The cytotoxicity and mechanisms of 1,2-naphthoquinone thiosemicarbazone and its metal derivatives against MCF-7 human breast cancer cells

We have investigated the antitumor functions and mechanisms of 1,2-naphthoquinone-2-thiosemicarbazone (NQTS) and its metal complexes (Cu(2+), Pd(2+), and Ni(2+)) against MCF-7 human breast cancer cells. The cells were dosed with these complexes at varying concentrations, and cell viability was measured by a sulforhodamine B (SRB) method. To study mechanisms of action, the complexes were incubated with topoisomerase II (topo II) and supercoiled DNA, linear DNA, nicked open DNA, and relaxed DNA were detected by agarose gel electrophoresis. The results revealed that these complexes are effective antitumor chemicals in inhibiting MCF-7 cell growth, with Ni-NQTS being the most effective among the complexes studied. Our data also indicated that Ni-NQTS is more effective than the commercial antitumor drug, etoposide, based on IC(50) values. The mechanistic study of action showed that metal complexes of NQTS, NQ, and NQTS can only stabilize the single-strand nicked DNA, but not double-strand breakage intermediates. In addition, metal derivatives of these ligands, but not the parent NQ and NQTS, exerted an antagonizing effect on topoisomerase II activity. In summary, chemicals with or without metal derivatives might possess different chemical-topoisomerase II-DNA interactions.     

Evaluation of Natural and Synthetic 1,4‐naphthoquinones as Inhibitors of Monoamine Oxidase

Previous reports have documented that 1,4‐naphthoquinones act as inhibitors of the monoamine oxidase (MAO) enzymes. In particular, fractionation of the extracts of cured tobacco leafs has led to the characterization of 2,3,6‐trimethyl‐1,4‐naphthoquinone, a non‐selective MAO inhibitor. To derive structure–activity relationships for MAO inhibition by the 1,4‐naphthoquinone class of compounds, this study investigates the human MAO inhibitory activities of fourteen structurally diverse 1,4‐naphthoquinones of natural and synthetic origin. Of these, 5,8‐dihydroxy‐1,4‐naphthoquinone was found to be the most potent inhibitor with an IC₅₀ value of 0.860 μm for the inhibition of MAO‐B. A related compound, shikonin, inhibits both the MAO‐A and MAO‐B isoforms with IC₅₀ values of 1.50 and 1.01 μm , respectively. It is further shown that MAO‐A and MAO‐B inhibition by these compounds is reversible by dialysis. In this respect, kinetic analysis suggests that the modes of MAO inhibition are competitive. This study contributes to the discovery of novel MAO inhibitors, which may be useful in the treatment for disorders such as Parkinson's disease, depressive illness, congestive heart failure and cancer.     

Recent developments of rebeccamycin analogues as topoisomerase I inhibitors and antitumor agents

DNA topoisomerases are essential for the survival of prokaryotic and eukaryotic organisms. Topoisomerases inhibitors, due to their capacity to induce DNA breaking, can exhibit interesting antitumor properties. While there are many potent antitumor agents which target topoisomerase II, relatively few families of specific topoisomerase I inhibitors have been identified. The present review describes a new family of topoisomerase I inhibitors, analogues of the bacterial metabolite rebeccamycin. These compounds possess an indolocarbazole chromophore onto which is attached a sugar residue. Important structure-activity relationships studies in this series have helped to understand the role of the carbohydrate moiety which is absolutely necessary for topoisomerase I poisoning, the influence of the stereochemistry (alpha or beta) of its linkage to indole, the influence of the functionalities and substitutions on the sugar moiety and on the aromatic framework have been investigated. In addition to their action on DNA, rebeccamycin analogues may inhibit the SR kinase activity of topoisomerase I and therefore constitute a unique family of topoisomerase I poisons quite different from the well known camptothecins.     

Mutation of cysteine residue 455 to alanine in human topoisomerase IIalpha confers hypersensitivity to quinones: enhancing DNA scission by closing the N-terminal protein gate

