Luteolin induced growth inhibition and apoptosis in hepatoma cells involving TGF-β and Fas/Fas-ligand signaling pathways    Free

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Anti-proliferative effects of flavonoids, such as luteolin and apigenin, were investigated on immortalized Chang liver cells, HepG2 (wild-type p53) cells and Hep3B (p53 deleted) cells. Luteolin showed more anti-proliferative effects on HepG2 and Hep3B cells than apigenin. However, luteolin and apigenin did not show any significant anti-proliferative effect on Chang liver cells. We thereafter used luteolin to study more detailed molecular mechanism involved in anti-proliferative effects on both cells. Luteolin-treated HepG2 and Hep3 cells showed typical apoptotic nuclear changes and the increase of a sub-G1 population. Flow cytometry analysis also revealed that luteolin induced G₁ phase arrest on HepG2 and Hep3B cells dose- and time-dependent manners. Cell cycle related proteins, such as cyclin D1, Cdk4, Cdk2 as well as retinoblastoma protein (pRb) and E2F were down-regulated in HepG2 cells, while the expression level of cyclin-dependent kinase inhibitor, p21^WAF1/CIP1, was increased in HepG2 cells after treatment with luteolin. However, Hep3B cells cells showed no significant induction of cyclin-dependent kinase inhibitor, p21^WAF1/CIP1, which is regulated by p53 and instead showed significant upregulation of the expression level of cyclin-dependent kinase inhibitor, p27 ^KIP1, which is regulated by TGF-β1 signaling pathway. To clarify the role of TGF-β1 signaling pathway in Hep3B cells, we examined the expression level of Smad4, which is a down stream regulator of the TGF-β1 signaling pathway, and identified the dose-dependent up-regulation of Smad4 proteins in Hep3B cells treated with luteolin. Fas/Fas-L signaling pathway was also involved at the apoptotic process in both cells treated with luteolin. Moreover, pro-caspase-3 was activated and showed the dose- and time-dependent cleavages of poly(ADP-ribose) polymerase (PARP) in both cells. Luteolin also modulated the ratio of Bax/Bcl-2 to induce apoptosis in both cells treated with luteolin. Finally we examined the expression levels of transcription factors, such as c-Jun and c-Myc, and early growth response-1 (Egr-1), and found out that these transcription factors were significantly up-regulated in luteolin-treated HepG2 cells, but not in Hep3B cells. Therefore, these results suggest that the state of p53 protein in human hepatocarcinoma cells might have a key role on the regulation of susceptibility to luteolin. These findings also suggest that luteolin may have a potential to be developed as an anti-cancer agent. (This work was supported by grant No. R01-2006-000-11117-0 from the Basic Research Program of the Korea Science & Engineering Foundation).