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The cytotoxicity of (−)-lomaiviticin A arises from induction of double-strand breaks in DNA

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Abstract

The metabolite (−)-lomaiviticin A, which contains two diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups, inhibits the growth of cultured human cancer cells at nanomolar–picomolar concentrations; however, the mechanism responsible for the potent cytotoxicity of this natural product is not known. Here we report that (−)-lomaiviticin A nicks and cleaves plasmid DNA by a pathway that is independent of reactive oxygen species and iron, and that the potent cytotoxicity of (−)-lomaiviticin A arises from the induction of DNA double-strand breaks (dsbs). In a plasmid cleavage assay, the ratio of single-strand breaks (ssbs) to dsbs is 5.3 ± 0.6:1. Labelling studies suggest that this cleavage occurs via a radical pathway. The structurally related isolates (−)-lomaiviticin C and (−)-kinamycin C, which contain one diazofluorene, are demonstrated to be much less effective DNA cleavage agents, thereby providing an explanation for the enhanced cytotoxicity of (−)-lomaiviticin A compared to that of other members of this family.

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Figure 1: Structures of the metabolites employed in this study and their proposed reaction pathways.
Figure 2: Analysis of nicked and cleaved plasmid pBR322 DNA by (−)-lomaiviticin A (1), (−)-lomaiviticin C (2) and (−)-kinamycin C (3).
Figure 3: Immunofluorescence imaging of γH2AX and 53BP1 foci in K562 cells treated with (−)-lomaiviticin A (1), (−)-lomaiviticin C (2) or (−)-kinamycin C (3).
Figure 4: Neutral comet unwinding assay of K562 cells treated with (−)-lomaiviticin A (1), (−)-lomaiviticin C (2) or (−)-kinamycin C (3).
Figure 5: Clonogenic survival curves and western blot analyses of cells treated with (−)-lomaiviticin A (1) or (−)-lomaiviticin C (2).
Figure 6: Relative reactivity studies and mechanistic pathways for the reduction of the diazofluorene.

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Acknowledgements

The authors dedicate this paper to the memory of their colleague Donald M. Crothers. Financial support from the National Institute of General Medical Sciences (R01GM090000, S.B.H.), the National Institute of Environmental Health Sciences (R01ES005775, P.M.G.), the National Cancer Institute (R01CA168733, P.M.G.), the National Science Foundation (Graduate Research Fellowship to C.M.W.), the Searle Scholars Program (S.B.H.) and Yale University (S.B.H.) is gratefully acknowledged. S.B.H. acknowledges early-stage investigator awards from the David and Lucile Packard Foundation, the Alfred P. Sloan Foundation, the Camille and Henry Dreyfus Foundation, and the Research Corporation for Science Advancement. We thank T. Dymarz, J. Shen, D. Spiegel and T. Wang for assistance.

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L.C.C. and C.M.W. designed and performed the plasmid cleavage, immunofluorescence, comet and flow cytometry experiments. Z.L. and C.M.W. performed the in vitro reactivity studies. P.M.G. and D.A.H. designed, performed and analysed the clonogenic survival assays, pATM/pChk2/pATR/pChk1 western blots and comet assay employing BRCA2-deficient cells. S.B.H. conceived and designed the study, analysed the data and composed the manuscript.

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Correspondence to Seth B. Herzon.

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Colis, L., Woo, C., Hegan, D. et al. The cytotoxicity of (−)-lomaiviticin A arises from induction of double-strand breaks in DNA. Nature Chem 6, 504–510 (2014). https://doi.org/10.1038/nchem.1944

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