Effects and mechanisms of silibinin on human hepatoma cell lines.
28 Oct 2007-World Journal of Gastroenterology (Baishideng Publishing Group Inc)-Vol. 13, Iss: 40, pp 5299-5305
TL;DR: It is demonstrated that silibinin significantly reduced the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells and increased acetylation of histone H3 and H4, indicating a possible role of altered histone acetylations in silib inin-reduced HCC cell proliferation.
Abstract: AIM: To investigate in vitro effects and mechanisms of silibinin on hepatocellular carcinoma (HCC) cell growth.
METHODS: Human HCC cell lines were treated with different doses of silibinin. The effects of silibinin on HCC cell growth and proliferation, apoptosis, cell cycle progression, histone acetylation, and other related signal transductions were systematically examined.
RESULTS: We demonstrated that silibinin significantly reduced the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells. Silibinin-reduced HuH7 cell growth was associated with significantly up-regulated p21/CDK4 and p27/CDK4 complexes, down-regulated Rb-phosphorylation and E2F1/DP1 complex. Silibinin promoted apoptosis of HuH7 cells that was associated with down-regulated survivin and up-regulated activated caspase-3 and -9. Silibinin's anti-angiogenic effects were indicated by down-regulated metalloproteinase-2 (MMP2) and CD34. We found that silibinin-reduced growth of HuH7 cells was associated with increased activity of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and decreased p-Akt production, indicating the role of PTEN/PI3K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a possible role of altered histone acetylation in silibinin-reduced HCC cell proliferation.
CONCLUSION: Our results defined silibinin's in vitro anti-HCC effects and possible mechanisms, and provided a rationale to further test silibinin for HCC chemoprevention.
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TL;DR: It is emphasized how increased understanding of the chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide unique and yet unexplored novel and highly effective chemopresventive strategies for reducing the health burden of cancer and other diseases in humans.
429 citations
TL;DR: The protective effects of silymarin and its major active constituent, silibinin, studied in various tissues, suggest a clinical application in cancer patients as an adjunct to established therapies, to prevent or reduce chemotherapy as well as radiotherapy-induced toxicity.
369 citations
Cites background from "Effects and mechanisms of silibinin..."
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...Silibinin also promotes apoptosis of human hepatoma HuH7 cells by down-regulating survivin and up-regulating activated caspase-3 and -9 [31]....
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...In other studies, silibinin significantly upregulated p21/CDK4 and p27/CDK4 complexes and down-regulated Rb-phosphorylation and E2F1/DP1 complex thereby inhibiting human hepatoma HuH7 cell growth [31]....
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TL;DR: A review of available studies on the effects of the purified product silybin on liver cells or on experimentally induced liver damage, and in patients with liver disease indicates that the bioavailability of slybin phytosome is higher than that of silymarin and is less influenced by liver damage.
Abstract: Herbal products are increasingly used, mainly in chronic liver disease. Extracts of milk thistle, Silymarin and silybin, are the most prescribed natural compounds, with different indications, but with no definitive results in terms of clinical efficacy. This review analyzes the available studies on the effects of the purified product silybin, both as a free and a conjugated molecule, on liver cells or on experimentally induced liver damage, and in patients with liver disease. We searched PUBMED for articles pertaining to the in vitro and in vivo effects of silybin, its antifibrotic, anti-inflammatory, and antioxidant properties, as well as its metabolic effects, combined with the authors' own knowledge of the literature. Results indicate that the bioavailability of silybin phytosome is higher than that of silymarin and is less influenced by liver damage; silybin does not show significant interactions with other drugs and at doses < 10 g/d has no significant side effects. Experimental studies have clearly demonstrated the antifibrotic, antioxidant and metabolic effects of silybin; previous human studies were insufficient for confirming the clinical efficacy in chronic liver disease, while ongoing clinical trials are promising. On the basis of literature data, silybin seems a promising drug for chronic liver disease.