Several quinone-based metabolites of industrial and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein, and previous studies suggest that they increase levels of enzyme-associated DNA strand breaks by at least two potential mechanisms. Quinones act directly on the DNA cleavage-ligation equilibrium of topoisomerase II by inhibiting the rate of ligation. They also block the N-terminal gate of the protein, thereby stabilizing topoisomerase II in its "closed clamp" form and trapping DNA in the central annulus of the enzyme. It has been proposed that this latter activity enhances DNA cleavage by increasing the population of enzyme molecules with DNA in their active sites, but a causal relationship has not been established. In order to more fully characterize the mechanistic basis for quinone action against topoisomerase II, the present study characterized the sensitivity of human topoisomerase IIalpha carrying a Cys455-->Ala mutation (top2alphaC455A) toward quinones. Cys455 was identified as a site of quinone adduction by mass spectrometry. The mutant enzyme was approximately 1.5-2-fold hypersensitive to 1,4-benzoquinone and the polychlorinated biphenyl quinone 4'Cl-2,5pQ, but it displayed wild-type sensitivity to traditional topoisomerase II poisons. The ability of 1,4-benzoquinone to inhibit DNA ligation mediated by top2alphaC455A was similar to that of wild-type topoisomerase IIalpha. However, the quinone induced approximately 3 times the level of clamp closure with the mutant enzyme. These findings strongly support the hypothesis that the ability of quinones to block the N-terminal gate of the type II enzyme contributes to their actions as topoisomerase II poisons.     

Stimulation by gamma-carboline derivatives (simplified analogues of antitumor ellipticines) of site specific DNA cleavage by calf DNA topoisomerase II

gamma-Carbolines are tricyclic aromatic compounds which intercalate into DNA base pairs and exhibit significant cytotoxic and antitumor activities. These compounds which are structurally related to ellipticine by deletion of an aromatic ring, induce DNA breaks in cultured L1210 cells. Since the mechanism of cytotoxic activity of ellipticines involves DNA topoisomerase II, this enzyme might also be a target for gamma-Carbolines. We have tested this hypothesis using an in vitro system containing purified enzyme and pBR322 DNA. The ability of nine derivatives to stabilize the DNA-enzyme covalent complex was studied and compared to their cytotoxicity. The four less cytotoxic compounds do not induce cleavable complex to a significant extent. In contrast, the two most cytotoxic gamma-Carbolines are the most efficient stabilizers of the cleavable complex. The last three compounds exhibit an intermediate cytotoxicity and cleavage activity. In the presence of gamma-Carbolines, cleavage occurs predominantly at a single site in pBR322 which is one of the cleavage sites observed with ellipticines. The cleavage position was determined at the nucleotide level. The increased DNA cleavage specificity observed with gamma-Carbolines suggests that a tricyclic system is as efficient as ellipticines for DNA topoisomerase II cleavage at DNA sequences involved specifically in cytotoxic response. The data presented support the hypothesis that DNA topoisomerase II is a target involved in the mechanisms of action of antitumor gamma-Carbolines.     

Design, synthesis, and evaluation of dibenzo[c,h][1,6]naphthyridines as topoisomerase I inhibitors and potential anticancer agents

Indenoisoquinoline topoisomerase I (Top1) inhibitors are a novel class of anticancer agents. Modifications of the indenoisoquinoline A, B, and D rings have been extensively studied in order to optimize Top1 inhibitory activity and cytotoxicity. To improve understanding of the forces that stabilize drug-Top1-DNA ternary complexes, the five-membered cyclopentadienone C-ring of the indenoisoquinoline system was replaced by six-membered nitrogen heterocyclic rings, resulting in dibenzo[c,h][1,6]naphthyridines that were synthesized by a novel route and tested for Top1 inhibition. This resulted in several compounds that have unique DNA cleavage site selectivities and potent antitumor activities in a number of cancer cell lines.     

Synthesis and antitumor activity of fused tetracyclic quinoline derivatives. 1

Several fused tri- and tetracyclic quinolines (I and II) with [2-methoxy-4-[(methylsulfonyl)amino]phenyl]amino or [3-(N,N-dimethylamino)propyl]amino side chains were prepared, and their DNA intercalative properties, KB cytotoxicity, antitumor activity (P388 leukemia), and ability to induce topoisomerase II dependent DNA cleavage were investigated. Some compounds having both intercalative ability and KB cytotoxicity were found to be inactive in vivo. However, a positive correlation was seen between the ability to induce topoisomerase II dependent DNA cleavage and antitumor activity in vivo. The indeno- (13a), benzofuro- (21a), and benzothieno- (22a) quinoline derivatives exhibited potent antitumor activities in vitro and in vivo, comparable to those of m-AMSA. They also intercalate DNA and induce topoisomerase II dependent DNA cleavage. Extended screening of 13a showed it to be active against solid tumors such as M5076 sarcoma, B16 melanoma, and colon 38 carcinoma.     

