The activation of signal transducer and activator of transcription 3 (STAT3) has been linked with the proliferation, survival, invasion, and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Agents that can suppress STAT3 activation have potential for the prevention and treatment of HCC. In this study, we tested an agent, β-escin, for its ability to suppress STAT3 activation. We found that β-escin, a pentacyclic triterpenoid, inhibited both constitutive and interleukin-6-inducible STAT3 activation in a dose- and time-dependent manner in HCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2. Vanadate treatment reversed the β-escin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that β-escin induced the expression of tyrosine phosphatase Src homology phosphatase 1 that correlated with the down-regulation of constitutive STAT3 activation. β-Escin also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1, and vascular endothelial growth factor. Finally, β-escin inhibited proliferation and also substantially potentiated the apoptotic effects of paclitaxel and doxorubicin in HCC cells. Overall, these results suggest that β-escin is a novel blocker of STAT3 activation that may have potential in the suppression of proliferation and chemosensitization in HCC.
Hepatocellular carcinoma (HCC) is one of the most common solid tumors, ranking fifth in incidence and third in mortality worldwide (Bruix et al., 2004). Although epidemiologic studies have shown that chronic viral infections and hepatotoxic agents are the major risk factors, the molecular pathogenesis of HCC is quite complex with the involvement of several oncogenes and tumor suppressor genes (Thorgeirsson et al., 2006). Surgery remains the first choice for treatment of HCC; however, tumor size, hepatic functional reserve, and/or portal hypertension all may restrict surgical ablation (Kerr and Kerr, 2009). Currently, first-line drugs used for HCC include doxorubicin, fuorouracil, cisplatin, and mitomycin, but most of them are nonselective cytotoxic molecules with significant side effects (Kerr and Kerr, 2009). Hence, novel agents that are cheap, nontoxic, and efficacious are urgently needed.
STAT proteins were originally discovered as latent cytoplasmic transcription factors more than a decade ago (Ihle, 1996). There are seven known mammalian STAT proteins, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, which are involved in cell proliferation, differentiation, and apoptosis (Yu and Jove, 2004; Aggarwal et al., 2009). One STAT family member, STAT3, is often constitutively active in many human cancer cells, including multiple myeloma, leukemia, lymphoma, and solid tumors (Yu et al., 2009). STAT3 can also be activated by certain interleukins (e.g., IL-6) and growth factors [e.g., epidermal growth factor (EGF)]. Upon activation, STAT3 undergoes phosphorylation-induced homodimerization, leading to nuclear translocation, DNA binding, and subsequent gene transcription. The phosphorylation is mediated through the activation of nonreceptor protein tyrosine kinases called Janus-like kinase (JAK). JAK1, JAK2, JAK3, and tyrosine protein kinase 2 have been implicated in the activation of STAT3 (Yue and Turkson, 2009). In addition, the role of c-Src kinase has been shown in STAT3 phosphorylation (Ihle, 1996). Among the STATs, STAT3 is perhaps the most intimately linked to tumorigenesis and is often constitutively activated in many human cancer cells including HCC (Niwa et al., 2005). Moreover, STAT3 has been implicated as a promising target for HCC therapy, because inhibition of STAT3 induces growth arrest and apoptosis of human HCC cells (Sun et al., 2008).
One potential source of STAT3 inhibitors is agents derived from natural sources, because approximately 70% of all anticancer drugs being used in clinical therapy are isolated from natural sources or bear a close structural relationship to compounds of natural origin (Newman, 2008). We describe here the identification of a compound derived from the seeds of horse chestnut (Aesculus hippocastanum) called β-escin or aescin, which has potential in the prevention and treatment of cancer. β-Escin is a pentacyclic triterpenoid that has been previously reported to exhibit antiedematous, anti-inflammatory, and anticarcinogenic properties in various disease models (Sirtori, 2001). For example, it was found that β-escin sodium can inhibit the growth of various tumor cell lines [a human oral mucosal cell line (KB cells), a mice liver cancer cell line (H22), and a mice sarcoma cell line (S180)] and their transplant tumors (Guo et al., 2003). It has also been reported that β-escin can suppress colonic aberrant crypt foci formation in rats and inhibit growth of colon cancer cells (Patlolla et al., 2006). Moreover, this pentacyclic triterpenoid was recently found to exhibit significant antitumor effects in human hepatocellular carcinoma both in vitro and in vivo (Zhou et al., 2009). These reports suggest that β-escin may be a suitable candidate for cancer treatment.
