Evidence from studies with intact mammalian cells that merbarone and bis(dioxopiperazine)s are topoisomerase II poisons

A Chinese hamster V79 cell-based assay for detection of topoisomerase II (topo II) poisons and catalytic inhibitors has been applied to study two bis(dioxopiperazine)s (ICRF-187 and ICRF-154) and a structurally distinct but related compound, merbarone. All three compounds have been previously characterized as being catalytic inhibitors of DNA topo II based primarily on in vitro studies with purified enzymes. The present studies indicate, to the contrary, that all three compounds are very potent DNA clastogens in V79 cells, by virtue of their ability to produce micronuclei, the formation of which is strongly antagonized under conditions in which DNA topo II is rendered catalytically inactive. None of the compounds could be demonstrated to possess catalytic inhibitory activity in intact V79 cells under the conditions tested. These studies provide biological evidence that bis(dioxopiperazine)s are capable of functional topo II poisoning in intact mammalian cells.     

DNA topoisomerase II rescue by catalytic inhibitors: A new strategy to improve the antitumor selectivity of etoposide

The nuclear enzyme DNA topoisomerase II (topo II) is the target of important antitumor agents such as etoposide. Recent work has classified topo II targeting drugs into either topo II poisons that act by stabilizing enzyme-DNA cleavable complexes leading to DNA breaks, or topo II catalytic inhibitors that act at stages in the catalytic cycle of the enzyme where both DNA strands are intact and, therefore, do not cause DNA breaks. Accordingly, catalytic inhibitors are known to abrogate DNA damage and cytotoxicity caused by topo II poisons. In this commentary, we have focused on the possibilities of enabling high-dose therapy with the topo II poison etoposide by protection of normal tissue with catalytic inhibitors, analogous to folinic acid rescue in high-dose methotrexate treatment. Thus, we have demonstrated recently that ( + )-1,2-bis(3,5-dioxopiperazinyl-1-yl) propane (ICRF-187) enabled a 3- to 4-fold dose escalation of etoposide in mice. Two high-dose etoposide models are described, namely use of the weak base chloroquine in tumors with acidic extracellular pH and targeting of CNS tumors with protection of normal tissue by the bisdioxopiperazine ICRF-187. In conclusion, high supralethal doses of topo II poisons in combination with catalytic inhibitor protection form a new strategy to improve the antitumor selectivity of etoposide and other topo II poisons. Such an approach may be used to overcome problems with drug resistance and drug penetration.     

Induction of apoptosis by dexrazoxane (ICRF-187) through caspases in the absence of c-jun expression and c-Jun NH2-terminal kinase 1 (JNK1) activation in VM-26-resistant CEM cells.

Dexrazoxane (ICRF-187) is an inhibitor of the catalytic activity of DNA topoisomerase II (topo II) that does not stabilize DNA-topo II covalent complexes. Here, we examined cytotoxic signaling by ICRF-187 in human leukemic CEM cells and a teniposide (VM-26)-resistant subline, CEM/VM-1. Treatment of CEM and CEM/VM-1 cells with ICRF-187 induced apoptotic cell death characterized by internucleosomal DNA fragmentation, nuclear condensation, and induction of at least caspase-3- and -7-like protease activities (but not caspase 1). Treatment of these cells with Z-Asp-2,6-dichlorobenzoyloxymethyl-ketone, a potent inhibitor of apoptosis, inhibited ICRF-187-induced DEVD-specific caspase activity and apoptosis in a concentration-dependent manner. ICRF-187-induced apoptosis in CEM cells was associated with transient induction of c-jun and activation of c-Jun NH2-terminal kinase 1 (JNK1). However, CEM/VM-1 cells, which were 3-fold more sensitive than CEM cells to ICRF-187 due to a decrease in topo II activity, exhibited ICRF-187-induced apoptosis in the absence of c-jun induction and JNK1 activation. These results indicate that catalytic inhibition of topo II by ICRF-187 leads to apoptosis through at least a caspase-3- and -7-like protease-dependent mechanism and suggest that c-jun and JNK1 are not required in ICRF-187-induced apoptosis in CEM cells.     

