Silymarin, a defined mixture of natural
flavonoid, has recently been shown to have potent
cancer chemopreventive efficacy against colon
carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in
silymarin, namely
silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon
carcinoma HT-29 cells.
Silibinin treatment of cells at 50-100 microg/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression,
silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21
protein as well as
mRNA levels, and decreased CDK2, CDK4,
cyclin E and
cyclin D1 protein levels together with an inhibition in CDK2 and CDK4
kinase activities. In other studies, we observed that G2/M arrest by
silibinin was associated with a decrease in cdc25C, cdc2/p34 and
cyclin B1 protein levels, as well as cdc2/p34
kinase activity. In the studies assessing
biological fate of
silibinin-treated cells,
silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of
silibinin treatment. Interestingly,
silibinin-induced apoptosis in HT-29 cells was independent of
caspases activation, as all
caspases inhibitor did not reverse
silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in
caspases activity increase and
caspases and PARP cleavage as well as a lack in
cytochrome c release in cytosol following
silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that
silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of
silibinin against
colon cancer. Together, these results identify molecular mechanisms of
silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon
carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in
colon cancer prevention and intervention.