Isolation and Biochemical Characterization of a New Topoisomerase I Inhibitor from Ocotea leucoxylon
Bing‐Nan ZhouRandall K. JohnsonMichael R. MatternXiangyang WangSidney M. HechtHans T. BeckAlonzo OrtizDavid G. I. Kingston
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In a continuation of our search for potential tumor inhibitors from plants, we found that a crude extract from Ocotea leucoxylon showed selective activity typical of inhibitors of the enzyme topoisomerase I in a yeast assay for DNA-damaging agents. Using a bioassay-directed fractionation approach, the major bioactive compound was isolated and identified as the known aporphine alkaloid dicentrinone (4); the inactive alkaloid dicentrine (3) was also isolated. Compound 4 showed selective bioactivity against the rad52 repair-deficient yeast strain RS322 (IC12 49 μg/mL) and was inactive against the rad52- and topo1-deficient strain RS321 (IC12 > 2000 μg/mL) and against the repair-proficient strain RJ03 (IC12 > 2000 μg/mL). Biochemical studies with recombinant human topoisomerase I indicated that dicentrinone (4) is an inhibitor of the human enzyme. Colony formation studies suggest that it is weakly cytotoxic, but that its mechanism of toxicity differs from that of camptothecin and its derivatives.Keywords:
Camptothecin
A bstract : The sole target for the anticancer drug camptothecin (CPT) is the type I topoisomerase. The drug poisons the topoisomerase by slowing the religation step of the reaction, thereby trapping the enzyme in a covalent complex on the DNA. In addition, CPT has been shown to inhibit plasmid DNA relaxation in vitro . The structural bases for these two activities of CPT are explored in relation to the recently published crystal structure of the enzyme with bound DNA.
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Camptothecin, a plant alkaloid with antitumor activity, has been shown to be a potent inhibitor of nucleic acid synthesis and a strong inducer of DNA strand breaks in mammalian cells. Previous studies have shown that camptothecin inhibits purified mammalian DNA topoisomerase I by trapping a reversible enzyme-DNA "cleavable complex" (Hsiang et al., J. Biol. Chem., 260: 14873-14878, 1985). Our present studies, using L1210 cells and SV40-infected monkey cells, have shown that camptothecin-induced strand breaks are protein linked. The linked protein is most likely DNA topoisomerase I as revealed by immunoblot analysis, using antibodies against purified mammalian DNA topoisomerase I. Brief heating of camptothecin-treated cells to 65 degrees C resulted in a rapid reduction of the number of protein-linked DNA breaks. Reversal of the camptothecin-induced topoisomerase I-DNA complex by heat was also observed in an in vitro system by using purified mammalian DNA topoisomerase I. Our results suggest that camptothecin interferes with DNA topoisomerase I both in cultured mammalian cells and in the purified system by trapping a reversible enzyme-DNA cleavable complex.
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A camptothecin-resistant subline of P388 leukemia (P388/CPT) was developed by repeated transplantation of P388 cells in mice treated with therapeutic doses of camptothecin. In mice bearing the resistant tumor, a maximally tolerated dose of camptothecin produced no net reduction in tumor cell burden, in contrast to a 5-log cell kill in the parental P388 (P388/S). The IC50 of camptothecin, as determined by colony formation assays of cultured cells, was 8 times greater for the cloned P388/CPT cell line than for P388/S. P388/CPT cells were not cross-resistant to other antineoplastic agents, including topoisomerase II inhibitors. The type I topoisomerases purified from P388/CPT and P388/S cells were identical with respect to molecular weight, specific activity, in vitro camptothecin sensitivity, and DNA cleavage specificity. Camptothecin induced fewer protein-associated DNA single-strand breaks in the resistant cells than in the wild-type P388 cells. Topoisomerase I mRNA, immunoreactivity, and extractable enzymatic activity were 2-4 times lower for P388/CPT cells than for P388/S cells. As resistance to camptothecin developed, topoisomerase I extractable activity decreased, concomitant with an increase in topoisomerase II extractable activity. Furthermore, the appearance of camptothecin resistance was associated with specific rearrangements of the topoisomerase I gene. These results suggest that development of resistance to inhibitors of topoisomerase I can occur by down-regulation of the target enzyme, thus reducing the production of lethal enzyme-mediated DNA damage. The enhanced topoisomerase II activity in these cells suggests that resistance to camptothecin may be overcome by co-treatment with topoisomerase II inhibitors.