295 citations
TL;DR: The aim of this review is to examine scientific studies concerning the effects derived from silymarin/silybin use in chronic liver diseases, cirrhosis and hepatocellular carcinoma.
Abstract: Silymarin is the extract of Silybum marianum, or milk thistle, and its major active compound is silybin, which has a remarkable biological effect. It is used in different liver disorders, particularly chronic liver diseases, cirrhosis and hepatocellular carcinoma, because of its antioxidant, anti-inflammatory and antifibrotic power. Indeed, the anti-oxidant and anti-inflammatory effect of silymarin is oriented towards the reduction of virus-related liver damages through inflammatory cascade softening and immune system modulation. It also has a direct antiviral effect associated with its intravenous administration in hepatitis C virus infection. With respect to alcohol abuse, silymarin is able to increase cellular vitality and to reduce both lipid peroxidation and cellular necrosis. Furthermore, silymarin/silybin use has important biological effects in non-alcoholic fatty liver disease. These substances antagonize the progression of non-alcoholic fatty liver disease, by intervening in various therapeutic targets: oxidative stress, insulin resistance, liver fat accumulation and mitochondrial dysfunction. Silymarin is also used in liver cirrhosis and hepatocellular carcinoma that represent common end stages of different hepatopathies by modulating different molecular patterns. Therefore, the aim of this review is to examine scientific studies concerning the effects derived from silymarin/silybin use in chronic liver diseases, cirrhosis and hepatocellular carcinoma.
270 citations
Cites background from "Effects and mechanisms of silibinin..."
...proved that silybin significantly reduces HuH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells growth by increasing cyclin-dependent kinase inhibitor p21 and p27/cyclin-dependent kinase (CDK) 4 complexes, by reducing retinoblastoma protein (Rb)-phosphorylation and transcription factor E2F1/transcription factor dimerization Partner (DP) 1 complex, as well as by promoting induction of codifying genes for caspase 3–9 and reducing the levels of survivin, whose sovraexpression is associated with a reduction of cell death [140,141]....
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TL;DR: Detailed mechanistic analyses revealed that silibinin targets signaling molecules involved in the regulation of epithelial-to-mesenchymal transition, proteases activation, adhesion, motility, invasiveness as well as the supportive tumor-microenvironment components, thereby inhibiting metastasis.
Abstract: Cancer is a major health problem around the world. Research efforts in the last few decades have been successful in providing better and effective treatments against both early stage and localized cancer, but clinical options against advanced metastatic stage/s of cancer remain limited. The high morbidity and mortality in most of the cancers are attributed to their metastatic spread to distant organs. Due to its extreme clinical relevance, metastasis has been extensively studied and is now understood as a highly complex biological event that involves multiple steps including acquisition of invasiveness by cancer cells, intravasation into circulatory system, survival in the circulation, arrest in microvasculature, extravasation, and growth at distant organs. The increasing understanding of molecular underpinnings of these events has provided excellent opportunity to target metastasis especially through nontoxic and biologically effective nutraceuticals. Silibinin, a popular dietary supplement isolated from milk thistle seed extracts, is one such natural agent that has shown biological efficacy through pleiotropic mechanisms against a variety of cancers and is currently in clinical trials. Recent preclinical studies have also shown strong efficacy of silibinin to target cancer cell’s migratory and invasive characteristics as well as their ability to metastasize to distant organs. Detailed mechanistic analyses revealed that silibinin targets signaling molecules involved in the regulation of epithelial-to-mesenchymal transition, proteases activation, adhesion, motility, invasiveness as well as the supportive tumor-microenvironment components, thereby inhibiting metastasis. Overall, the long history of human use, remarkable nontoxicity, and preclinical efficacy strongly favor the clinical use of silibinin against advanced metastatic cancers.
