negative attribute of the TOP1 activity to genome stability,the detrimental effect of the TOP1-induced DNA lesions on cell survival has made this enzyme a prime target for cancer therapies to kill fast-growing cancer cells.In addition,cumulative evidence supports a direct role of TOP1 in promoting transcriptional progression independent of its topoisomerase activity.The involvement of TOP1 in transcriptional regulation has recently become a focus in developing potential new treatments for a subtype of autism spectrum disorders.Clearly,the impact of TOP1 on human health is multifold.In this review,we will summarize our current understandings on how TOP1 contributes to human diseases and how its activity is targeted for disease treatments.