Because of the critical role of STAT3 in HCC survival, proliferation, invasion, and angiogenesis, we investigated whether β-escin can mediate its effects in part through the suppression of the STAT3 pathway. We found that β-escin can indeed suppress both constitutive and inducible STAT3 expression in HCC cells. This inhibition decreased cell survival and down-regulated expression of proliferative, antiapoptotic, and angiogenic gene products, leading to the suppression of proliferation, induction of apoptosis, and enhancement of the response to the apoptotic effects of doxorubicin and paclitaxel in HCC cells.
Materials and Methods
β-Escin or aescin (Sirtori, 2001) for our experiments was kindly supplied by Wuxi Gorunjie Technology Co., Ltd. (Jiangsu, China). Hoechst 33342, MTT, Tris, glycine, NaCl, SDS, bovine serum albumin, AG490 [(E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide], EGF, doxorubicin, and paclitaxel were purchased from Sigma-Aldrich (St. Louis, MO). The caspase inhibitor zVAD-FMK was obtained from Promega (Madison, WI). β-Escin was dissolved in dimethylsulfoxide as a 10 mM stock solution and stored at 4°C. Further dilution was done in cell culture medium. RPMI medium 1640, fetal bovine serum (FBS), 0.4% trypan blue vital stain, and antibiotic–antimycotic mixture were obtained from Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies to STAT3 and STAT5 and mouse monoclonal antibodies against phospho-STAT3 (Tyr705) and phospho-STAT5, phospho-Akt, Akt, Bcl-2, Bcl-xL, cyclin D1, survivin, Mcl-1, SHP1, VEGF, procaspase-3, cleaved caspase-3, and PARP were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to phospho-specific Src (Tyr416), Src, phospho-specific JAK1 (Tyr1022/1023), JAK1, phospho-specific JAK2 (Tyr1007/1008), and JAK2 were purchased from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit-horseradish peroxidase (HRP) conjugate and goat anti-mouse HRP were purchased from Sigma-Aldrich. Bacteria-derived recombinant human IL-6 was purchased from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel).
Human hepatocellular carcinoma cell lines HepG2 and PLC/PRF5 were obtained from the American Type Culture Collection (Manassas, VA). The human hepatoma HUH-7 cell line was provided by K.M.H.. Wild-type and STAT3 knockout mouse fibroblasts were a kind gift from Dr. Valeria Poli, University of Turin, Turin, Italy. HepG2, HUH-7, wild-type. and STAT3 knockout mouse fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 1× antibiotic–antimycotic solution with 10% FBS. PLC/PRF5 cells were cultured in DMEM containing 1× penicillin-streptomycin solution, nonessential amino acids, sodium pyruvate, and l-glutamine with 10% FBS.
For detection of phopho-proteins, β-escin-treated whole-cell extracts were lysed in lysis buffer [20 mM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.01 mg/ml aprotinin, 0.005 mg/ml leupeptin, 0.4 mM phenylmethylsulfonyl fluoride, and 4 mM NaVO4]. Lysates were then spun at 14,000 rpm for 10 min to remove insoluble material and resolved on a 7.5% SDS-PAGE gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with anti-STAT antibodies (1:1000) overnight at 4°C. The blot was washed, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence (ECL; GE Healthcare, Little Chalfont, Buckinghamshire, UK).
To detect STAT3-regulated proteins and PARP, HepG2 cells (2 × 106/ml) were treated with β-escin for the indicated times. The cells were then washed and extracted by incubation for 30 min on ice in 0.05 ml of buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% NP-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 μg/ml benzamidine, 1 mM dithiothreitol, and 1 mM sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Whole-cell extract protein (30 μg) was resolved on 12% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with antibodies against survivin, Bcl-2, Bcl-xl, cyclin D1, VEGF, procaspase-3, and PARP and then detected by chemiluminescence (ECL; GE Healthcare).
