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Potential conflict of interest: Nothing to report.
Epithelial-mesenchymal transition (EMT) is a physiological process that has been recognized to occur during the progression of an increasingly large number of human diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. The activation of transforming growth factor β (TGF-β) signaling is considered a critical event during EMT, and efforts have been made to screen small molecules that interfere with the TGF-β signaling pathway during EMT. Here we report the identification of sorafenib, a clinical agent that inhibits TGF-β signaling. When applied to AML12 cells and primary hepatocytes, sorafenib strikingly suppressed TGF-β1-induced EMT and apoptosis. Additionally, sorafenib inhibited TGF-β1-induced signal transducer and activator of transcription 3 phosphorylation. We further present in vitro evidence that sorafenib ameliorates the proapoptotic and profibrotic effects of TGF-β1 in mouse primary hepatocytes, suggesting that this drug exerts a protective effect on hepatocytes and has therapeutic potential for the treatment of liver fibrosis. (HEPATOLOGY 2011;)
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The epithelial-mesenchymal transition (EMT) is a dynamic cellular program in which polarized epithelial cells lose epithelial properties, undergo morphological changes, and acquire mesenchymal characteristics.1 This phenotypic change generates functionally distinct cell types and an increased capacity for cell migration. Aside from the essential role this transition plays in embryonic development, EMT has also been well studied in the context of physiological and pathological events such as tumor metastasis, wound healing, and organ fibrosis.2 Fibrosis is a progressive pathologic process characterized by excessive accumulation of extracellular matrix (ECM) proteins in response to injury or disease. An increasing number of distinct cytokines have been found to be involved in the initiation of EMT in many tissues. Among these mediators, transforming growth factor-β (TGF-β) is considered to act as a master switch.3
Members of the TGF-β superfamily are multifunctional cytokines that play critical roles in a variety of biological events, including embryogenesis, organogenesis, and certain human diseases.4, 5 TGF-β triggers EMT primarily via a canonical Smad-dependent mechanism, which requires two types of receptor kinases and a family of signal transducers called R-Smads (Smad2 and Smad3). Upon phosphorylation, R-Smads form complexes with a common partner (Smad4) and subsequently translocate into the nucleus to regulate the transcription of target genes responsible for EMT, such as Smad7, Snail, and collagen I.6–8 In the liver, injuries caused in a variety of different ways result in a rapid response involving of TGF-β synthesis and secretion, predominantly in hepatic stellate cells (HSCs). Subsequently, TGF-β induces quiescent mature hepatocytes to undergo EMT and apoptosis. EMT-derived hepatic myofibroblasts proliferate and up-regulate their production of fibrillar collagens with a resultant increase in the deposition of fibrotic matrix.9–11 Thus, strategies aimed at disrupting TGF-β production and/or blocking signal transduction using particular proteins or small molecules have important theoretical and practical implications for producing effective treatments for liver fibrosis, cirrhosis, portal hypertension, and liver cancers.
Over the past 20 years the successful development of small chemicals that disrupt several fundamental signaling pathways has signified a paradigm shift in medical therapy.12 Sorafenib (Nexavar) is a potent multikinase inhibitor that targets both Raf and a number of tyrosine kinases, including vascular endothelial growth factor R2 (VEGF-R2), platelet-derived growth factor (PDGF) receptor β, and VEGF receptor 3.13 Among similar compounds, sorafenib has progressed the furthest in clinical development and has been approved in several countries worldwide for the treatment of renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC).14, 15
In addition to its established clinical benefits for patients with a broad range of tumor types, recent studies in rats have demonstrated that sorafenib has potential utility in the treatment of portal hypertension and cirrhosis.16, 17 However, a detailed understanding of the underlying molecular mechanism remains elusive. In the current study we identified a new function for sorafenib as an effective inhibitor of TGF-β signaling. When applied to AML12 cells and mouse primary hepatocytes, sorafenib profoundly suppressed TGF-β1-induced EMT and apoptosis. Moreover, sorafenib inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3). We further demonstrated that sorafenib reduced the expression levels of proapoptotic and profibrotic genes in mouse primary hepatocytes, suggesting a potential therapeutic use of this drug in the treatment of liver fibrosis.
ECM, extracellular matrix; EMT, Epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; RCC, renal cell carcinoma; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor-β.
Materials and Methods
Reagents and Antibodies.
Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Sorafenib (Nexavar, BAY 43-9006) is manufactured by Bayer Pharmaceuticals (West Haven, CT, USA). Primary antibodies against E-cadherin, p-Smad2 (Ser465/467), Smad2, Snail, p-STAT3 (Tyr705), and STAT3 were purchased from Cell Signaling Technology (Beverly, MA). The mouse monoclonal antibody against ZO-1 and the rabbit polyclonal antibody against p-Smad3 (Ser423/425) were purchased from Invitrogen (Carlsbad, CA). The rabbit polyclonal antibody against fibronectin and the mouse monoclonal antibodies against α-SMA, β-actin, β-tubulin, and collagen type I were purchased from Sigma-Aldrich (St. Louis, MO). The rabbit polyclonal antibody against Smad3 was kindly provided by Dr. Ye-Guang Chen (Tsinghua Univ., P.R. China). Other primary antibodies described in this article including anti-PARP, anti-Smad7, and anti-vimentin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
C57BL/6 mice weighing 23-25 g were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences. During the study, all animals received humane care and had free access to food and water, in compliance with relevant guidelines. All procedures were approved by the Laboratory Animal Care and Use Committees of Shanghai Institutes for Biological Sciences.
Cell Culture and Mouse Hepatocyte Isolation.
AML12 (alpha mouse liver 12) cells were obtained from ATCC (Manassas, VA) and cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with 10% fetal bovine serum (FBS), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone at 37°C with 5% CO2.
Mouse primary hepatocytes were isolated using a two-step in situ collagenase perfusion method. Briefly, the hepatic portal vein was cannulated in situ, perfused with calcium- and magnesium-free Earle's balanced salt solution (EBSS) for 15 minutes, followed by 0.5 mg/mL of type IV collagenase dissolved in EBSS at 37°C until the liver capsule was incised. After perfusion, the thick fibrous connective tissue was discarded and filtered cell suspensions were harvested. To avoid contamination of hepatocytes with stellate cells, we used an additional purification step as described.18 Primary hepatocytes were then collected by centrifugation and seeded in DMEM containing 10% FBS (Biochrom, Berlin, Germany). The viability of the freshly isolated hepatocytes was determined by trypan blue exclusion and cell samples with viability greater than 90% were used in the subsequent assays.
DNA Transfection and Luciferase Assay.
AML12 cells were seeded into 24-well plates and transiently transfected with 100 ng of (CAGA)12-Lux reporter, which encodes 12 copies of the CAGA canonical Smad DNA-binding sequence. Cells were cotransfected with 5 ng of pRL-SV40 plasmid expressing Renilla luciferase per well as an internal control. At 24 hours posttransfection, cells were incubated in serum-free medium supplemented with TGF-β1 (5 ng/mL) for an additional 12 hours prior to harvesting. Luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) and normalized to Renilla luciferase activity in each sample. All assays were performed in triplicate and the data are shown as mean values ± standard error (SE) of at least three independent experiments.
To detect the expression levels of epithelial and mesenchymal markers, AML12 cells treated as indicated were lysed in 200 μL of lysis buffer19 and subjected to western blot analysis. Approximately 50 μg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and incubated with appropriate antibodies, as indicated in the figure legends. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham).
DNA Fragmentation Assay.
AML12 cells treated as indicated were collected and lysed in lysis buffer containing 10 mM Tris-Cl (pH 8.0), 25 mM EDTA (pH 8.0), 0.25% Triton X-100, and 5 mg/mL RNase A. After incubation on ice for 30 minutes, cells were scraped and centrifuged at 12,000g for 15 minutes. The supernatant was incubated with proteinase K at 56°C overnight and subsequently extracted with a 1:1 mixture of phenol and chloroform. DNA fragments were precipitated in two volumes of cold ethanol and a one-tenth volume of 3 M sodium acetate and were separated by 1.5% agarose gel electrophoresis. The gel was stained with ethidium bromide and visualized under ultraviolet light.
Flow Cytometry Analysis.
AML12 cells from each group were collected and stained with FITC-labeled Annexin V and propidium iodide (PI) according to the manufacturer's instructions (Biovision). The percentage of apoptotic cells was measured with a FACScan flow cytometer.
Measurement of Caspase-3 Activity.
