Inhibition of DMXAA-induced tumor necrosis factor production in murine splenocyte cultures by NF-kappaB inhibitors.

The induction of cytokine synthesis within tumor tissue is a key component of the antivascular action of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in murine tumors. We previously showed that DMXAA alone induced only low amounts of tumor necrosis factor (TNF) in cultured spleen cells, but the addition of suboptimal concentrations of lipopolysaccharide (LPS) provided a costimulatory signal that resulted in 6-10-fold increase in secreted TNF. In this study we investigated the molecular pathway involved, and showed that the addition of NF-kappaB inhibitors salicylate and parthenolide reduced the levels of TNF secreted into the culture supernatants induced with DMXAA (10 microg/ml) alone or in combination with LPS (10 microg/ml). Results from gene arrays, confirmed with RT-PCR, showed that the TNF gene was not upregulated with DMXAA alone, and was only slightly increased above the level of significance when LPS was added simultaneously. This contrasted with secreted TNF protein levels, which increased 5- and 48-fold, respectively, above that in untreated cultures with DMXAA alone or in combination with LPS. In addition to TNF, protein arrays showed IL-6, IL-10, MIP-1alpha, MIP-2, and RANTES were also secreted following treatment with 10 microg/ml DMXAA alone, and IL-4, IFN-gamma, MCP-5, and TIMP-1 were additionally induced using a higher dose of 300 microg/ml DMXAA. The drug is currently showing promise in phase II combination trials, and these studies suggest that DMXAA-induced TNF production in the splenocyte cultures was not due to increased expression of the TNF gene, but through effects on NF-kappaB-dependent posttranscriptional regulation.