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Deregulated activation of oncogenic transcription factors such as signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in proliferation and survival of hepatocellular carcinoma (HCC). Thus, agents which can inhibit STAT3 activation may have an enormous potential for treatment of HCC patients. Hence, in the present report, we investigated the effect of ascochlorin (ASC), an isoprenoid antibiotic on STAT3 activation cascade in various HCC cell lines and orthotopic mouse model. We observed that ASC could substantially inhibit both constitutive and IL‐6/EGF inducible STAT3 activation as well as reduce its DNA binding ability. ASC increased the expression of protein inhibitor of activated STAT3 (PIAS3) which could bind to STAT3 DNA binding domain and thereby down‐regulate STAT3 activation. Deletion of PIAS3 gene by siRNA abolished the ability of ASC to inhibit STAT3 activation and induce apoptosis in HCC cells. ASC also modulated the expression of diverse STAT3‐regulated oncogenic gene products. Finally, when administered intraperitoneally, ASC also inhibited tumor growth in an orthotopic HCC mouse model and reduced STAT3 activation in tumor tissues. Overall our results indicate that ASC mediates its anti‐tumor effects predominantly through the suppression of STAT3 signaling cascade, and can form the basis of novel therapy for HCC patients.
Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and the third most common cause of cancer mortality in the world. The treatment outcome of HCC is far from satisfactory as this malignancy has a dismal 5‐year survival rate of approximately 10% (Altekruse et al., 2009). Chemotherapeutic drugs, such as cisplatin, doxorubicin and fluorouracil are most commonly used treatment options, especially for patients with unresectable HCC tumors. However, because of poor response rates to chemotherapy, severe toxicities and high recurrence rates, the mean survival time for advanced stage disease is only 6 months (Thomas and Zhu, 2005). Thus, more effective agents are urgently required to clinically combat this lethal malignancy.
Recent evidences suggest that STAT family proteins, especially STAT3, play a crucial role in inducing as well as maintaining a pro‐carcinogenic inflammatory microenvironment, promoting malignant transformation and cancer progression (Mantovani et al., 2008; Yu et al., 2009). Phosphorylation at tyrosine residue (Tyr705) is critical for STAT3 activation, which contributes to its dimerization that is a prerequisite for nucleus entry and DNA binding (Subramaniam et al., 2013b). Activation of STAT3 is the most frequently mediated by receptor tyrosine kinases such as JAK1 and JAK2, but it also can be activated by other mechanism(s), including reversible acetylation and non‐receptor tyrosine kinases such as Src which is upstream of STAT3 and commonly overexpressed in a variety of solid tumor cells (Heinrich et al., 2003; Levy and Darnell, 2002). Cytokine‐induced STAT3 activation is a transient event in normal cells, because this activity can be controlled by negative feedback loops, and which in turn is regulated by diverse proteins including those of protein inhibitor of activated STAT (PIAS) family, especially PIAS3, that bind specifically to STAT3 and abrogate its activity (Chung et al., 1997).
However, in tumor cells STAT3 is often found to be constitutively activated through other diverse mechanism(s) as well (He and Karin, 2011). Persistent activation of STAT3 has also been found in the majority of HCC patient tissues but not in surrounding normal liver tissues and has been closely associated with poor prognosis (Calvisi et al., 2006). Accumulating evidences have demonstrated that STAT3 is an attractive molecular target both for cancer treatment and prevention (Siveen et al., 2014). For example, phosphopeptides and their derivative peptidomimetics have been designed to block STAT3 dimerization and DNA‐binding activity (Turkson et al., 2004, 2001). Furthermore, AG490, a Janus kinase (JAK) specific inhibitor, significantly inhibited the expression of p‐STAT3, and subsequently reduced invasion and adhesion of human pancreatic cancer cells (Huang et al., 2006). Thus, blockage of STAT3 activation by using diverse pharmacological agents can be considered as an important therapeutic strategy for HCC management.
One potential source of STAT3 inhibitors is agents derived from natural sources, because approximately 74.8% (131/175) of all anti‐cancer drugs approved either were isolated from natural sources or mimicked them in one form or another (1981–2010) (Newman and Cragg, 2012). ASC an isoprenoid antibiotic, isolated from a culture broth of the phytopathogenic fungus, Ascochyta viciae, has been found to exhibit diverse antiviral and antifungal activities (Tamura et al., 1968). In addition to its reported antimicrobial effects, natural and synthetic derivatives of ASC can suppress hypertension (Hosokawa et al., 1981), induce immunomodulation (Magae et al., 1986), and ameliorate type I as well as type II diabetes (Hosokawa et al., 1985). Interestingly, few recent studies have indicated that ASC may also exhibit anti‐cancer activities in various tumor cells, primarily mediated through activation of p53 and inhibition of the mitochondrial cytochrome bc1 complex and activator protein‐1 (AP‐1), leading to the suppression of the extra‐cellular enzyme, matrix metalloproteinase‐9 (MMP9) (Hong et al., 2005; Jeong and Chang, 2010; Jeong et al., 2009). However, the detailed molecular mechanism(s) through which ASC exhibits its anti‐cancer effects still remains to be elucidated.
