Effects of Soy Isoflavones on Apoptosis Induction and G2-M Arrest in Human Hepatoma Cells Involvement of Caspase-3 Activation, Bcl-2 and Bcl-XL Downregulation, and Cdc2 Kinase Activity

Abstract

Genistein, biochanin-A, and daidzein, the predominant soy isoflavones, have been reported to lower the risk of cancer, but it is not known whether they protect against human hepatoma cancer. This study was designed to investigate their effects on cell growth, the cell cycle, and apoptosis induction in the human hepatoma cell lines, HepG2, Hep3B, Huh7, PLC, and HA22T. Genistein, biochanin-A, and daidzein inhibited growth of all five lines in a dose-dependent manner. DNA fragmentation studies and the TUNEL assay demonstrated that isoflavones caused tumor cell death by induction of apoptosis. Activation of caspase-3 and cleavage of the caspase-3 substrate, poly(ADP-ribose)polymerase, was seen in hepatoma cells after 24 hours' exposure to isoflavones. In addition, isoflavone cytotoxicity correlated with downregulation of Bcl-2 and Bcl-XL expression. Synergistic effects of the three isoflavones were observed on cell growth inhibition, apoptosis induction, and anti-apoptotic protein expression. Flow cytometry showed that genistein, but not biochanin-A or daidzein, induced progressive and sustained accumulation of hepatoma cancer cells in the G2/M phase as a result of inhibition of Cdc2 kinase activity. Coapplication of caffeine prevented this cell cycle arrest, but not apoptosis, showing that cell cycle arrest was not necessary for apoptosis. Furthermore, the isoflavones combination also had a significant tumor-suppressive effect in nude mice. These results suggest that isoflavones might be promising agents for the treatment of human hepatoma.