Labeling of Oxidizable Proteins With a Photoactivatable Analog of the Antitumor Agent DMXAA: Evidence for Redox Signaling in Its Mode of Action

Neoplasia. 2010 Sep;12(9):755-65. doi: 10.1593/neo.10636.

Abstract

The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-alpha production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology
  • Biomarkers, Tumor / analysis
  • Biomarkers, Tumor / chemistry
  • Biomarkers, Tumor / metabolism
  • Cells, Cultured
  • Electrophoresis, Gel, Two-Dimensional
  • Endothelial Cells / chemistry
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Neovascularization, Pathologic / metabolism
  • Neovascularization, Pathologic / pathology
  • Oxidation-Reduction
  • Photoaffinity Labels / chemistry
  • Photoaffinity Labels / metabolism
  • Photoaffinity Labels / pharmacology*
  • Photochemistry
  • Proteins / analysis
  • Proteins / chemistry
  • Proteins / metabolism*
  • RNA, Small Interfering / pharmacology
  • Signal Transduction / drug effects
  • Spleen / cytology
  • Spleen / drug effects
  • Spleen / metabolism
  • Spleen / physiology
  • Staining and Labeling / methods*
  • Xanthones / chemistry
  • Xanthones / pharmacology*

Substances

  • Antineoplastic Agents
  • Biomarkers, Tumor
  • Photoaffinity Labels
  • Proteins
  • RNA, Small Interfering
  • Xanthones
  • vadimezan