Evodiamine, a dual catalytic inhibitor of type I and II topoisomerases, exhibits enhanced inhibition against camptothecin resistant cells.
DNA topoisomerases are nuclear enzymes that are the targets for several anticancer drugs. In this study we investigated the antiproliferative activity against human leukaemia cell lines and the effects on topoisomerase 1 and 11 of evodiamine, which is a quinazolinocarboline alkaloid isolated from the fruit of a traditional Chinese medicinal plant, Evodia rutaecarpa. We report here the anti-proliferative activity against human leukaemia cells K562, THP-1, CCRF-CEM and CCRF-CEM/C1 and the inhibitory mechanism on human topoisomerases I and II, important anticancer drugs targets, of evodiamine. Evodiamine failed to trap [Topo-DNA] complexes and induce any detectable DNA damage in cells, was unable to bind or intercalate DNA, and arrested cells in the [G.sub.2] /M phase. The results suggest evodiamine is a dual catalytic inhibitor of topoisomerases I and II, with [IC.sub.50] of 60.74 and 78.81 [micro]M, respectively. The improved toxicity towards camptothecin resistant cells further supports its inhibitory mechanism which is different from camptothecin, and its therapeutic potential.
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Topoisomerases have become important drug targets for cancer therapy treatments (Teicher 2008; Pommier and Cushman 2009; Larsen et al. 2003). They are enzymes that can relieve torsional stress developed in cellular DNA molecules. Topoisomerase I (Topo I) acts by generating a transient single-strand break in the DNA double helix and it is associated with all DNA processes including tracking systems and maintaining genomic integrity during the cell cycle process through two trans-esterification reactions breaking and rejoining DNA strands (Wang 1996; Chen and Liu 1994; Champoux 2001). Topoisomerase II (Topo II) acts by generating a transient double-strand break in a reaction which requires ATP, and it also participates in DNA processes including separation of daughter chromosomes, recombination and chromosome condensation (Wang 2002). Initially the DNA strand is broken by the enzyme forming a topoisomerase-DNA covalent complex (cleavage complex) leaving a DNA terminus with a hydroxyl group on the sugar. Then, the deoxyribose hydroxyl group at the broken end attacks the enzyme-DNA linkage, forming the DNA phosphodiester linkage and freeing the enzyme for the next round of reactions (Wang 1994). When a nucleic acid tracking system, such as a replication or transcription complex, attempts to traverse the cleavage complex, it converts this transient enzyme/DNA interaction to a permanent double-strand break as if the topoisomerase was trapped in a [topoisomerase-DNA]-drug ternary complex (Liu 1989; Pommier et al. 1998). Cleavage complexes are therefore potentially deleterious to the cell. Agents that increase the level of topoisomerase DNA cleavage complexes are identified as topoisomerase poisons, which stabilise the covalent Topo-DNA cleavage complexes formed in the trans-esterification reaction and prevent the re-ligation of DNA.
In contrast to the topoisomerase poisons, compounds that interfere with any of the other steps in the catalytic cycle are classified as catalytic inhibitors. Catalytic topoisomerase inhibitors do not generate DNA strand breaks and they act at a step upstream of DNA cleavage. The catalytic Topo II inhibitors include a variety of compounds that interfere with the binding between DNA and topoisomerase, stabilise noncovalent DNA topoisomerase complexes, or inhibit Topo II-ATP binding (Larsen et al. 2003).
Topoisomerase inhibitor-based cancer drugs at present in clinical use include Irinotecan for colorectal cancer (Wiseman and Markham 1996), Hycamtin for lung cancer (Riemsma et al. 2010) and Novantrone for leukaemia (Mauro et al. 2002; Willmore et al. 2011). Both Irinotecan and Hycamtin (Topotecan) are derivatives of camptothecin (CPT) and target Topo I enzyme as Topo I poisons. Novantrone is a synthetic anthracenedione that acts as a Topo II inhibitor and a DNA intercalator. In addition to the search for CPT derivatives as topoisomerase inhibitors, many non-CPT derivatives have been synthesised, such as indolocarbazoles and indenoisoquinolines, some of which are now in clinical evaluation (Long and Balasubramanian 2000; Liu 1989). Semisynthetic Topo II poisons, amsacrine (m-AMSA) and etoposide, stabilise the covalent Topo II-DNA cleavage complex on both DNA strands. Dual poisons of both Topo I and II have also been developed, for example aclarubicin and intopolincine (Hajji et al. 2005; Riou et al. 1993).