Reduction of genistein clastogenicity in Chinese hamster V79 cells by daidzein and other flavonoids.

A broad spectrum of health benefits has been ascribed to soy products. These products contain soy protein and relatively high levels of polyphenolic compounds known as flavonoids. While they are the most likely candidates for biological activity, flavonoids as a class, and of specific interest, genistein, are well known to be genotoxic due to their ability to "poison" cellular DNA topoisomerase II (topo II) resulting in stable chromosome breakage and mutation and raising questions about the long term health effects associated with chronic flavonoid exposure. Interestingly, some flavonoids, such as biochanin, galangin and daidzein, are catalytic topo II inhibitors (not poisons) and actually antagonize the clastogenicity of topo II poisons. It is shown in the present paper that flavonoids possessing catalytic topo II inhibitory activity, strongly antagonize the clastogenicity of genistein in Chinese hamster V79 cells. Importantly, one of these, daidzein, is a major constituent of marketed soy products. It is conjectured that the potential human clastogenic risk of soy products containing genistein might be mitigated or abolished due to the presence of daidzein or other flavonoids in those products.     

Structural determinants of the catalytic inhibition of human topoisomerase IIα by salicylate analogs and salicylate-based drugs

We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.3) that preferentially targets the alpha isoform by interfering with topo II-mediated DNA cleavage. Many pharmaceuticals and compounds found in foods are salicylate-based. We have now investigated whether these are also catalytic inhibitors of topo II and the structural determinants modulating these effects. We have determined that a number of hydroxylated benzoic acids attenuate doxorubicin-induced DNA damage signaling mediated by the ATM protein kinase and inhibit topo II decatenation activity in vitro with varying potencies. Based on the chemical structures of these and other derivatives, we identified unique properties influencing topo II inhibition, including the importance of substitutions at the 2′- and 5′-positions. We extended our findings to a number of salicylate-based pharmaceuticals including sulfasalazine and diflunisal and found that both were effective at attenuating doxorubicin-induced DNA damage signaling, topo II DNA decatenation and they blocked stabilization of doxorubicin-induced topo II cleavable complexes in cells. In a manner similar to salicylate, we determined that these agents inhibit topo II-mediated DNA cleavage. This was accompanied by a concomitant decrease in topo II-mediated ATP-hydrolysis. Taken together, these findings reveal a novel function for the broader class of salicylate-related compounds and highlight the need for additional studies into whether they may impact the efficacy of chemotherapy regimens that include topo II poisons.     

Topoisomerase inhibitors as anticancer agents: a patent update

Introduction: Topoisomerases (topos) are nuclear enzymes that resolve topological problems associated with DNA during various genetic processes. The essential role of topos in vital processes of the cell, their elevated level in solid tumors and cell death due to their inhibition make topos inhibitors as a potent class of antineoplastic agents.

Areas covered: This review specifically summarizes patents embracing topo I, topo I and II inhibitors. The review covers topos inhibitors which are structurally close to camptothecin (CPT), natural products such as lamellarins and synthetic trisubstituted pyridines. It largely focuses on chemical entities developed by systematic structure–activity relationship (SAR) studies of natural benzo[c]phenanthridine (nitidine) and synthetic protoberberine (coralyne) established as antineoplastic agents targeting topo(s). In addition, indenoisoquinolines and evodiamines initially discovered through COMPARE analysis and receptor-based virtual screening (VS) respectively have been discussed.

Expert opinion: Along with conventional techniques, computer-aided VS, molecular modeling and docking studies have been applied for drug design, discovery and development. Computer-aided tools provide a rational way to explain pharmacological activities of topos inhibitors under study. Comparative study of crystal structures of topo I/II-DNA-drug ternary complex and use of appropriate pharmacological screening methods will lead to potential anticancer drugs in the coming days.     

