Author Affiliations: Institut de Recherche Experimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy (Ezhilarasan D, Evraerts J, Sokal E and Najimi M), and Louvain Drug Research Institute, Toxicology and Cancer Biology Research Group, PMNT Unit (Sid B and Calderon PB), Universite Catholique de Louvain, 1200, Brussels, Belgium; Facultad de Ciencias de la Salud, Universidad Arturo Prat, Iquique, Chile (Calderon PB); Department of Pharmacology and Environmental Toxicology, Food and Hepatotoxicology Laboratory, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600 113, India (Karthikeyan S)

Corresponding Author: Mustapha Najimi, PhD, Institut de Recherche Experimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Universite Catholique de Louvain, Avenue Mounier, 52, Box B1.52.03, 1200, Brussels, Belgium (Tel: +3227645283; Fax: +3227645258; Email: mustapha.najimi@uclouvain.be)

 

© 2017, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(16)60166-2

Published online December 28, 2016.

 

 

Contributors: ED and NM designed the research, analyzed the data and wrote the paper. ED, EJ and SB performed the research. CPB, KS, SE and NM contributed analytic tools and scientific discussion. NM is the guarantor.

Funding: None.

Ethical approval: The protocol and experiments were approved by the ethical committees of the St-Luc Hospital and faculty of Medicine of Université Catholique de Louvain, Brussels, Belgium.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect.

 

METHODS: The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 µmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay.

 

RESULTS: Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells.

 

CONCLUSION: The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

 

(Hepatobiliary Pancreat Dis Int 2017;16:80-87)

 

KEY WORDS: silibinin; hepatic stellate cells; in vitro; cell cycle arrest; proliferation

Liver disease is one of the major causes of morbidity and mortality worldwide, affecting humans of all ages. According to WHO, liver fibrosis is a significant health problem with a worldwide mortality attributable to cirrhosis and primary liver cancer of around 1.5 millions death per year.^[1] Liver fibrosis is the common final pathway of virtually all chronic inflammatory liver injuries. Hepatic stellate cells (HSCs) are known as the primary source of the extracellular matrix (ECM) components in liver fibrosis.^[2] Following liver injury of any etiology, HSCs undergo a response known as “activation”, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts.^[3]

 

HSC activation is a remarkably pleiotropic tightly programmed response occurring in a reproducible sequence. This sequence consists of “initiation” and “perpetuation” steps.^[4] Initiation encompasses rapid changes in gene expression and phenotype that render the cells response to cytokines and other local stimuli. Perpetuation involves key phenotypic responses mediated by increased cytokine effects and remodeling of ECM. Discrete phenotype responses of stellate cell perpetuation include proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, retinoid loss, and cytokine release.^[4] Proliferation of activated HSCs and their increased synthesis of ECM are essential steps in the initiation and progression of liver fibrosis in chronic liver diseases. Hence, targeting the anti-proliferative effect of HSCs is clearly an appropriate therapeutic approach for liver fibrosis.

 

Plant derived antioxidant substances have been evaluated as a potential therapeutic approach for liver fibrosis. Curcumin,^[5] caffeine,^[6] and salvianolic acid B^[7] have been found to suppress the proliferation of cultured HSCs. Silibinin (SBN), a standard extract isolated from the medicinal plant Silybum marianum (L.) (milk thistle), is the major bioactive component of silymarin. The most abundant flavonolignans present in SBN are the diastereoisomers silybin A and silybin B.^[8] Silymarin and SBN have been used for decades as a herbal remedy and as a hepatoprotectant in acute and chronic liver disease settings.^[9] Several in vitro, in vivo and clinical studies have demonstrated the antioxidant and hepatoprotective effects of silymarin and its major active constituent SBN in alcoholic or nonalcoholic chronic liver disease models.^[10-15] Moreover, SBN has been found to be virtually nontoxic in both chronic and acute toxicity studies, and no LD₅₀ has been reported for these flavonolignans in vivo, which further underscores its promise in chronic liver diseases and cancer prevention.^[16] Several studies were also emphasized regarding the manifold inhibitory effects of SBN on cell proliferation, cell cycle regulation, and apoptosis induction in several cancer cell lines of different origins.^[17-22] Although these studies imply that SBN has a suppressive effect on cell cycle, its influence on the mechanisms of cell cycle arrest in HSC remains unclear.