In this study, we specifically investigated whether ASC could suppress the growth of HCC in vitro and in vivo by down‐regulating STAT3 signaling pathway, which plays a pivotal role in HCC initiation and progression. Our results indeed indicate for the first time that ASC could effectively abrogate both constitutive and inducible STAT3 activation in HCC cells through modulating upstream kinases and PIAS3 expression. Interestingly, this isoprenoid antibiotic also down‐regulated expression of proliferative, anti‐apoptotic as well as invasive gene products, leading to the suppression of proliferation, migration/invasion and induction of apoptosis in HCC cells. Alongside the effects of ASC in vitro, we also found that ASC significantly suppressed the growth of human HCC cells in an orthotopic mouse model and abrogated STAT3 activation in tumor tissues.
ASC was kindly provided by Dr. Junji Magae at Magae Bioscience Institute (Japan). ASC was dissolved in dimethylsulfoxide as a 50 mmol/L stock solution and stored at 4 °C. Further dilution was done in cell culture medium. DAPI, MTT, Tris, glycine, Sodium orthovanadate (90% titration), NaCl, SDS, BSA, IL‐6 and EGF were purchased from Sigma (St. Louis, MO). DMEM, 10% FBS were obtained from Life Technologies (Carlsbad, CA). Rabbit or mouse monoclonal antibodies against phospho‐STAT3 (Ser 727), STAT3, PARP, XIAP, SHP‐1, SHP‐2, PTEN, PTP1B, PIAS3, Bak, Bcl‐2, cyclin D1, Bid, MMP‐9, survivin, Mcl‐1, caspase‐3, caspase‐8 and caspase‐9 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phospho‐STAT3 (Tyr 705), phospho‐specific Src (Tyr 416), Src, phospho‐specific JAK2 (Tyr 1007/1008), JAK2, phospho‐specific JAK1 (Tyr 1022/1023), JAK1, phospho‐specific STAT5 (Tyr 694), and STAT5 were purchased from Cell Signaling Technology (Beverly, MA). The siRNA for PIAS3 (sc‐37005) and scrambled control (sc‐37007) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti‐rabbit–horse radish per‐oxidase (HRP) conjugate and goat anti‐mouse HRP were purchased from Sigma–Aldrich (St. Louis, MO). Bacteria‐derived recombinant human IL‐6 was purchased from ProSpec‐Tany Techno Gene Ltd.
HepG2, Hep3B and Huh7 were obtained from American Type Culture Collection. All the HCC cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 1X antibiotic‐antimycotic solution with 10% FBS. HCCLM3 was a kind gift of Professor Zhao‐You Tang at the Liver Cancer Institute (Zhongshan Hospital, Fudan University, Shanghai). HCCLM3 were cultured in high glucose DMEM containing 1X antibiotic‐antimycotic solution with 10% FBS.
Whole‐cell extracts were lysed in lysis buffer (250 mM NaCl, 20 mM HEPES, 2 mM EDTA (pH 8.0), 0.5 mM EGTA, 0.1% Triton X‐100, 1.5 μg/mL aprotinin, 1.5 μg/mL leupeptin, 1 mM PMSF, and 1.5 mM Na3VO4). Lysates were then spun at 13,300 rpm for 10 min to remove insoluble material and resolved on a 10% SDS gel. After electrophoresis, the proteins were electro‐transferred to a nitrocellulose membrane (Biorad), blocked with Blocking One (Nacalai Tesque, Inc.), and probed with primary antibodies of interest overnight at 4 °C. The blot was washed, exposed to HRP‐conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence (ECL; Amersham Pharmacia Biotech).
HCC cells were seeded in the culture‐insert (Ibidi) until fully confluent. A cell‐free gap of 500 μm was created after removing the Culture‐Insert. After incubation with 50 μM ASC for 8 h, the medium was changed to medium with or without CXCL12. After migration for 24 h, the wounds were stained and observed using bright field microscopy.
An in vitro cell invasion assay was performed using Bio‐Coat Matrigel invasion assay system (BD Biosciences, San Jose, CA), as described previously (Manu et al., 2013).
To determine the effect of ASC on STAT3 DNA binding activity, we performed DNA binding assay using TransAM STAT3 transcription factor assay kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions and as described previously (Subramaniam et al., 2013a).
HepG2 cells were plated in 8 chamber slides in DMEM containing 10% FBS and allowed to adhere for overnight. After treatment with 50 μM ASC for 8 h, the cells were fixed with cold acetone for 15 min, washed with PBS and blocked with 5% normal goat serum for 1 h. The cells were incubated with rabbit polyclonal anti‐human STAT3 (dilution, 1:100). After overnight incubation, the slides were washed and then incubated with goat anti‐rabbit IgG‐Alexa 488 (dilution, 1:100) for 1 h and counterstained for nuclei with DAPI (50 ng/ml) for 5 min. Stained slides will be mounted with mounting medium (Sigma–Aldrich) and analyzed under a fluorescence microscope (Olympus DP 70, Japan).
