Oula C Mansour1, Abraham Nudelman2, Ada Rephaeli3, Don R Phillips1, Suzanne M Cutts4, Benny J Evison5,6. 1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia. 2. Chemistry Department, Bar-Ilan University, 52900, Ramat-Gan, Israel. 3. Sackler Faculty of Medicine, Felsenstein Medical Research Center, Tel-Aviv University, 49100, Petach-Tikva, Israel. 4. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia. s.cutts@latrobe.edu.au. 5. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia. benny.evison@nyrada.com. 6. Nyrada Inc, Suite 2, Level 3, 828 Pacific Highway, Gordon, NSW, 2072, Australia. benny.evison@nyrada.com.
Abstract
PURPOSE: Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. METHODS: In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. RESULTS: Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10-40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. CONCLUSIONS: The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.
PURPOSE: Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. METHODS: In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. RESULTS: Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10-40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. CONCLUSIONS: The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.
Authors: Belinda S Parker; Trevor Buley; Ben J Evison; Suzanne M Cutts; Greg M Neumann; Magdy N Iskander; Don R Phillips Journal: J Biol Chem Date: 2004-02-12 Impact factor: 5.157
Authors: Michal Entin-Meer; Ada Rephaeli; Xiaodong Yang; Abraham Nudelman; Scott R VandenBerg; Daphne Adele Haas-Kogan Journal: Mol Cancer Ther Date: 2005-12 Impact factor: 6.261
Authors: Ada Rephaeli; Michal Entin-Meer; Dikla Angel; Nataly Tarasenko; Tal Gruss-Fischer; Irena Bruachman; Don R Phillips; Suzanne M Cutts; Daphne Haas-Kogan; Abraham Nudelman Journal: Invest New Drugs Date: 2006-09 Impact factor: 3.850
Authors: Oula C Mansour; Benny J Evison; Brad E Sleebs; Keith G Watson; Abraham Nudelman; Ada Rephaeli; Damian P Buck; J Grant Collins; Rebecca A Bilardi; Don R Phillips; Suzanne M Cutts Journal: J Med Chem Date: 2010-10-14 Impact factor: 7.446
Authors: Elias Péan; Beatriz Flores; Ian Hudson; Jan Sjöberg; Kristina Dunder; Tomas Salmonson; Christian Gisselbrecht; Edward Laane; Francesco Pignatti Journal: Oncologist Date: 2013-04-24
Authors: P De Isabella; M Palumbo; C Sissi; G Capranico; N Carenini; E Menta; A Oliva; S Spinelli; A P Krapcho; F C Giuliani Journal: Mol Pharmacol Date: 1995-07 Impact factor: 4.436
Authors: Ben J Evison; Oula C Mansour; Ernesto Menta; Don R Phillips; Suzanne M Cutts Journal: Nucleic Acids Res Date: 2007-05-05 Impact factor: 16.971