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.     

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.     

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.     

The human topoisomerase I damage response plays a role in apoptosis

Previous studies have shown that human topoisomerase I cleavage complexes form as a response to various DNA damages in vivo, the so called human topoisomerase I "damage response". It was suggested that this damage response may play a role in DNA repair as well as in apoptosis, but only very few investigations have been done and the significance of the damage response still remains unclear. Here we demonstrate that human topoisomerase I cleavage complexes induced by high doses of UV irradiation are highly stable for up to 48 h. Furthermore, we show that human topoisomerase I cleavage complexes correlate with apoptosis. However, at low UV doses the cleavage complex level was very low and the complexes were repaired. Surprisingly, we found that high levels of stable cleavage complexes were not only found in UV-irradiated cells but also in untreated cells that underwent apoptosis. A possible role of human topoisomerase I in apoptosis is discussed.     

Contributions of the D-Ring to the activity of etoposide against human topoisomerase IIα: potential interactions with DNA in the ternary enzyme--drug--DNA complex

Etoposide is a widely prescribed anticancer drug that stabilizes covalent topoisomerase II-cleaved DNA complexes. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. Interactions between human topoisomerase IIα and etoposide in the binary enzyme--drug complex appear to be mediated by substituents on the A-, B-, and E-rings of etoposide. These protein--drug contacts in the binary complex have predictive value for the actions of etoposide within the ternary topoisomerase IIα--drug--DNA complex. Although the D-ring of etoposide does not appear to contact topoisomerase IIα in the binary complex, etoposide derivatives with modified D-rings display reduced cytotoxicity against murine leukemia cells [Meresse, P., et al. (2003) Bioorg. Med. Chem. Lett. 13, 4107]. This finding suggests that alterations in the D-ring may affect etoposide activity toward topoisomerase IIα in the ternary enzyme--drug--DNA complex. Therefore, to address the potential contributions of the D-ring to the activity of etoposide, we characterized drug derivatives in which the C13 carbonyl was moved to the C11 position (retroetoposide and retroDEPT) or the D-ring was opened (D-ring diol). All of the D-ring alterations decreased the ability of etoposide to enhance DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. They also weakened etoposide binding in the ternary enzyme--drug--DNA complex and altered sites of enzyme-mediated DNA cleavage. On the basis of these findings, we propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex.     

Cell killing and DNA damage induced in cultured mammalian cells by some tetrahydrobenzopsoralenquinones

The mechanism of action of two tetrahydrobenzopsoralenquinones: 4-methyl-tetrahydrobenzopsoralenquinone (compound 3) and 4-hydroxymethyltetrahydrobenzopsoralenquinone (compound 4) was studied in mammalian cells. These agents differ structurally from earlier benzo and tetrahydrobenzopsoralen derivatives 4-hydroxymethylbenzopsoralen (compound 1) and 4-hydroxymethyltetrahydrobenzopsoralen (compound 2) by the replacement of the benzopyranone with a quinonepyranone. In this study, we evaluated the antiproliferative activity of such derivatives in normal human lymphocytes and CHO cells cultivated in vitro. Compound 4 showed a noticeable antiproliferative activity. Studying the induction of chromosomal aberrations and of SCEs, we demonstrated that compound 4 has a clastogenic effect on mammalian cells. By means of DNA filter elution and protein precipitation techniques we evaluated the DNA damage produced by the tested compounds. Some experiments performed in presence of a DNA synthesis inhibitor showed that ongoing DNA synthesis is involved in cell killing by derivative 4. All data obtained suggest that compound 4 can interfere with the activity of topoisomerase II. Catalytic studies carried out with purified topoisomerase II and bacteriophage DNA confirmed this hypothesis.     