HepG2 cells (600–800 cells/well) were seeded in 6‐well plate for 24 h, and then treated with various concentrations of ASC. After incubation for 72 h, the cells were washed by PBS and cultured in normal medium for two weeks. At the end of time point, colonies were washed with PBS, fixed with methanol and thereafter stained with 1% crystal violet solution. Colonies with >50 cells were counted under microscope.
The anti‐proliferative effects of ASC against various HCC cells were determined by the MTT dye uptake method as described previously (Ramachandran et al., 2012). Briefly, the cells (7 × 103) were seeded in a 96‐well plate overnight, and then treated with or without different concentrations of ASC for indicated time intervals at 37 °C. Thereafter, 20 μL MTT solution (5 mg/mL in PBS) was added to each well. After 2 h incubation at 37 °C, 0.1 mL lysis buffer (20% SDS, 50% dimethylformamide) was added after removal of the medium and incubation at 37 °C for 1 h; and then the optical density (OD) at 570 nm was measured by Tecan plate reader.
Cellular DNA fragmentation was detected using cell death detection ELISAPLUS kit according to the manufacturer's protocol (Roche Molecular Biochemicals, Mannheim, Germany) and as described previously (Shanmugam et al., 2014).
Apoptosis of cells was also determined by TUNEL enzyme kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer's instruction.
To determine the effect of ASC on STAT3 transcriptional activity, the STAT3 luciferase reporter assay was performed as described previously (Rajendran et al., 2011).
HepG2 cells were plated in each well of six‐well plates and allowed to adhere for 24 h. On the day of transfection, 4 μL of lipofectamine Life Technologies (Carlsbad, CA) was added to 50 nM PIAS3 siRNA in a final volume of 100 μL of culture medium. After 48 h of transfection, cells were treated with ASC, and whole‐cell extracts were prepared to analyze the expression of PIAS3, phospho‐STAT3, STAT3, and PARP by Western blot analysis.
Total cellular RNA was extracted from untreated and ASC‐treated cells by using Trizol reagent Life Technologies (Carlsbad, CA) as described previously (Shanmugam et al., 2014). The expression of PIAS3 was analyzed using QIAGEN One Step RT‐PCR kit with GAPDH as an internal control. The RT‐PCR reaction mixture contained 10 μL of 5X QIAGEN OneStep RT‐PCR buffer, 1 μg of total RNA, 0.6 μM each of forward and reverse primers, 2 μL of dNTP mix and 2 μL of QIAGEN OneStep RT‐PCR enzyme mix in a final volume of 50 μL. The reaction was performed at 50 °C for 30 min, 95 °C for 5 min, 95 °C for 1 min, 61 °C for 1 min, and 72 °C for 1 min for 33 cycles with a final extension at 72 °C for 10 min. PCR products were run on 1% agarose gel containing 1X Gel red nucleic acid gel stain from Biotium (Hayward, CA). Stained bands were visualized under UV light and photographed. The primer sequences for PIAS3 mRNA were as follows: 5′‐ATTGACTGCTGACCCTGACA‐3′ (forward) and, 5′‐GGGACAGCGAAGTTTCCATA‐3′ (reverse). The primer sequences for GAPDH were 5′‐CCACAGTCCATGCCATCAD‐3′ (forward) and 5′‐TCCACCACCCTGTTGCTGTA‐3′ (reverse).
Real‐time polymerase chain reaction for cyclin D1, survivin and XIAP genes was performed as described previously (Shanmugam et al., 2014).
All procedures involving animals were reviewed and approved by SingHealth Institutional Animal Care and Use Committee. In order to define suitable treatment doses, a pharmacokinetics study of ASC at 2.5 mg/kg (i.p.) was conducted. The blood samples were collected at 10 min, 30 min, 1, 2, 4, 6, and 8 h post dose. A sensitive liquid chromatography‐tandem mass spectrometry method was developed and validated for determination of serum concentrations of ASC. Eight week‐old athymic balb/c nude female mice (Biolasco, Taiwan) were implanted orthotopically with approximately 1 mm3 of human HCCLM3_Luc2 tumor stably expressing firefly luciferase. Tumor growth was monitored by IVIS 200 Bioluminescence Imaging System (Xenogen Corp., Alameda, CA). Once increasing bioluminescence tumor signals were detected in the mice liver, mice were randomly assigned to the treatment groups. Mice received intraperitoneal injections of 2.5 mg/kg or 5 mg/kg of ASC on day 0, 1, 2, 3, 13, 15, 17, 20, 22, 24, 27, 29 and 31. Animals were euthanized at day 34 after first therapeutic dose injection, and tumors were harvested for subsequent analysis. For imaging, mice were given i.p. injections of 150 mg/kg D‐luciferin (Xenogen) 10 min before imaging. To quantitate tumor burden, bioluminescence signals were calculated from the imaging data using the Living Image software 3.2 (Xenogen) according to manufacturer's protocol.
