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
Isabelle
I
Centre de Recherche en Oncologie Expérimentale, Institut de Recherche Pierre Fabre, France.
Créancier
Laurent
L
Vispé
Stéphane
S
Annereau
Jean-Philippe
JP
Barret
Jean-Marc
JM
Pouny
Isabelle
I
Samson
Arnaud
A
Aussagues
Yannick
Y
Massiot
Georges
G
Ausseil
Frédéric
F
Bailly
Christian
C
Kruczynski
Anna
A
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D010936:Plant Extracts / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000502:physiology; D012632:Secosteroids / Q000494:pharmacology*; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C498702:PMAIP1 protein, human; D010936:Plant Extracts; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D012632:Secosteroids; D025801:Ubiquitin; C033270:physalin B; D046988:Proteasome Endopeptidase Complex