Evodiamine, a characteristic quinazolinocarbolin alkaloid from Evodia rutaecarpa, has been reported to inhibit the invasion and metastasis of tumours and induces cell death in several types of cancer cell lines including human acute leukaemia CCRF-CEM cells ([IC.sub.50] 4.53 [micro]M) (Adams et al. 2007), human androgen independent prostate cancer PC-3 cells ([IC.sub.50] 1.53 [micro]M) (Huang et al. 2005), human breast cancer MCF-7 cells ([IC.sub.50] 6.02 [micro]M) (Chan et al. 2009), human melanoma A375-S2 cells ([IC.sub.50] 15 [micro]M) (Zhang et al. 2003), and murine fibrosarcoma L929 cells ([IC.sub.50] 20.3 [micro]M) (Zhang et al. 2004). In addition, it has also been reported that evodiamine caused the mitotic arrest and a consequent apoptosis in CCRF-CEM cells through the enhancement of polymerised tubulin levels (Huang et al. 2004). So far there is only one report on the inhibitory activity of evodiamine on human Topo I indicating that evodiamine stabilises the Topo I-DNA-cleavable complex (Chan et al. 2009). There is no report on its inhibition characteristics of Topo II or its cytotoxicity on CPT-resistant cells. In this study we aim to understand further the mechanism of evodiamine inhibition towards Topo I and II enzymes by comparing its activities with the known inhibitors CPT for Topo I, and m-AMSA and etopo-side for Topo II (Fig. 1). Here, we have carried out studies including anti-proliferation against human leukaemia cell lines, K562, THP-1 CCRF-CEM and CCRF-CEM/C1 (a camptothecin resistant cell line), topoisomerase relaxation and decatenation assays, measurement of DNA strand breaks using the comet assay, DNA-intercalating and footprinting assays, and cell cycle analysis.
Materials and methods
Camptothecin, etoposide and amsacrine were obtained from Sigma-Aldrich (Dorset, UK) and prepared (10 mM) in dimethyl-sulphoxide (DMSO). All solvents used for HPLC and mass spectrometry were HPLC grade and obtained from Fisher Scientific (Loughborough, UK). Water used in the extraction and HPLC analysis was from a Millipore Simplicity 185 Purification System (Walford, UK). All the other chemicals and solvent were laboratory grade and used without further purification.
E. rutaecarpa was collected in 1999 from Taiping county, Fujian province, PR China, and was authenticated by Dr. Lihong Wu, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SUTCM), PR China. A voucher specimen was deposited in the herbarium of Institute of Chinese Materia Medica, SUTCM. Evodiamine was isolated from the fruit of E. rutaecarpa adopting a literature method (Liu et al. 2005). Evodiamine was fully characterised by NMR ([.sup.1]H, [.sup.13]C, 2D-HSQC, 2D-HMBC) and mass spectra. NMR spectra were obtained using a Bruker Avance 500 NMR spectrometer (Bruker, Coventry, UK). MS spectra were measured using a Waters Q-Tof Micro mass spectrometer (Waters, MA, USA). The data were compared with those in the literature (Bergman and Bergman 1985). The purity was >98% as determined by HPLC using standard compound from Shanghai R&D Center for Standardization of Traditional Chinese Medicines (Shanghai, PR China).
Cell culture and cytotoxicity assay
Human K562 myelogenous leukaemia cells, THP-1 acute monocytic leukaemia cells, CCRF-CEM leukaemic lymphoblast cells and CCRF-CEM/C1 CPT-resistant leukaemic lymphoblast cells were obtained from the American Type Culture Collection (ATCC, UK). Cells were grown at 37 [degrees] C in a humidified atmosphere containing 5% [CO.sub.2] in RPMI-1640 medium with 10% of fetal bovine serum and 2 mM of glutamine. The viability of cells was determined using the MTT [3-(4,5-dimethyl thiazol-2y1)-2,5-diphemyltetrazolium bromide (Sigma, Dorset, UK) assay as previously described (Mosmann 1983).
Topoisomerase relaxation assay
Topoisomerase relaxation assays and decatenation assays were carried out as previously described (Peixoto et al. 2010). Supercoiled pBR322 DNA (0.3 [micro]g) was incubated with 1 U of human Topo I (Inspiralis, Norwich, UK) in 30 [micro]l of relaxation buffer (20 mM Tris-HCI pH 7.5, 200 mM NaCl, 0.25 mM EDTA and 5% glycerol) at 37 [degrees] C for 30 min in the presence of varying concentrations of the compounds under study. 100 [micro]M of Topo I poison CPT was used as a positive control.
