Silibinin

The extract of milk thistle seed is commonly referred to as silymarin. Contained within silymarin are specific flavonoids called silibinin. Silymarin is a collective name for a mixture of flavonoids or, more technically, flavolignans. Both flavonoids and lignans are often loosely grouped together with isoflavonoids (soy), phytosterols and coumestans into a category of compounds known as phytoestrogens. Structural and chemical similarities between flavonoids and human steroid hormones, and thyroid hormone are particularly intriguing. Molecular biologists have begun studying the complex hormone-like actions of flavonoids, including the modulation of various enzyme activities and signaling pathways. Eventually this may lead to a fuller understanding of how diets rich in phytoestrogens help protect against a variety of cancers, including breast cancer, prostate cancer and colon cancer.

However, the most valuable use of milk thistle-derived silymarin is as a liver supporter to prevent and treat cirrhosis, chronic hepatitis, and gall bladder problems. It modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. A recent study suggested that silymarin may help patients with type II diabetes by assisting in blood sugar control.

Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells.

Cancer: Breast cancer

Action: Anti-inflammatory, chemotherapy, Myeloid-derived suppressor cells

Silibinin inhibits accumulation of myeloid-derived suppressor cells and tumor growth of murine breast cancer

Forghani P, Khorramizadeh MR & Waller EK. (2014). Cancer Medicine. Volume 3, Issue 2, pages 215–224, April 2014 DOI: 10.1002/cam4.186

Myeloid-derived suppressor cells (MDSC)s increase in blood and accumulate in the tumor microenvironment of tumor-bearing animals, contributing to immune suppression in cancer. Silibinin, a natural flavonoid from the seeds of milk thistle, has been developed as an anti-inflammatory agent and supportive care agent to reduce the toxicity of cancer chemotherapy. The goals of this study were to evaluate the effect of silibinin on MDSCs in tumor-bearing mice and antitumor activity of silibinin in a mouse model of breast cancer. 4T1 luciferase-transfected mammary carcinoma cells were injected into in the mammary fat pad female BALB/c mice, and female CB17-Prkdc Scid/J mice. Silibinin treatment started on day 4 or day 14 after tumor inoculation continued every other day.

Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Flow cytometry measured total leukocytes, CD11b+ Gr-1+ MDSC, and T cells in the blood and tumors of tumor-bearing mice. The effects of silibinin on 4T1 cell viability in vitro were measured by BLI. Treatment with silibinin increased overall survival in mice harboring tumors derived from the 4T1-luciferase breast cancer cell line, and reduced tumor volumes and numbers of CD11b+Gr-1+ MDSCs in the blood and tumor, and increased the content of T cells in the tumor microenvironment.

Silibinin failed to inhibit tumor growth in immunocompromised severe combined immunodeficiency mice, supporting the hypothesis that anticancer effect of silibinin is immune-mediated. The antitumor activity of silibinin requires an intact host immune system and is associated with decreased accumulation of blood and tumor-associated MDSCs.

Silibinin inhibits TPA-induced cell migration and MMP-9 expression in thyroid and breast cancer cells.

Oh SJ, Jung SP, Han J, Kim S, Kim JS, Nam SJ, Lee JE, Kim JH.

Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Gangnam-gu, Seoul 135- 710, Republic of Korea.

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis, cell migration and invasion. Herein, we investigated the effects of silibinin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration and MMP-9 expression in thyroid and breast cancer cells. Our results revealed that the levels of MMP-9 mRNA and protein expression were significantly increased by TPA but not MMP-2 in TPC-1 and MCF7 cells. To verify the regulatory mechanism of TPA-induced MMP-9 expression, we treated TPC-1 and MCF7 cells with the MEK1/2 inhibitor, UO126, and TPA-induced MMP-9 expression was significantly decreased. We also found that TPA-induced cell migration and MMP-9 expression was significantly decreased by silibinin. In addition, TPA-induced phosphorylation of MEK and ERK was also inhibited by silibinin. Taken together, we suggest that silibinin suppresses TPA-induced cell migration and MMP-9 expression through the MEK/ERK-dependent pathway in thyroid and breast cancer cells.

Silibinin and cancer

The last decade has brought many additional discoveries about the use of Silibinin to prevent breast cancer and prostate cancer.

