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Luteolin, a flavonoid, is abundant in seeds of perilla, a common plant for the Japanese traditional diet. Recently, luteolin has been shown to have anti-tumor effects, including a pro-apoptotic effect, on cancer cells. However, precise molecular mechanisms of the luteolin-induced apoptosis have not yet been fully understood. Thus, the AIMs of this study were to evaluate the pro-apoptotic effect of luteolin on human hepatoma cells both in vitro and in vivo, and to determine molecular mechanisms of the effect, focusing on the Fas/STAT3 signaling. Methods: HLF and HAK-1B hepatoma cells were used. Expressions of cleaved caspases, poly ADP-ribose polymerase (PARP), Fas, p-STAT3 were examined by Western blot. The STAT3 overexpression was achieved by transfection of the STAT3 cDNA into HLF cells. The ubiquitination assay was used to examine p-STAT3 degradation. Anti-serine-p-CDK5 and anti-tyrosine-p-CDK5 antibodies were used to evaluate phosphorylation status of CDK5. Both subcellular localization and expression levels in immunoreactive Fas and p-STAT3 were observed by confocal laser scanning microscopy. The growth-inhibitory effect of luteolin on subcutaneously-transplanted HAK-1B tumors was evaluated in nude mice. Results: A significant proliferation-inhibitory effect of luteolin was found on the hepatoma cells, showing 43.6% reduction in cell number in the luteolin-treated cells at the concentration of 10 μM for 3 days. A clear apoptosis was induced within 12 hours at the concentration of 50 μM, showing cleavages for caspase-7, caspase-3, and PARP. Of interest, luteolin induced a membranous Fas expression in the HLF cells, leading to their enhanced sensitivity to apoptosis when treated by the anti-Fas antibody (clone CH-11). Consistent with the increase in Fas expression, a drastic attenuation in phosphorylation of STAT3, a negative regulator for Fas transcription, was identified within 30 min in the luteolin-treated cells. The down-regulation of STAT3 was, at least in part, due to an accelerated ubiquitin-dependent proteolysis. An overexpression of STAT3 led to resistance to luteolin, suggesting that the STAT3 signaling pathway was a critical target of luteolin. Because STAT3 has recently been shown to be phosphorylated by CDK5 (PNAS 2004), we investigated the phosphorylation status of CDK5 in luteolin-treated cells, demonstrating a selective decrease in the tyrosine-p- CDK5 expression. In the animal experiments, luteolin significantly inhibited the growth of transplanted tumors. Conclusions: We demonstrated a pro-apoptotic effect of luteolin in human hepatoma cells, and first identified that this effect was critically attributable to the up-regulation of Fas expression, which was triggered by the down-regulation of p-STAT3 expression through its ubiquitin-dependent degradation and/or the inactivation of CDK5.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]