Flavonoid, Silibinin, Inhibits Proliferation and Promotes Cell-Cycle Arrest of Human Colon Cancer


      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.

      Materials and methods

      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 μg/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. G2-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.

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        • Kvasnicka F.
        • Biba B.
        • Sevcik R.
        • et al.
        Analysis of the active components of silymarin.
        J Chromatogr. 2003; 990: 239
      1. Creighton University School of Medicine: Complementary and alternative medicine.
        Milk thistle. 2005; (5 Oct (
        • Rainone F.
        Milk Thistle.
        Am Fam Physician. 2005; 72: 1285
      2. National Toxicology Programϵsummary of data for chemical selection.
        Executive summary—ilk Thistle/Silymarin/Silybin (84604-20-6/ 65666-07-1/ 22888-70-6). 1998 (Aug. (
        • National Cancer Institute—Clinical Trials
        Phase II.
        2005 (13 July. (
        • Tyagi A.K.
        • Singh R.P.
        • Agarwal C.
        • et al.
        Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth inhibition, G2-M arrest, and apoptosis.
        Clin Cancer Res. 2002; 8: 3512
        • Sharma Y.
        • Agarwal C.
        • Singh A.K.
        • et al.
        Inhibitory effect of silibinin on ligand binding to erbB1 and associated mitogenic signaling, growth, and DNA synthesis in advanced human prostate carcinoma cells.
        Mol Carcinog. 2001; 30: 224
        • Singh R.P.
        • Dhanalakshmi S.
        • Tyagi A.K.
        • et al.
        Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor binding protein-3 levels.
        Cancer Res. 2002; 62: 3063
        • Tyagi A.
        • Agarwal C.
        • Harrison G.
        • et al.
        Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase3 and PARP cleavages.
        Carcinogenesis. 2004; 25: 1711
        • Zi X.
        • Feyes D.K.
        • Agarwal R.
        Anticarcinogenic effect of a flavanoid antioxidant, Silymarin, in human breast cancer cells MDA-MB 468: Induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin dependent kinases and associated cyclins.
        Clin Cancer Res. 1998; 4: 1055
        • Scambia R.
        • De Vincenzo F.O.
        Antiproliferative effect of silybin on gynecological malignancies: Synergism with cisplatin and doxorubicin.
        Eur J Cancer. 1996; 32A: 877
        • Kohno H.
        • Tanaka T.
        • Kawabata K.
        • et al.
        Silymarin, a naturally occurring polyphenolic antioxidant flavinoid inhibits azoxymethane-induced colon carcinogenesis in male F344 rats.
        Int J Cancer. 2002; 101: 461
        • Agarwal C.
        • Singh R.
        • Dhanalakshmi S.
        • et al.
        Silibinin up-regulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells.
        Oncogene. 2003; 22: 8271
        • Yang S.H.
        • Lin J.K.
        • Chen W.S.
        • et al.
        Anti-angiogenic effect of silymarin on colon cancer LoVo cell line.
        J Surg Res. 2003; 113: 133
        • Volate S.R.
        • Davenport D.M.
        • Muga S.J.
        • et al.
        Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin, curcumin, silymarin, ginseng, and rutin).
        Carcinogenesis. 2005; 26: 1450
      3. Centers for Disease Control and Prevention—CDC Colorectal Cancer. 2005 (
        • Normanno N.
        • Bianco C.
        • Damiano V.
        • et al.
        Growth inhibition of human colon carcinoma combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides.
        Clin Cancer Res. 1996; 2: 601
        • Wang D.
        • Li W.
        • Jiang W.
        • et al.
        Autocrine TGFA expression in the regulation of initiation of human colon carcinoma growth.
        J Cell Physiol. 1998; 177: 387
        • Comoglio A.
        • Leonaduzzi G.
        • Carini R.
        • et al.
        Studies on the antioxidant and free radical scavenging properties of IdB1016, a new flavinolignan complex.
        Free Rad Res Commun. 1990; 11: 109
        • Muzes G.
        • Deak G.
        • Lang I.
        Effect of bioflavonoid silymarin on the in vitro activity and expression of superoxide dismutase enzyme.
        Acta Physiol Hung. 1991; 78: 3
        • Grana X.
        • Reddy P.
        Cell cycle control in mammalian cells: Role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CDKIs).
        Oncogene. 1995; 11: 211
        • Bhatiaa N.
        • Zhaoa J.
        • Wolf D.
        Inhibition of human carcinoma cell growth and DNA synthesis by silibinin, an active constituent of milk thistle: Comparison with silymarin.
        Cancer Lett. 1999; 147: 77
        • Sinicrope F.A.
        • Gill S.
        Role of cyclooxygenase-2 in colorectal cancer.
        Cancer Metastasis Rev. 2004; 23: 63
        • Schwartz G.K.
        • Shah M.A.
        Targeting the cell cycle: A new approach to cancer therapy.
        J Clin Oncol. 2005; 23: 9408
        • Awwad R.A.
        • Sergina N.
        • Yang H.
        • et al.
        The role of transforming growth factor α in determining growth factor independence.
        Cancer Res. 2003; 63: 4731
        • Sawhney R.S.
        • Sharma B.
        • Humphrey L.E.
        • et al.
        Integrin α2 and extracellular signal-regulated kinase are functionally linked in highly malignant autocrine transforming growth factor-α driven colon cancer cells.
        J Biol Chem. 2003; 278: 19861
        • Agarwal R.
        • Agarwal C.
        • Ichikawa H.
        • et al.
        Anticancer potential of silymarin: From bench to bed side.
        Anticancer Res. 2006; 26: 4457