A recent in vitro study by Zi and Agarwal (1999) found that silibinin was able to arrest cell growth in prostate cancer lines, probably through inhibiting various kinase enzymes. Silibinin helped arrest cell growth in the early phase of the cell cycle, known as G1. The researchers found a 20% increase in G1 cell population when the culture was treated with silibinin. It is well known that potent flavonoids have an anti- proliferative effect on tumor tissue.

Another study found that silibinin inhibits proliferation in both drug-sensitive and drug-resistant breast cancer and ovarian cancer lines. The suggested mechanism of action involves silibinin's ability to bind to nuclear type II estrogen receptors, which are thought to mediate the anti- proliferative effects of flavonoids (Scambia, 1996). Comparing the properties of silymarin and silibinin, two investigators, Zhao and Agarwal (1999) state:

"Studies from our laboratory have shown that silibinin, the major active constituent of silymarin, has comparable [to silymarin] inhibitory effects towards human prostate, breast and cervical carcinoma cell growth, DNA synthesis and cell viability, and is as strong an antioxidant as silymarin."

Can silibinin slow aging? The authors of a recent study (Onat, 1999) concluded that silymarin's and silibinin's anti- proliferative mechanism of action is not yet fully known, but it may involve modulating signal transduction pathways. These signaling pathways are involved in aging, atherosclerosis and cancer. Compounds that can inhibit excess proliferation involved in aging-related disorders are of great clinical interest. Onat found that both alpha tocopherol and silibinin had a similar inhibitory action on the proliferation of skin fibroblasts. Insofar as excess fibroblast proliferation is one of the phenomena of aging, silibinin could become one of the agents used to slow the aging of the skin.

Cardiovascular health and the brain

An early German study (Schriewer and Rauen, 1977) showed that silibinin dose-dependently inhibits the biosynthesis of cholesterol in vitro. This has been confirmed by more recent studies (reviewed by Skottova 1998). Another interesting effect is faster removal of low-density lipoproteins by the liver in the presence of silymarin. Studies have also shown that silymarin and silibinin inhibit the development of diet-related excess cholesterol levels in rats. Supplementing the diet with silymarin or silibinin resulted in an increase in HDL levels and a decrease in liver cholesterol content.

Silibinin's effect on diabetes

Silibinin is of considerable interest in the treatment of diabetes, since preliminary evidence indicates that it may prove helpful in normalizing the action of insulin. A Chinese study found that rats subjected to heat injury showed elevated blood glucose and high insulin levels due to stress- induced insulin resistance. The function of insulin receptors in the liver was shown to be impaired. Treatment with silibinin significantly enhanced the binding of insulin to the receptors (Tang 1991).

Silibinin was also found to help normalize pancreatic function in the presence of cyclosporine A, an immunosuppressive drug that is damaging to the pancreas This included a lowering of insulin secretion without raising serum glucose, possibly indicating that silibinin improves insulin sensitivity.

Schonfeld and colleagues (Schonfeld 1997) suggest that silibinin should be investigated as a potential treatment for Type II diabetics, who overproduce insulin due to insulin resistance. The authors also suggest that the protective effect of silibinin on the pancreas is non-specific, and is probably due to its antioxidant and membrane-stabilizing properties. Very likely, silibinin protects the pancreas not only against cyclosporine A, but also against alcohol and other toxins, and against free radicals in general.

Silibinin Shows Potent Antiviral Activity in Hepatitis C Patients Who Are Non- responders to Pegylated Interferon/Ribavirin

Silibinin is the main flavonoid extracted from Silybum marianum. This compound has been used in several cultures for the treatment of a variety of liver diseases without a precise knowledge of its mode of action. Because of its potential anti-oxidative action, pre-treatment with silibinin may improve response to interferon. Furthermore, silibinin inhibits viral replication in HCV laboratory replicon systems (Gastroenterology 132:1925. 2007).

In the current study, presented at the 43rd annual meeting of the European Association for the Study of the Liver (EASL) recently in Milan, researchers at the Medical University of Vienna in Austria discovered that silibinin is a potent antiviral agent active against the hepatitis C virus (HCV) in humans.

The study included 16 non-responders to full dose pegylated interferon alfa-2a (Pegasys) plus ribavirin combination therapy. Most (12) were men, the mean age was 50 years, 14 had HCV genotype 1, 2 had genotype 4, and 13 had advanced (F3/F4) fibrosis. Participants were pre- treated with 10 mg/kg/day intravenous (IV) silibinin (Legalon Sil) for 7 days before starting treatment with 180 mcg/week Pegasys plus 1000-1200 mg/day weight-based ribavirin, while also continuing on 140 mg thrice-daily silymarin (Legalon), an oral milk thistle product. HCV RNA was quantified using the TaqMan assay at baseline, after 7 infusions of silibinin, and every 2 weeks while taking pegylated interferon/ribavirin.

Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-κB and Signal Transducers and Activators of Transcription 3

Potential antiangiogenic mechanism of silibinin in advanced lung tumor cells in A/J mice. Silibinin inhibits the production and secretion of cytokines and interleukins via inhibition of TAMs. Furthermore, silibinin inhibits the activation of transcription factors (NF-κB/STAT/HIF-1α) but induces the expression of Ang-2/Tie-2 to inhibit the angiogenesis in urethane-induced advanced lung tumors in A/J mice. Events involved in driving angiogenesis (green) and the effect of silibinin (red).

The latency period for lung tumor progression offers a window of opportunity for therapeutic intervention. Herein, we studied the effect of oral silibinin (742 mg/kg body weight, 5 d/wk for 10 weeks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibinin strongly decreased both tumor number and tumor size, an antitumor effect that correlates with reduced antiangiogenic activity. Silibinin reduced microvessel size (50%, P < 0.01) with no change in the number of tumor microvessels and reduced (by 30%, P < 0.05) the formation of nestin-positive microvessels in tumors. Analysis of several proteins involved in new blood vessel formation showed that silibinin decreased the tumor expression of interleukin-13 (47%) and tumor necrosis factor-α (47%), and increased tissue inhibitor of metalloproteinase-1 (2-fold) and tissue inhibitor of metalloproteinase-2 (7-fold) expression, without significant changes in vascular endothelial growth factor levels. Hypoxia- inducible factor-1α expression and nuclear localization were also decreased by silibinin treatment. Cytokines secreted by tumor cells and tumor-associated macrophages regulate angiogenesis by activating nuclear factor-κB (NF-κB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-κB (ser276, 38%; P < 0.01) and STAT-3 (ser727, 16%; P < 0.01) in tumor cells and decreased the lung macrophage population. Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased by silibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression by antiangiogenic mechanisms seem to be mediated by decreased tumor-associated macrophages and cytokines, inhibition of hypoxia-inducible factor-1α, NF-κB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.

(Cancer Prev Res January 1, 2009 vol.2 no.1 Pp.74-83)

Silibinin modulates TNF-α and IFN-γ mediated signaling to regulate COX2 and iNOS expression in tumorigenic mouse lung epithelial LM2 cells

Tyagi A, Agarwal C, Dwyer-Nield LD, Singh RP, Malkinson AM, Agarwal R. Mol Carcinog. 2012 Oct;51(10):832-42. doi: 10.1002/mc.20851. Epub 2011 Aug 31.

Silibinin inhibits mouse lung tumorigenesis in part by targeting tumor microenvironment. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) can be pro- or anti-tumorigenic, but in lung cancer cell lines they induce pro-inflammatory enzymes cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS). Accordingly, here we examined mechanism of silibinin action on TNF-α + IFN-γ (hereafter referred as cytokine mixture) elicited signaling in tumor-derived mouse lung epithelial LM2 cells. Both signal transducers and activators of the transcription (STAT)3 (tyr705 and ser727) and STAT1 (tyr701) were activated within 15 min of cytokine mixture exposure, while STAT1 (ser727) activated after 3 h. Cytokine mixture also activated Erk1/2 and caused an increase in both COX2 and iNOS levels. Pretreatment of cells with a MEK, NF-κB, and/or epidermal growth factor receptor (EGFR) inhibitor inhibited cytokine mixture-induced activation of Erk1/2, NF-κB, or EGFR, respectively, and strongly decreased phosphorylation of STAT3 and STAT1 and expression of COX2 and iNOS. Also, janus family kinases (JAK)1 and JAK2 inhibitors specifically decreased cytokine-induced iNOS expression, suggesting possible roles of JAK1, JAK2, Erk1/2, NF-κB, and EGFR in cytokine mixture-caused induction of COX2 and iNOS expression via STAT3/STAT1 activation in LM2 cells. Importantly, silibinin pretreatment inhibited cytokine mixture-induced phosphorylation of STAT3, STAT1, and Erk1/2, NF-κB-DNA binding, and expression of COX2, iNOS, matrix metalloproteinases (MMP)2, and MMP9, which was mediated through impairment of STAT3 and STAT1 nuclear localization. Silibinin also inhibited cytokine mixture-induced migration of LM2 cells. Together, we showed that STAT3 and STAT1 could be valuable chemopreventive and therapeutic targets within the lung tumor microenvironment in addition to being targets within tumor itself, and that silibinin inhibits their activation as a plausible mechanism of its efficacy against lung cancer

Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by silibinin: involvement of protein kinase C

Kang SN1, Lee MH, Kim KM, Cho D, Kim TS. Biochem Pharmacol. 2001 Jun 15;61(12):1487-95.

The effect of silibinin, an active component of Silybum marianum, on cellular differentiation was investigated in the human promyelocytic leukemia HL-60 cell culture system. Treatment of HL-60 cells with silibinin inhibited cellular proliferation and induced cellular differentiation in a dose-dependent manner. Cytofluorometric analysis and morphologic studies indicated that silibinin induced differentiation of HL-60 cells predominantly into monocytes. Importantly, strongly synergistic induction of differentiation into monocytes was observed when silibinin was combined with 5 nM 1α,25-dihydroxyvitamin D3 [1,25-(OH)2D3], a well-known differentiation inducer of HL-60 cells into the monocytic lineage. Silibinin enhanced protein kinase C (PKC) activity and increased protein levels of both PKCα and PKCβ in 1,25-(OH)2D3-treated HL-60 cells. PKC and extracellular signal-regulated kinase (ERK) inhibitors significantly inhibited HL-60 cell differentiation induced by silibinin alone or in combination with 1,25-(OH)2D3, indicating that PKC and ERK may be involved in silibinin-induced HL-60 cell differentiation

Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to antiviral combination therapy.

P Ferenci, TM Scherzer, H Hofer et al. Gastroenterology. 2008 Nov;135(5):1561-7. doi: 10.1053/j.gastro.2008.07.072.

Silibinin is the main flavonoid extracted from Silybum marianum. This compound has been used in several cultures for the treatment of a variety of liver diseases without a precise knowledge of its mode of action. Because of its potential anti-oxidative action, pre-treatment with silibinin may improve response to interferon. Furthermore, silibinin inhibits viral replication in HCV laboratory replicon systems (Gastroenterology 132:1925. 2007).

In the current study, presented at the 43rd annual meeting of the European Association for the Study of the Liver (EASL) recently in Milan, researchers at the Medical University of Vienna in Austria discovered that silibinin is a potent antiviral agent active against the hepatitis C virus (HCV) in humans.

The study included 16 non-responders to full dose pegylated interferon alfa-2a (Pegasys) plus ribavirin combination therapy. Most (12) were men, the mean age was 50 years, 14 had HCV genotype 1, 2 had genotype 4, and 13 had advanced (F3/F4) fibrosis. Participants were pre-treated with 10 mg/kg/day intravenous (IV) silibinin (Legalon Sil) for 7 days before starting treatment with 180 mcg/week Pegasys plus 1000-1200 mg/day weight-based ribavirin, while also continuing on 140 mg thrice-daily silymarin (Legalon), an oral milk thistle product. HCV RNA was quantified using the TaqMan assay at baseline, after 7 infusions of silibinin, and every 2 weeks while taking pegylated interferon/ribavirin.

Results

Within 1 week, IV silibinin monotherapy lead to a decrease in HCV viral load in all patients (baseline: 6.2 MIU/mL; day 8: 0.96; P=0.00005).

The mean log decline in viral load was 0.82.

Mean ALT decreased from 162 to 118 (P=0.004).

At the time of the abstract presentation, viral kinetics were available only for 3 patients.

Viral load began to decrease started on day 3 of silibinin monotherapy and showed a linear decline over the next days.

Treatment was well tolerated.

While on antiviral treatment, HCV RNA tended to decline further (week 2: 0.81; week 4: 0.96; both P<0.001 vs baseline).

However, viral load started to increase again at week 8 (log drop: 0.69).

After 12 weeks, 5 patients had a log drop greater than 2.

Conclusion

These preliminary findings suggest that silibinin has marked antiviral activity against HCV, and this effect was unrelated to the action of interferon/ribavirin, according to the investigators.

They concluded that, "This drug may be a useful for treatment of chronic hepatitis C, especially in non-responders. The potential of silibinin for combination with new antivirals needs to be explored [further]."

References

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