Effects of the proteasome inhibitor bortezomib in rat hepatoma

Abstract

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Objectives: The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. This study measured the effects of the proteasome inhibitor bortezomib on rat hepatoma cells.

Methods: MH3924A rat hepatoma cells were cultured with different concentrations of Bortezomib (1nM-10µM) for 12, 24 or 48 hours and the uptake of 3H thymidine (TdR) FDG and 14C-aminoisobutyric acid (AIB), respectively, was measured. In addition, apoptosis of the tumor cell lines was determind by YOPRO-1 staining. Furthermore, transient transfection with reporter constructs consisting of the NFkB regulatory region and the luciferase gene were done and compared to unspecific reporter constructs (HSVtk minimal promoter, AP1 binding site, CMV promoter, CRE promoter).

Results: After incubation with 10 nM Bortezomib for 24h the TdR uptake as well as the cell number decreased to 5%. A cytotoxicity assay showed similar results. The AIB uptake increased after 12 h incubation (by 50%) and decreased after 24 h incubation in a dose-dependent manner (by 60%), whereas the FDG uptake was not significantly changed. Furthermore, we found a significant decrease in NFkB induction, whereas the constructs with CRE, AP1 and CMV showed no change.

Conclusions: According to these in vitro data, bortezomib is effective in rat hepatoma which occurs at least partly by inhibition of NFkB activity. Changes in amino acid transport may be used for therapy monitoring.