NVP-LBH589, a novel histone deacetylase inhibitor, activates caspase-3 and induces apoptosis of human tumor cells in vivo

4013 NVP-LBH589 is a potent inhibitor of histone deacetylases. The compound inhibits proliferation of human tumor cells in culture, and causes regressions of established human tumor xenografts in athymic nude mice. These effects are associated with the induction of expression of a cyclin-dependent kinase inhibitor, p21 WAF-1 . Here we report that the compound activates caspase-3-dependent apoptotic pathway in vitro and in vivo . Treatment of human tumor cells with the NVP-LBH589 resulted in the cleavage of the pro-caspase-3 and generation of the active form of the enzyme as determined by the IHC staining. The presence of the functional caspase-3 was also verified by a functional reporter assay. We have used previously described construct where luciferase is flanked by Estrogen Responsive (ER) Elements linked to the enzyme by the caspase-3 recognition sequence. This construct was stably transfected into D54 human glioma cell line. When caspase-3 is inactive in these cells, ER Elements inhibit luciferase-dependent light emission. Caspase-3 activation results in the cleavage and separation of the ER elements, and in light emission by resulting free luciferase. The D54 reporter cell line was encapsulated in hollow fibers, the fibers were implanted subcutaneously into athymic, nude mice, animals were treated systemically with the NVP-LBH589, the fibers were retrieved, and light emission was quantified with the CCD IVIS camera. The role of p21 WAF-1 expression in activation of caspase-3-dependent apoptotic pathway will be also discussed.