5 U of the enzyme was used to study the intercalating ability of compounds based on the Topo I relaxation assay (Peixoto et al. 2010).
The ability of compounds to stabilise the covalent DNA enzyme reaction intermediate were evaluated by incubating supercoiled pBR322 DNA (0.3 [micro]g) with 10 U of human Topo II for the relaxation assay. After incubation with enzyme, the reaction was further incubated with 0.1 mg/ml proteinase K and 0.2% SDS for 30 min at 37 [degrees] C.
Topoisomerase II decatenation assay
kDNA (200 ng) was incubated with 1 U of human Topo II (Inspirails, Norwich, UK) in assay buffer (50 mM Tris HCI pH 7.5, 125 mM NaCl, 10 mM Mg[Cl.sub.2], 5 mM DTT and 100 [micro] g/ml albumin) with 1 mM of ATP at 37 [degrees] C for 30 min in the presence of different concentrations of the compounds under study. The known Topo II poison, m-AMSA, was used as a positive control.
Topoisomerase catalytic reactions were stopped by adding 18 [micro]l of stop buffer (40% sucrose, 100 mM Tris pH7.5, 10 mM disodium EDTA and 0.05% bromophenol blue). DNA samples were extracted with a mixture of chloroform/iso amyl alcohol (24:1) and run on a 1% agarose gel at 4 V/cm in a TAE buffer for 15 h. Gels were stained with ethidium bromide (0.5 [micro]g/ml in distilled water) for 1 h and destained with distilled water for 30 min. Similar experiments were performed using ethidium bromide (0.5 [micro]g/ml) containing agarose gel to achieve separation of nicked and relaxed species. Gel were visualised under UV light and photographed. For quantitative determinations, the integrated intensities of the ethidium bromide fluorescence of the bands (supercoiled form for relaxation assay and minicircled form for the decatenation assay) were quantified and calculated using image) software from Wayne Rasband (Washington, USA).
Single cell gel electrophoresis (Comet) assay
K562 cells (2 ml, 2.5 x [10.sup.4] cells/ml) in exponential growth were treated with various concentrations of compounds under study for 1 h. DMSO (1%) treated cells were taken as a negative control. The single cell gel electrophoresis (comet) assay developed to allow visualisation of DNA strand break damage in individual cells was performed as previously described in detail (Spanswick et al. 2010).
Cell cycle analysis
K562 cells (2 ml, 5 x [10.sup.5] cells/ml) in exponential growth were treated with different concentrations (20, 40 and 80 [micro]M) of compounds under study for 1 h and then re-suspended in 2 ml of RPMI-1640 medium for a time course (0 h, 8 h, 24 h, 32 h, 48 h and 72 h). At the end of each time point, cells were centrifuged (1500 rpm, 5 min) and the pellets were suspended in 1 ml of PBS. Absolute ethanol (3 ml) was added to the cells with vortex for fixation. All the cells were stored at -20 [degrees] C until analysis. On the day of analysis, cells were centrifuged (2000 rpm, 10 min), washed with PBS, suspended in 500 [micro]l of PI solution (50 [micro]g/ml propidium iodide, 100 [micro]g/ml RNase A and 0.05% Tritin X-100), and incubated at 37 [degrees] C for 30 min. After incubation, 3 ml of PBS was added, and the cells were pelleted (1500 rpm, 5 min), suspended in PBS at a final cell density of 5 x [10.sup.5] cells/ml and transferred to a Falcon 2054 tube (BD Bioscience, UK) for flow cytometry analysis. Analysis of 20,000 events was done on a CyAn ADP flow cytometer from Beckman Coulter (High Wycombe, UK) using Summit 4.3 version and ungated data were gathered within 1 h.
Data and statistical analysis
All data are presented as mean [+ or -] standard deviation (SD) from at least three separate experiments. The [IC.sub.50] curves were estimated by plotting percentage of viability from the triplicate treatment versus concentration. The [IC.sub.50] value was defined and calculated using the 4 full parameter equation as defined by the Grafit software 5.0.4 version from Erithacus Software (Surrey, UK). Statistical calculations of the data were performed using an unpaired Student's t-test. Statistical significance was set at p < 0.05.
