G(alpha)12/13 inhibition enhances the anticancer effect of bortezomib through PSMB5 downregulation.
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) (Galpha(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)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.
Yang
Yoon Mee
YM
Department of Pharmacy, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea.
Lee
Sanghwan
S
Nam
Chang Won
CW
Ha
Ji Hee
JH
Jayaraman
Muralidharan
M
Dhanasekaran
Danny N
DN
Lee
Chang Ho
CH
Kwak
Mi-Kyoung
MK
Kim
Sang Geon
SG
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000473:pathology; D002460:Cell Line; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008113:Liver Neoplasms / Q000188:drug therapy / Q000473:pathology; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis
D000970:Antineoplastic Agents; D001897:Boronic Acids; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011719:Pyrazines; D012333:RNA, Messenger; D000069286:Bortezomib; C088874:PSMB5 protein, human; D046988:Proteasome Endopeptidase Complex; D044365:GTP-Binding Protein alpha Subunits, G12-G13