Immunocytochemistry for STAT3 Localization.
HepG2 cells were plated in chamber slides in DMEM containing 10% FBS and allowed to adhere for 24 h. The next day, the cells were fixed with cold acetone for 10 min, washed with phosphate-buffered saline, and blocked with 5% normal goat serum for 1 h. The cells were then incubated with rabbit polyclonal anti-human STAT3 antibody (dilution, 1/100). After overnight incubation, the cells were washed and then incubated with goat anti-rabbit IgG-Alexa 594 (1/100) for 1 h and counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. Stained cells were mounted with mounting medium (Sigma-Aldrich) and analyzed under an fluorescence microscope (DP 70; Olympus, Tokyo, Japan).
STAT3 Luciferase Reporter Assay.
PLC/PRF5 cells were plated in 90 six-well plates with 1 × 104 per well in DMEM containing 10% FBS. The STAT3-responsive elements linked to a luciferase reporter gene were transfected with wild-type or dominant-negative STAT3-Y705F (STAT3F). These plasmids were a kind gift from Dr. Bharat B. Aggarwal, M.D. Anderson Cancer Center, Houston, TX. Transfections were done according to the manufacturer's protocols by using Fugene-6 (Roche Diagnostics, Indianapolis, IN). At 24 h after transfection, cells were pretreated with β-escin for 4 h and then stimulated with EGF for an additional 2 h before being washed and lysed in luciferase lysis buffer (Promega). Luciferase activity was measured with a luminometer and a luciferase assay kit (Promega) and normalized to β-galactosidase activity. All luciferase experiments were done in triplicate and repeated three or more times.
The antiproliferative effect of β-escin against HCC cells was determined by the MTT dye uptake method as described previously (Bhutani et al., 2007). In brief, the cells (5 × 103/ml) were incubated in triplicate in a 96-well plate in the presence or absence of the indicated concentrations of β-escin in a final volume of 0.2 ml for different time intervals at 37°C. Thereafter, 20 μl of MTT solution (5 mg/ml in phosphate-buffered saline) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of lysis buffer (20% SDS, 50% dimethylformamide) was added, incubation was continued overnight at 37°C, and the optical density at 570 nm was measured with a Tecan (Durham, NC) plate reader.
Apoptosis of cells was also determined by Live/Dead assay (Invitrogen) that measures intracellular esterase activity and plasma membrane integrity as described previously (Bhutani et al., 2007). In brief, 1 × 106 cells were incubated with β-escin/doxorubicin/paclitaxel alone or in combination for the indicated time intervals at 37°C. Cells were stained with the Live/Dead reagent (5 μM ethidium homodimer, 5 μM calcein-acetoxymethyl ester) and then incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Olympus DP 70).
Statistical analysis was performed by one-way analysis of variance. Probability (p) values less than 0.05 were considered statistically significant.
We investigated the effect of β-escin on constitutive and IL-6-inducible STAT3 activation in HCC cells. We also evaluated the effect of β-escin on various mediators of cellular proliferation, cell survival, and apoptosis. The structure of β-escin is shown in Fig. 1A. The dose and duration of β-escin used to modulate STAT3 activation did not affect cell viability, indicating that down-regulation of STAT3 was not caused by cell killing (data not shown).
β-Escin Inhibits Constitutive STAT3 Phosphorylation in HepG2 Cells.
The ability of β-escin to modulate constitutive STAT3 activation in HCC cells was investigated. HepG2 cells were incubated with different concentrations of β-escin for 6 h, whole-cell extracts were prepared, and the phosphorylation of STAT3 was examined by Western blot analysis using antibodies that recognize STAT3 phosphorylation at Tyr705. As shown in Fig. 1B, β-escin inhibited the constitutive activation of STAT3 in HepG2 cells in a dose-dependent manner, with maximum inhibition occurring at approximately 30 μM. β-Escin had no effect on the expression of STAT3 protein (Fig. 1B, bottom). As shown in Fig. 1C, the inhibition was time-dependent, with maximum inhibition occurring at approximately 4 to 6 h, again with no effect on the expression of STAT3 protein (Fig. 1C, bottom).