213 citations
References
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TL;DR: It is demonstrated that phosphatidylinositol 3-kinase (PI3K) and its downstream targets serine/threonine kinases AKT1 and AKT2 (AKT), are required for the full induction of p21 in A2780 cells treated with cisplatin or paclitaxel and suggested that this pathway contributes to cell cycle regulation promoted by p53 in response to drug-induced stress.
Abstract: The cyclin-dependent kinase inhibitor p21WAF1/CIP1/SD11 (p21) plays a crucial role in DNA repair, cell differentiation, and apoptosis through regulation of the cell cycle. A2780 human ovarian carcinoma cells, which are sensitive to cisplatin and paclitaxel, express wild-type p53 and exhibit a p53-mediated increase in p21 in response to the chemotherapeutic agents. Here, we demonstrate that phosphatidylinositol 3-kinase (PI3K) and its downstream targets serine/threonine kinases AKT1 and AKT2 (AKT), are required for the full induction of p21 in A2780 cells treated with cisplatin or paclitaxel. Inactivation of the PI3K/AKT signal transduction pathway either by its specific inhibitor LY294002 or by expression of dominant negative AKT inhibited p21 expression but had no inhibitory effect on the expression of the proapoptotic protein BAX by cisplatin and paclitaxel treatment. In addition, overexpression of wild-type or constitutively active AKT in A2780 cells sustained the regulation of p21 induction or increased the level of p21 expression, respectively. Experiments with additional ovarian carcinoma cell lines revealed that PI3K is involved in the expression of p21 induced by cisplatin or paclitaxel in OVCAR-10 cells, which have wild-type p53, but not in OVCAR-5 cells, which lack functional p53. These data indicate that the PI3K/AKT signal transduction pathway mediates p21 expression and suggest that this pathway contributes to cell cycle regulation promoted by p53 in response to drug-induced stress. However, inactivation of PI3K/AKT signaling did not result in significant alteration of the drug sensitivity of A2780 cells, suggesting that the cell death induced by cisplatin or paclitaxel proceeds independently of cell protective effects of PI3K and AKT.
142 citations
TL;DR: The data suggest that silymarin and/or its major active constituent silibinin could be an effective agent for both prevention and intervention of human skin cancer.
Abstract: This study reports in vivo therapeutic efficacy of silymarin against skin tumors with mechanistic rationale. 7,12-Dimethylbenz[a]anthracene-12-O-tetradecanoyl-phorbol-13-acetate (DMBA-TPA)-induced established skin papilloma (tumor)-bearing SENCAR mice were fed with 0.5% silymarin in AIN-93M-purified diet (w/w), and both tumor growth and regression were monitored during 5 weeks of feeding regimen. Silymarin feeding significantly inhibited (74%, P < 0.01) tumor growth and also caused regression (43%, P < 0.01) of established tumors. Proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling immunohistochemical staining of tumors showed that silymarin decreases proliferation index by 48% (P < 0.001) and increases apoptotic index by 2.5-fold (P < 0.001), respectively. Skin tumor growth inhibition and regression by silymarin were also accompanied by a strong decrease (P < 0.001) in phospho-ERK1/2 levels in tumors from silymarin-fed mice compared with controls. In the studies evaluating bioavailability and physiologically achievable level of silymarin (as silibinin) in plasma, skin tumor, skin, liver, lung, mammary gland and spleen, we found 10, 6.5, 3.1, 13.7, 7.7, 5.9 and 4.4 microg silibinin/ml plasma or per gram tissue, respectively. In an attempt to translate these findings to human skin cancer and to establish biological significance of physiologically achievable level, effect of plasma concentration of silibinin was next examined in human epidermoid carcinoma A431 cells. Silibinin treatment of cells in culture at 12.5, 25 (plasma level) and 50 microM doses resulted in 30-74% (P < 0.01-0.001) growth inhibition and 7-42% death of A431 cells in a dose- and time-dependent manner; apoptosis was identified as a cell death response by silibinin. Similar silibinin treatments also resulted in a significant decrease in phospho-mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1/2 (MAPK/ERK1/2) levels, but an up-regulation of stress-activated protein kinase/jun NH(2)-terminal kinase (SAPK/JNK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) activation in A431 cells. The use of MEK1 inhibitor, PD98059, showed that inhibition of ERK1/2 signaling, in part, contributes to silibinin-caused cell growth inhibition. Together, the data suggest that an inhibition of ERK1/2 activation and an increased activation of JNK1/2 and p38 by silibinin could be possible underlying molecular events involved in inhibition of proliferation and induction of apoptosis in A431 cells. These data suggest that silymarin and/or its major active constituent silibinin could be an effective agent for both prevention and intervention of human skin cancer.
141 citations
TL;DR: The results for the first time identify the biological efficacy of silibinin against HCC cells, suggesting the importance of conducting further investigations in preclinical HCC models, especially on in vivo efficacy, to support the clinical usefulness ofsilibin in against hepatocellular carcinoma.
Abstract: Purpose: Hepatocellular carcinoma (HCC) is one of the most common recurrent malignancies, for which, currently, there is no effective therapy. Considering the antihepatotoxic activity of silibinin, a widely used drug and supplement for various liver disorders, together with its strong preventive and anticancer efficacy against various epithelial cancers, we investigated the efficacy of silibin against human HCC cells. Experimental Design: Silibinin effects were examined on growth, cytotoxicity, apoptosis, and cell cycle progression in two different HCC cell lines, HepG2 (hepatitis B virus negative; p53 intact) and Hep3B (hepatitis B virus positive; p53 mutated). At molecular level, cell cycle effects of silibinin were assessed by immunoblotting and in-bead kinase assays. Results: Silibinin strongly inhibited growth of both HepG2 and Hep3B cells with a relatively stronger cytotoxicity in Hep3B cells, which was associated with apoptosis induction. Silibinin also caused G1 arrest in HepG2 and both G1 and G2-M arrests in Hep3B cells. Mechanistic studies revealed that silibinin induces Kip1/p27 but decreases cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)-2, and CDK4 levels in both cell lines. In Hep3B cells, silibinin also reduced the protein levels of G2-M regulators. Furthermore, silibinin strongly inhibited CDK2, CDK4, and CDC2 kinase activity in these HCC cells. Conclusion: Together, these results for the first time identify the biological efficacy of silibinin against HCC cells, suggesting the importance of conducting further investigations in preclinical HCC models, especially on in vivo efficacy, to support the clinical usefulness of silibinin against hepatocellular carcinoma in addition to its known clinical efficacy as an antihepatotoxic agent.
136 citations
"Effects and mechanisms of silibinin..." refers background or result in this paper
...Silibinin at IC25 and IC50 doses for HuH7 cells also resulted in reduced growth of HepG2, Hep3B, and PLC/PRF/5 cells, confirming the previous reports [21, 22 ]...
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...Varghese, et al reported that silibinin increases levels of p27 in human hepatoma cells [ 22 ]...
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...effects [21, 22 ] provided us with a rationale to further define the related effects and mechanisms of HCC chemoprevention....
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...As shown in Figure 1B, silibinin also significantly inhibited the growth of HepG2, Hep3B, and PLC/PRF/5 human HCC cell lines, indicating a wide spectrum of silibinin's inhibitory effects on human HCC cell growth, as previously reported [21, 22 ]...
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...Varghese et al reported silibinin's anti-HCC effects [21, 22 ]...
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TL;DR: It is suggested that silibinin modulates CDKI-CDK-cyclin cascade and activates caspase 3 causing growth inhibition and apoptotic death of human TCC cells, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silib inin in bladder cancer pre-clinical models.