After being treated as indicated in Fig. 5E, primary hepatocytes in 60-mm dishes were harvested and caspase-3 activity was detected by measuring the absorbance at 405 nm according to the manufacturer's instructions (Biovision).
As described,19, 20 cells grown on coverslips were fixed in 4% paraformaldehyde and stained with the appropriate primary antibodies, followed by Cy2-conjugated antimouse IgG or Cy3-conjugated antirabbit IgG (Jackson ImmunoResearch). Nuclei were stained with DAPI.
Total RNA isolation and reverse transcription were performed essentially as described.21 Using SYBR Green PCR Master Mix (Toyobo), real-time qPCR analysis was run on the MX3000p system (Stratagene). Each measurement was repeated at least in triplicate and normalized to the corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) content values. The optimized primers used for real-time PCR are listed in Supporting Table 1.
In Vitro Cell Migration.
The ability of cell migration was evaluated using a Transwell system (Corning Coster, Cambridge, MA), which allows cells to migrate through a polycarbonate membrane (8-μm pore size). Briefly, the upper compartment was filled with DMEM containing 1% FBS, and the lower chamber contained DMEM plus 10% FBS. Cells were treated as indicated in Supporting Fig. S3, and then seeded in the upper compartment of the Transwell chamber and cultured for an additional 12 hours at 37°C. Nonmigrated cells on the upper surface of the filter membrane were removed and migrated cells attached to the bottom surface of the filter membrane were fixed in methanol, stained with Giemsa, and counted in five random fields.
All assays were performed in triplicate. Data are presented as mean values ± SE. Comparisons were made using Student t test. For all analyses, a two-sided P < 0.05 was considered to indicate statistical significance.
Identification of Sorafenib as a Novel Inhibitor of TGF-β Signaling.
Given the prominent role of TGF-β in EMT and fibrogenesis, a number of strategies for blocking TGF-β signaling have been proposed.22–24 Small molecules targeting this signaling cascade have great therapeutic potential. To identify such candidates, a drug library screen was performed using (CAGA)12-Lux, a luciferase reporter that is activated in response to a wide range of TGF-β1 concentrations (Supporting Fig. S1). Interestingly, the activity of this reporter could be inhibited by treatment with sorafenib, but not with other clinical agents (Fig. 1A). The inhibitory effect of sorafenib on TGF-β-dependent gene transcription of the (CAGA)12-Lux reporter was dose-dependent (Fig. 1B). To further investigate the intracellular signal transduction mechanism, we treated cells with increasing doses of sorafenib under TGF-β1 stimulation. As shown in Fig. 1C, sorafenib abrogated TGF-β-mediated phosphorylation of Smad2 and Smad3, again in a dose-dependent manner. Moreover, sorafenib reduces the nuclear localization of phosphorylated Smad2/3 (Supporting Fig. S2A), which are the central mediators of the TGF-β signaling pathway.5, 6 We next examined whether treatment with sorafenib impaired the endogenous expression of Smad7, a target gene that is transiently induced by TGF-β1 through a negative feedback mechanism.4–6 Indeed, the application of sorafenib markedly decreased the expression of Smad7 mRNA (Fig. 1D). Experiments similar to those shown in Fig. 1B were repeated in HEK 293T, NIH 3T3, and HeLa cells with essentially the same results (data not shown), indicating that sorafenib acts as an effective inhibitor of TGF-β signaling regardless of cell type.
Sorafenib Suppresses TGF-β1-Induced EMT and Apoptosis in AML12 Cells.
These findings prompted us to assess the impact of sorafenib on TGF-β-mediated physiological events. TGF-β signaling is involved in the tight control of both apoptosis and EMT in hepatocytes8, 18, 25, 26; therefore, we speculated that sorafenib may play a role in concomitant TGF-β-induced EMT and apoptosis in hepatocytes. To test this hypothesis we used mouse AML12 cells, a normal cell line that exhibits typical hepatocyte features, such as peroxisomes and bile canaliculi-like structures.27 To date, this cell line has been used extensively as an ideal model to assess TGF-β-induced EMT on the resultant cellular phenotypes and gene expression profiles in vitro.8, 18, 24 When exposed to TGF-β1 for 48 hours, AML12 cells underwent EMT, in which cells lost their epithelial honeycomb-like morphology and obtained a spindle-like shape (Fig. 2A). Along with these morphological alterations, the expression levels of two epithelial markers (the adherens junction protein E-cadherin and the tight junction protein ZO-1) were decreased, whereas the expression levels of the intermediate filament proteins fibronectin and vimentin were up-regulated (Fig. 2B). As expected, treatment of AML12 cells with sorafenib reversed TGF-β1-induced EMT as shown by cellular phenotypic changes (Fig. 2A) and expression profiles of EMT markers (Fig. 2B). We also treated cells with increasing doses of sorafenib under TGF-β1 stimulation. As shown in Fig. 2C,D, sorafenib mediated cellular resistance to EMT in a dose-dependent manner. Furthermore, TGF-β1 induced an increase of cell migration in mouse hepatocytes, which was also be repressed by sorafenib (Supporting Fig. S3).