Solid tumors from control and ASC‐treated mice were fixed with 10% phosphate buffered formalin. Tumors were cut and embedded in paraffin. And then tissues were deparaffinized in xylene, dehydrated in graded alcohol, and finally hydrated in water. Antigen retrieval was carried out by boiling the slide in 10 mmol/L sodium citrate (pH 6.0) for 30 min. Immunohistochemistry was done following manufacturer's instructions (Dako LSAB kit). Briefly, endogenous peroxidases were quenched with 3% hydrogen peroxide. Non‐specific binding was blocked by incubation in the blocking reagent in the LSAB kit (Dako), according to the manufacturer's instructions. Sections were incubated overnight with primary antibodies as follows: anti‐p‐STAT3, anti‐CD31, anti‐Ki67, and anti‐cleaved‐caspase‐3 (each at 1:100 dilution). Slides were subsequently washed several times in Tris‐buffered saline with 0.1% Tween 20 and were incubated with biotinylated linker for 30 min, followed by incubation with streptavidin conjugate provided in LSAB kit (Dako), according to the manufacturer's instructions. Immunoreactive species were detected using 3, 30‐diamino‐benzidine tetrahydrochloride as a substrate. Sections were counterstained with Gill's hematoxylin and mounted under glass cover slips. Images were taken using an Olympus BX51 microscope (magnification: 20×). Positive cells (brown) were quantitated using the Image‐Pro plus 6.0 software package (Media Cybernetics, Inc.).
Data are expressed as the mean ± S.E.M. In all figures, vertical error bars denote the S.E.M. The significance of differences between groups was evaluated by Student's t‐test or one way analysis of variance, (ANOVA) test. A p‐value of less than 0.05 was considered statistically significant.
In this study, we analyzed the potential effect of ASC (chemical structure shown in Figure 1A) on STAT3 activation and on various markers of cellular proliferation, survival, and apoptosis in HCC cell lines and an orthotopic mouse model.
ASC inhibits constitutively active STAT3 in HCC cells. (A) The chemical structure of ASC. (B) ASC suppresses phosphorylation of STAT3 at tyrosine 705 in HepG2 cells at does and time dependent. HepG2 cells (2 × 106 mL−1) ...
The ability of ASC to modulate constitutive STAT3 activation in HCC cells was first determined by Western blot analysis using specific antibodies against p‐STAT3 (Tyr 705) residue. As shown in Figure 1B, ASC substantially suppressed the phosphorylation of STAT3 in a dose‐ and time‐dependent manner, and the maximum inhibition was observed with 50 μM at 10 h. We further found that various HCC cell lines including (HepG2, HCCLM3 and Huh7) displayed constitutively active STAT3 and this expression was abrogated upon treatment with 50 μM ASC for 8 h (Figure 1C). Because STAT3 also undergoes phosphorylation at Ser 727 for its transcriptional activation, we next investigated whether ASC has an effect on this phosphorylation and found that this isoprenoid antibiotic had little effect on STAT3 activation at Ser 727 residue (Figure 1D, upper panel). We also analyzed whether ASC also affects STAT5 phosphorylation in HCC cells. Under the conditions in which ASC inhibited STAT3 (Tyr705) phosphorylation, it had minimal effect on STAT5 phosphorylation (Figure 1D, lower panel), thereby indicating its specificity towards p‐STAT3 (Tyr 705) residue.
As nuclear translocation is critical for the function of transcription factors (Subramaniam et al., 2013b), we next studied the effect of ASC on nuclear translocation of STAT3 by immunocytochemistry analysis. The data reveals relatively strong expression of STAT3 in nuclei of untreated cells, however only reduced expression of STAT3 was observed in the nuclei of ASC‐treated cells. Similar inhibitory effects were noted when HepG2 cells were treated with 100 μM pharmacological JAK2 inhibitor AG490 for 8 h (Figure 2A, left panel). Moreover, the expression of STAT3 in nuclear and cytoplasmic was also analyzed by western blot analysis and results obtained were consistence with immunocytochemistry data (Figure 2A, right panel). Overall, our data indicates that ASC can also abrogate nuclear translocation of STAT3 in HepG2 cells.
(A) ASC reduces translocation of STAT3 to the nucleus. Immunocytochemistry was performed as described as described under “Materials and methods”. After treatment with 100 μM AG490 for 8 h, it was used as a positive ...
To further investigate whether ASC could inhibit the STAT3‐DNA binding activity in HCC cells, ELISA‐based TransAM STAT3 assay kit was used. Analysis of nuclear extracts prepared from both HepG2 and HCCLM3 cells using ELISA based TransAM STAT3 assay kit showed that ASC abrogated STAT3‐DNA binding activity in a time‐dependent manner (Figure 2B). Inhibition of STAT3‐DNA binding activity was found in HCCLM3 cells as early as 2 h after treatment with 50 μM ASC, whereas in HepG2 cells it was observed only after 6 h. These results suggest that ASC could abrogate the DNA binding ability of STAT3 in HCC cells.
Whether ASC could also inhibit the transcriptional activity of STAT3 was examined by luciferase reporter gene assay in HepG2 cells. As shown in Figure 2C, the transcriptional activity of STAT3 was suppressed by ASC in a dose‐dependent manner with the maximal inhibition observed with 50 μM concentration. These data suggest that ASC can indeed abrogate STAT3 activation at multiple steps in HCC cells.
