Indenoindolone derivatives as topoisomerase II–inhibiting anticancer agents

Based on known heterocyclic topoisomerase II inhibitors and anticancer agents, various indenoindolone derivatives were predicted as potential topoisomerase II–inhibiting anticancer agents. They are hydrazones, (thio)semicarbazones, and oximes of indenoindolones, and indenoindolols. These derivatives with suitable substitutions exhibited potent specific inhibition of human DNA TopoIIα while not showing inhibition of topoisomerase I and DNA intercalation, despite the fact that parent indenoindolones are known poor/moderate inhibitors of topoisomerase II. The potent topoisomerase II inhibitor indenoindolone derivatives exhibited good anticancer activities compared to etoposide and 5-fluorouracil, and relatively low toxicity to normal cells. These derivatizations of indenoindolones were found to result in enhancement of anticancer activities.     

Antitumor agents 216. Synthesis and evaluation of paclitaxel-camptothecin conjugates as novel cytotoxic agents

Five conjugates (16-20) composed of a paclitaxel and a camptothecin derivative joined by an imine linkage were synthesized and evaluated as cytotoxic agents and as inhibitors of DNA topoisomerase I. All of the conjugates were potent inhibitors of tumor cell replication with improved activity relative to camptothecin. Significantly, compounds 16-18 were more active than paclitaxel and camptothecin against HCT-8 (colon adenocarcinoma) cell replication, and the spectrum of activity was different from a simple mixture of paclitaxel and camptothecin. All of the conjugates were significantly less potent than camptothecin as inhibitors of human topoisomerase I in vitro with 16, 18, and 19 showing only marginal activity at 50 microM. Based on activity against drug-resistant cell line replication, one could conclude that the conjugates are simply acting as 'weak taxanes', but the spectrum of activity, particularly against MCF-7 and HCT-8, strongly suggests that a novel mechanism of action has been achieved through conjugation.     

Cytotoxicity and DNA topoisomerase inhibitory activity of benz[f]indole-4,9-dione analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 microg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 microg/ml)) compared to colon (IC50>20.0 microg/ml) and stomach (IC50>20.0 microg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

Novel DNA Topoisomerase IIα Inhibitors from Combined Ligand- and Structure-Based Virtual Screening

DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, “poisons”, binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the β-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the β-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors.     

6-Arylamino-7-chloro-quinazoline-5,8-diones as novel cytotoxic and DNA topoisomerase inhibitory agents

A series of 6-arylamino-7-chloro-quinazoline-5,8-diones were prepared and evaluated for their in vitro cytotoxicity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer). The preliminary structure-activity relationship has been described for providing further development of potent antitumor agents. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities have been assessed.     

Cell cycle effects of the DNA topoisomerase inhibitors camptothecin and m-AMSA in lymphoblastoid cell lines from patients with Fanconi anemia

Patients with the autosomal recessive disorder Fanconi anemia (FA) present with progressive pancytopenia, skeletal abnormalities and a predisposition to leukemia. In addition to elevated rates of spontaneous chromosome aberrations occurring in cultured fibroblasts and lymphoblastoid cell lines, an increased susceptibility to DNA cross-linking agents and oxygen has been found. To explain this hypersensitivity to clastogenic agents a defective function of DNA topoisomerase I or II could be invoked, a suggestion which is supported by the co-localization of the DNA topoisomerase I gene and a putative FA gene to chromosome 20q. In order to investigate the function of DNA topoisomerases in FA, the sensitivity of lymphoid B-cell lines derived from FA patients and control cell lines to inhibitors of DNA topoisomerases I and II was compared using continuous bromodeoxyuridine labeling and bivariate Hoechst/ethidium bromide flow cytometry. Both agents inhibited cell proliferation mainly by arresting cells in the G2 phase of the cell cycle. However, no difference was found in sensitivity towards both DNA topoisomerase inhibitors between control and FA cell lines.     

Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC₅₀<20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC₅₀=0.4 μg/ml)) compared to colon (IC₅₀>20.0 μg/ml) and stomach (IC₅₀>20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

Synthesis and biological evaluation of histone deacetylase and DNA topoisomerase II-Targeted inhibitors

HDAC inhibitors enable histones to maintain a high degree of acetylation. The resulting looser state of chromatin DNA may increase the accessibility of DNA drug targets and consequently improve the efficiency of anticancer drugs targeting DNA, such as Topo II inhibitors. A novel class of nucleoside-SAHA derivatives has been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities toward histone deacetylases and Topo II, and their cytotoxicities in cancer cell lines, were evaluated. Among the synthesized hybrid compounds, compound 16b showed the potent HDAC inhibitory activity at a low nanomolar level and exhibited antiproliferative activity toward cancer cell lines including MCF-7 (breast), HCT-116 (colon), and DU-145 (prostate) cancer cells at a low micromolar level. Moreover, compound 16a showed HDAC6-selectivity 20-fold over HDAC1.     

Overproduction of topoisomerase II in an ataxia telangiectasia fibroblast cell line: comparison with a topoisomerase II-overproducing hamster cell mutant.

