Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells

Charles S. Bestwick*, Lesley Milne, Anne Marie Dance, Gaela Cochennec, Gillian Cruickshank, Eflamm Allain, Lynda Constable, Susan J. Duthie, Paul Kong Thoo Lin

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Bisnaphthalimides are DNA intercalators of potential use as chemotherapeutics but for which the range of mechanism of action is only gradually being elucidated. Using human promyelocytic HL-60 cells, we extend characterization of the cytotoxicity of bisnaphthalimidopropylspermidine (BNIPSpd) and examine the relationship with caspase-activity. Within 4 h exposure, BNIPSpd (1–10 μM) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6 h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12 h, active caspase expression increased and was sustained with 5 and 10 μM BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24 h, 5 and 10 μM BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1 μM BNIPSpd exposure also increased hypodiploid DNA content by 48 h. Polyamine levels decreased by 24 h BNIPSpd exposure. The pan-caspase inhibitor, z-VAD-fmk, significantly decreased DNA degradation (hypodiploid DNA and DNA ladders) and cytotoxicity. Despite this, cell growth and viability remained significantly impaired. We propose that BNIPSpd cytotoxicity arises through DNA damage and not polyamine depletion and that cytotoxicity is dominated by but not dependent upon caspase driven apoptosis.

Original languageEnglish
Pages (from-to)342-350
Number of pages9
JournalToxicology in Vitro
Volume52
Early online date23 Jul 2018
DOIs
Publication statusPublished - 1 Oct 2018

Fingerprint

Spermidine
HL-60 Cells
Cytotoxicity
Caspases
DNA
Polyamines
Apoptosis
Ladders
Cell membranes
Intercalating Agents
Caspase Inhibitors
bisnaphthalimidopropylspermidine
Phosphatidylserines
DNA Fragmentation
DNA Damage
Cell growth
Cell Survival
Cell Membrane
Membranes
Growth

Keywords

  • Apoptosis
  • Bisnaphthalimides
  • Caspase-inhibition
  • Cytotoxicity
  • Genotoxicity
  • HL-60 cells

ASJC Scopus subject areas

  • Toxicology

Cite this

Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells. / Bestwick, Charles S.; Milne, Lesley; Dance, Anne Marie; Cochennec, Gaela; Cruickshank, Gillian; Allain, Eflamm; Constable, Lynda; Duthie, Susan J.; Thoo Lin, Paul Kong.

In: Toxicology in Vitro, Vol. 52, 01.10.2018, p. 342-350.

Research output: Contribution to journalArticle

Bestwick, CS, Milne, L, Dance, AM, Cochennec, G, Cruickshank, G, Allain, E, Constable, L, Duthie, SJ & Thoo Lin, PK 2018, 'Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells', Toxicology in Vitro, vol. 52, pp. 342-350. https://doi.org/10.1016/j.tiv.2018.06.023
Bestwick, Charles S. ; Milne, Lesley ; Dance, Anne Marie ; Cochennec, Gaela ; Cruickshank, Gillian ; Allain, Eflamm ; Constable, Lynda ; Duthie, Susan J. ; Thoo Lin, Paul Kong. / Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells. In: Toxicology in Vitro. 2018 ; Vol. 52. pp. 342-350.
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abstract = "Bisnaphthalimides are DNA intercalators of potential use as chemotherapeutics but for which the range of mechanism of action is only gradually being elucidated. Using human promyelocytic HL-60 cells, we extend characterization of the cytotoxicity of bisnaphthalimidopropylspermidine (BNIPSpd) and examine the relationship with caspase-activity. Within 4 h exposure, BNIPSpd (1–10 μM) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6 h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12 h, active caspase expression increased and was sustained with 5 and 10 μM BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24 h, 5 and 10 μM BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1 μM BNIPSpd exposure also increased hypodiploid DNA content by 48 h. Polyamine levels decreased by 24 h BNIPSpd exposure. The pan-caspase inhibitor, z-VAD-fmk, significantly decreased DNA degradation (hypodiploid DNA and DNA ladders) and cytotoxicity. Despite this, cell growth and viability remained significantly impaired. We propose that BNIPSpd cytotoxicity arises through DNA damage and not polyamine depletion and that cytotoxicity is dominated by but not dependent upon caspase driven apoptosis.",
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AU - Bestwick, Charles S.

AU - Milne, Lesley

AU - Dance, Anne Marie

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AU - Cruickshank, Gillian

AU - Allain, Eflamm

AU - Constable, Lynda

AU - Duthie, Susan J.

AU - Thoo Lin, Paul Kong

N1 - The authors wish to thank Viv Buchan of the Rowett Institute Analytical Division for polyamine analysis and gratefully acknowledges the Robert Gordon University (PKTL, LC, CSB, SJD), The Scottish Government (Rural and Environment Science and Analytical Services [RESAS] Division; CSB, SJD, LM) and the Royal Society of Chemistry (PKTL) for financial support.

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N2 - Bisnaphthalimides are DNA intercalators of potential use as chemotherapeutics but for which the range of mechanism of action is only gradually being elucidated. Using human promyelocytic HL-60 cells, we extend characterization of the cytotoxicity of bisnaphthalimidopropylspermidine (BNIPSpd) and examine the relationship with caspase-activity. Within 4 h exposure, BNIPSpd (1–10 μM) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6 h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12 h, active caspase expression increased and was sustained with 5 and 10 μM BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24 h, 5 and 10 μM BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1 μM BNIPSpd exposure also increased hypodiploid DNA content by 48 h. Polyamine levels decreased by 24 h BNIPSpd exposure. The pan-caspase inhibitor, z-VAD-fmk, significantly decreased DNA degradation (hypodiploid DNA and DNA ladders) and cytotoxicity. Despite this, cell growth and viability remained significantly impaired. We propose that BNIPSpd cytotoxicity arises through DNA damage and not polyamine depletion and that cytotoxicity is dominated by but not dependent upon caspase driven apoptosis.

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