Lithium‐mediated downregulation of PKB/Akt and cyclin E with growth inhibition in hepatocellular carcinoma cells

Abstract

We studied in vitro effects of glycogen synthase kinase 3b (GSK3b)-inhibitor lithium on the growth of hepatocellular carcinoma (HCC) cells. Lithium induced strong growth inhibition (>70%) in 75% (n = 9 of 12) of cell lines, apparently independent from the status of major genes that are mutated in HCC including p53, p16 INK4a , b-catenin and Axin1. Comparative studies with a growth-sensitive Huh7 and growth-resistant Hep40 cell lines showed that lithium induces growth arrest in Huh7 cells but not in Hep40 cells. Lithium induced the accumulation of N-terminally phosphorylated inactive form of GSK3b with concomitant increase in b-catenin and b-catenin/TCF transcriptional activity in both cell lines. This suggests that lithium-mediated HCC growth inhibition is independent of its well-known stimulatory effect on Wnt-b-catenin signaling. The main differences between Huh7 and Hep40 responses to lithium treatment were observed at the levels PKB/Akt and cyclin E proteins. Lithium induced depletion of both proteins in growth-sensitive Huh7, but not in growth-resistant Hep40 cells. PKB/Akt and Cyclin E are 2 major proteins that are known to be constitutively active in HCC. The targeting of both proteins with lithium may be the main reason why most HCC cells are responsive to lithium-mediated growth inhibition, independent of their p53, retinoblastoma and Wnt-b-catenin pathways. The exploration of molecular mechanisms involved in lithium-mediated growth inhibition in relation with PKB/Akt and cyclin E downregulation may provide new insights for therapy of liver tumors. ' 2005 Wiley-Liss, Inc.Key words: hepatoma; lithium; cyclin E; b-catenin; PKB/Akt; glycogen synthase kinase-3bLithium is the main therapeutic agent for the treatment of bipolar disorders, but its mechanism of action is poorly understood. 1 Mechanistic studies have shown that lithium affects profoundly the embryonic development and tissue patterning in various organisms. 2 The effects of lithium on the embryonic development of Xenopus is quite similar to the effects observed upon experimental activation of Wnt-b-catenin pathway. 2,[3][4][5][6] This similarity is due to the inhibitory effects of lithium on the activity of glycogen synthase kinase-3b (GSK-3b) enzyme, 7-9 which plays a key role in the regulation of Wnt-b-catenin signaling pathway. In the absence of Wnt signals, GSK-3b-mediated N-terminal phosphorylation is required for ubiquitin-mediated degradation of b-catenin. 10,11 Exposure of cells to either Wnt ligands or lithium treatment results in the inhibition of GSK-3b activity, whereby leading to the cytoplasmic accumulation of b-catenin. Stabilized b-catenin forms nuclear complexes with a family of lymphoid enhancer/T-cell transcription factor (TCF) proteins, leading to the up-regulation of TCF target genes. [12][13][14][15] The wnt-b-catenin pathway is one of the most commonly activated signaling pathways in different tumor types, including hepatocellular carcinoma (HCC). The aberrant activation of this pathway may occur via different mechanisms, ...

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