 

Our previous study has demonstrated that SBN is not cytotoxic to LX-2 cells, inhibits proliferation and does not induce apoptosis and cellular senescence.^[23] Hence, in the present study, we examined the possible reasons for anti-proliferative effects of SBN on human hepatic stellate LX-2 cell line and explored the related signaling pathways. The potential effects of SBN on cyclin dependant kinase inhibitors (CDKIs) proteins i.e., Cip1/p21, Kip1/p27, p53, Akt and their downstream targets signaling pathways were particularly investigated.

 

 

Silibinin (C₂₅H₂₂O₁₀; CAS No. 22888-70-6) was purchased from Sigma Chemical Co. (Belgium). It is a mixture of two diastereisomers (silybin A and silybin B).

 

LX-2 cells were kindly provided by Dr. S. L. Friedman, Mount Sinai School of Medicine, New York, USA. These cells are derived from normal human HSC that are spontaneously immortalized. LX-2 cells exhibit the typical features of HSCs in primary culture as for instance the expression of desmin, glial acidic fibrillary protein, and the response to platelet-derived growth factor BB and TGF-β1. LX-2 cells express α-smooth muscle actin (α-SMA) under all culture conditions.^[24] The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% of fetal bovine serum (Gibco, Belgium) and 1% penicillin-streptomycin (Life Technologies, Belgium). Cell cultures were maintained at 37 �� in a fully humidified atmosphere containing 5% CO₂. Those used in the present study were from passages 6-10 after thawing.

 

SBN was dissolved in 0.1% DMSO (v/v) according to Agarwal et al.^[25] LX-2 cells were plated at 10 000 cells/cm². Twenty-four hours later, cells were fed with fresh expansion culture medium supplemented with different final concentrations of SBN (10, 50 and 100 µmol/L) or the corresponding volumes of the vehicle. After 24 and 96 hours of incubation, cells were collected after 0.05% trypsin (Life Technologies) application. Total cell number was determined by counting each sample in triplicate using a KOVA Glasstic® Slide 10 under Leica DMIL inverted microscope. Viability was also evaluated by the trypan blue dye exclusion assay.

 

LX-2 cells were seeded at density of 6×10⁵ cells in 6-well plates. After 24 hours, plated LX-2 cells were fed with fresh expansion culture medium supplemented or not with different final concentrations of SBN. After 24 and 96 hours of SBN incubation, cells were detached/collected using 0.05% trypsin for intracellular ATP analysis. The assay was performed using Perkin Elmer luminescence kit by luminescence method. This method was based on the luciferase and luciferin reaction, which determines the number of viable cells present in the well, based on ATP levels. Briefly, 50 µL of lysis buffer was added to the plate containing 1×10⁵ LX-2 cells suspended in 100 µL of Iscove’s medium without phenol red. The plate was shaken for 5 minutes on ice and 50 µL of luciferase substrate was added and incubated for 15 minutes on ice. The emitted luminescence was recorded using Perkin Elmer 2030 multi label reader.

 

LX-2 cells (10 000/cm²) were plated on glass cover slips in 24-well culture dishes. After 24 hours post-plating, cells were incubated in the presence of increasing doses of SBN for 96 hours. At the end of exposure, the cells were washed twice with D-PBS and then fixed using 4% formalin in PBS for 20 minutes at room temperature. After fixation, cells were washed twice with PBS and incubated with hydrogen peroxide (3.3%) for 3 minutes to inactivate the endogenous peroxidase activity. Thereafter, fixed cells were permeabilized after 10 minutes incubation with D-PBS containing 1% Triton X-100 at room temperature. Permeabilized cells were washed twice with D-PBS and incubated with blocking solution (1% BSA in PBS) for one hour at room temperature. Primary antibody of monoclonal mouse Ki-67 (Dako, Denmark) diluted at 1:150 in 0.1% BSA was incubated with the cells for one hour at room temperature. After three washes with D-PBS, cells were incubated with 200 µL of HRP labeled anti-mouse secondary antibody (Dako, Carpintaria, CA) for 45 minutes at room temperature. After three washes with D-PBS, detection was performed after 5 minutes incubation with liquid DAB and substrate chromogen (Dako, Denmark). Counterstaining was performed using Mayer’s hematoxylin for 10 minutes. Preparations were then mounted for microscopic analysis (DMIL, Leica, Belgium).

