G alpha(12/13) inhibition enhances the anticancer effect of bortezomib through PSMB5 downregulation

Abstract

Bortezomib is a proteasome inhibitor approved for anticancer therapy. However, variable sensitivity of tumor cells exists in this therapy probably due to differences in the expression of proteasome subunits. G alpha(12/13) serves modulators or signal transducers in diverse pathways. This study investigated whether cancer cells display differential sensitivity to bortezomib with reference to G alpha(12/13) expression, and if so, whether G alpha(12/13) affects the expression of proteasome subunits and their activities. Bortezomib treatment exhibited greater sensitivities in Huh7 and SNU886 cells (epithelial type) than SK-Hep1 and SNU449 cells (mesenchymal type) that exhibited higher levels of G alpha(12/13). Overexpression of an active mutant of G alpha(12) (G alpha(12)QL) or G alpha(13) (G alpha(13)QL) diminished the ability of bortezomib to induce cytotoxicity in Huh7 cells. Moreover, transfection with the minigene that disturbs G protein-coupled receptor-G protein coupling (CT12 or CT13) increased it in SK-Hep1 cells. Consistently, MiaPaCa2 cells transfected with CT12 or CT13 exhibited a greater sensitivity to bortezomib. Evidence of G alpha(12/13)'s antagonism on the anticancer effect of bortezomib was verified in the reversal by G alpha(12)QL or G alpha(13)QL of the minigenes' enhancement of cytotoxity. Real-time polymerase chain reaction assay enabled us to identify PSMB5, multicatalytic endopeptidase complex-like-1, and proteasome activator subunit-1 repression by CT12 or CT13. Furthermore, G alpha(12/13) inhibition enhanced the ability of bortezomib to repress PSMB5, as shown by immunoblotting and proteasome activity assay. Moreover, this inhibitory effect on PSMB5 was attenuated by G alpha(12)QL or G alpha(13)QL. In conclusion, the inhibition of G alpha(12/13) activities may enhance the anticancer effect of bortezomib through PSMB5 repression, providing insight into the G alpha(12/13) pathway for the regulation of proteasomal activity.