In mature hepatocytes, TGF-β signaling is responsible for the inhibition of cell proliferation and the induction of cell apoptosis.28, 29 In AML12 cells, TGF-β1 not only triggers EMT but also simultaneously induces apoptosis, which are concurrent but distinct responses in the same cell type. As measured by a fluorescence-activated cell sorter (FACS), the apoptotic response of AML12 cells was only apparent after treatment with TGF-β1 for 24 hours, and ≈50% of cells underwent apoptosis upon TGF-β1 stimulation for 48 hours. We then examined whether sorafenib could block TGF-β1-induced apoptosis. As expected, treatment with sorafenib impeded the apoptosis of AML12 cells in a dose-dependent manner (Fig. 3A). In line with these results, sorafenib also protected cells from apoptosis, as evaluated by assays characterizing DNA fragmentation, chromatin condensation, and nuclear disintegration, and the protective effects became pronounced when cells were treated with 10 μM sorafenib (Fig. 3B,C). Further experiments revealed that sorafenib treatment blocked the cleavage of poly (ADP-ribose) polymerase (PARP), a ubiquitous DNA-binding protein that is considered a robust and reliable marker of apoptosis (Fig. 3D). Collectively, these data show that sorafenib maintains the epithelial properties of mouse hepatocytes and counteracts TGF-β1-induced EMT and apoptosis in AML12 cells.
Sorafenib Blocks the Phosphorylation of STAT3 Induced by TGF-β1.
In addition to the TGF-β/Smad signaling, crosstalk between cellular signaling pathways is believed to be responsible for the underlying molecular mechanisms of EMT. Members of the STAT family, particularly STAT3, have been shown to be essential signal transducers during EMT via their transcriptional activities.30 Because STAT3 was activated in response to a TGF-β stimulus,31 we investigated whether sorafenib blocked TGF-β1-induced EMT and apoptosis through the regulation of STAT3 activation. To address this possibility we first validated the expression profile of STAT3 phosphorylation during hepatocyte EMT. As shown in Fig. 4A,B, TGF-β1 stimulated STAT3 phosphorylation at Tyr705 in a dose- and time-dependent manner. After treatment of AML12 cells with increasing doses of sorafenib for 24 hours, STAT3 phosphorylation was substantially reduced (Fig. 4C). These results are further supported by our observations of the subcellular localization of STAT3, which was distributed in the nuclear compartments of untreated cells, suggesting a role of STAT3 as a transcriptional activator. When cells were treated with sorafenib, STAT3 proteins were predominantly localized in the cytoplasm, rather than accumulating in the nucleus (Fig. 4D). Taken together, our results indicate that sorafenib blocks TGF-β-induced EMT and cell migration by mediating STAT3 dephosphorylation in mouse hepatocytes.
Sorafenib Inhibits TGF-β1-Induced EMT in Primary Hepatocytes.