 

LX-2 cells were lysed in RIPA buffer (20 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1% NP-40, 0.1% SDS, 1% deoxycholate sodium and 0.1% protease and phosphatase inhibitor cocktails, Sigma-Aldrich, Belgium). After extraction, protein concentration was estimated by the Bradford method (Biorad Laboratories) with BSA as standard. Total protein extracts were subjected to SDS-PAGE and electroblotted onto nitrocellulose membranes (Hybond™-ECL, Amersham, CA). The membranes were blocked with 5% BSA, probed overnight at 4 �� with primary antibodies and 2 hours at room temperature with corresponding secondary antibodies. Immunoreactive bands were detected by enhanced chemiluminescence with protein A-horseradish peroxidase and the SuperSignal chemiluminescent system (Pierce, Rockford, IL). Details of primary and corresponding secondary antibodies used for this study are given in the Table.

 

Mammalian sirtuins are involved in several cell biological functions including survival, resistance to stress and apoptosis. Sirtuin activity was analyzed using the SIRT-Glo™ luminescent assay (Ref: G6450) according to the manufacturer’s instructions. Control and treated LX-2 cells were trypsinized, counted and seeded at 10⁵ cells/100 µL of SIRT-Glo buffer per well of a 96-well plate (in duplicate). One hundred µL of SIRT-Glo reagent were added and the plate was shaken for 2 minutes at room temperature. The cell suspensions were incubated for 45 minutes at room temperature during the sirtuin activities deacetylates the luminogenic peptide substrate. Such deacetylation is measured using a coupled enzymatic system in which a protease, contained in the Developer Reagent, cleaves the peptide from aminoluciferin. The produced luminescence, which is proportional to SIRT-deacetylase activity, was measured using a Victor3 luminometer (Perkin Elmer). Substrate without cells and cells with no enzyme were used as negative controls.

 

The data were subjected to one-way analysis of variance (ANOVA) and Newman Keuls’ multiple comparison tests were performed to assess the significance of differences in the means of various treatment groups, using Graph pad prism software. The values were tabulated and presented as mean±SEM. A P value <0.05 was considered statistically significant.

 

 

SBN treated LX-2 cells showed no difference in cell viability in comparison with both naïve and vehicle-treated LX-2 cells either at different time points or variable concentrations. More than 90% of the LX-2 cells remained viable and they were observed as culture activated and acquired the initial myofibroblast-like characteristic features (Fig. 1A). SBN treatment induced a dose- and time- dependent drop in the number of LX-2 cells as compared to both naïve and vehicle-treated cells (Fig. 1B). The counted cells in SBN 10, 50 and 100 µmol/L for 24 hours treated groups represent 77%±7%, 67%±5%, and 59%±7% of the control groups, respectively (Fig. 1B). These percentages after 96 hour incubation were 79%±13%, 70%±14%, and 54%±12% of the control groups, respectively (Fig. 1B). Intracellular ATP assay showed that SBN did not affect intracellular ATP level in comparison with controls (Fig. 1C).

 

Western blotting demonstrated that SBN treated LX-2 cells significantly increased p53 and p27 expression at 24 hours in comparison with controls (Fig. 2). The maximum effect of SBN on protein induction was observed in cells treated with 100 µmol/L of SBN (Fig. 2). Conversely, no significant changes were observed in the levels of both p21 and survivin (inhibitor of apoptosis) protein levels at the times analyzed. No significant changes of these proteins were noticed after 96 hours. This is in line with the earlier activation of the above-mentioned proteins and its impact on the progression of cell cycle arrest.

 

An immunocytochemical analysis on Ki-67, a good marker of cell cycle arrest in proliferating cells, showed dose-dependent decrease in Ki-67 in 96 hours of SBN treated cells as compared to naïve and vehicle treated groups (Fig. 3A). Quantification of Ki-67 protein nuclear expression confirmed the significant concomitant decrease in the percentage of Ki-67 positively immunostained cells and an increase of the negatively immunostained cells (Fig. 3B). It should be underlined that a maximum effect was observed at 100 µmol/L SBN. These results pointed out the ability of SBN to induce a cell cycle arrest in proliferating LX-2 cells. There was no significant change in the Ki-67 expression after 24 hours of SBN treatment in LX-2 cells.

