Induction of STAT and NFkappaB activation by the antitumor agents 5,6-dimethylxanthenone-4-acetic acid and flavone acetic acid in a murine macrophage cell line

Abstract

The antitumor agents flavone-8-acetic acid (FAA) and its dose-potent analogue 5,6-dimethylxanthenone-4-acetic acid (DMXAA), currently in clinical trials, have a novel mechanism of action that is mediated through their ability to induce a spectrum of cytokines. Since NFkappaB and STAT transcription factors participate in the regulation of a number of genes involved in immune and cytokine responses, we investigated whether these transcription factors were activated in the ANA-1 murine macrophage cell line by DMXAA and FAA compared with lipopolysaccharide (LPS), a bacterial component that induces an overlapping spectrum of cytokines. Activation of STAT1 and STAT3 was observed distinctly 4 hr after DMXAA and FAA stimulation. DMXAA and FAA induced NFkappaB translocation with slower kinetics of activation compared with LPS. STAT activation by DMXAA and FAA was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. The ANA-1 cells produced high titres of interferons (IFNs) in the culture supernatant after stimulation with DMXAA and FAA, and the addition of antibodies to IFNalpha/beta inhibited STAT activation, indicating that IFNs mediated STAT activation. NFkappaB activation, on the other hand, was not inhibitable with cycloheximide or with antibodies to IFNalpha/beta. NFkappaB activation appeared to be a direct action of the anticancer agents, whereas activation of the STAT proteins was due, in part, to the high titres of IFNs induced. These results demonstrate the significance of the IFN response in initiating the cascade of secondary events that may contribute to the overall antitumor efficacy of DMXAA and FAA in murine models.