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

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.

5,6-Dimethylxanthenone-4-acetic Acid (DMXAA) Activates Stimulator of Interferon Gene (STING)-dependent Innate Immune Pathways and Is Regulated by Mitochondrial Membrane Potential

“…The Antioxidant DPI Diminishes DMXAA-mediated Signaling in Primary Macrophages-It has been shown previously that the antioxidant NAC inhibits DMXAA-mediated expression of cytokine genes such as Tnf in the macrophage cell line RAW 264.7 (43), suggesting that ROS could be regulating signaling in this pathway. Potential sources of cellular ROS in professional phagocytic cells are NADPH oxidases.…”

“…N-Acetylcysteine (NAC), the precursor of the cellular antioxidant glutathione, can be added to analyze the affects of oxidative stress. Previously, it has been shown that NAC treatment of the macrophage cell line RAW 264.7 decreases the expression of numerous cytokine genes such as Il6 and Tnf in response to DMXAA (43). This data suggests that antioxidants might also influence the expression of IFN-␤ in response to DMXAA.…”

“…It is important to note that any time pharmacologic agents play a prominent role in an experiment, off-target effects must be taken into account. In the RAW 264.7 macrophage cell line, it has been demonstrated that addition of the antioxidant NAC inhibited production of many cytokines such as TNF-␣, IL-6, and IL-1␤ in response to stimulation with DMXAA (43). This indicates that ROS could potentially be modulating the signaling pathway mediated by DMXAA.…”

Identification of human-selective analogues of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

“…The NF- κ B signalling pathway (Joseph et al , 1999; Woon et al , 2003; Wang et al , 2006), the TBK1-IRF-3 signalling axis (Roberts et al , 2007), the NOD signalling pathway (Cheng et al , 2010), and at least three members of the MAPK superfamily (Sun et al , 2011) are involved in one or more of the pleiotrophic effects of DMXAA in mice. Studies from our own laboratory showed that more than thirty oxidisable proteins were photoaffinity labelled with an azido-analogue of DMXAA in cellular extracts from murine leukocytes, implicating a role for redox signalling (Palmer et al , 2007; Brauer et al , 2010). The yet unidentified cellular enzymes that catalyse the first-step, one-electron oxidation of DMXAA to form the benzyl radical initiating the generation of reactive oxygen species and redox signalling could also be regarded as biochemical target(s) for this class of compounds.…”

The Chemotherapeutic Agent DMXAA as a Unique IRF3-Dependent Type-2 Vaccine Adjuvant

“…It can activate several inflammatory cell signaling pathways, including extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases, and cytosolic nucleotide-binding oligomerization domain 1 and 2-like receptors [4], [5]. In addition, DMXAA is a strong inducer of reactive oxygen species (ROS) [6]. The most striking immunogenic feature of DMXAA is its induction of immediate and predominant type-I-IFN [7].…”