Effect of β-Escin on STAT3 Phosphorylation Is Specific.
Whether β-escin affects the activation of other STAT proteins in HepG2 cells was also investigated. Under the conditions where β-escin completely inhibited STAT3 phosphorylation, it did not alter either the levels of constitutively phosphorylated STAT5 or the expression of STAT5 proteins (Fig. 1D).
β-Escin Depletes Nuclear Pool of STAT3 in HCC Cells.
Because nuclear translocation is central to the function of transcription factors and it is not certain whether phosphorylation is mandatory for nuclear transport of STAT3 and its oncogenic functions (Brierley and Fish, 2005), we determined whether β-escin suppresses nuclear translocation of STAT3. Figure 1E clearly demonstrates that β-escin inhibited the translocation of STAT3 to the nucleus in HepG2 cells.
β-Escin Inhibits Inducible STAT3 Phosphorylation in HCC Cells.
Because IL-6 induces STAT3 phosphorylation (Moran et al., 2008), we determined whether β-escin could inhibit IL-6-induced STAT3 phosphorylation. HUH-7 cells, which lack constitutively active STAT3, were treated with IL-6 for different times and then examined for phosphorylated STAT3. IL-6 induced phosphorylation of STAT3 as early as 5 min, with maximum phosphorylation observed at 30 to 60 min (Fig. 2A). IL-6 also induced phosphorylation of STAT3 in a dose-dependent manner with initial activation observed at a dose of 5 ng/ml (Fig. 2B). In HUH-7 cells incubated with β-escin for the different times, IL-6-induced STAT3 phosphorylation was suppressed by β-escin in a time-dependent manner. Exposure of cells to β-escin for 3 to 4 h was sufficient to completely suppress IL-6-induced STAT3 phosphorylation (Fig. 2C).
β-Escin Inhibits IL-6-Inducible Akt Phosphorylation in HCC Cells.
Activated Akt has been shown to play a critical role in the mechanism of action of IL-6. Moreover, activation of Akt has also been linked with STAT3 activation (Chen et al., 1999). We also examined whether β-escin could modulate IL-6-induced Akt activation. Treatment of HUH-7 cells with IL-6 induced phosphorylation of Akt, and treatment of cells with β-escin suppressed the activation in a time-dependent manner (Fig. 2D). Under these conditions, β-escin had no effect on the expression of Akt protein.
β-Escin Suppresses EGF-Induced STAT3-Dependent Reporter Gene Expression.
Our above results showed that β-escin inhibited the phosphorylation and nuclear translocation of STAT3. We next determined whether β-escin affects STAT3-dependent gene transcription. When PLC/PRF5 cells transiently transfected with the pSTAT3-Luc construct were stimulated with EGF, STAT3-mediated luciferase gene expression was found to be substantially increased. Dominant-negative STAT3 blocked this increase, indicating specificity. When the cells were pretreated with β-escin, EGF-induced STAT3 activity was inhibited in a dose-dependent manner (Fig. 2E).
β-Escin Suppresses Constitutive Activation of c-Src.
STAT3 has also been reported to be activated by soluble tyrosine kinases of the Src kinase families (Schreiner et al., 2002). Hence, we determined whether β-escin affects constitutive activation of Src kinase in HepG2 cells. We found that β-escin suppressed the constitutive phosphorylation of c-Src kinases (Fig. 3A). The levels of nonphosphorylated Src kinases remained unchanged under the same conditions.
β-Escin Suppresses Constitutive Activation of JAK1 and JAK2.
STAT3 has been reported to be activated by soluble tyrosine kinases of the Janus family (JAKs) (Ihle, 1996), so we determined whether β-escin affects constitutive activation of JAK1 in HepG2 cells. We found that β-escin suppressed the constitutive phosphorylation of JAK1 (Fig. 3B). The levels of nonphosphorylated JAK1 remained unchanged under the same conditions (Fig. 3B, bottom). To determine the effect of β-escin on JAK2 activation, HepG2 cells were treated for different time intervals with β-escin, and phosphorylation of JAK2 was analyzed by Western blot. As shown in Fig. 3C, JAK2 was constitutively active in HepG2 cells, and pretreatment with β-escin suppressed this phosphorylation in a time-dependent manner.
