Abstract 24: Labeling of oxidizable proteins with a photoactivatable analog of the antitumor agent DMXAA and evidence for redox signaling in its mode of action

Abstract

Abstract DMXAA (5,6-dimethylxanthenone-4-acetic acid) is in late stages of clinical development as a tumor vascular disrupting agent. The signalling pathway(s) and molecular target(s) for this drug are still undefined. As an approach towards identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA is used in the studies here to probe for cellular binding proteins. Unexpectedly, over 20 proteins were photo-affinity labelled from the cytosolic protein extracts from murine spleen cells and the RAW 264.7 murine macrophage-like cell line. While no protein domain, fold or binding site for a specific ligand was found to be shared by all the candidate proteins, almost all were noted to be oxidisable proteins, implicating a role for redox cycling in the action of DMXAA. Consistent with this hypothesis, we showed that DMXAA caused an accumulative increase in concentrations of reactive oxygen species (ROS) in RAW 264.7 cells over the first two hours. This increase in ROS was suppressed in the presence of the anti-oxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. SiRNA knock-down in RAW 264.7 cells of one of the photolabelled proteins, superoxide dismutase 1, an ROS scavenger resulted in an increase in tumour necrosis factor-alpha production in response to DMXAA compared with controls treated with transfection reagent alone, or with unrelated siRNA molecules. Knock-down of two other photoaffinity labelled proteins which do not have direct ROS scavenging properties, thioredoxin and protein disulfide isomerase 6, had no significant effect on tumour necrosis factor-alpha production. The results from these three lines of study have led us to speculate on a role of modulation of redox signalling in cytokine induction by DMXAA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 24.