Uptake of the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and activation of NF-kappaB in human tumor cell lines.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer drug synthesized in this laboratory and currently in clinical trial, induces tumor vascular damage in vivo that is mediated primarily by cytokine synthesis by host cells. Although its pharmacology and antitumor activity have been extensively studied, little is known of its action on tumor cell lines. We measured [3H]DMXAA uptake in the Raji, Daudi, Jurkat, ECV304, NZM12, HL60, and K562 human tumor lines using velocity centrifugation through silicon oil layers, and also measured NF-kappaB activation by electrophoretic mobility shift assays. All lines accumulated [3H]DMXAA, and uptake by ECV304 cells was rapid, pH dependent (greater uptake at pH 6.5), similar at 4 degrees C and 37 degrees C, and unaffected by the addition of 5 mM sodium azide. The uptake ratio was 4.5-fold at a low drug concentration (4 microM) and decreased significantly (P < 0.01) to 4.0 as the external drug concentration was increased to 0.7 mM, providing evidence of saturability. [3H]DMXAA interacted weakly with isolated cytoplasmic proteins, as measured by equilibrium dialysis, providing a basis for the observed cellular uptake. Uptake was slightly reduced by addition of a less potent analogue, flavone acetic acid, or of an inactive analogue, 8-methylxanthenone-4-acetic acid, suggesting competition for binding sites. The Raji, Daudi, Jurkat. and ECV304 lines showed evidence of activation of the NF-kappaB transcription factor in response to DMXAA, but the identity of the NF-kappaB subunits translocated to the nucleus varied according to the line. The results are consistent with the hypothesis that DMXAA is taken up rapidly into cells by passive diffusion and binds to cellular proteins. The observed activation of NF-kappaB in some lines suggests that the effects of DMXAA on tumor cells, as well as host cells, must be considered in understanding its antitumoraction.