Inhibition of DNA topoisomerases II and/or I by pyrazolo[1,5-a]indole derivatives and their growth inhibitory activities

DNA topoisomerases (topos) I and II are molecular targets of several potent anticancer agents. Thus inhibitors of these enzymes are potential candidates or model compounds for anticancer drugs. We found some of the totally synthetic pyrazolo[1,5-a]indole derivatives, GS-2, -3, and -4, to be strong inhibitors of topo II, and GS-5 was found to be a dual inhibitor of topos I and II (IC(50) values were in the range of 10-30 microM). Because of the DNA-intercalating activity of these compounds affecting supercoil structure of closed circular DNA, the method of evaluation of topo I inhibition designed for such compounds by Pommier et al. (Nucleic Acids Res 15:6713-6731, 1987) was employed. Results showed that only GS-5 with a hydroxyl group at position C-6 was found to be a strong inhibitor of topo I with an IC(50) of approximately 10 microM. Inhibition of topo I and/or topo II by these compounds does not involve significant accumulation of DNA-topo I/II cleavable complexes, demonstrating that they are not topo poisons but catalytic inhibitors. In the "band depletion" analysis for in vivo targeting of topo I and II, these compounds were shown to suppress depletion of intracellular free enzymes by the topo poisons etoposide and/or camptothecin, indicating that they do target topo I and/or II in living cells. These compounds also exhibit moderate to strong growth-inhibitory activity in panels of human cancer cell lines. This study shows pyrazolo[1,5-a]indole derivatives to be a novel group of anticancer chemotherapeutic agents with single or dual catalytic inhibitory activities against topo I and topo II.     

Catalytic inhibition of DNA topoisomerase IIalpha by sodium azide

It has been demonstrated previously that sodium azide reduces the clastogenicity of several DNA topoisomerase II (topo II) poisons in cultured mammalian cells. These studies suggested that azide may be a catalytic topo II inhibitor. Azide interferes with mitochondrial production of ATP and is also known to inhibit cellular ATPases. Since topo II requires ATP for catalytic activity (enzyme turnover), it seemed likely that interference with ATP levels or ATP catabolism was the underlying mechanism of topo II inactivation; however, this has not been examined in living cells under conditions where the endogenous topo II is active on genomic DNA. The present studies were carried out to verify that azide inhibits endogenous topo II in cells. We show that azide blocks both decatenation and relaxation activity of purified topo II in a concentration dependent manner and reduces topoII/DNA covalent complex formation in cells. From these studies, it is concluded that sodium azide catalytically inactivates topo II via an ATP-sensitive process.     

Characterisation of cytotoxicity and DNA damage induced by the topoisomerase II-directed bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells

Background

Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison.

Results

By utilizing a panel of Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PK_cs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced γH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells.

Conclusion

Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.     

Synthesis, DNA binding, topoisomerase inhibition and cytotoxic properties of 2-chloroethylnitrosourea derivatives of hoechst 33258

A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.     

Formation and longevity of idarubicin-induced DNA topoisomerase II cleavable complexes in K562 human leukaemia cells