 

The protein expression of Akt (a G1 phase related protein) was a dose-dependent decrease in LX-2 cells exposed to SBN. The dose-dependent effect was more pronounced after 96 hours as compared to vehicle treated cells (Fig. 4). Akt phosphorylation (phospho-Thr308, phospho-Ser473) was not detectable in 24 hours. The phosphorylated Akt was positive at 96 hours in all studied groups. SBN at 100 µmol/L significantly decreased Akt and phosphorylated Akt (Fig. 4).

 

Foxo3A, the downstream target of Akt, was increased by SBN (50 µmol/L) at 24 hours only (Fig. 4). SBN (100 µmol/L) significantly decreased pSer-Foxo3A after 96 hours treatment (Fig. 4).

 

Sirtuin activity was significantly increased in LX-2 cells treated with SBN for 96 hours as compared to controls (Fig. 5). This effect was more prominent at the highest concentration of SBN used. The sirtuin activity was not significantly changed after 24 hours of SBN treatment in LX-2 cells.

 

 

The inhibition of HSC proliferation is one of the promising therapeutic strategies to prevent the progression of liver fibrosis in chronic liver diseases.^[26] Several studies were documented the anti-proliferative effect of SBN through cell cycle arrest.^{[19, 25, 27]} In liver context, it has been previously reported an ameliorative effect of SBN on repeated dimethylnitrosamine-induced liver fibrosis in rats.^[10] Therefore, we hypothesized that SBN might modulate liver fibrosis development via an inhibition of the activated HSCs proliferation. Hence, in this study, we demonstrated the anti-proliferative effect of SBN in dose-dependant manner in immortalized human LX-2 HSCs with no alteration of cell viability. We also deciphered the pathways targeted by SBN to block LX-2 cell cycle.

 

Our results clearly show a retard active proliferation of LX-2 cells by SBN which is in line with previously reported effects of this compound. Indeed, SBN has been proposed to exhibit anti-proliferative effect by different mechanisms including stimulation of cell cycle arrest, apoptosis and cellular senescence.^{[28, 29]} These effects were mainly observed in proliferating cancer cell lines. In our study, we mainly investigated the mechanisms involved in inhibiting the proliferation of the human non-tumor LX-2 cells.

 

Promoting the clearance of activated HSCs by enhancing their cell death by apoptosis is considered as one of the effective strategy towards the mitigation of liver fibrosis.^[30] SBN exposure has been reported to induce cytotoxicity in various cell lines.^{[31, 32]} In contrast to these reports, we did not observe a marked cytotoxicity by SBN in LX-2 cells. In addition, in SBN treated LX-2 cells we did not notice any significant change in survivin expression whatever the investigated SBN concentrations. Survivin has been reported to inhibit apoptotic cell death in several in vitro and in vivo studies.^[33] These effects are in agreement with the absence of cell death as determined by flow cytometry using annexin V/PI staining even after 96 hours treatment (data not shown). Altogether, these data clearly indicate that SBN treatment does not have any effect on inducing apoptosis of LX-2 cells. Moreover, the discrepancy in cytotoxic response to SBN exposure in the current study as compared to previous reports could be due to different SBN doses and cell types as well.^[28]

 

We thereafter analyzed if the observed SBN effect is due to an inhibition of LX-2 cell proliferation. To further delineate whether the anti-proliferative effect of LX-2 cells upon SBN treatment was mainly through cell cycle arrest, the effect of SBN on proteins which are pertinent to G1 phase of cell cycle was investigated. In cell cycle progression, activation of Akt downstream signaling pathway plays a pivotal role in stimulation of HSCs proliferation, survival and migration.^{[4, 34, 35]} It is also reported that Akt plays an important role in proliferation of rat primary HSCs.^[36] In the present investigation, we revealed that SBN exposure caused down-regulation of Akt (Akt, Akt-Thr308 and Akt-Ser473) protein expressions at 96 hours of SBN treatment. This suggests that SBN could inhibit cell proliferation even in the earlier downstream process of cell cycle progression by altering the Akt signaling. Our data are in line with other studies showing that inhibition of Akt expression occurs following SBN treatment in various in vitro models other than HSCs.^{[37, 38]}

 

