In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h]isoquinoline-1, 3-diones with substitutions at the 6 and 7 positions (azonafides)

Craig A. Mayr, Salah M. Sami, Robert T Dorr

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These agents differ structurally from amonafide by the replacement of the naphthalene chromophore with an anthracene chromophore, the lack of a primary amine moiety in the 5 position, and substitutions at the 6 and 7 positions on the anthracene nucleus. In this study, five analogs with potent growth inhibitory activity and with low cardiotoxicity were chosen. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro and continuous drug exposure revealed IC50 values in CHO cells in the nanomolar range. Intracellular scanning laser confocal microscopy of these drug-treated CHO cells showed that all analogs are able to enter cell nuclei with varying nuclear/cytoplasmic distribution: the more potent dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted parent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl substituted) inhibited DNA and RNA synthesis when assayed immediately after a 1 h exposure. In contrast, analogs 47 and 104 required 24 h post-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkaline elution techniques, each analog also produced DNA single- and double-stranded breaks, as well as DNA-protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand damage in CHO cells at their IC90 concentrations, whereas the three other compounds with lower growth inhibitory potency produced marked and roughly equivalent DNA damage at equitoxic concentrations. Gel shift analysis ot SV40 DNA exposed to the compounds demonstrated that these agents do not directly induce DNA strand breaks. However, catalytic studies with purified human topoisomerase II (Topo II) and plasmid DNA demonstrated that these drugs inhibit this enzyme. These results suggest that the azonafides inhibit Topo II to cause protein-associated strand breaks and impaired DNA and RNA synthesis. However, other mechanisms may also be operant, especially with the more potent dimethylamino ethyl substituted analogs.

Original languageEnglish (US)
Pages (from-to)245-256
Number of pages12
JournalAnti-Cancer Drugs
Volume8
Issue number3
DOIs
StatePublished - 1997

Fingerprint

Type II DNA Topoisomerase
Cricetulus
DNA Damage
Ovary
amonafide
DNA
DNA Breaks
RNA
Pharmaceutical Preparations
Single-Stranded DNA
Electrophoretic Mobility Shift Assay
Growth
Cell Nucleus
Confocal Microscopy
Inhibitory Concentration 50
Amines
Plasmids
Coloring Agents
azonafide
In Vitro Techniques

Keywords

  • alkaline elution
  • anthracene
  • azonafide
  • intercalation
  • strand breaks

ASJC Scopus subject areas

  • Pharmacology
  • Cancer Research
  • Oncology

Cite this

@article{07b767b86c2542708bcd1abdac8a8c0b,
title = "In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h]isoquinoline-1, 3-diones with substitutions at the 6 and 7 positions (azonafides)",
abstract = "The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These agents differ structurally from amonafide by the replacement of the naphthalene chromophore with an anthracene chromophore, the lack of a primary amine moiety in the 5 position, and substitutions at the 6 and 7 positions on the anthracene nucleus. In this study, five analogs with potent growth inhibitory activity and with low cardiotoxicity were chosen. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro and continuous drug exposure revealed IC50 values in CHO cells in the nanomolar range. Intracellular scanning laser confocal microscopy of these drug-treated CHO cells showed that all analogs are able to enter cell nuclei with varying nuclear/cytoplasmic distribution: the more potent dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted parent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl substituted) inhibited DNA and RNA synthesis when assayed immediately after a 1 h exposure. In contrast, analogs 47 and 104 required 24 h post-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkaline elution techniques, each analog also produced DNA single- and double-stranded breaks, as well as DNA-protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand damage in CHO cells at their IC90 concentrations, whereas the three other compounds with lower growth inhibitory potency produced marked and roughly equivalent DNA damage at equitoxic concentrations. Gel shift analysis ot SV40 DNA exposed to the compounds demonstrated that these agents do not directly induce DNA strand breaks. However, catalytic studies with purified human topoisomerase II (Topo II) and plasmid DNA demonstrated that these drugs inhibit this enzyme. These results suggest that the azonafides inhibit Topo II to cause protein-associated strand breaks and impaired DNA and RNA synthesis. However, other mechanisms may also be operant, especially with the more potent dimethylamino ethyl substituted analogs.",
keywords = "alkaline elution, anthracene, azonafide, intercalation, strand breaks",
author = "Mayr, {Craig A.} and Sami, {Salah M.} and Dorr, {Robert T}",
year = "1997",
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T1 - In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h]isoquinoline-1, 3-diones with substitutions at the 6 and 7 positions (azonafides)

AU - Mayr, Craig A.

AU - Sami, Salah M.

AU - Dorr, Robert T

PY - 1997

Y1 - 1997

N2 - The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These agents differ structurally from amonafide by the replacement of the naphthalene chromophore with an anthracene chromophore, the lack of a primary amine moiety in the 5 position, and substitutions at the 6 and 7 positions on the anthracene nucleus. In this study, five analogs with potent growth inhibitory activity and with low cardiotoxicity were chosen. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro and continuous drug exposure revealed IC50 values in CHO cells in the nanomolar range. Intracellular scanning laser confocal microscopy of these drug-treated CHO cells showed that all analogs are able to enter cell nuclei with varying nuclear/cytoplasmic distribution: the more potent dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted parent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl substituted) inhibited DNA and RNA synthesis when assayed immediately after a 1 h exposure. In contrast, analogs 47 and 104 required 24 h post-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkaline elution techniques, each analog also produced DNA single- and double-stranded breaks, as well as DNA-protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand damage in CHO cells at their IC90 concentrations, whereas the three other compounds with lower growth inhibitory potency produced marked and roughly equivalent DNA damage at equitoxic concentrations. Gel shift analysis ot SV40 DNA exposed to the compounds demonstrated that these agents do not directly induce DNA strand breaks. However, catalytic studies with purified human topoisomerase II (Topo II) and plasmid DNA demonstrated that these drugs inhibit this enzyme. These results suggest that the azonafides inhibit Topo II to cause protein-associated strand breaks and impaired DNA and RNA synthesis. However, other mechanisms may also be operant, especially with the more potent dimethylamino ethyl substituted analogs.

AB - The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These agents differ structurally from amonafide by the replacement of the naphthalene chromophore with an anthracene chromophore, the lack of a primary amine moiety in the 5 position, and substitutions at the 6 and 7 positions on the anthracene nucleus. In this study, five analogs with potent growth inhibitory activity and with low cardiotoxicity were chosen. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro and continuous drug exposure revealed IC50 values in CHO cells in the nanomolar range. Intracellular scanning laser confocal microscopy of these drug-treated CHO cells showed that all analogs are able to enter cell nuclei with varying nuclear/cytoplasmic distribution: the more potent dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted parent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl substituted) inhibited DNA and RNA synthesis when assayed immediately after a 1 h exposure. In contrast, analogs 47 and 104 required 24 h post-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkaline elution techniques, each analog also produced DNA single- and double-stranded breaks, as well as DNA-protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand damage in CHO cells at their IC90 concentrations, whereas the three other compounds with lower growth inhibitory potency produced marked and roughly equivalent DNA damage at equitoxic concentrations. Gel shift analysis ot SV40 DNA exposed to the compounds demonstrated that these agents do not directly induce DNA strand breaks. However, catalytic studies with purified human topoisomerase II (Topo II) and plasmid DNA demonstrated that these drugs inhibit this enzyme. These results suggest that the azonafides inhibit Topo II to cause protein-associated strand breaks and impaired DNA and RNA synthesis. However, other mechanisms may also be operant, especially with the more potent dimethylamino ethyl substituted analogs.

KW - alkaline elution

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KW - azonafide

KW - intercalation

KW - strand breaks

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