As cytokines like IL‐6 can also induce transient STAT3 activation (Subramaniam et al., 2013b), we next examined whether ASC could also negatively regulate IL‐6 induced STAT3 phosphorylation. First, we noticed that the STAT3 phosphorylation level could be substantially increased upon IL‐6 treatment in a dose‐ and time‐dependent manner in Hep3B cells that display relatively low basal STAT3 activation (Figure 3A). Thereafter, following pre‐treatment with ASC for different time intervals, IL‐6 at a concentration of (25 ng/mL) was added to Hep3B cells for 20 min. Interestingly, we found that IL‐6 induced STAT3 phosphorylation was substantially prevented after ASC treatment in a time‐dependent manner (Figure 3B). These results suggest that ASC could also reduce inducible STAT3 activation in HCC cells.
ASC prevents IL‐6/EGF inducible STAT3 activation in HCC cells. (A) IL‐6 increases phosphorylation of STAT3 in time‐ and does‐dependent in Hep3B cells. Hep3B cells were treated with IL‐6 at indicated concentrations ...
Our above results clearly indicated that ASC could indeed inhibit IL‐6‐induced phosphorylation of STAT3. We next determined whether ASC can also affect EGF‐induced STAT3‐dependent gene transcription in HCC cells. We found that when Hep3B cells were transiently transfected with the pSTAT3‐Luc construct and thereafter stimulated with EGF (100 ng/ml) for 2 h, STAT3‐mediated luciferase gene expression was significantly increased. Conversely, when cell were pre‐treated with ASC, the EGF‐induced STAT3 activity was inhibited in a dose‐dependent manner. The maximum inhibition was again found at 50 μM concentration (Figure 3C).
Activation of STAT3 is frequently mediated by receptor tyrosine kinase JAK1 and JAK2 and non‐receptor tyrosine kinases such as Src (Levy and Darnell, 2002). We next investigated whether ASC could modulate constitutive activation of JAK1 and JAK2 in HepG2 cells. The results indicated that substantial reduction of JAK1 and JAK2 activation could be noted in a time‐dependent manner after treatment with ASC (Figure 4A). Furthermore, we determined whether ASC can also suppress constitutive activation of Src kinase in HCC cells. Figure 4A clearly shows that ASC can also abrogate the constitutive activation of Src kinase in a time‐dependent manner and the levels of non‐phosphorylated Src remained unchanged under the similar treatment conditions.
ASC modulates the expression of upstream regulators of STAT3 signaling pathway. (A) ASC inhibits phosphorylation of JAK1, JAK2 and Src in HepG2 cells. HepG2 cells were treated with 50 μM ASC for 4, 6, 8 and 10 h. Cells were lysed ...
Whether ASC could also abrogate IL‐6‐induced JAK1, JAK2 and Src phosphorylation in Hep3B cells was examined by western blot analysis. When cells were stimulated with IL‐6, the phosphorylation of JAK1, JAK2 and Src was found to be substantially increased. However, when the cells were pre‐treated with ASC, this increase could be abrogated in a time‐dependent manner (Figure 4B). Exposure of cells to ASC for 4 h was sufficient to substantially suppress IL‐6‐induced JAK1, JAK2 and Src activation in Hep3B cells (Figure 4B).
Next, we attempted to elucidate the potential effect of ASC on the negative regulators of STAT3 signaling cascade namely protein inhibitors of activated‐STAT 3 (PIAS3) (Chung et al., 1997) and protein phosphatases such as SHP‐1, SHP‐2 (He and Karin, 2011), PTEN (Sun and Steinberg, 2002) and PTP1B (Johnston and Grandis, 2011) in HCC cells which have been reported to be closely associated with dephosphorylation of STAT3. First, treatment of HepG2 cells with the broad‐acting tyrosine phosphatase inhibitor sodium pervanadate prevented the ASC‐induced inhibition of STAT3 activation in a dose dependent manner (Figure S1). This data indicated that tyrosine phosphatases may be involved in ASC‐induced inhibition of STAT3 activation. However, the expression of SHP‐1, SHP‐2, PTEN and PTP1B proteins was not substantially modulated upon ASC treatment (Figure 4C). Interestingly, we observed that treatment with 50 μM ASC substantially increased the expression of PIAS3 protein in HepG2 cells in a time‐dependent manner with maximum expression observed at 8–10 h (Figure 4C). Whether modulation of PIAS3 by ASC is also regulated at the transcriptional level was studied as well. We observed that treatment of ASC also induced the expression of PIAS3 mRNA in a time‐dependent manner (Figure 4D). Overall, these finding suggest that the induction of PIAS3 expression may mediate the negative regulation of STAT3 activation caused by ASC in HCC cells.