Ataxia telangiectasia (AT) cell lines are characterised by their hypersensitivity to ionizing radiation and bleomycin, and their failure to inhibit DNA synthesis after DNA damage. A recent report [Singh et al. (1988) Nucl. Acids Res. 16, 3919-3929] indicated that a reduction in topoisomerase II (topo II) activity was a feature of AT lymphoblast cell lines. We have studied the possible role of DNA topoisomerases in determining the phenotype of an AT fibroblast cell line. AT5BIVA cells are sensitive to the topo II inhibitors etoposide (VP16) and amsacrine (m-AMSA), compared to normal human fibroblasts (MRC5-V1 and VA13). AT5BIVA cells express a 3-fold higher level of topo II protein than MRC5-V1 cells, and 6-fold higher than VA13. This is reflected in elevated topo II activity in AT5BIVA cells. Untransformed AT5BI cells also show elevated topo II activity compared to untransformed normal cells. The extent of overproduction of topo II in AT5BIVA cells is comparable with that seen in a mutant Chinese hamster cell line, ADR-1, which is similarly hypersensitive to both bleomycin and topo II inhibitors. However, ADR-1 cells show neither hypersensitivity to ionizing radiation nor abnormal inhibition of DNA synthesis following DNA damage. Topo II overproduction per se does not appear sufficient to generate an "AT-like" phenotype. AT5BIVA cells express a reduced level of topoisomerase I (topo I) and are hypersensitive to the topo I inhibitor, camptothecin. ADR-1 cells express a normal level of topo I, indicating that a reduction in the level of topo I is not the inevitable consequence of an elevation in topo II.     

Topoisomerase I-mediated DNA cleavage as a guide to the development of antitumor agents derived from the marine alkaloid lamellarin D: triester derivatives incorporating amino acid residues

The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure-activity relationship study in the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8, 14 and 20 of 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-6-one pentacyclic planar chromophore typical of the parent alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisomerase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine or alanine residues, or a related amino side chain, stabilize topoisomerase I-DNA complexes. The DNA cleavage sites detected at T downward arrow G or C downward arrow G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with camptothecin which stimulates topoisomerase I-mediated cleavage at T downward arrow G only. In the DNA relaxation and cleavage assays, the corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5-6 double bond in the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temperature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian (IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and LoVo-Dox cells resistant to doxorubicin) cancer cells (but not with HT29 colon carcinoma cells), the most cytotoxic compounds correspond to the most potent topoisomerase I poisons. The observed correlation between cytotoxicity and topoisomerase I inhibition strongly suggests that topoisomerase I-mediated DNA cleavage assays can be used as a guide to the development of superior analogues in this series. LAM-D is the lead compound of a new promising family of antitumor agents targeting topoisomerase I and the amino acid derivatives appear to be excellent candidates for a preclinical development.     

In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols

Several herbal teas contain bioactive compounds that have been associated with a lower risk of chronic diseases including cancer. The aim of this study was to evaluate the chemopreventive activity of tea aqueous extracts and selected constituent pure polyphenols using a battery of in vitro marker systems relevant for the prevention of cancer. The effects of (-) epigallocatechin gallate (EGCG), quercetin (Q), gallic acid (GA), green tea (GT, Camellia sinensis), ardisia tea (AT, Ardisia compressa) and mate tea (MT, Ilex paraguariensis) extracts were tested. Cytotoxicity, TPA-induced ornithine decarboxylase (ODC) and quinone reductase (QR) activities were evaluated in vitro using HepG2 cells. The topoisomerase inhibitory activity was also tested, using the Saccharomyces cerevisiae yeast system. Results suggest that MT, AT and GT are cytotoxic to the HepG2 cells, with MT demonstrating dominant cytotoxicity. EGCG showed greater cytotoxicity than Q and GA against HepG2 cells. The greatest inhibition (82%) of TPA-induced ODC activity was shown by Q, with 25 microM (IC50 = 11.90 microM). Topoisomerase II, but not topoisomerase I, was the cellular target of MT, AT, EGCG, Q and GA, which acted mainly as true catalytic inhibitors. The cytotoxic activity and the inhibition of topoisomerase II may contribute to the overall chemopreventive activity of AT and MT extracts. Ardisia and mate teas may thus share a public health potential as chemopreventive agents.     

Identification of topoisomerases as molecular targets of cytosporolide C and its analog

Cytosporolide (Cytos) A–C, isolated from the fungus Cytospora sp., have anti-microbial activity, but their molecular targets in mammalian cells are unknown. We have previously reported the total synthesis of Cytos A by biomimetic hetero-Diels-Alder reaction. In this study, to examine the novel bioactivity of Cytos, we synthesized Cytos C and measured cell growth-inhibiting activities of 7 compounds, including Cytos A and C, in several human cancer cell lines. Among these compounds, Cytos C and tetradeoxycytosporolide A (TD-Cytos A), a model compound for the synthesis of Cytos A, had anti-proliferative effects on cancer cells, and TD-Cytos A exhibited stronger activity than Cytos C. In vitro topoisomerase-mediated DNA relaxing experiments showed that TD-Cytos A inhibited the activities of topoisomerase I and II, whereas Cytos C targeted only topoisomerase I. These data suggest that the anti-proliferative activities of Cytos correlate with the inhibition of topoisomerases and implicated TD-Cytos A as a novel anti-cancer drug that suppresses the activities of topoisomerase I and II.     