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium,cobalt, and nickel

The alkaline comet assay is able to identify in individual cells DNA strand breaks associated with different processes. Topoisomerase inhibitors, some of which are used as chemotherapeutic agents, stabilise topoisomerase-DNA cleavable complexes by stimulating DNA strand cleavage and inhibiting religation. This can result in the activation of stress-associated signalling pathways, inducing cell cycle arrest and activation of the biochemical cascade of apoptosis. The aim of our study was to assess the ability of the comet assay to detect stabilisation of cleavable complexes and induction of apoptosis by two topoisomerase II inhibitors, etoposide and ellipticine, and two topoisomerase I inhibitors, camptothecin and topotecan. The study was carried out on Chinese hamster ovary (CHO) cells, DC3F cells and DC3F/C-10, its camptothecin-resistant counterpart. The comet assay was able to identify stabilised cleavable complexes through the presence of DNA strand breaks after 1h treatment that disappeared within 24h after drug removal. Kinetics studies allowed to discriminate between these early DNA damages and DNA fragmentation related to apoptosis characterised by reappearance of DNA strand breaks 48h after treatment.     

Antitumor agents. Part 230: C4'-esters of GL-331 as cytotoxic agents and DNA topoisomerase II inhibitors

Three C4'-acyl derivatives (5-7) of GL-331 (4) were synthesized and evaluated for cytotoxic and DNA topoisomerase II (topo II) inhibitory activity. All three compounds were cytotoxic against KB and KB-7d cells. Compounds 5 and 7, but not 6, were potent inhibitors of the DNA topoisomerase II in vitro and this relative activity ranking was maintained for cytotoxicity, in vitro cell growth inhibition, and ability to induce cellular double-strand DNA breaks. These results provided new information on the structure-activity relationships of the C4' molecular area of 4-analogues.     

Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase alpha and beta and human DNA topoisomerase II, with IC(50) values of 25, 94, and 10 microM, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed.     

Biological activity of bis-benzimidazole derivatives on DNA topoisomerase I and HeLa, MCF7 and A431 cells

Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H(2) receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.     

Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase IIα

It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to further characterize xanthone mechanism and generate compounds with potential as anticancer drugs, we synthesized a series of derivatives in which position 3 was substituted with different polyamine chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent topoisomerase IIα inhibitors. Although xanthone derivatives inhibit topoisomerase IIα-catalyzed ATP hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity, competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation active site of topoisomerase IIα and inhibit the catalytic activity of the enzyme by interfering with the DNA strand passage step.     

Cleavage of DNA induced by 9-anilinoacridine inhibitors of topoisomerase II in the malaria parasite Plasmodium falciparum

Due to resistance by Plasmodium falciparum, the most virulent strain of the four species of human malaria parasites, to most currently used antimalarial drugs, development of new effective antimalarials is urgently needed. Derivatives of 9-anilinoacridine, an antitumor drug, have been shown to inhibit P. falciparum growth in culture and to inhibit parasite DNA topoisomerase II activity in vitro. Using KCl-SDS precipitation assay to detect the presence of protein-DNA complexes within parasite cells, an indicator of DNA topoisomerase II inactivation, derivatives containing 3,6-diNH(2) substitutions with 1'-electron donating (NMe(2), CH(2)NMe(2), NHSO(2)Me, OH, OMe), and 1'-electron withdrawing (SO(2)NH(2)) groups produced protein-DNA complexes. However, the antimalarial pyronaridine, 9-anilinoazaacridine, did not generate protein-DNA complexes, although it was capable of inhibiting P. falciparum DNA topoisomerase II activity in vitro. These results should prove useful in future designs of novel antimalarial compounds directed against parasite DNA topoisomerase II.     

Topoisomerase II and the etiology of chromosomal translocations

Acute leukemias with balanced chromosomal translocations, protean morphologic and immunophenotypic presentations but generally shorter latency and absence of myelodysplasia are recognized as a complication of anti-cancer drugs that behave as topoisomerase II poisons. Translocations affecting the breakpoint cluster region of the MLL gene at chromosome band 11q23 are the most common molecular genetic aberrations in leukemias associated with the topoisomerase II poisons. These agents perturb the cleavage-religation equilibrium of topoisomerase II and increase cleavage complexes. One model suggests that this damages the DNA directly and leads to chromosomal breakage, which may result in untoward DNA recombination in the form of translocations. This review will summarize the evidence for topoisomerase II involvement in the genesis of translocations and extension of the model to acute leukemia in infants characterized by similar MLL translocations.     

