Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosi
June 27th, 2008 | by admin |Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosis.
The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.
Vandenberghe I, Créancier L, Vispé S, Annereau JP, Barret JM, Pouny I, Samson A, Aussagues Y, Massiot G, Ausseil F, Bailly C, Kruczynski A.
Centre de Recherche en Oncologie Expérimentale, Institut de Recherche Pierre Fabre, France.