Anti-proliferation study of evodiamine against human leukaemia cell lines K562 and THP-1
An MTT-based assay was used to evaluate the anti-proliferative activities of evodiamine, m-AMSA and etoposide against the K562 and THP-1 cells. The viabilities of THP-1 cells during 3 days continuous treatments are shown in Fig. 2A, showing that evodiamine and the positive control m-AMSA, at a concentration of 5 [micro]g/ml, exhibited anti-proliferative activity against THP-1 cells compared with control (p <0.05).
Subsequently, the [IC.sub.50] values of evodiamine on the proliferation of human leukaemia THP-1 and K562 cell lines were compared with two known anti-tumour drugs, m-AMSA and etoposide, after a 1 h treatment. As shown in Fig. 2B, evodiamine was more effective against the K562 cell line with an [IC.sub.50] value of 34.43 [micro]M compared to the known topoisomerase 11 poison etoposide ([IC.sub.50] 73.76 [micro]M), but was less active than m-AMSA ([IC.sub.50] 0.91 and 34.73 [micro]M for K562 and THP-1, respectively) in both cell lines.
Effects of evodiamine on topoisomerase activity
The ability of evodiamine to inhibit topoisomerase activity was examined by measuring the relaxation of supercoiled plasmid pBR322 DNA by Topo I in the absence and presence of ethidium bromide (EtBr), and the decatenation of kinetoplast DNA by Topo II. The gels without EtBr allow the detection of compounds inhibiting DNA relaxation by Topo I, while EtBr containing gels indicate whether or not the inhibiting activities of evodiamine might be due to the effects on the Topo I-DNA cleavage complexes. Evodiamine exhibited Topo I inhibitory activities since the relaxation of supercoiled DNA was totally inhibited (Fig. 3A). On the other hand, CPT did not unwind DNA to any detectable extent at 100 [micro]M, and the intensity of the nicked band was increased (Fig. 3B), which indicated the formation of the enzyme-DNA cleavage complex with one broken DNA strand. Furthermore, evodiamine did not show any evidence of increasing the intensity of nicked band in the EtBr containing gel comparing to that of CPT (Fig. 3B) and thus indicating evodiamine is not acting as a Topo I poison.
Evodiamine also showed Topo II inhibitory activity and was more effective as an inhibitor compared with a well-known Topo II poison, m-AMSA (Fig. 3C). Catenated kDNA (90%) remained in the well containing 100 [micro]M evodiamine (Fig. 3C, lane 4), while only 17.8% of catenated kDNA was in the well after incubating with 100 [micro]M of m-AMSA (Fig. 3C, lane 3).
The inhibition of human Topo I and Topo II by evodiamine was further evaluated by the Topo I relaxation assay and Topo II decatenation assay, respectively, in a concentration-dependant manner (Fig. 3). Evodiamine was identified as a dual inhibitor against both human Topo I and II enzymes with [IC.sub.50] 60.74 [micro]M and 78.81 [micro]M, respectively, and is a better inhibitor than m-AMSA ([IC.sub.50] 146.72 [micro]M) of Topo II. Mechanistic studies were therefore carried out to investigate which type of inhibitor evodiamine was acting as.
Effect of evodiamine on Topo 11-mediated DNA cleavage
Topo II poisons such as m-AMSA are known to stabilise the cleavage complex that leads to DNA strand breaks, while catalytic inhibitors such as aclarubicin can protect cells against Topo II poison-induced DNA damage. We tested whether evodiamine could act as a poison and stimulate formation of DNA cleavage complexes of Topo II, using the Topo II cleavage assay, with 100 [micro]M m-AMSA as a positive control (Fig. 4, lane 3). In contrast to m-AMSA, which stimulated formation of cleavage complexes as indicated by the band of linearised plasmid DNA (Fig. 4, lane 3), evodiamine had no effect on Topo II cleavage activity even at a concentration of 100 [micro]M (Fig. 4, lane 4) and Topo II activity was only partially inhibited at the high concentration of enzyme (10U). Therefore, evodiamine was identified as a topoisomerase catalytic inhibitor, which inhibits the catalytic activity of topoisomerase rather than inducing the formation of a cleavage complex.
Interaction between evodiamine and DNA
During agarose gel electrophoresis, plasmid pBR322 the drug-free DNA is negatively supercoiled and migrates through the gel as a single band, but its profile changes significantly in the presence of an intercalating agent (EtBr). At low concentrations of EtBr, the DNA relaxes and migrates through the agarose gel slower than the negative supercoiled DNA (Fig. 5, lanes 4 and 5). As the concentration increases, the DNA molecules wind in the oppposite direction and generate positive supercoiled DNA. When the DNA is fully positively supercoiled, it migrates as a single band with a mobility close to that of the negatively supercoiled DNA (Fig. 5, lane 7). Therefore, a typical intercalating agent should show a charactersitic profile of decreased then increased migration with increasing concentration.