Synthesis,antitumor activity and DNA binding features of benzothiazolyl and benzimidazolyl substituted isoindolines

In this paper novel isoindolines substituted with cyano and amidino benzimidazoles and benzothiazoles were synthesized as new potential anti-cancer agents. The new structures were evaluated for antiproliferative activity, cell cycle changes, cell death, as well as DNA binding and topoisomerase inhibition properties on selected compounds. Results showed that all tested compounds exerted antitumor activity, especially amidinobenzothiazole and amidinobenzimidazole substituted isoindolin-1-ones and benzimidazole substituted 1-iminoisoindoline that showed antiproliferative effect in the submicromolar range. Moreover, the DNA-binding properties of selected compounds were evaluated by biophysical and biochemical approaches including thermal denaturation studies, circular dichroism spectra analyses and topoisomerase I/II inhibition assays and results identified some of them as strong DNA ligands, harboring or not additional topoisomerase II inhibition and able to locate in the nucleus as determined by fluorescence microscopy. In conclusion, we evidenced novel cyano- and amidino-substituted isoindolines coupled with benzimidazoles and benzothiazoles as topoisomerase inhibitors and/or DNA binding compounds with potent antitumor activities.     

Mithramycin inhibits etoposide resistance in glucose-deprived HT-29 human colon carcinoma cells

Physiological cell conditions such as glucose deprivation and hypoxia play roles in the development of drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha, rendering cells resistant to topo II target drugs such as etoposide. Thus, targeting tumor-specific conditions such as a low glucose environment may be a novel strategy in the development of anticancer drugs. On this basis, we established a novel screening program for anticancer agents with preferential cytotoxic activity in cancer cells under glucose-deprived conditions. We recently isolated an active compound, AA-98, from Streptomyces sp. AA030098 that can prevent stress-induced etoposide resistance in vitro. Furthermore, LC-MS and various NMR spectroscopic methods identified AA-98 as mithramycin, which belongs to the aureolic acid group of antitumor compounds. We found that mithramycin prevents the etoposide resistance that is induced by glucose deprivation. The etoposide-chemosensitive action of mithramycin was just dependent on strict low glucose conditions, and resulted in the selective cell death of etoposide-resistant HT-29 human colon cancer cells.     

Centromere fragmentation is a common mitotic defect of S and G2 checkpoint override

DNA damaging agents, including those used in the clinic, activate cell cycle checkpoints, which blocks entry into mitosis. Given that checkpoint override results in cell death via mitotic catastrophe, inhibitors of the DNA damage checkpoint are actively being pursued as chemosensitization agents. Here we explored the effects of gemcitabine in combination with Chk1 inhibitors in a panel of pancreatic cancer cell lines and found variable abilities to override the S phase checkpoint. In cells that were able to enter mitosis, the chromatin was extensively fragmented, as assessed by metaphase spreads and Comet assay. Notably, electron microscopy and high-resolution light microscopy showed that the kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). Cell lines that were unable to override the S phase checkpoint were able to override a G₂ arrest induced by the alkylator MMS or the topoisomerase II inhibitors doxorubicin or etoposide. Interestingly, checkpoint override from the topoisomerase II inhibitors generated fragmented kinetochores (MUGs) due to unreplicated centromeres. Our studies show that kinetochore and centromere fragmentation is a defining feature of checkpoint override and suggests that loss of cell viability is due in part to acentric genomes. Furthermore, given the greater efficacy of forcing cells into premature mitosis from topoisomerase II-mediated arrest as compared with gemcitabine-mediated arrest, topoisomerase II inhibitors maybe more suitable when used in combination with checkpoint inhibitors.     

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level.The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.     

EGCG synergizes the therapeutic effect of irinotecan through enhanced DNA damage in human colorectal cancer cells

Irinotecan is a kind of alkaloid with antitumour activity, but its low solubility and high toxicity limit its application. Epigallocatechin-3-gallate (EGCG) is one of the main bioactive components in tea. The epidemiological investigation and animal and cell experiments show that EGCG has a preventive and therapeutic effect on many kinds of tumours. Here, colorectal cancer cells RKO and HCT116 were employed, and the CCK8 proliferation test was used to screen the appropriate concentration of EGCG and irinotecan, and the effects of single and/or combined drugs on migration, invasion, DNA damage, cell cycle and autophagy of tumour cells were investigated. The results showed that EGCG combined with irinotecan (0.5 μmol L⁻) not only had a stronger inhibitory effect on tumour cells than EGCG or irinotecan alone but also prevented tumour cell migration and invasion. EGCG alone did not cause DNA damage in colorectal cancer cells, but its combination with irinotecan could induce S or G2 phase arrest by inhibiting topoisomerase I to cause more extensive DNA damage. EGCG also induced apoptosis by promoting autophagy with irinotecan synergistically. These results indicated that EGCG in combination with irinotecan could be a promising strategy for colorectal cancer.