Tyrosine Phosphatases Are Involved in β-Escin-Induced Inhibition of STAT3 Activation.
Because protein tyrosine phosphatases have also been implicated in STAT3 activation (Han et al., 2006) we determined whether β-escin-induced inhibition of STAT3 tyrosine phosphorylation could be caused by activation of a protein tyrosine phosphatase (PTP). Treatment of HepG2 cells with the broad-acting tyrosine phosphatase inhibitor sodium pervanadate prevented the β-escin-induced inhibition of STAT3 activation (Fig. 3D). This suggests that tyrosine phosphatases are involved in β-escin-induced inhibition of STAT3 activation.
β-Escin Induces the Expression of SHP1 in HCC Cells.
SHP1 is a Src homology 2-containing tyrosine phosphatase involved in the suppression of a variety of cytokine signals, including STAT3 (Calvisi et al., 2006). We therefore examined whether β-escin can modulate expression of SHP1 in HepG2 cells. Cells were incubated with different concentrations of β-escin for 4 h, and whole-cell extracts were prepared and examined for SHP1 protein by Western blot analysis. As shown in Fig. 3E, β-escin induced the expression of SHP1 protein in HepG2 cells in a dose-dependent manner, with maximum expression at 15 to 30 μM. This stimulation of SHP1 expression by β-escin correlated with down-regulation of constitutive STAT3 activation in HepG2 cells (Fig. 1B).
β-Escin Down-Regulates the Expression of Cyclin D1, Bcl-2, Bcl-xL, Survivin, and VEGF.
STAT3 activation has been shown to regulate the expression of various gene products involved in cell survival, proliferation, angiogenesis, and chemoresistance (Aggarwal et al., 2009). We found that expression of the cell cycle regulator cyclin D1, the antiapoptotic proteins Bcl-2, Bcl-xL, survivin, and Mcl-1, and the angiogenic gene product VEGF, all of which have been reported to be regulated by STAT3, were modulated by β-escin treatment. Their expression decreased in a time-dependent manner, with maximum suppression observed at approximately 24 h (Fig. 4).
β-Escin Inhibits the Proliferation of HCC Cells in a Dose- and Time-Dependent Manner.
Because β-escin down-regulated the expression of cyclin D1, the gene critical for cell proliferation, we investigated whether β-escin inhibits the proliferation of HCC cells by using the MTT method. β-Escin inhibited the proliferation of HepG2, HUH-7, and PLC/PRF5 cells in a dose- and time-dependent manner (Fig. 5A).
β-Escin Activates Caspase-3 and Induces Cleavage of PARP.
Whether suppression of constitutively active STAT3 in HepG2 cells by β-escin leads to apoptosis was also investigated. In HepG2 cells treated with β-escin there was a time-dependent cleavage of pro-caspase-3 and increased expression of cleaved-caspase-3 (Fig. 5B). Activation of downstream caspase-3 led to the cleavage of 116-kDa PARP protein into a 85-kDa fragment (Fig. 5C). Moreover, the treatment with broad-spectrum caspase inhibitor zVAD-FMK prevented β-escin-induced apoptosis as examined by Western blot for PARP cleavage (Fig. 5D). These results clearly suggest that β-escin induces caspase-3-dependent apoptosis in HepG2 cells.
β-Escin Potentiates the Apoptotic Effect of Doxorubicin and Paclitaxel in HepG2 Cells.
Among chemotherapeutic agents, doxorubicin, an anthracycline antibiotic, and paclitaxel, a mitotic inhibitor, have been used for HCC treatment (Jin et al., 2010). We examined whether β-escin can potentiate the effect of these drugs. HepG2 cells were treated with β-escin together with either doxorubicin or paclitaxel, and then apoptosis was measured by the Live/Dead assay. As shown in Fig. 6, β-escin substantially enhanced the apoptotic effects of doxorubicin from 18 to 60% and paclitaxel from 15 to 45%.