As indicated in Fig. 5, both CPT and evodiamine did not give the intercalator specific profile with increasing concentrations when incubated with 5 U of human Topo I. In addition, DNase I footprinting did not provide any evidence of evodiamine binding to DNA, since no change in DNase I cleavage was observed at concentrations up to 100 [micro]M of evodiamine (data not shown).
Effect of evodiamine on cell cycle distribution in K562 cells
To investigate the effect of evodiamine on cell cycle progression, K562 cells were exposed to evodiamine for 1 h, and then grown in drug-free medium. Cell cycle distribution was determined by flow cytometric analysis. As shown in Fig. 6, evodiamine clearly induced [G.sub.2]/M arrest in exponentially growing cells treated with 20 [micro]M. The arrest was observed by 24 h and persisted for a further 48 h. [G.sub.2]/M arrest was demonstrated previously as an effect of topoisomerase 11 inhibitors, such as etoposide (Lu et al. 2009).
We next evaluated the possibility that the cytotoxicity of evodiamine in K562 cells was not attributable to a high level of DNA damage. The potential of evodiamine to induce DNA strand breaks in K562 cells was assessed using the single cell gel electrophoresis (Comet) assay with the Topo I poison CPT as a positive control. No strand breaks were induced by evodiamine from 1 to 100 [micro]M, whereas 10 [micro]M CPT-treated K562 cells exhibited highly damaged DNA (Fig. 7). The Topo I poison CPT stabilises the enzyme-DNA cleavage complex, which leads to strand breaks in chromosomal DNA resulting in cell death. Consistent with the observation in both Topo I and ll DNA assays in the presence of evodiamine, evodiamine did not cause any significant DNA damage in cells. The mechanism of topoisomerase inhibition by evodiamine was therefore considered as catalytic for both Topo 1 and II.
Effect of evodiamine on CPT-resistant cells
Evodiamine has enhanced effective inhibitory activity (Fig. 8A) when incubated with lower concentrations of enzyme, and the activity is reduced with increasing amounts of enzyme. This unusual result has led to us investigate the potential of evodiamine as an inhibitor of CPT-resistant cells. It has been reported that the level of Topo enzymes is decreased in the resistant cells (Kapoor et al. 1995). Anti-proliferation assays of evodiamine on human leukaemic lymphoblast CCRF-CEM cells and CPT-resistant CCRF-CEM/C1 cells were performed and the [IC.sub.50] were 4.70 [+ or -] 0.34 [micro]M and 2.92 [+ or -] 0.12 [micro]M respectively (Fig. 8B). Indeed, evodiamine has enhanced anti-proliferative effect on CPT-resistant cells which were >40 fold resistant to CPT.
Topoisomerase inhibitors are clinically used for leukaemia, colorectal and lung cancer. Evodiamine has previously been reported to have anti-invasive and anti-metastatic effects on Lewis lung carcinoma (LLC) and colon 26-L5 carcinoma with [IC.sub.50] values of 2.4 [micro]M and 3.7 [micro]M, respectively (Ogasawara et al. 2002). In addition, it has also been reported that evodiamine only inhibited normal human mammary epithelial cell (H184B5H5/M10) proliferation at 10 [micro]M on day 4 (Chen et al. 2010). Therefore, the aims of this study were to evaluate the anti-proliferative activity of evodiamine in leukaemia and CPT-resistant cell lines, and to further understand the mechanism of its inhibitory activity on both human Topo I and II enzymes. Our anti-proliferation results on CCRF-CEM cells ([IC.sub.50] of 4.70 [+ or -] 0.34 [micro]M) agree well with the data reported by Adams et al. (2007), but not with that by Huang et al. (2004) ([IC.sub.50] 0.57 [+ or -] 0.05 [micro]M). The differences were likely due to the duration of treatment of cells with evodiamine. Our data were recorded after 1 h treatment rather than 24 h, as reported by Huang et al. However, our data agreed that evodiamine induced apoptotic cell death with a cell cycle arrest at the [G.sub.2]/M phase. Evodiamine was demonstrated to have cytotoxicity against both K562 and THP-1, but m-AMSA (K562, [IC.sub.50] 0.91 [micro]M) and etoposide (THP-1, IC50 9.61 [micro]M) still out-performed evodiamine in either assay.
