Probing the dynamics of doxorubicin-DNA intercalation during the initial activation of apoptosis by fluorescence lifetime imaging microscopy (FLIM)

PLoS One. 2012;7(9):e44947. doi: 10.1371/journal.pone.0044947. Epub 2012 Sep 18.

Abstract

Doxorubicin is a potent anthracycline antibiotic, commonly used to treat a wide range of cancers. Although postulated to intercalate between DNA bases, many of the details of doxorubicin's mechanism of action remain unclear. In this work, we demonstrate the ability of fluorescence lifetime imaging microscopy (FLIM) to dynamically monitor doxorubicin-DNA intercalation during the earliest stages of apoptosis. The fluorescence lifetime of doxorubicin in nuclei is found to decrease rapidly during the first 2 hours following drug administration, suggesting significant changes in the doxorubicin-DNA binding site's microenvironment upon apoptosis initiation. Decreases in doxorubicin fluorescence lifetimes were found to be concurrent with increases in phosphorylation of H2AX (an immediate signal of DNA double-strand breakage), but preceded activation of caspase-3 (a late signature of apoptosis) by more than 150 minutes. Time-dependent doxorubicin FLIM analyses of the effects of pretreating cells with either Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)-hydrazine (a histone acetyltransferase inhibitor) or Trichostatin A (a histone deacetylase inhibitor) revealed significant correlation of fluorescence lifetime with the stage of chromatin decondensation. Taken together, our findings suggest that monitoring the dynamics of doxorubicin fluorescence lifetimes can provide valuable information during the earliest phases of doxorubicin-induced apoptosis; and implicate that FLIM can serve as a sensitive, high-resolution tool for the elucidation of intercellular mechanisms and kinetics of anti-cancer drugs that bear fluorescent moieties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / metabolism
  • Antineoplastic Agents / pharmacology
  • Apoptosis / drug effects*
  • Caspase 3 / metabolism
  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism
  • DNA / metabolism*
  • Doxorubicin / metabolism*
  • Doxorubicin / pharmacology*
  • Enzyme Activation / drug effects
  • Extracellular Space / drug effects
  • Extracellular Space / metabolism
  • HeLa Cells
  • Humans
  • Intercalating Agents / metabolism
  • Intercalating Agents / pharmacology
  • Kinetics
  • Microscopy, Fluorescence*
  • Time Factors

Substances

  • Antineoplastic Agents
  • Intercalating Agents
  • Doxorubicin
  • DNA
  • Caspase 3

Grant support

This study was supported by grants MED-099-PP-04 and NM-099-PP-01 from the National Health Research Institutes of Taiwan, and grant NSC 99–2113-M-400–001-MY3 and the Biomedical NanoImaging Core Facility of the National Nanoscience and Nanotechnology Program from the National Science Council of Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.