STAT3 Deletion Reduces β-Escin-Induced Apoptosis.
We next determined the apoptotic effect of β-escin on STAT3 gene-deleted mouse embryonic fibroblasts that lack activation of STAT3. Apoptotic effects of β-escin were measured through esterase staining (Live and Dead Assay). Results shown in Fig. 6B indicate that β-escin-induced apoptosis was effectively abolished in the STAT3 gene-deleted fibroblasts (18%) compared with 40% in wild-type fibroblasts. These results suggest that induction of apoptosis is mediated through the suppression of STAT3 by β-escin.
The aim of this study was to determine whether β-escin exerts its anticancer effects in HCC cells through the abrogation of the STAT3 signaling pathway. We found that this triterpene suppressed constitutive and IL-6-inducible STAT3 activation in human HCC cells in parallel with the inhibition of c-Src, JAK1, and JAK2 activation. β-Escin also down-regulated the expression of STAT3-regulated gene products including cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1, and VEGF. It caused the inhibition of proliferation, induced apoptosis as evident by PARP cleavage, and also potentiated the apoptotic effects of doxorubicin and paclitaxel in HCC cells.
We report for the first time that β-escin could suppress both constitutive and inducible STAT3 activation in HCC cells and that these effects were specific to STAT3, because β-escin had no effect on STAT5 phosphorylation. In comparison, higher dose (100–30 μM) and longer exposure (24 versus 6 h) of AG490, a rationally designed JAK2 inhibitor (Meydan et al., 1996), were needed to completely suppress STAT3 phosphorylation in HCC cells (Fuke et al., 2007). The effects of β-escin on STAT3 phosphorylation correlated with the suppression of upstream protein tyrosine kinases c-Src, JAK1, and JAK2. Previous studies have indicated that Src and JAK1 kinase activities cooperate to mediate constitutive activation of STAT3 (Garcia et al., 2001). Our findings suggest that β-escin may block cooperation of Src and JAKs involved in tyrosyl phosphorylation of STAT3. How β-escin inhibits IL-6-induced STAT3 activation was also investigated. The roles of JAK2, mitogen-activated protein kinase, and Akt have been implicated in IL-6-induced STAT3 activation (Chen et al., 1999). We found that IL-6-induced Akt activation was also suppressed by β-escin. We also observed that β-escin suppressed nuclear translocation and EGF induced reporter activity of STAT3. Thus, the recent reported antitumor effects of β-escin on human hepatocellular carcinoma (Zhou et al., 2009) could be caused by inhibition of STAT3 signaling pathway as described here.
STAT3 phosphorylation plays a critical role in the proliferation and survival of tumor cells (Yue and Turkson, 2009). Several types of cancer, including head and neck cancers (Song and Grandis, 2000), multiple myeloma (Bhardwaj et al., 2007), lymphomas, and leukemia (Zhang et al., 2002), also have constitutively active STAT3. The suppression of constitutively active STAT3 in HCC cells raises the possibility that this novel STAT3 inhibitor might also inhibit constitutively activated STAT3 in other types of cancer cells. Previously, it has been reported that β-escin can also suppress NF-κB activation in the brain of rats and different human tumor cells (Xiao and Wei, 2005; Harikumar et al., 2010). Whether suppression of STAT3 activation by β-escin is linked to the inhibition of NF-κB activation is not clear. However, a recent report indicated that STAT3 prolongs NF-κB nuclear retention through acetyltransferase p300-mediated RelA acetylation, thereby interfering with NF-κB nuclear export (Lee et al., 2009). Thus it is possible that suppression of STAT3 activation may mediate inhibition of NF-κB activation by β-escin.
