title: 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.
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Romy
authors.initials: R
authors.lastname: Brauer
authors.forename: Liang-Chuan S
authors.initials: LC
authors.lastname: Wang
authors.forename: See-Tarn
authors.initials: ST
authors.lastname: Woon
authors.forename: David J A
authors.initials: DJ
authors.lastname: Bridewell
authors.forename: Kimiora
authors.initials: K
authors.lastname: Henare
authors.forename: Dieter
authors.initials: D
authors.lastname: Malinger
authors.forename: Brian D
authors.initials: BD
authors.lastname: Palmer
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
authors.forename: Claudine
authors.initials: C
authors.lastname: Kieda
authors.forename: Sofian M
authors.initials: SM
authors.lastname: Tijono
authors.forename: Lai-Ming
authors.initials: LM
authors.lastname: Ching
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology; D010084:Oxidation-Reduction; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010777:Photochemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*; D044004:Xanthones / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D019920:Photoaffinity Labels; D011506:Proteins; D034741:RNA, Small Interfering; D044004:Xanthones; C066668:vadimezan
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