Abstract: Bladder cancer is the fourth and eighth most common cancer in men and women in the USA, respectively. Flavonoid phytochemicals are being studied for both prevention and therapy of various human malignancies including bladder cancer. One such naturally occurring flavonoid is silibinin isolated from milk thistle. Here, we assessed the effect of silibinin on human bladder transitional cell carcinoma (TCC) cell growth, cell cycle modulation and apoptosis induction, and associated molecular alterations, employing two different cell lines representing high-grade invasive tumor (TCC-SUP) and high-grade TCC (T-24) human bladder cancer. Silibinin treatment of these cells resulted in a significant dose- and time-dependent growth inhibition together with a G(1) arrest only at lower doses in TCC-SUP cells but at both lower and higher doses in T-24 cells; higher silibinin dose showed a G(2)/M arrest in TCC-SUP cells. In other studies, silibinin treatment strongly induced the expression of Cip1/p21 and Kip1/p27, but resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins involved in G(1) progression. Silibinin treatment also showed an increased interaction between cyclin-dependent kinase inhibitors (CDKIs)-CDKs and a decreased CDK kinase activity. Further, the G(2)/M arrest by silibinin in TCC-SUP cells was associated with a decrease in pCdc25c (Ser216), Cdc25c, pCdc2 (Tyr15), Cdc2 and cyclin B1 protein levels. In additional studies, silibinin showed a dose- and a time-dependent apoptotic death only in TCC-SUP cells that was associated with cleaved forms of caspase 3 and poly(ADP-ribose) polymerase. Together, these results suggest that silibinin modulates CDKI-CDK-cyclin cascade and activates caspase 3 causing growth inhibition and apoptotic death of human TCC cells, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in bladder cancer pre-clinical models.
129 citations
"Effects and mechanisms of silibinin..." refers background in this paper
.... Studies indicated that silibinin induces apoptosis in several malignant cell lines [13,14, 18 ]...
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..., bladder [ 18 ,19] and lung cancers...
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TL;DR: Plasma levels of these enzymes may have potential as a noninvasive indicator of invasion or metastasis in colorectal cancer or as a marker of disease status during follow‐up following curative surgery.
Abstract: Matrix metalloproteinases, and notably the gelatinases MMP-2 and MMP-9, have important roles in tumour invasion, metastasis and angiogenesis. Our study investigates the distribution of MMP-2 and MMP-9 in colorectal cancer, the correlation with plasma levels, changes following surgical resection and whether plasma levels reflect clinical staging and disease course. MMP-2 and MMP-9 expression in 48 colorectal tumours and 13 adenomatous polyps was analysed by RT-PCR, immunohistochemistry, and quantified by ELISA of tumour lysates. Concentrations of MMP-2 and MMP-9 in plasma samples from these patients and 36 other patients who underwent curative resections were measured by ELISA prior to and 6-12 months after surgery. MMP-2 expression was significantly increased in colorectal cancer tissues compared to matched normal colon as measured by ELISA. Active MMP-2 was localised by immunohistochemistry to regions where tumour cells invaded the muscularis with little staining in more superficial areas. Plasma MMP-2 levels were also significantly elevated in patients with colorectal cancer, with significant reductions following curative resections at all stages. Similarly, MMP-9 expression was significantly increased in colorectal cancer tissues, predominantly in the tumour stroma. Plasma levels of MMP-9 were significantly elevated at all stages in colorectal cancer patients and a significant reduction was seen following curative resections. With both MMP-2 and MMP-9, the strongest correlation with clinical staging in colorectal cancer was represented by the total plasma concentration of the enzymes, both falling to within the normal range following curative surgery. Plasma levels of these enzymes may therefore have potential as a noninvasive indicator of invasion or metastasis in colorectal cancer or as a marker of disease status during follow-up.
123 citations
"Effects and mechanisms of silibinin..." refers background or methods in this paper
...protein expression. MMP-2 and MMP-9 have been used as markers for angiogenesis and malignant invasion [ 45 ]...
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...and MMP-2 and MMP-9, which are markers associated with angiogenesis as well as metastatic invasion [ 45 ]...
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