We also found evidence that the β-escin-induced inhibition of STAT3 activation involves a protein tyrosine phosphatase. Numerous PTPs have been implicated in STAT3 signaling including SHP1, SH-PTP2, TC-PTP, PTEN, PTP-1D, CD45, PTP-ε, LMW, and PTP (Kunnumakkara et al., 2009). SHP1 is implicated in the negative regulation of JAK/STAT signaling pathways (Calvisi et al., 2006), and it has been found that loss of SHP1 may contribute to the activation of JAK or STAT proteins in cancer (Wu et al., 2003). Indeed, we observed that β-escin stimulates the expression of SHP1 protein in HCC cells, which correlated with the down-regulation of constitutive STAT3 phosphorylation. Whether it also affects other putative inhibitors such as suppressor of cytokine signaling (SOCS1) and protein inhibitors of activated STAT3 (PIAS3) requires further investigation.
We also found that β-escin suppressed the expression of several STAT3-regulated genes, including proliferative (cyclin D1) and antiapoptotic gene products (Bcl-2, Bcl-xL, survivin, and Mcl-1) and angiogenic gene product (VEGF). β-Escin has been reported previously to induce growth arrest at the G1–S phase in human colon cancer HT29 cells (Patlolla et al., 2006). Because cyclin D1 has been closely linked with G1–S-phase arrest, down-regulation of cyclin D1 as shown here may mediate this effect. Mcl-1 is highly expressed in tumor cells (Epling-Burnette et al., 2001), and Niu et al. (2002) reported that inhibition of STAT3 by a Src inhibitor results in down-regulation of expression of the Mcl-1 gene in melanoma cells. In addition, activation of STAT3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells (Gritsko et al., 2006). Bcl-2 and Bcl-xL can also block cell death induced by a variety of chemotherapeutic agents, in parallel with an increase in chemoresistance (Seitz et al., 2010). Thus, the down-regulation of the expression of Bcl-2, Bcl-xL, survivin, and Mcl-1 probably is linked with β-escin's ability to induce apoptosis in HCC cells as evident by activation of caspase-3 and cleavage of PARP. Furthermore, we observed that apoptotic effects of β-escin were abolished in STAT3-deleted cells, thus strengthening our hypothesis that antiproliferative and proapoptotic effects of β-escin are mediated through the abrogation of the STAT3 signaling pathway. The down-modulation of VEGF expression as reported here can explain the antiangiogenic effects of this triterpene as described previously in rat aortic disk assay (Zhao et al., 2007).
Doxorubicin and paclitaxel are commonly used chemotherapeutic drugs for the treatment of HCC (Jin et al., 2010). We further demonstrate that β-escin substantially potentiates the apoptotic effect of doxorubicin and paclitaxel in HCC cells as evident by esterase staining and can be used in combination with existing chemotherapeutic drugs. Several studies in animals suggest that β-escin is very well tolerated and has potential against inflammatory diseases and cancers (Sirtori, 2001). β-Escin has also shown satisfactory evidence for a clinically significant activity in chronic venous insufficiency, hemorrhoids, and postoperative edema and is currently in clinical trials in HIV patients (Sirtori, 2001; Grases et al., 2004). We contend that the apparent pharmacologic safety of β-escin and its ability to down-regulate the expression of several genes involved in cell proliferation, survival, and invasion provides a sufficient rationale for testing β-escin in patients for treatment of HCC and other cancers harboring active STAT3.
This work was supported by the Department of Research and Technology, National University of Singapore [Grant R-184-000-161-112] and the National Medical Research Council of Singapore [Grant R-184-000-168-275] (to G.S.). A.P.K. was supported by the National Medical Research Council of Singapore [Grant R-364-000-085-275] and the Cancer Science Institute of Singapore, Experimental Therapeutics I Program [Grant R-713-001-011-271]. K.M.H. was supported by the National Medical Research Council of Singapore [Grant NMRC/IBG/NCC/2009].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- signal transducer and activator of transcription 3
- hepatocellular carcinoma
- fetal bovine serum
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
- Janus-activated kinase
- polyacrylamide gel electrophoresis
- epidermal growth factor
- vascular endothelial growth factor
- Src homology phosphatase 1
- poly(ADP-ribose) polymerase
- protein tyrosine phosphatase
- Dulbecco's modified Eagle's medium
- horseradish peroxidase
- benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone
- nuclear factor-κB.
- Received January 5, 2010.
- Accepted April 7, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics