PS-341 is a novel, highly selective and potent proteasome inhibitor, which showed cytotoxicity against some tumor cells. Its anti-tumor activity has been suggested to be associated with modulation of the expression of apoptosis-associated proteins, such as p53, p21^WAF/CIP1, p27^KIP1, NF-κ, Bax and Bcl-2. c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3β(GSK-3β are important modulators of apoptosis. However, their role in PS-341-induced apoptosis is unclear. This study was undertaken to elucidate the role of JNK and GSK-3β in the PS-341-induced apoptosis in lung cancer cells.

NCI-H157 and A549 cells were used in the experiments. The cell viability was assayed using the MTT assay and apoptosis was evaluated by proteolysis of PARP. The JNK activity was measured by an in vitro immuno complex kinase assay and by phosphorylation of endogenous c-Jun. The protein expression was evaluated by Western blot analysis. Dominant negative JNK1 (DN-JNK1) and GSK-3βwere overexpressed using plasmid and adenovirus vectors, respectively.

PS-341 reduced the cell viability via apoptosis, activated JNK and increased the c-Jun expression. Blocking of the JNK activation by overexpression of DN-JNK1, or pretreatment with SP600125, suppressed the apoptosis induced by PS-341. The activation of caspase 3 was mediated by JNK activation. Blocking of the caspase 3 activation suppressed PS-341-induced apoptosis. PS-341 activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but its blockade enhanced the PS-341-induced cell death via apoptosis. GSK-3βwas inactivated by PS-341 via the PI3K/Akt pathway. Overexpression of constitutively active GSK-3β enhanced PS-341-induced apoptosis; in contrast, this was suppressed by dominant negative GSK-3β(DN-GSK-3β. Inactivation of GSK-3β by pretreatment with lithium chloride or the overexpression of DN-GSK-3β suppressed both the JNK activation and c-Jun up-regulation induced by PS-341.

The JNK/caspase pathway is involved in PS-341-induced apoptosis, which is negatively regulated by the PI3K/Akt-mediated inactivation of GSK-3β in lung cancer cells.