Glycogen synthase kinase-3 inhibitors augment TRAIL-induced apoptotic death in human hepatoma cells.

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC.

Dysregulation of glycogen synthase kinase-3beta signaling in hepatocellular carcinoma cells.

It has been reported that upstream components of the insulin-like growth factor (IGF) signaling axis could be overexpressed during hepatocarcinogenesis in humans and rodents. However, the signal transduction pathways activated downstream have been poorly studied. Here, we examined whether glycogen synthase kinase-3beta (GSK-3beta) could be a target in human hepatoma cell lines and transgenic ASV mice with hepatic expression of the SV40 large T antigen. In HuH7, Mahlavu, and Hep3B cells, basal levels of GSK-3beta(Ser9) phosphorylation were strongly elevated, indicating that GSK-3beta was inhibited. GSK-3beta phosphorylation was insensitive to exogenous IGFs and was blocked with an IGF-1 receptor-neutralizing antibody in Mahlavu and Hep3B cells. By using LY294002 and ML-9, which act as phosphatidylinositol 3-kinase (PI3-K) and Akt inhibitors, respectively, we showed that GSK-3beta phosphorylation required PI3-K activation in both cell lines whereas downstream Akt activation was required only in Mahlavu cells. However, in the 2 cell lines, GSK-3beta(Ser9) phosphorylation was controlled by protein kinase C (PKC)zeta because it was blocked by an inhibitory PKCzeta peptide. The blockage of GSK-3beta phosphorylation markedly inhibited glycogen synthesis and decreased beta-catenin expression. In addition, the overexpression of a constitutively active GSK-3beta reduced AP-1-mediated gene transcription in Hep3B cells. Finally, we observed that reexpression of IGF-2 in tumoral livers from ASV mice was associated with a marked phosphorylation of GSK-3beta. In conclusion, our results identify GSK-3beta as a molecular target of the constitutive activation of the IGF axis in in vitro and in vivo models of hepatocarcinogenesis. Persistent phosphorylation of GSK-3beta could be critical for regulation of glycogen metabolism and cell growth in hepatoma cells.