Activation of Foxo transcription factors has been shown to regulate HSC cell cycle via an activation of Akt pathway.^{[39, 40]} The activation of Foxo transcription factors also enhanced the expression of p21 protein, which causes cell cycle arrest in the G1 phase.^{[41, 42]} Adachi et al^[39] has shown that p27 protein is also a crucial downstream target of the Foxo transcription factor, which control both HSC proliferation and differentiation. Our data demonstrated that SBN caused an increase in the expression of Foxo3A and its phosphorylated protein pSer-Foxo3A only at 24 hours treatment with highest concentration (50 µmol/L) used in this study. This discrepancy indicate that SBN treatment, especially at high dose levels induced cell cycle arrest and block the progression of proliferating LX-2 cells.

 

Similarly, SBN increased p27 protein expression in dose-dependent manner in LX-2 cells. These results show that the inhibition of Akt expression by SBN is positively correlated to an up-regulation of the transcription factor Foxo3A as well as CDK1 protein p27 in LX-2 cells. The cell cycle progression is basically controlled by activation of several CDKs which is critically regulated by p21 and p27 proteins as well as by the tumor suppressor proteins i.e., p53.^[43] Experiment in rat primary HSCs demonstrated that an increase in the expression of p53 causes an enhancement of p21 protein expression.^[44] In the present investigation, SBN treatment caused a dose-dependent increase in the expression of p53 protein. However, the expression of p21 remains unchanged. The increased expression of p53 could have also contributed towards the inhibition of cyclin E binding with CDK2 leading to cell cycle arrest. The mechanism of SBN induced p53 protein up-regulation is unknown and studies on these lines are warranted. These results demonstrated that inhibition of Akt could possibly inhibit its downstream signaling and LX-2 cell proliferation via cell cycle arrest involving up regulation of p53 and p27.

 

Data from above experiments suggest that SBN 24-hour treatment of LX-2 cells inhibit the early G1 phase of Akt downstream signaling thereby it could have enhanced the Foxo3A transcription factors. This enhanced transcription factor expression might have induced the p27 expression which might have caused cell cycle arrest. The Ki-67 protein is a proliferation index and is normally expressed at its peak during all the phases of cell cycle except G0 and early G1 phases.^[45] The absence of Ki-67 protein in quiescent cells has created great interest because it serves as a marker of cell proliferation.^[46] The dose-dependent decrease in the expression of Ki-67 as well as the number of negatively stained LX-2 cells exposed to SBN is a clear indication to show that these cells have not achieved their proliferative state and they probably remain quiescent.

 

Recently, role of sirtuin has been implicated in liver fibrosis. A recent study reported that sirtuin ameliorates liver fibrosis by inducing the reversion of the activated stellate cell and apoptosis in LX-2 cells.^[47] In the present study, SBN increased the activity of sirtuin in LX-2 cells. Further, studies also indicate that sirtuin can act as a tumor suppressor. Sirtuin suppressed intestinal tumorigenesis and colon cancer growth in a β-catenin-driven mouse model of colon cancer.^[48] In view of the above scenarios, it is suggested that the observed anti-proliferative effect of SBN in LX-2 cells could be via the increase of sirtuin expression in this study. However, it has been apparent that the effects of sirtuin on various cell lines are not entirely clear and still a subject of controversy.

 

In conclusion, our present study on the potential effect of SBN on LX-2 cell cycle arrest have shown that SBN inhibits Akt and it phosphorylated protein expression through an induced earlier expression of the transcription factor Foxo3A and its phosphorylation as well as an enhanced protein expression of p27 and p53 and activity of sirtuin. This reflects the complex regulatory pathways that SBN may provoke LX-2 cell cycle arrest (Fig. 6).

 

 

1 World Health Organization: Global burden of disease 2010 estimates. Available from: http://www.who.int/healthinfo/global_burden_disease/en/index.html.