It has been reported that TGF-β1 can induce an EMT state in mature hepatocyte in vitro.8, 18 Therefore, we assessed the effects of sorafenib on TGF-β1-induced EMT in mouse primary hepatocytes. To rule out potential contamination with nonparenchymal cells, the purity of the hepatocytes was determined by albumin staining. About 95% of cells isolated from 8-week-old C57BL/6 mice were albumin-positive (data not shown). Meanwhile, we confirmed that the expression of α-smooth muscle actin (α-SMA), a marker of activated HSCs, was absent in both TGF-β1-treated and sorafenib-treated primary hepatocytes (Supporting Fig. S4). Consistent with the results described above for AML12 cells, sorafenib treatment blunted TGF-β1-dependent reporter activity in mouse primary hepatocytes (Fig. 5A). Moreover, sorafenib abrogated the reduction in the expression levels of E-cadherin and ZO-1 and the augmentation of fibronectin expression (Fig. 5B). On the other hand, coimmunofluorescence staining for E-cadherin, ZO-1, and fibronection revealed that sorafenib reversed TGF-β1-induced EMT in primary hepatocytes (Fig. 5C and Supporting Fig. S5). Because Snail is a zinc-finger transcriptional repressor that has been shown to be an immediate-early response gene for TGF-β during EMT,7 we then examined whether sorafenib regulated the transcription of Snail. As shown in Fig. 5B,D, both the messenger RNA (mRNA) and protein levels of Snail were rapidly induced by TGF-β1 and were remarkably decreased after treatment with sorafenib. Indeed, in addition to Snail, an increasing number of genes have been shown to orchestrate EMT.32 We further showed that sorafenib negatively regulated the expression of other EMT-related transcription factors, including Twist, Zeb1, and Zeb2, but only had a subtle effect on the mRNA level of Slug (Fig. 5D).
Sorafenib Ameliorates the Proapoptotic and Profibrotic Effects of TGF-β1 in Cultured Primary Hepatocytes.
Based on numerous studies, TGF-β has been recognized as a proapoptotic and profibrotic master cytokine in hepatocytes3, 9-11; therefore, we hypothesized that sorafenib may potentially exert both antiapoptotic and antifibrotic effects by disrupting TGF-β signaling. To test this hypothesis we first confirmed the protective effect of sorafenib in blocking apoptosis in primary hepatocytes. As shown in Fig. 6A, caspase-3 activity was attenuated when cells were treated with sorafenib. Further experiments demonstrated that exposure of primary hepatocytes to sorafenib eventually led to a significant decrease in the expression of proapoptotic genes, such as Bad, Bax, and Caspase 3 (Fig. 6B), indicating that this drug prevents hepatocytes from undergoing apoptosis.
Because primary hepatocytes may also contribute to the production of ECM,8 we subsequently assessed the effects of sorafenib on collagen production in vitro. In response to external TGF-β1 stimulation, primary hepatocytes up-regulate the production of fibrotic matrix components, including procollagen type I (col I), procollagen type III (col III), and collagen IV α1. Interestingly, these changes were substantially attenuated after treatment with sorafenib (Fig. 6C), suggesting an antifibrotic role of sorafenib in counteracting ECM accumulation. This effect was further supported by real-time qPCR analysis assessing gene expression profiles of sorafenib-treated hepatocytes, which revealed a profound decrease in the expression of Timp-3, a tissue inhibitor of metalloproteinases that is expressed only in hepatocytes.33 Likewise, the expression of the potent profibrotic factors TGF-β1 and CCN2 (connective tissue growth factor) were reduced by ≈44% to 58% after sorafenib treatment (Fig. 6C). Taken together, these results clearly provide in vitro evidence that sorafenib exerts both antiapoptotic and antifibrotic effects against TGF-β signaling in mouse hepatocytes.
In 2005, sorafenib became the first oral agent approved for the treatment of patients with advanced RCC. Previous reports have largely focused on the role of sorafenib in tumor progression and apoptosis through blocking multiple receptor tyrosine kinases.13-15, 34 In this study we uncovered a novel capacity of sorafenib to antagonize TGF-β signaling and, consequently, to counteract TGF-β1-induced concomitant EMT and apoptosis in mouse hepatocytes. We observed that sorafenib treatment significantly decreased Smad2/3 phosphorylation (Fig. 1C and Supporting Fig. S2) and the expression of TGF-β target genes, such as CCN2, ColIa1, and Smad7 (Figs. 1D, 5D, 6C), raising the possibility that sorafenib may directly or indirectly modify key proteins in the TGF-β signaling pathway. Smad proteins are crucial intracellular transducers of TGF-β-mediated EMT in liver cells8; therefore, the inhibitory effect of sorafenib on TGF-β-induced EMT and apoptosis is related to the inhibition of TGF-β1-dependent Smad2/3 transcriptional activity. Because TGF-β can also promote EMT during carcinogenesis and enhance the migratory and invasive properties of tumor cells,35 inhibition of EMT and cell migration by sorafenib may provide a possible explanation for its effects in terms of tumor control and reduced cancer metastasis.