Our results clearly demonstrate ASC could significantly increase expression of PIAS3 protein, which is possibly associated with observed decrease in STAT3 phosphorylation following ASC treatment. To explore this hypothesis, HepG2 cells were transfected with siRNA against PIAS3 to inhibit PIAS3 expression and then treated with or without 50 μM ASC for 8 h. As observed by Western blot analysis, ASC‐induced PIAS3 expression was effectively inhibited in the cells transfected with PIAS3 siRNA but not in those treated with the scrambled siRNA (Figure 4E). We next analyzed whether the suppression of PIAS3 expression by siRNA blocks the inhibitory effect of ASC on STAT3 activation in HCC cells. Interestingly, we noted that ASC did not substantially suppress STAT3 activation in the cells transfected with PIAS3 siRNA (Figure 4E). However, in cells transfected with scrambled siRNA, ASC caused substantial downregulation of STAT3 activation. Thus these results indicate the important role of PIAS3 in suppression of STAT3 phosphorylation by ASC.
The effect of ASC on the viability of HCC cell lines was first investigated by MTT assay. The results showed that ASC could inhibit the viability of three different HCC cell lines tested (HepG2, HCCLM3 and Huh7) in a time and dose dependent manner (Figure 5A). We next investigated, by in vitro clonogenic assay, whether ASC could also decrease long term survival ability of HCC cells. Only a very small number of highly stained and tightly packed clones were observed in 50 μM ASC treated cells which was only one sixth of the total number observed in untreated group (Figure 5B).
ASC inhibits the cell viability, invasion and migration in HCC cells. (A) ASC inhibits the viability of HCC cells. MTT assays were performed in HepG2, HCCLM3 and Huh7 cells. Cell viability was determined as percentage of the vehicle control. (B) ASC ...
These findings indicate that the viability of HCC cells is significantly inhibited upon ASC treatment.
In addition to proliferation, activated STAT3 signaling may also positively correlate with increased HCC metastasis and invasiveness (Manu et al., 2013; Zhang et al., 2002). Whether down regulation of STAT3 by ASC also correlated with its ability to inhibit cellular migration was examined using an in vitro wound healing assay. We found that treatment with ASC significantly suppressed CXCL12 induced migration of both HepG2 and HCCLM3 cells at 50 μM concentration (Figure 5C). To further elucidate the effect of ASC on CXCL12‐induced cellular invasion, we employed an in vitro matrigel invasion assay. As shown in Figure 5D, HepG2 and HCCLM3 cells moved to another side faster under the influence of CXCL12 and this effect was significantly abolished upon treatment with ASC.
It has been shown that abrogation of persistent STAT3 activation also could induce apoptosis (Mora et al., 2002; Rahaman et al., 2002). To investigate the potential effects of ASC on cellular apoptosis, PI staining was first conducted to examine the changes in sub G1 percentage. As shown in Figure 6A, the fraction of cells with sub G1 DNA content was substantially increased in a time‐dependent manner in both HepG2 and Huh7 cells after exposure to 50 μM ASC for 24, 48 and 72 h. Additionally ASC induced apoptosis was further confirmed by TUNEL and ELISA based DNA fragmentation assays. Increased TUNEL positive cells were observed after ASC treatment for 24 and 48 h (Figure 6B). Also, in HepG2 cells treated with ASC there was a time‐dependent increase in the percentage of DNA fragmentation as determined by Cell Death Detection ELISAPLUS kit (Figure 6C).
ASC induces substantial apoptosis in HCC cells. (A) The sub G1 ratio in HCC cells treated with ASC was examined using flow cytometry. Cells were exposed to 50 μM ASC at indicated times, after which cell were harvested and stained with ...
We next analyzed whether ASC‐induced apoptosis might also involve caspases using western blot analysis and representative blots are shown in Figure 6D. Treatment of HepG2 cells with 50 μM ASC resulted in cleavage of procaspase‐3 as evident by the appearance of 19 and 17 kDa intermediate bands in a time dependent manner. Caspase‐3 is an executioner caspase that can be activated by mitochondrial pathway involving activation of caspase‐9 or a death receptor mediated cascade involving caspase‐8 (Fan et al., 2005). As shown in Figure 6D, both caspase‐8 and 9 were activated after ASC treatment in HepG2 cells in a time‐dependent manner. These observations suggest the possible involvement of both caspase‐8 and caspase‐9 in ASC‐mediated cleavage of caspase‐3.
Activation of downstream caspase‐3 can lead to the cleavage of a 116 kDa PARP protein into 85 kDa fragments (Tewari et al., 1995). As shown in Figure 6E, ASC could cause a dramatic increase in the expression of PARP cleavage products. We next determined whether the suppression of PIAS3 expression by siRNA could abrogate the observed pro‐apoptotic effect of ASC on HCC cells. Results shown in Figure 6E clearly indicate that the observed effect of ASC on PARP cleavage was substantially abolished in the cells transfected with PIAS3 siRNA, whereas treatment with scrambled siRNA had minimal effect.