2 Trautwein C, Friedman SL, Schuppan D, Pinzani M. Hepatic fibrosis: concept to treatment. J Hepatol 2015;62:S15-24. PMID: 25920084

3 Puche JE, Saiman Y, Friedman SL. Hepatic stellate cells and liver fibrosis. Compr Physiol 2013;3:1473-1492. PMID: 24265236

4 Mann DA, Marra F. Fibrogenic signalling in hepatic stellate cells. J Hepatol 2010;52:949-950. PMID: 20395006

5 Zhang F, Zhang Z, Chen L, Kong D, Zhang X, Lu C, et al. Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells. J Cell Mol Med 2014;18:1392-1406. PMID: 24779927

6 Wang H, Guan W, Yang W, Wang Q, Zhao H, Yang F, et al. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway. PLoS One 2014;9:e92482. PMID: 24682220

7 Yu F, Lu Z, Chen B, Wu X, Dong P, Zheng J. Salvianolic acid B-induced microRNA-152 inhibits liver fibrosis by attenuating DNMT1-mediated Patched1 methylation. J Cell Mol Med 2015;19:2617-2632. PMID: 26257392

8 National Toxicology Program. Toxicology and carcinogenesis studies of milk thistle extract (CAS No. 84604-20-6) in F344/N rats and B6C3F1 mice (Feed Studies). Natl Toxicol Program Tech Rep Ser 2011;(565):1-177. PMID: 21685957

9 Gazák R, Walterová D, Kren V. Silybin and silymarin--new and emerging applications in medicine. Curr Med Chem 2007;14: 315-338. PMID: 17305535

10 Ezhilarasan D, Karthikeyan S, Vivekanandan P. Ameliorative effect of silibinin against N-nitrosodimethylamine-induced hepatic fibrosis in rats. Environ Toxicol Pharmacol 2012;34:1004-1013. PMID: 22986105

11 Trappoliere M, Caligiuri A, Schmid M, Bertolani C, Failli P, Vizzutti F, et al. Silybin, a component of sylimarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells. J Hepatol 2009;50:1102-1111. PMID: 19398228

12 Ezhilarasan D, Karthikeyan S. Silibinin alleviates N-nitrosodimethylamine-induced glutathione dysregulation and hepatotoxicity in rats. Chin J Nat Med 2016;14:40-47. PMID: 26850345

13 Kren V, Walterová D. Silybin and silymarin--new effects and applications. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2005;149:29-41. PMID: 16170386

14 Lucena MI, Andrade RJ, de la Cruz JP, Rodriguez-Mendizabal M, Blanco E, Sánchez de la Cuesta F. Effects of silymarin MZ-80 on oxidative stress in patients with alcoholic cirrhosis. Results of a randomized, double-blind, placebo-controlled clinical study. Int J Clin Pharmacol Ther 2002;40:2-8. PMID: 11841050

15 Gu HR, Park SC, Choi SJ, Lee JC, Kim YC, Han CJ, et al. Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells. Clin Mol Hepatol 2015;21:49-59. PMID: 25834802

16 Roy S, Kaur M, Agarwal C, Tecklenburg M, Sclafani RA, Agarwal R. p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells. Mol Cancer Ther 2007;6:2696-2707. PMID: 17938263

17 Varghese L, Agarwal C, Tyagi A, Singh RP, Agarwal R. Silibinin efficacy against human hepatocellular carcinoma. Clin Cancer Res 2005;11:8441-8448. PMID: 16322307

18 Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS. Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol Carcinog 2004;40:143-149. PMID: 15224346

19 Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages. Carcinogenesis 2004;25:1711-1720. PMID: 15117815

20 Yang SH, Lin JK, Chen WS, Chiu JH. Anti-angiogenic effect of silymarin on colon cancer LoVo cell line. J Surg Res 2003;113: 133-138. PMID: 12943822

21 Singh RP, Tyagi AK, Zhao J, Agarwal R. Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis. Carcinogenesis 2002;23:499-510. PMID: 11895866

22 Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate cancer prevention. Mol Cancer Ther 2002;1:525-532. PMID: 12479270

23 Ezhilarasan D, Karthikeyan S, Sokal E, Najimi M. Silibinin treatment to hepatic stellate cells: an exploration of apoptosis and cellular senescence. J Pharm Res 2014;8:1126-1131.