Effects of benzene metabolites on DNA cleavage mediated by human topoisomerase II alpha: 1,4-hydroquinone is a topoisomerase II poison

Although benzene induces leukemias in humans, the compound is not believed to generate chromosomal damage directly. Rather, benzene is thought to act through a series of phenolic- and quinone-based metabolites, especially 1,4-benzoquinone. A recent study found that 1,4-benzoquinone is a potent topoisomerase II poison in vitro and in cultured human cells [Lindsey et al. (2004) Biochemistry 43, 7363-7374]. Because benzene is metabolized to multiple compounds in addition to 1,4-benzoquinone, we determined the effects of several phenolic metabolites, including catechol, 1,2,4-benzenetriol, 1,4-hydroquinone, 2,2'-biphenol, and 4,4'-biphenol, on the DNA cleavage activity of human topoisomerase II alpha. Only 1,4-hydroquinone generated substantial levels of topoisomerase II-mediated DNA scission. DNA cleavage with this compound approached levels observed with 1,4-benzoquinone (approximately 5- vs 8-fold) but required a considerably higher concentration (approximately 250 vs 25 microM). 1,4-Hydroquinone is a precursor to 1,4-benzoquinone in the body and can be activated to the quinone by redox cycling. It is not known whether the effects of 1,4-hydroquinone on human topoisomerase II alpha reflect a lower reactivity of the hydroquinone or a low level of activation to the quinone. The high concentration of 1,4-hydroquinone required to increase enzyme-mediated DNA cleavage is consistent with either explanation. 1,4-Hydroquinone displayed attributes against topoisomerase II alpha, including DNA cleavage specificity, that were similar to those of 1,4-benzoquinone. However, 1,4-hydroquinone consistently inhibited DNA ligation to a greater extent than 1,4-benzoquinone. This latter result implies that the hydroquinone may display (at least in part) independent activity against topoisomerase II alpha. The present findings are consistent with the hypothesis that topoisomerase II alpha plays a role in the initiation of specific types of leukemia that are induced by benzene and its metabolites.     

Selective acetylenic 'suicide' and reversible inhibitors of monoamine oxidase types A and B.

1 A number of aromatic-N-propargyl (acetylenic) compounds and indoleamines were tested for their inhibitory action on monoamine oxidase (MAO) type A and type B using the substrates 5-hydroxytryptamine (5-HT), beta-phenylethylamine (PEA) and dopamine. 2 Structure activity studies with aromatic-N-propragyl (acetylenic) derivatives have shown that MAO inhibitory potency is least dependent on the aromatic portion of the compounds. N-methylated propargyl derivatives are the most active and replacement of the methyl group with a higher alkyl or aromatic group results in significant reduction of activity. The triple bond in the N-propargyl portion is absolutely essential for activity and must be beta-to the nitrogen. It is the acetylenic group that gives these compounds their irreversible MAO inhibitory property. 3 The present study has indicated that since the acetylenic compounds resemble the enzyme substrates the distance between the aromatic ring and the N-propargyl terminal is crucial in designating the type A or type B MAO inhibitory property. For MAO type A inhibition, a distance equivalent to at least three carbon units is required, while for the inhibition of the B type enzyme this distance can be 1 or 2 carbon units. 4 The compounds AGN-1133 and AGN-1135 show most promise in Parkinson's disease or as anti-depressants because of their irreversible selective type B MAO inhibition in vitro and in vivo. 5 A number of indoleamine derivatives were found to be reversible selective type A inhibitors.     

Recent advances in the discovery and development of quinolones and analogs as antitumor agents

Compounds that interact with DNA or microtubules by multiple mechanisms and cause diverse cytotoxic lesions are potential targets for anticancer drug development. Accordingly, a relatively new approach to the rational design of antitumor agents is based on the quinolone class of antibacterials. Their mechanism of antibacterial action involves inhibition of DNA gyrase, and numerous new quinolones do exhibit antitumor activity. Thus, these new quinolone structures display a novel mode of action for the quinolone class as antitumor agents. The potential for quinolones to be used as topoisomerase II inhibitors, as well as antimitotic agents, is reviewed with a focus on recent discoveries and development of antitumor quinolones, especially related work in the author's laboratory.     

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds

The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.     

Non-camptothecin DNA topoisomerase I inhibitors in cancer therapy

Human DNA topoisomerase I is the target of camptothecins, which have been recently introduced in the clinic for cancer chemotherapy. The discovery of novel non-camptothecin inhibitors is facilitated by the availability of biochemical and cellular assays for testing topoisomerase I activity. Among the non-camptothecin inhibitors, the indolocarbazoles (NB-506 and J-107088) are the most advanced in their development, and are in clinical trials. A number of indenoisoquinolines and minor groove binders (benzimidazoles) have been reported recently. Their antitumor activity is promising for further development. The potential binding site(s) of topoisomerase I inhibitors in the enzyme I-DNA complex is discussed.     

Antitumor agents. 194. Synthesis and biological evaluations of 4-beta-mono-, -di-, and -trisubstituted aniline-4'-O-demethyl-podophyllotoxin and related compounds with improved pharmacological profiles.

As a continuation of our structure-activity relationship studies, several new 4-beta-substituted 4'-O-demethyl-4-desoxypodophyllotoxins bearing mono-, di-, or trisubstituted anilines have been synthesized and evaluated as inhibitors of DNA topoisomerase II and tumor cell growth in tissue culture. Selected compounds were further evaluated as cytotoxic agents using a clonogenic survival assay. The target compounds include 4'-O-demethyl-4beta-[(4' '-(benzimidazol-2' '-yl)anilino]-4-desoxypodophyllotoxin (21), 4'-O-demethyl-4beta-(-)-(4' '-camphanamido-anilino)-4-desoxypodophyllotoxin (25), 4-beta-disubstituted-anilino-4'-demethyl-4-desoxypodophyllotoxins (18-20, 26), 4-alpha-disubstituted-anilino-4'-demethyl-4-desoxypodophyllotoxin (27), 4-beta-trisubstituted-anilino-4'-demethyl-desoxypodophyllotoxin (22, 23), and 4'-O-demethyl-4beta-[4' '-(benzimidazol-2' '-yl)amino]-4-desoxypodophyllotoxin (24). Among the target series, 19, 21, and 24 displayed significant growth inhibitory action against a panel of tumor cell lines including human epidermoid carcinoma of the nasopharynx (KB) and its etoposide-resistant (KB7B) and vincristine-resistant (vin20c KB) subclones, lung carcinoma (A549), human ileocecal carcinoma (HCT-8), human kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), and human malignant melanoma (SK-MEL-2) cells. Compounds 19, 21, 24, and 25 were "cleavable-complex"-forming DNA topoisomerase II inhibitors with either improved or similar activity compared with the prototype drug etoposide (VP-16). Compound 21 was the most active analogue, being 10-fold more potent than etoposide in both cell killing and topoisomerase II inhibition in vitro assays. Using mouse models of antitumor activity, 21 was effective against (P388/0) leukemia but not against the growth of a (MCF7) mammary tumor.     

Current status and perspectives in the development of camptothecins

Camptothecins are cytotoxic agents with a wide spectrum of antitumor activity. The unique mechanism of action, the impressive preclinical efficacy and the clinical success of irinotecan and topotecan have stimulated intensive efforts to identify novel analogues. The development of novel camptothecins was recently rationalized on the basis of the detailed knowledge of mechanism of drug-target interaction and was aimed to overcome the major limitations of these drugs (i.e. lactone ring instability and reversibility of topoisomerase I-DNA cleavage complexes). The development of novel series of analogues (7-substituted camptothecins, silatecans and homocamptothecins) resulted in identification of promising compounds, which are currently in clinical development. Considering the lack of precise correlations between preclinical activity and clinical efficacy of camptothecins, the potential advantages of novel analogs in clinical therapy remains to be documented. However, a rational basis for drug selection and development is now provided by the recognition of major limitations of these agents and by a detailed knowledge of multiple interactions between drug, cellular target and serum albumin. Inhibition of the nuclear enzyme DNA topoisomerase I has proven to be a promising strategy in the design of antitumor agents, in spite of a limited cellular basis of selectivity in cytotoxic action of camptothecins (i.e., overexpression of the target enzyme in tumor cells, and increased sensitivity of proliferating cells). The interest in topoisomerase I as a therapeutic target promoted various efforts to identify other chemotypes effective as topoisomerase inhibitors and chemical/modelling efforts to rationally design specific analogs among known inhibitors. Additional approaches, including drug delivery/formulation, optimization of dose/schedule and route of administration, are expected to improve the therapy with camptothecins and other inhibitors.     

Novel human topoisomerase I inhibitors, topopyrones A, B, C and D. I. Producing strain, fermentation, isolation, physico-chemical properties and biological activity

In the course of a screening program for specific inhibitors of human topoisomerase I using a recombinant yeast, we have discovered four new active compounds. All four compounds were isolated from the culture broth of a fungus, Phoma sp. BAUA2861, and two of them were isolated from the culture broth of a fungus, Penicillium sp. BAUA4206. We designated these compounds as topopyrones A, B, C and D. Topopyrones A, B, C and D selectively inhibited recombinant yeast growth dependent on expression of human topoisomerase I with IC50 values of 1.22, 0.15, 4.88 and 19.63 ng/ml, respectively. The activity and selectivity of topopyrone B were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds, however they did not inhibit human DNA topoisomerase II. Topopyrones A, B, C and D were cytotoxic to all tumor cell lines when tested in vitro. Topopyrone B has potent inhibitory activity against herpesvirus, especially varicella zoster virus (VZV). It inhibited VZV growth with EC50 value of 0.038 microg/ml, which is 24-fold stronger than that of acyclovir (0.9 microg/ml). Topopyrones A, B, and C were inhibitory to Gram-positive bacteria.     

Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity

Several camptothecin derivatives containing a modified hydroxy lactone ring have been synthesized and evaluated for inhibition of topoisomerase I and cytotoxicity to mammalian cells. Each of the groups of the hydroxy lactone moiety, the carbonyl oxygen, the ring lactone oxygen, and the 20-hydroxy group, were shown to be critical for enzyme inhibition. For example the lactol, lactam, thiolactone, and 20-deoxy derivatives did not stabilize the covalent DNA-topoisomerase I complex. With a few exceptions, those compounds that did not inhibit topoisomerase I were not cytotoxic to mammalian cells. Two cytotoxic derivatives that did not inhibit topoisomerase I were shown to produce non-protein-associated DNA single-strand breaks and are likely to have a different mechanism of action. One of these compounds was tested for antitumor activity and was found to be inactive. The present findings, as well as other reports that the hydroxy lactone ring of camptothecin is critical for antitumor activity in vivo, correlate with the structure-activity relationships at the level of topoisomerase I and support the hypothesis that antitumor activity is related to inhibition of this target enzyme.     

Promotion or suppression of experimental metastasis of B16 melanoma cells after oral administration of lapachol

Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] is a vitamin K antagonist with antitumor activity. The effect of lapachol on the experimental metastasis of murine B16BL6 melanoma cells was examined. A single oral administration of a high toxic dose of lapachol (80-100 mg/kg) 6 h before iv injection of tumor cells drastically promoted metastasis. This promotion of metastasis was also observed in T-cell-deficient mice and NK-suppressed mice. In vitro treatment of B16BL6 cells with lapachol promoted metastasis only slightly, indicating that lapachol promotes metastasis primarily by affecting host factors other than T cells and NK cells. A single oral administration of warfarin, the most commonly used vitamin K antagonist, 6 h before iv injection of tumor cells also drastically promoted the metastasis of B16BL6 cells. The promotion of metastasis by lapachol and warfarin was almost completely suppressed by preadministration of vitamin K3, indicating that the promotion of metastasis by lapachol was derived from vitamin K antagonism. Six hours after oral administration of lapachol or warfarin, the protein C level was reduced maximally, without elongation of prothrombin time. These observations suggest that a high toxic dose of lapachol promotes metastasis by inducing a hypercoagulable state as a result of vitamin K-dependent pathway inhibition. On the other hand, serial oral administration of low non-toxic doses of lapachol (5-20 mg/kg) weakly but significantly suppressed metastasis by an unknown mechanism, suggesting the possible use of lapachol as an anti-metastatic agent.     

Maleimide is a potent inhibitor of topoisomerase II in vitro and in vivo: a new mode of catalytic inhibition

Maleimide, N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM) were identified as potent catalytic inhibitors of purified human topoisomerase IIalpha, whereas the ring-saturated analog succinimide was completely inactive. Catalytic inhibition was not abrogated by topoisomerase II mutations that totally abolish the effect of bisdioxopiperazine compounds on catalytic inhibition, suggesting a different mode of action by these maleimides. Furthermore, in DNA cleavage assay maleimide and NEM could antagonize etoposide-induced DNA double-strand breaks. Consistently, maleimide could antagonize the effect of topoisomerase II poisons in three different in vivo assays: 1) In an alkaline elution assay maleimide protected against etoposide-induced DNA damage. 2) In a band depletion assay maleimide reduced etoposide-induced trapping of topoisomerase IIalpha and beta on DNA. 3) In a clonogenic assay maleimide antagonized the cytotoxicity of etoposide and daunorubicin on four different cell lines of human and murine origin. at-MDR cell lines with reduced nuclear topoisomerase IIalpha content are fully sensitive to maleimide, indicating that it is not a topoisomerase II poison in vivo. Our finding that topoisomerase II is sensitive to maleimide, NMM, and NEM but insensitive to succinimide demonstrates a strict requirement for the unsaturated ring bond for activity. We suggest that the observed antagonism in vitro and in vivo is caused by covalent modification of topoisomerase II cysteine residues reducing the amount of catalytically active enzyme sensitive to the action of topoisomerase II poisons.