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Camptothecin was recently identified as an inhibitor of mammalian topoisomerase I. Similar to inhibitors of topoisomerase II, camptothecin produces DNA single-strand breaks (SSB) and DNA-protein cross-links (DPC) in mammalian cells. However, their one-to-one association, expected for trapped topoisomerase complexes, has not previously been demonstrated. We have studied camptothecin-induced SSB and DPC in Chinese hamster DC3F cells and their isolated nuclei, using the DNA alkaline elution technique. It was found that the SSB and DPC frequencies detected following camptothecin treatment depend upon the conditions used for lysis. When lysis was with sodium dodecyl sulfate, the observed frequencies of SSB and DPC were 2- to 3-fold greater than when sodium dodecyl sarkosinate (Sarkosyl) was used. In either case, the SSB:DPC ratio was close to 1. All of the camptothecin-induced SSB were protein linked, as indicated by the absence of DNA elution under nondeproteinizing conditions. DNA cleavage assays with purified topoisomerase I also indicated that the weaker Sarkosyl detergent fails to trap all of the enzyme-DNA complexes. In contrast, lysis conditions had little effect on levels of SSB or DPC produced by 4'-(9-acridinylamino)-methanesulfon-m-anisidide, suggesting that trapping of topoisomerase II complexes occurs equally well with either detergent. In experiments using isolated nuclei, it was found that the camptothecin-induced SSB, in contrast to trapped topoisomerase II complexes, can form and reverse within minutes at 4 degrees C. The activity of camptothecin at low temperature was also seen with purified topoisomerase I. These results support the hypothesis that the SSB and DPC induced by camptothecin in mammalian cells are due to an action on topoisomerase I.
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A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.
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Abstract Topoisomerases are ubiquitous enzymes that solve topological problems due to DNA (deoxyribonucleic acid) supercoiling occurring during the replication, transcription, recombination and chromatin remodelling processes. Human topoisomerase IB (Topo IB) is the selective target of camptothecin, a natural compound from which two powerful anticancer drugs, topotecan and irinotecan, are produced. Camptothecin acts as an interfacial inhibitor interacting with both the enzyme and DNA stabilising the covalent enzyme–DNA complex, and slowing down the religation of the broken DNA strands brings cells to death. Topo IB is also important for transcription of genes involved in neurodevelopment, and its inhibition during critical stages of brain development may be responsible for neurodevelopmental disorders. Key Concepts Topoisomerases are crucial enzymes essential in the relaxation of supercoiled DNA. Human topoisomerase IB cuts a single DNA strand forming a transient enzyme–DNA cleavage complex. Camptothecin is a natural compound of which human topoisomerase IB is the only cellular target. Camptothecin can trap the enzyme–DNA cleavage complex bringing cells to death. Two camptothecin derivatives have been approved by the US Food and Drug Administration: topotecan for ovarian and lung cancers and irinotecan for colorectal cancer. Camptothecin and its derivatives act as interfacial inhibitors interacting with both the enzyme and the DNA. Single topoisomerase mutation may induce resistance to camptothecin. New non‐camptothecin derivatives are under development. The cytotoxic activity of camptothecin increases in the presence of other compounds inhibiting enzymes involved in DNA repair. Inhibition of topoisomerase IB has also a strong influence on the modulation expression of groups of genes associated with autism.
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The antitumor agent coralyne and a number of structural analogues were found to be inhibitors of DNA topoisomerase I and were characterized biochemically. Several of these analogues stabilized the covalent binary complex formed between calf thymus topoisomerase I and pSP64 plasmid DNA; coralyne and 5,6-dihydrocoralyne had the greatest potency as inhibitors in this assay. In common with camptothecin, the effects of coralyne and 5,6-dihydrocoralyne were reversed in the presence of increasing salt concentration or temperature, consistent with the interpretation that both functioned mechanistically in a fashion analogous to camptothecin. The sequence specificity of DNA cleavage by coralyne and 5,6-dihydrocoralyne was also studied in comparison with camptothecin using a 471-bp DNA duplex as a substrate for topoisomerase I. Seven sites of cleavage were apparent, four of which were shared in common by coralyne, 5,6-dihydrocoralyne and camptothecin. Coralyne and 5,6-dihydrocoralyne produced cleavage at one sequence, 5'-TCTC decreases GTAA=3', that was not apparent in the presence of camptothecin; correspondingly, two cleavage bands appeared only when camptothecin was present. Coralyne and 5,6-dihydrocoralyne also inhibited topoisomerase I-mediated relaxation of supercoiled plasmid DNA. Coralyne was the most potent inhibitor of DNA relaxation; the effects of camptothecin and 5,6-dihydrocoralyne were roughly equal. At high concentrations, coralyne completely suppressed the formation of the topoisomerase I-DNA covalent binary complex.
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