Idarubicin (IDA) is an anthracycline used during treatment of acute myelogenous leukaemia (AML) and is clinically important because of its potency and lipophilicity (compared to the related compounds daunorubicin and doxorubicin). These drugs target DNA topoisomerase II (topo II), a nuclear enzyme that regulates DNA topology. Topo II poisoning leads to the trapping of an intermediate in the enzyme's cycle termed the "cleavable complex." This study aims to increase understanding of drug interactions by use of the "TARDIS" (trapped in agarose DNA immunostaining) assay to measure drug-induced topo II cleavable complexes in individual cells treated with anthracyclines. Mammalian cells contain two isoforms of topo II (alpha and beta) and the TARDIS assay enables visualisation of isoform-specific complexes. Drug-treated cells were embedded in agarose, lysed and incubated with anti-topo II antibodies to microscopically detect topo IIalpha or beta complexes. Results for K562 cells (at clinically relevant concentrations) showed that IDA and idarubicinol, its metabolite, formed mainly topo IIalpha cleavable complexes, the level of which decreases at doses > 1 microM for IDA. Our data suggest that this decrease is due to catalytic inhibition by IDA at these doses. Doxorubicin formed low levels of topo IIalpha complexes and negligible topo IIbeta complexes. In cytotoxicity studies, IDA and idarubicinol were equipotent, but both were more potent than daunorubicin and doxorubicin. We showed for the first time that there was a persistent increase in levels of topo IIalpha cleavable complexes after removal of IDA, suggesting that its greater effectiveness may be associated with both the longevity and high levels of these complexes.     

Inhibition of DNA topoisomerases I and II,and growth inhibition of human cancer cell lines by a marine microalgal polysaccharide

We have previously reported purification of an extracellular polysaccharide GA3P, D-galactan sulfate associated with L-(+)-lactic acid, produced by a toxic marine microalga Dinoflagellate Gymnodinium sp. A(3) (GA3), and induction thereby of apoptosis on human myeloid leukemia K562 cells. In the present report, we show that the GA3P is a potent inhibitor of DNA topoisomerase (topo) I and topo II, irrespective of the presence or absence of the lactate group. Dextran sulfate also showed similar level of inhibition of topo I and topo II. We also demonstrated that, unlike camptothecin (CPT) or teniposide (VM-26), the inhibition of topo I or topo II by the polysaccharide does not involve accumulation of DNA-topo I/II cleavable complexes, clearly showing that they are not topo poisons but catalytic inhibitors with dual activity. Furthermore, the polysaccharide, when added to the reaction mixture with CPT or VM-26, inhibited stabilization of cleavable complex induced by the latter compounds. In addition, when added to the reaction mixture after the formation of the cleavable complexes by topo poisons, CPT for topo I and VM-26 for topo II, either GA3P or dextran sulfate diminished the amount of the complexes already accumulated, i.e. reversal of the reaction. These results suggest that the polysaccharides bind to the enzymes with high affinities, and that, as for topo I/II inhibition, the GA3P shares a common mechanism with dextran sulfate. As examined in vitro with a human cancer cell line panel, GA3P exhibited significant cytotoxicity against a variety of cancer cells. These findings show that the polysaccharide GA3P would prove to be a potential anticancer chemotherapeutic agent with dual activity of topo I and topo II catalytic inhibition.     

Dual topoisomerase I/II inhibitors in cancer therapy

While the majority of topoisomerase (topo) inhibitors show selectivity against either topo I or topo II, a small class of compounds can act against both enzymes. These can be divided into three classes. The first and largest class comprise drugs that bind to DNA by intercalation and include the clinically-evaluated acridine DACA, the benzopyridoindole intoplicine, the indenoquinolinone TAS-103, the benzophenazine XR11576, and the pyrazoloacridine NSC 366140. The second category comprises hybrid molecules, prepared by physically linking separate inhibitors of topo I and topo II, or by linking pure topo inhibitors to other DNA-interactive carriers. While several derivatives (e.g., camptothecin-epipodophyllotoxin and ellipticine-distamycin hybrids) have been prepared, there have been no detailed studies. The third category are less well defined as a structural class, but apparently recognize structural motifs that are present in both topo I and II enzymes. These include a series of benzoisoquinolinium quaternary salts such as NK 109, and more interestingly modified versions of classical topo I or topo II inhibitors; e.g., the modified camptothecin BN 80927 and the modified epipodophyllotoxin tafluposide (F-11782). There is as yet no detailed understanding of the factors that result in selective or dual inhibition, but structure-activity studies in several classes show that structural changes can influence topo I/II selectivity. DNA intercalation mode also appears to play a part. The basis for the high antitumor activity of some topo inhibitors is not yet understood but may depend on the complex pattern of activities that include both inhibition and poisoning of the two enzymes.     

Probing the role of linker substituents in bisdioxopiperazine analogs for activity against wild-type and mutant human topoisomerase II alpha

The bisdioxopiperazines are catalytic inhibitors of eukaryotic type II DNA topoisomerases capable of trapping these enzymes as a salt-stable closed-clamp complex on circular DNA. The various bisdioxopiperazine analogs differ from each other because of structural differences in the linker connecting the two dioxopiperazine rings. Although the composition of this linker region has been found to be important for potency, the structural basis for this is largely unknown. To elucidate the role of the linker region in drug action, we have analyzed the effect of different linker substituents in otherwise identical analogs by studying their interaction with wild-type and mutant human topoisomerase II alpha. Two mutations, L169I and R162Q, displayed differential sensitivity toward closely related analogs, suggesting that the linker region in these compounds plays a highly specific role in protein drug interaction. The finding that the L169I mutation, which probably represents a subtle structural change, was sufficient to confer resistance further emphases the importance of this region of the protein for bisdioxopiperazine inhibition of topoisomerase II. Comparing the sensitivity profiles of different bisdioxopiperazines against wild-type and mutant proteins with that of mitindomide, we observed a spectrum of sensitivity closely resembling that of ICRF-154, a bisdioxopiperazine with no linker substituents. We discuss the implications of these observations for the understanding of the mechanism of bisdioxopiperazine action on topoisomerase II.     

Characterization of the biological and biochemical activities of F 11782 and the bisdioxopiperazines, ICRF-187 and ICRF-193, two types of topoisomerase II catalytic inhibitors with distinctive mechanisms of action

F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.     

Guanidine-reactive agent phenylglyoxal induces DNA damage and cancer cell death

BackgroundDNA-damaging compounds (e.g., alkylating agents, cytotoxic antibiotics and DNA topoisomerase poisons) are the most widely used anticancer drugs. The inability of tumor cells to properly repair some types of DNA damage may explain why specific DNA-damaging drugs can selectively kill tumor cells. Phenylglyoxal is a dicarbonyl compound known to react with guanidine groups such as that of the DNA base guanine, therefore suggesting that phenylglyoxal could induce DNA damage and have anticancer activity.MethodsCellular DNA damage was measured by the alkaline comet assay and the γH2AX focus assay. Formation of topoisomerase I- and topoisomerase II-DNA complexes was assessed by the TARDIS assay, an immunofluorescence technique that employs specific antibodies to DNA topo I or topo II to detect the protein covalently bound to the DNA in individual cells. Cell growth inhibition and cytotoxicity were determined by XTT, MTT and clonogenic assays. Apoptosis was assessed by the Annexin V flow cytometry assay.ResultsPhenylglyoxal induced cellular DNA damage and formation of high levels of topoisomerase I- and topoisomerase II-DNA complexes in cells. These topoisomerase-DNA complexes were abolished by catalase pretreatment and correlated well with the induction of apoptosis. Phenylglyoxal-induced cell death was partially prevented by catalase pretreatment and was higher in lung cancer cells (A549) than in normal lung fibroblasts (MRC5). Mammalian cell lines defective in nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ) were more sensitive to phenylglyoxal than parental cells; this suggests that phenylglyoxal may induce bulky distortions in the shape of the DNA double helix (which are repaired by the NER pathway) and DNA double-strand breaks (which are repaired by HR and NHEJ).ConclusionsThis report shows that phenylglyoxal is a new DNA-damaging agent with anticancer activity, and suggests that tumor cells with defects in NER, HR and NHEJ may be hypersensitive to the cytotoxic activity of phenylglyoxal.     

DNA-binding activity and cytotoxicity of Pt-berenil compounds in MDA-MB-231 and MCF-7 breast cancer cells

The compounds of formula [Pt2Cl4(berenil)2]Cl4 and [Pt2Cl2(NH3)2(berenil)2]Cl4 were examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than cisplatin. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds show strong specificity for AT base pairs. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than cisplatin. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase II (topo II) inhibitors in plasmid relaxation assays.     