24 Xu L, Hui AY, Albanis E, Arthur MJ, O’Byrne SM, Blaner WS, et al. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 2005;54:142-151. PMID: 15591520

25 Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, et al. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 2003;22:8271-8282. PMID: 14614451

26 Friedman SL. Liver fibrosis -- from bench to bedside. J Hepatol 2003;38:S38-53. PMID: 12591185

27 Feng W, Cai D, Zhang B, Lou G, Zou X. Combination of HDAC inhibitor TSA and silibinin induces cell cycle arrest and apoptosis by targeting survivin and cyclinB1/Cdk1 in pancreatic cancer cells. Biomed Pharmacother 2015;74:257-264. PMID: 26349994

28 Zhang Y, Li Q, Ge Y, Chen Y, Chen J, Dong Y, et al. Silibinin triggers apoptosis and cell-cycle arrest of SGC7901 cells. Phytother Res 2013;27:397-403. PMID: 22619007

29 Deep G, Agarwal R. Antimetastatic efficacy of silibinin: molecular mechanisms and therapeutic potential against cancer. Cancer Metastasis Rev 2010;29:447-463. PMID: 20714788

30 Wright MC, Issa R, Smart DE, Trim N, Murray GI, Primrose JN, et al. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats. Gastroenterology 2001;121:685-698. PMID: 11522753

31 Li L, Gao Y, Zhang L, Zeng J, He D, Sun Y. Silibinin inhibits cell growth and induces apoptosis by caspase activation, down-regulating survivin and blocking EGFR-ERK activation in renal cell carcinoma. Cancer Lett 2008;272:61-69. PMID: 18723275

32 Deep G, Singh RP, Agarwal C, Kroll DJ, Agarwal R. Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin. Oncogene 2006;25:1053-1069. PMID: 16205633

33 Mobahat M, Narendran A, Riabowol K. Survivin as a preferential target for cancer therapy. Int J Mol Sci 2014;15:2494-2516. PMID: 24531137

34 Cheng GZ, Zhang W, Wang LH. Regulation of cancer cell survival, migration, and invasion by Twist: AKT2 comes to interplay. Cancer Res 2008;68:957-960. PMID: 18281467

35 Kim TH, Woo JS, Kim YK, Kim KH. Silibinin induces cell death through reactive oxygen species-dependent downregulation of notch-1/ERK/Akt signaling in human breast cancer cells. J Pharmacol Exp Ther 2014;349:268-278. PMID: 24472723

36 Reif S, Lang A, Lindquist JN, Yata Y, Gabele E, Scanga A, et al. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression. J Biol Chem 2003;278:8083-8090. PMID: 12502711

37 Chen PN, Hsieh YS, Chiou HL, Chu SC. Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chem Biol Interact 2005;156:141-150. PMID: 16169542

38 Mallikarjuna G, Dhanalakshmi S, Singh RP, Agarwal C, Agarwal R. Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling. Cancer Res 2004;64:6349-6356. PMID: 15342425

39 Adachi M, Osawa Y, Uchinami H, Kitamura T, Accili D, Brenner DA. The forkhead transcription factor FoxO1 regulates proliferation and transdifferentiation of hepatic stellate cells. Gastroenterology 2007;132:1434-1446. PMID: 17408630

40 Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 2000;404:782-787. PMID: 10783894

41 Nakae J, Kitamura T, Kitamura Y, Biggs WH 3rd, Arden KC, Accili D. The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Dev Cell 2003;4:119-129. PMID: 12530968

42 Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW, et al. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol Cell Biol 2000;20:9138-9148. PMID: 11094066

43 Hofseth LJ, Hussain SP, Harris CC. p53: 25 years after its discovery. Trends Pharmacol Sci 2004;25:177-181. PMID: 15116721

44 Saile B, Matthes N, El Armouche H, Neubauer K, Ramadori G. The bcl, NFkappaB and p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells. Eur J Cell Biol 2001;80:554-561. PMID: 11561906

45 Suciu C, Muresan A, Cornea R, Suciu O, Dema A, Raica M. Semi-automated evaluation of Ki-67 index in invasive ductal carcinoma of the breast. Oncol Lett 2014;7:107-114. PMID: 24348830

46 Bullwinkel J, Baron-Lühr B, Lüdemann A, Wohlenberg C, Gerdes J, Scholzen T. Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells. J Cell Physiol 2006;206:624-635. PMID: 16206250

47 Wu Y, Liu X, Zhou Q, Huang C, Meng X, Xu F, et al. Silent information regulator 1 (SIRT1) ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion. Toxicol Appl Pharmacol 2015;289:163-176. PMID: 26435214

48 Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, Campbell J, et al. The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth. PLoS One 2008;3:e2020. PMID: 18414679