Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80262, USA.

Significant emphasis is being placed on combination chemotherapy of cancer using cytotoxic agents and naturally occurring chemopreventive agents, having different mechanisms of action with non-overlapping toxicity. In this regard, here we assessed whether a cancer preventive agent silibinin synergizes the therapeutic potential of doxorubicin (Dox), cisplatin or carboplatin, the chemotherapeutic drugs, in both estrogen-dependent and -independent human breast carcinoma, MCF-7 and MDA-MB468 cells, respectively. When tested alone, each of the four agents showed growth inhibition in both the cell lines in a dose- and a time-dependent manner. Based on their growth inhibitory effects, several combinations of silibinin (25-100 microM) with Dox (10-75 nM), cisplatin (0.2-2 microg/ml) or carboplatin (2-20 microg/ml) were next assessed for their synergistic, additive and/or antagonistic efficacy towards cell growth inhibition and apoptotic death. The strongest synergistic effects for cell growth inhibition [combination index (CI) 0.35 for MCF-7 and 0.45 for MDA-MB468 cells] were evident at a silibinin dose of 100 microM plus 25 nM Dox, in both the cell lines. Most of the CIs for other combinations of these three drugs with silibinin also suggested strong synergistic effects for cell growth inhibition in both MCF-7 and MDA-MB468 cells. In quantitative apoptosis studies, combination of silibinin with Dox resulted in much stronger apoptotic death compared to each agent alone in both cell lines. In case of silibinin combination with cisplatin, it showed no additional apoptotic effect in either cell line. Similarly, silibinin plus carboplatin combination showed stronger apoptotic effect only in MCF-7 cells. Together, these results suggest a possible synergism between silibinin and conventional cytotoxic agents for breast cancer treatment, and warrant further in vivo studies in pre-clinical breast cancer models.1: Cancer Lett. 1999 Dec 1;147(1-2):77-84. Links

Center for Cancer Causation and Prevention, AMC Cancer Research Center, Denver, CO 80214, USA.

Several studies from our laboratory have shown the cancer chemopreventive and anti-carcinogenic effects of silymarin, a flavonoid antioxidant isolated from milk thistle, in long-term tumorigenesis models and in human prostate, breast and cervical carcinoma cells. Since silymarin is composed mainly of silibinin with small amounts of other stereoisomers of silibinin, in the present communication, studies were performed to assess whether the cancer preventive and anti-carcinogenic effects of silymarin are due to its major component silibinin. Treatment of different prostate, breast, and cervical human carcinoma cells with silibinin resulted in a highly significant inhibition of both cell growth and DNA synthesis in a time-dependent manner with large loss of cell viability only in case of cervical carcinoma cells. When compared with silymarin, these effects of silibinin were consistent and comparable in terms of cell growth and DNA synthesis inhibition, and loss of cell viability. Based on the comparable results of silibinin and silymarin, we suggest that the cancer chemopreventive and anti-carcinogenic effects of silymarin reported earlier are due to the main constituent silibinin.: Clin Cancer Res. 1998 Apr;4(4):1055-64. Links

Department of Dermatology, Case Western Reserve University, Cleveland, Ohio 44106, USA.

There is an increasing interest in identifying potent cancer preventive and therapeutic agents against breast cancer. Silymarin, a flavonoid antioxidant isolated from milk thistle, exerts exceptionally high to complete anticarcinogenic effects in tumorigenesis models of epithelial origin. In this study, we investigated the anticarcinogenic effect of silymarin and associated molecular mechanisms, using human breast carcinoma cells MDA-MB 468. Silymarin treatment resulted in a significantly high to complete inhibition of both anchorage-dependent and anchorage-independent cell growth in a dose- and time-dependent manner. The inhibitory effects of silymarin on cell growth and proliferation were associated with a G1 arrest in cell cycle progression concomitant with an induction of up to 19-fold in the protein expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21. Following silymarin treatment of cells, an incremental binding of Cip1/p21 with CDK2 and CDK6 paralleled a significant decrease in CDK2-, CDK6-, cyclin D1-, and cyclin E-associated kinase activity with no change in CDK2 and CDK6 expression but a decrease in G1 cyclins D1 and E. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against breast cancer and that this effect possibly involves an induction of Cip1/p21 by silymarin, which inhibits the threshold kinase activities of CDKs and associated cyclins, leading to a G1 arrest in cell cycle progression.1: Eur J Cancer. 1996 May;32A(5):877-82. Links

Department of Gynaecology, Catholic University, Rome, Italy.

The aim of this study was to test the antiproliferative activity of silybin, a flavonoid, on human ovarian and breast cancer cell lines. Since flavonoids are thought to act through Type II oestrogen binding sites (Type II EBS), silybin binding to Type II EBS was also examined. Silybin, used in concentrations from 0.1 to 20 microM, exerted a dose-dependent growth inhibitory effect on OVCA 433, A2780 parental and drug-resistant ovarian cancer cells, and MCF-7 doxorubicin (DOX)-resistant breast cancer cells (IC50 = 4.8-24 microM). Both L and D diastereoisomers of silybin were effective in inhibiting A2780 WT cell growth (IC50 = 14 and 20 microM, respectively). Flow cytometry revealed that silybin decreased the percentage of cells in the S and G2-M phases of the cell cycle with a concomitant increase in cells in the G0-G1 phase. Silybin was able to compete with [3H]E2 for nuclear but not cytosolic Type II EBS. Its affinity parallels its efficacy in inhibiting cell proliferation. Furthermore, silybin (0.1 and 1 microM) potentiates the effect of cisplatin (CDDP) (0.1-1 micrograms/ml) in inhibiting A2780 WT and CDDP-resistant cell growth. Similar results were obtained on MCF-7 DOX-resistant cells when silybin (0.1 microM) was associated with doxorubicin (0.1-10 micrograms/ml). As assessed by the Berembaum isobole method, the effect of silybin-CDDP and silybin-DOX combinations results in a synergistic action. Using the 'stem cell assay' described by Hamburger and Salmon [Science 1977, 197, 461-463], we found that silybin exerted a dose-dependent inhibition of clonogenic efficiency of cells derived from three ovarian tumours (IC50 = 7.4, 4 and 6.4 microM, respectively). Since CDDP and DOX are the two most commonly used drugs for gynaecological tumours, the clinical application of silybin is currently under investigation in our institute.