Characterization of a Chinese hamster ovary cell line with acquired resistance to the bisdioxopiperazine dexrazoxane (ICRF-187) catalytic inhibitor of topoisomerase II

A Chinese hamster ovary (CHO) cell line highly resistant to the non-cleavable complex-forming topoisomerase II inhibitor dexrazoxane (ICRF-187, Zinecard^®) was selected. The resistant cell line (DZR) was 1500-fold resistant (IC₅₀ = 2800 vs 1.8 μM) to continuous dexrazoxane exposure. DZR cells were also cross-resistant (8- to 500-fold) to other bisdioxopiperazines (ICRF-193, ICRF-154, and ICRF-186), and somewhat cross-resistant (4- to 14-fold) to anthracyclines (daunorubicin, doxorubicin, epirubicin, and idarubicin) and etoposide (8.5-fold), but not to the other non-cleavable complex-forming topoisomerase II inhibitors suramin and merbarone. The cytotoxicity of dexrazoxane to both cell lines was unchanged in the presence of the membrane-active agent verapamil. DZR cells were 9-fold resistant to dexrazoxane-mediated inhibition of topoisomerase II DNA decatenation activity compared with CHO cells (IC₅₀ = 400 vs 45 μM), but were only 1.4-fold (IC₅₀ = 110 vs 83 μM) resistant to etoposide. DZR cells contained one-half the level of topoisomerase II protein compared with parental CHO cells. However, the specific activity for decatenation using nuclear extract topoisomerase II was unchanged. Etoposide (100 μM)-induced topoisomerase II-DNA complexes in DZR cells and isolated nuclei were similarly one-half the level found in CHO cells and in isolated nuclei. However, the ability of 500 μM dexrazoxane to inhibit etoposide (100 μM)-induced topoisomerase II-DNA covalent complexes was reduced 4- to 6-fold in both DZR cells and nuclei compared with CHO cells and nuclei. In contrast, there was no differential ability of aclarubicin or merbarone to inhibit etoposide-induced topoisomerase II-DNA complexes in CHO compared with DZR cells and isolated nuclei. It was concluded that the DZR cell line acquired its resistance to dexrazoxane mainly through an alteration in the topoisomerase II target.     

A novel peroxisome proliferator-activated receptor alpha/gamma agonist, BPR1H0101, inhibits topoisomerase II catalytic activity in human cancer cells

Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used clinically for treating diabetes mellitus and cancer. 2-Methyl-2[(1-{3-phenyl-7-propylbenzol[d]isoxazol-6-yl}oxy)propyl]-1H-4-indolyl) oxy]propanoic acid (BPR1H0101) is a novel synthetic indole-based compound, discovered through research to identify new PPARgamma agonists, and it acts as a dual agonist for PPARgamma and PPARalpha. Isobologram analysis demonstrated that BPR1H0101 is capable of antagonistic interaction with the topoisomerase (topo) II poison, VP16. A study of its mechanism showed that BPR1H0101 could inhibit the catalytic activity of topo II in vitro, but did not produce detectable topo II-mediated DNA strand breaks in human oral cancer KB cells. Furthermore, BPR1H0101 could inhibit VP16-induced topo II-mediated DNA cleavage and ataxia-telangiectasia-mutated phosphorylation in KB cells. The results suggest that BPR1H0101 can interfere with the topo II reaction by inhibiting catalytic activity before the formation of the intermediate cleavable complex; consequently, it can impede VP16-induced topo II-mediated DNA cleavage and cell death. This is the first identified PPARalpha/gamma agonist that can serve as a topo II catalytic inhibitor, to interfere with VP16-induced cell death. The result might have relevance to the clinical use of the PPARalpha/gamma agonist in combination chemotherapy.