Impressively, sorafenib prevented hepatocytes from undergoing apoptosis induced by TGF-β1 (Figs. 3, Fig. 6), which differs entirely from its antiproliferative and proapoptotic activities in HSCs and most types of tumor cells.13, 35, 36 To our knowledge, the present study is the first demonstration that sorafenib can impede apoptosis. How could the same compound have two “faces” that can exert either antiapoptotic or proapoptotic effects in different cell types? One potential interpretation is that the variable cellular responses to sorafenib treatment depend on the diverse roles of TGF-β signaling. TGF-β is a potent inducer of growth inhibition and cell apoptosis in several cell types, including epithelial cells37; therefore it could make sense that sorafenib treatment counteracts cell apoptosis in normal hepatocytes (and perhaps other epithelial cells) by disrupting TGF-β signaling. On the other hand, tumor cells often show increased production of TGF-β, and considerable evidence documents its tumor-promoting role through its effects on the transdifferentiation and invasion of epithelial cells into the underlying mesenchyme during cancer progression.35, 37 In this case, the anticancer role of sorafenib is at least partly due to its interference with TGF-β signaling. Indeed, future studies are necessary to identify the underlying mechanisms responsible for the action of sorafenib and fully understand the seemingly puzzling role of this compound.
Recent studies in rats have demonstrated that sorafenib attenuates portal hypertension, cirrhosis, and liver fibrosis.16, 17, 36 However, very little is known regarding the mechanism underlying these effects. Our results largely extend these studies by identifying the mechanism for modulation of TGF-β signaling by sorafenib and highlighting the biological function of sorafenib in TGF-β-induced EMT and apoptosis in vitro. However, the in vivo evidence for hepatocyte EMT in carbon tetrachloride (CCl4)-induced liver fibrosis remains controversial.18, 38, 39 Because most studies have emphasized the activation of HSCs and apoptosis of hepatocytes in fibrogenesis,11, 33 it could be hypothesized that sorafenib suppresses TGF-β signaling in response to injury and subsequently this suppression of TGF-β signaling results in the inhibition of HSC activation and protects hepatocytes from apoptosis, leading to remarkable improvement of liver fibrosis. In fact, when mice exposed to CCl4 were treated with sorafenib, we observed that the TGF-β signal transduction in the liver was substantially repressed, together with the decrease in collagen production and hepatocytes apoptosis in vivo (unpubl. data). Based on these findings, sorafenib can be considered to be more than just an anticancer drug.
According to our current understanding, a variety of cytokines are involved in the pathological process of liver diseases, of which TGF-β is the most important inducer.3 Thus, studying TGF-β-induced EMT and apoptosis in mouse hepatocytes is very important for the development of new and efficacious therapies for fibrosis, cirrhosis, portal hypertension, and other liver diseases. In the past decade, several antifibrotic strategies have been successfully established based on the blockade or elimination of latent TGF-β signaling at various transduction steps. Several gene therapy approaches using dominant-negative TGF-β receptors and BMP-7 have been developed to prevent fibrosis in different tissues.22, 23 Similarly, ectopic overexpression of Smad7 in the hepatocytes of transgenic mice was shown to attenuate TGF-β signaling and thereby improve CCl4-induced liver fibrosis.24 In addition to these protein-based therapies, small molecules and biological agents that act on this signaling cascade have shown strong therapeutic potential in clinical settings. However, efficient and well-tolerated antifibrotic drugs are currently lacking. The present study provides a simple and efficient strategy for high-throughput screening of chemicals that interfere with TGF-β signaling. Aside from sorafenib, we have identified several small compounds that inhibit TGF-β signaling using this unbiased cellular screening model. Based on their down-regulation of TGF-β signaling, beneficial effects of these candidates on organ fibrosis could be expected. This expectation has been partially supported by in vivo animal studies showing antifibrotic effects on experimental hepatic, renal, and pulmonary fibrosis (unpubl. data). A more detailed set of such investigations are currently being performed.
In summary, our data provide in vitro evidence that sorafenib inhibits TGF-β signaling and suppresses TGF-β1-induced EMT and apoptosis in mouse hepatocytes.
We thank our colleagues Zheng Li, Jing Xie, Jiang-Sha Zhao, Shu-Yi Ji, and Xiao Hu for helpful discussions and technical assistance. We thank Dr. Ye-Guang Chen (Tsinghua Univ., P.R. China) for kindly providing Smad3 antibody.