Topoisomerase II poisoning and antineoplastic action by DNA-nonbinding diacetyl and dicarboxaldoxime derivatives of ferrocene

Topoisomerase II is a major molecular target for a number of DNA-binding anticancer drugs. In the present study, we report topoisomerase II inhibition and anticancer activity by four substituted ferrocene derivatives which do not bind to DNA. The first derivative, acetyl-substituted ferrocene (monoacetylferrocene), showed a minor inhibition of topoisomerase II activity along with a consequent inhibition of cancer cell proliferation. The second derivative (diacetylferrocene) showed a higher potency of action compared to the monosubstituted derivative. The third and fourth derivatives, with mono- and disubstituted carboxaldoxime groups (ferrocenecarboxaldoxime and ferrocenedicarboxaldoxime), showed a higher anticancer action and stronger topoisomerase II inhibition. To understand their molecular mechanism of action, cleavage assays were carried out to monitor the drug-induced, topoisomerase II mediated DNA cleavage. The results show that diacetylferrocene and ferrocenedicarboxaldoxime could form an enzyme-drug-DNA ternary complex, called a "cleavage complex," resulting in DNA cleavage. These results along with those of an immunoprecipitation assay indicate that the two compounds interact with topoisomerase II alone and poison its activity by trapping the enzyme and enzyme-cleaved DNA in the covalently closed cleavage complex. The formation of such a complex has numerous genetic implications, which ultimately results in neoplastic cell death.     

Apoptosis induction in HL-60 cells and inhibition of topoisomerase II by triterpene celastrol

Celastrol, which is a triterpene purified from Celastraceae plants, has anticancer and anti-inflammatory activities. In this study we investigated to clarify whether celastrol can induce apoptosis in a human leukemia HL-60 model system. Celastrol was found to induce apoptosis, and the rank order of the potency of celastrol and its derivatives to induce internucleosomal DNA fragmentation was found to be celastrol>cela-H>the other derivatives=vehicle control. Many anticancer agents are known to possess the ability to inhibit topoisomerase II, so the inhibitory activities of celastrol and its derivatives on topoisomerase II were also explored. The rank order of the inhibitory activity was found to be celastrol>etoposide>cela-H, indicating that the apoptosis-inducing activities of cela derivatives correspond to their inhibitory activities on topoisomerase II. These data suggested that celastrol may cause its effects such as anticancer activity by the mechanism of apoptosis along with topoisomerase II inhibition.     

Inhibition of human topoisomerase II by anti-neoplastic benzazolo[3,2-a]quinolinium chlorides

Previously we reported [20] that there is no correlation between the cytotoxic activity of four new structural analogs of the antitumor DNA intercalator 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ-2) and their interaction with DNA. In the present study, we present evidence suggesting that the molecular basis for the anti-proliferative activity of these drugs is the inhibition of topoisomerase II. The NBQ-2 derivatives inhibited the relaxation of supercoiled DNA plasmid pRYG mediated by purified human topoisomerase II. Inhibition of the decatenation of kinetoplast DNA mediated by partially purified topoisomerase II extracted from the human histiocytic lymphoma U937 (a cell line previously shown to be sensitive to the drugs) was also caused by these drugs. The potency of the benzazolo[3,2-a]quinolinium drugs against topoisomerase II in vitro was the following: 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-59) > 4-chlorobenzothiazolo[3,2-a]quinolinium chloride (NBQ-76) > 7-ethyl-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-48) > 7-benzyl-3-nitrobenzimidazolol[3,2-a]quinolinium chloride (NBQ-38). This rank of potency for topoisomerase II inhibition correlated very well with the cytotoxicity elicited by these drugs. Furthermore, significant levels of topoisomerase II/DNA cleavage complex induced by these drugs in vivo were detected when U937 cells were treated with NBQ-59 and NBQ-76 whereas NBQ-38 and NBQ-38 and NBQ-48 produced negligible amounts of the cleavage complex. Our results strongly suggest that topoisomerase II is the major cellular target of this family of compounds.     

Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.