Anti-oxidative extracts from the milk thistle plant (Silybum marianum) have been shown
to have antiproliferative effects in several tumor types. Silibinin is the primary
active component isolated from the crude seed extract, silymarin. It has been used
as a dietary supplement for hepatoprotection for over 2000 years. Silibinin has been
shown to be safe in multiple animal models and has had no significant adverse events
in human studies. We investigated the potential for this nontoxic flavolignan to inhibit
proliferation of human colon cancer.
Three well-characterized cell lines, Fet, Geo, and HCT116, were studied. The MTT cell-viability
assay was performed to study the effect of silibinin on proliferation. Fluorescence-activated
cell sorter (FACS) analysis was used to determine the effects of silibinin on cell
cycle and apoptosis. 4', 6'-diamidine-2'-phenylindole (DAPI) staining with confocal
microscopy was used to morphologically confirm these results. Poly ADP-ribose polymerase
(PARP) cleavage and expression levels of p21, p27, cyclins B1/D1, and CDK-2 were measured.
Cyclooxygenase-2 (COX-2) levels were also measured. The experiments were performed
in triplicate and reported as mean values with standard errors. Means were contrasted
using analysis of variance with Dunnet's correction for multiple testing. All statistical
testing was two-sided with a significance level of 5%.
The MTT assay revealed a strong dose-dependent inhibitory effect. Treatment with 75
microg/mL resulted in 50% inhibition of cell-viability (IC-50) in Fet and Geo lines
at 72 h. An IC50 dose of 40 ug/mL was obtained in HCT116, a poorly-differentiated
cell line, at 72 h. FACS analysis demonstrated statistically significant cell-cycle
arrest in all cell lines. G(2)-M phase arrests in Fet and Geo cell lines (P < 0.001)
and a G1 arrest in HCT116 (P = 0.005) were noted. Trivial increases in early apoptotic
rates (2% to 3%) for Geo and HCT116 were noted on FACS analysis via annexin V-propidium
iodide technique (P < 0.05), but no evidence for apoptosis was seen on Western blot
for PARP cleavage or DAPI. Cyclin B1/D1 and CDK-2 levels were inhibited. Increased
expression of cell cycle inhibitors, p21 or p27, was noted, and there was no effect
on COX-2 expression.
Silibinin significantly inhibits proliferation through cell-cycle arrest via inhibition
of cyclin-CDK promoter activity. Despite its antioxidant profile, there is no effect
on COX-2 expression. Apoptosis does not appear to be greatly increased in human colon
cancer cell lines Fet, Geo, and HCT116. Rather, inhibition of cell cycle regulatory
proteins plays a fundamental role in silibinin's mechanism of action, and this may
serve as a basis for combined use with conventional chemotherapeutics.