Evodiamine at 30-100 [micro]M inhibited human Topo 1 enzyme activity in the relaxation assay and Topo II activity in the decatenation assay. In neither assay was a [Topo-DNA]-evodiamine cleavage complex detected at 100 p.M. The results suggest evodiamine is neither a Topo I nor a Topo II poison. Recently, Chan et al. (2009) reported that evodiamine stabilised Topo 1-DNA cleavable complex as the inhibitory mechanism. Their results were not conclusive, however, in view of (a) only two concentrations of evodiamine being tested (1 and 3 [micro]M), (b) incomplete conversion of supercoiled DNA to the cleavage complex and (c) not using a positive control to give a clear indication of the enzyme activity.
To further our claims that evodiamine acts as a catalytic inhibitor for both Topo I and II, we examined whether DNA modifications occurred after treatment of evodiamine by using ethidium bromide as a DNA intercalator, comet assay for DNA strand break damage, and DNA footprint analysis. The results clearly indicate that there is no evidence of DNA interaction or damage induced by evodiamine. In addition, evodiamine does not inhibit the formation of cleavage complexes of Topo I and II with DNA by distorting its gross structure. This often occurs with drugs that intercalate or bind in the minor groove of DNA (Pommier et al. 1985a, b; Woynarowski et al. 1989). This is confirmed by the two independent DNA binding assays. We therefore propose evodiamine inhibits both Topo I and II enzymes binding to DNA.
In this study we noted that evodiamine enhanced effective inhibitory activity when incubated with lower concentrations of enzyme, and the activity was reduced with increasing amounts of enzyme. In contrast, the inhibitory activity of CPT increases when Topo I is over expressed (Sugimoto et al. 1990; Kanzawa et al. 1990). Previously it was reported that levels of DNA-protein complex formation resulting from cell exposure to CPT were found to be progressively reduced in the CPT-resistant cells (CEM/C1 and CEM/C2), despite equivalent CPT accumulation in the drug-sensitive and -resistant cells (Kapoor et al. 1995). It was concluded that the reduction was due to the decrease in Topo I catalytic activity associated with reduced cellular levels of both Topo I protein and mRNA. CPT is therefore not so effective when the concentration of Topo I is low in cells. In contrast, evodiamine gave an enhanced anti-proliferative activity in tumour cells with acquired resistance to CPT, confirming that CPT and evodiamine have different inhibitory mechanisms.
Evodiamine exhibited potent anti-proliferative activity against human leukaemia K562 (I[C.sub.50] 34.43 [micro]M), THP-1 (I[C.sub.50] 58.42 [micro]M), CCRF-CEM (I[C.sub.50] 4.70 [micro]M) and CCRF-CEM/C1 (a camptothecin resistant cell line, I[C.sub.50] 2.92 [micro]M). It is a dual catalytic inhibitor of both human Topo I and II with I[C.sub.50] 60.74 and 78.81 [micro]M respectively. Since evodiamine did not form [Topo-DNA]-evodiamine ternary complex at 100 [micro]M, it is identified as a catalytic inhibitor, i.e. it interferes with the enzyme catalytic cycle rather than stabilizing cleavage complexes. In addition, its enhanced anti-proliferative activity in CPT resistant cells has demonstrated its therapeutic potential as an anti-cancer drug.
Conflict of interest statement
None of the authors has any potential conflicts of interest.
Abbreviations: CPT, camptothecin; DMSO, dimethylsulphoxide; EtBr, ethidium bromide; m-AMSA, amsacrine; MTT, 3-(4,5-dimethyl thiazol-2yl)-2,5-diphemyltetrazolium bromide; Nck, nicked; Rely relaxed; Sc, supercoiled; Topo I, topoisomerase I; Topo II, topoisomerase II.
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Xiaobei Pan (a), Janet M. Hartley (b) John A. Hartley (b), Kenneth N. White (a), Zhengtao Wang (a), (c), S.W. Annie Bligh (a), *
(a.) Institute for Health Research and Policy, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK
(b.) Cancer Research UK Drug DNA Interactions Research Group, UCL Cancer Institute, University College London, Paul O'Gorman Building; London WC1E 6BT, UK
(c.) The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medico, Shanghai University of Traditional Chinese Medicine, 1200 Cailon Road. Zhangjiang Hi-tech Park, Shanghai 201210, China
* Corresponding author. Tel.: +44 207 133 2142; fax: +44 207 133 4149. E-mail address: a.bligh@Iondonmet.ac.uk (S.W.A. Bligh).
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