17DMAG inhibits NFkB activity in tumors in mice monitored by bioluminescence imaging

5729 The transcription factor NFkB is a key regulator of cellular activation, proliferation and apoptosis and recognized as an important target for inflammation and cancer therapies. Recently bioluminescence imaging has greatly facilitated the development of animal models of cancer, allowing sensitive detection of luciferase-expressing cancer cells in living mice. Here we have developed a method for detecting and quantifying changes in NFkB activity in vivo non-invasively by inoculating cancer cells, stable transfectant of firefly luciferase under the control of NFkB with IVIS 100 imaging system (Xenogen). There are several reports demonstrating geldanamycin inhibits TNF-induced NFkB activation in cultured cells. And its possible mechanism is explained by both the consequence of hsp90-inhibition and direct inhibition of NFkB-DNA binding. Since there are no reports demonstrating in vivo inhibition of NFkB by geldanamycin or its analogs, we investigated the effect of 17DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) on NFkB activity in tumors in mice by using our animal model of molecular imaging. To monitor NFkB activity in vivo, tumor xenografts are formed by injecting EBC-1/NFkB/Luc human lung cancer cells subcutaneously near the right hindlimb of nude mice. This luciferase was induced by extrinsic stimulation such as TNF-alpha or LPS. To neglect the non-specific anti-luciferase activity of test compounds, EBC-1/Luc cells, in which luciferase was consitutively expressed are also injected subcutaneously near the left hindlimb as a control. Once tumor mass has established, basal luciferase activity in the tumor was measured prior to 17DMAG administration on the day of its treatment. One hour after the drug treatment, mice were treated with TNF-alpha (0.12mg/kg, ip), then post-TNF imaging was monitored at 4h later. We found that TNF-alpha induced NFkB luciferase average 6 fold from basal level. After treatment with 17DMAG (30mg/kg, po), NFkB induction by measuring the specific luciferase activity was significantly decreased. Meanwhile, basal luciferase in the contralateral tumor was not impaired after 17DMAG treatment.Thus, We have developed a new animal model, which enables evaluation of pharmacodynamics of NFkB inhibitors and demonstrated that a geldanamycin derivative, 17DMAG has an inhibitory activity of NFkB in vivo.