STAT3 activation has been shown to regulate the expression of various gene products involved in proliferation, anti‐apoptosis, invasion, angiogenesis and chemoresistance (Subramaniam et al., 2013b). As shown in Figure 7A, expression of the cell cycle regulator protein cyclin D1, the anti‐apoptotic proteins Bcl‐2, Mcl‐1, survivin and XIAP, and the invasive gene product MMP‐9 were inhibited upon ASC treatment. The expression of pro‐apoptotic proteins (Bak and cleaved‐Bid) was up‐regulated in a time‐dependent manner, with maximum increase observed at around 48 h. We also found that mRNA expression of cyclin D1, survivin and XIAP was also reduced upon ASC treatment in a time‐dependent manner with maximum suppression observed at around 24 h (Figure 7B).
ASC modulates the expression of STAT3‐regulated gene products involved in proliferation, and survival. (A) HepG2 cells were treated with 50 μM ASC for 48 h. Cells were lysed for Western blot analysis by using antibodies ...
The results of pharmacokinetics study indicated that ASC showed a good exposure in mouse serum at 2.5 mg/kg with maximum concentration (Cmax) of 1272.3 nM (Figure 8A). It suggested that 2.5 and 5 mg/kg could be suitable dose range for evaluation of in vivo anticancer effect of ASC in an orthotopic mouse HCC model. We next analyzed the antitumor potential of ASC in vivo via intraperitoneal administration in an orthotopic model of human HCC using HCCLM3_Luc2 tumor stably‐expressing firefly luciferase. Orthotopic tumor growth was monitored non‐invasively using bioluminescence imaging. Prior to first therapeutic injection (10 days after tumor implantation), the growth of orthotopic tumors was monitored using bioluminescence imaging and it was found to be localized mainly at the liver (Figure 8B). Mice were treated with either vehicles (0.1% DMSO) alone (n = 9), 2.5 mg/kg (n = 7) or 5 mg/kg (n = 5) of ASC for five weeks. Bioluminescence images revealed that there was significant inhibition of tumor growth in the ASC‐treated group compared with the vehicle control group (Figure 8B). Differences in tumor burden at set time points were quantitated by measuring photon counts and expressed as tumor burden relative to photon counts before first therapeutic injection (Figure 8C). One‐way ANOVA indicated that ASC at 2.5 mg/kg and 5 mg/kg induced significant inhibition of tumor growth compared with the vehicle‐treated controls (Figure 8C). No significant changes in body weight were observed in ASC‐treated mice compared to control group (Figure 8D).
In vivo antitumor activities of ASC in an orthotopic human HCC tumor. (A) Pharmacokinetics analysis of ASC at 2.5 mg/kg following i.p. injection was performed. The blood samples were collected at 10 min, 30 min, 1, 2, ...
We further evaluated the effect of ASC on constitutive p‐STAT3 levels in HCC tumor tissues by immunohistochemical analysis and found that ASC can significantly inhibit the constitutive STAT3 activation in drug treated group as compared with the control group (Figure 8E). The effect of ASC was also analyzed on the expression of Ki‐67 (marker of proliferation), CD31 (marker of angiogenesis), and cleaved caspase‐3 (marker of apoptosis). As shown in Figure 8E, expression of various biomarkers including Ki‐67 as well as that of CD31 was downregulated, and that of caspase‐3 was significantly increased in ASC treated group as compared to the control group.
Persistent activation of STAT3 has been commonly observed in wide majority of HCC tissues (Calvisi et al., 2006), and is closely linked to the development of chemoresistance in patients (Sun and Karin, 2012). Given the pivotal role of STAT3 in HCC initiation and progression, the aim of this present study was to investigate whether ASC could exert its anticancer effects at least in part through the attenuation of the STAT3 signaling pathway in HCC cells and an orthotopic mouse model. We found that ASC treatment caused substantial inhibition of constitutive and inducible STAT3 activation concomitant with the blockade of c‐Src, JAK1 and JAK2 kinase activation and the induction of PIAS3 in human HCC cells. ASC could further modulate the expression of STAT3‐regulated gene products, significantly inhibit cell viability, migration as well as invasion and induce substantial apoptosis in HCC cells. The potential therapeutic efficacy of ASC was also analyzed in an orthotopic model of human HCC. Intraperitoneal injection of ASC resulted in a significant suppression of tumor progression and reduction in the expression of p‐STAT3 in ASC‐treated tumor tissues. Overall, our results clearly establish that ASC as a novel blocker of STAT3 activation in HCC.
We report for the first time that ASC could substantially suppress STAT3 activation in HCC cells in a dose and time‐dependent manner. Prior studies have indicated that STAT3 can be phosphorylated at both Ser 727 and Tyr 705 residues (Subramaniam et al., 2013b). However, STAT3 dimerization, translocation to the nucleus, and binding to DNA are primarily mediated through the phosphorylation at critical tyrosine site (Tyr705) (Bromberg and Darnell, 2000; Svinka et al., 2014). In this study, we observed that ASC could specifically inhibit STAT3 phosphorylation at Tyr 705 residue without affecting its activation at Ser 727 site, and this abrogation further decreased STAT3 nuclear translocation, DNA binding and transcriptional activities. IL‐6 is a major cytokine that has been reported to promote HCC development through activating STAT3 signaling cascade (He and Karin, 2011). Furthermore, we found that STAT3 activation induced by IL‐6 treatment was also prevented by ASC as early as 1 h following drug exposure in Hep3B cells. A prior study indicated that ASC could downregulate the expression of c‐Myc, which could be rapidly activated by STAT3 upon stimulation of the interleukin IL‐6 receptor or gp130 (Jeong and Chang, 2010; Kiuchi et al., 1999). Thus the reported inhibition of c‐Myc protein could be possibly explained by the suppressive effects of ASC on STAT3 phosphorylation as observed by us in the present report.
The underlying molecular mechanism(s) through which ASC inhibits STAT3 activation was also investigated in detail. STAT3 phosphorylation can be tightly controlled by growth factor receptor and non‐receptor tyrosine kinase‐induced signaling cascade (Levy and Darnell, 2002). In addition, STAT3 can directly interact with JAK1 and JAK2 kinases as scaffold, and this interaction leads to STAT3 phosphorylation at Tyr 705 (Hodge et al., 2005). We noted that ASC treatment caused substantial downregulation of both constitutive and inducible JAK1 and JAK2 phosphorylation levels in a time‐dependent manner that directly correlated with its observed STAT3 inhibitory effects in HCC cells. This is in agreement at least in part with a prior report that AZD1480, a JAK2 inhibitor, can signaling suppress STAT3 signaling and oncogenesis in different solid tumors (Hedvat et al., 2009). Besides causing abrogation of JAK1 and JAK2 activation, ASC also inhibited c‐Src phosphorylation which has also been implicated in STAT3 activation in tumor cells. Thus it is possible that ASC may directly abrogate the cooperation of Src and JAK1/2 proteins involved in tyrosine phosphorylation of STAT3. We also observed that ASC significantly inhibited EGF induced STAT3 reporter activity in HCC cells. This is also in agreement with a previous study in which ASC was reported to inhibit growth factor‐induced HIF‐1α activation and tumor‐angiogenesis through the suppression of EGFR/ERK/p70S6K signaling pathway in human cervical carcinoma cells (Jeong et al., 2012).
We also report for the first time that the ASC‐induced inhibition of STAT3 activation directly correlated with the induction of PIAS3 expression in HCC cells. Prior studies have reported that negative regulators of STAT3 signaling cascade such as PIAS3, which was originally identified as a specific inhibitor of STAT3 activation could significantly block the DNA binding activity of this transcription factor and also decrease dimer‐dependent gene transcription (Shuai, 2000). Moreover, loss of PIAS3 may contribute to the activation of STAT3 protein in various cancers and PIAS3 indeed has been shown to be inactive in diverse human tumors, including lymphoma (Zhang et al., 2002) and gastric carcinoma (Liu et al., 2011). In this report, we found that the ASC‐induced the expression of PIAS3 protein and mRNA in HCC cells, which correlated, with its ability to negatively regulate STAT3 phosphorylation. Transfection with PIAS3 siRNA reversed the STAT3 inhibitory effects of this antibiotic, thereby clearly implicating an important role of this protein in ASC‐induced downregulation of STAT3 activation. Interestingly, it has been previously reported that other important anticancer agents such as curcumin (Saydmohammed et al., 2010) and 8‐hydrocalamenene (Nam et al., 2014) can also exhibit tumor inhibition through enhancing PIAS3 expression in tumor cells. Thus, targeted induction of PIAS3 protein can also form a basis of important strategy to downmodulate deregulated STAT3 activation in tumor cells.
Our results also indicate that the expression of several STAT3‐regulated gene products including proliferative (cyclin D1), anti‐apoptotic (Bcl‐2, XIAP, Mcl‐1 and survivin), pro‐apoptotic (Bak and Bid) and invasive (MMP‐9) was modulated upon ASC treatment in HCC cells. The inhibition of cyclin D1 expression may account for its ability to induce G1 arrest in U2OS cells as reported recently (Jeong and Chang, 2010). Previous studies have already shown that constitutively active STAT3 can act as a marker of oncogenic transformation by preventing cancer cells from undergoing apoptosis (Bowman et al., 2000; Catlett‐Falcone et al., 1999; Siveen et al., 2014). Thus, the down‐regulation of the expression of Bcl‐2, XIAP, Mcl‐1 and survivin as well as activation of Bid is likely linked with the ability of ASC to induce apoptosis in HCC cells, as evident by activation of caspase‐8, ‐9, and ‐3 and cleavage of PARP. This is in contrast to a recent study in which a synthetic derivative of ASC, 4‐O‐carboxymethyl ascochlorin (AS‐6) was found to cause ER stress and induce autophagy in HCC cells (Kang et al., 2012). The downmodulation of MMP‐9 expression as shown here may also explain the anti‐invasive potential of ASC that needs further investigation.
The relevance of these interesting in vitro observations with ASC was also investigated under in vivo settings. Our data clearly shows that ASC significantly suppressed HCC growth in orthotopic mouse model, downregulated the expression of phospho‐STAT3, Ki‐67, as well as CD31 and increased the levels of cleaved caspase‐3 in treated group as compared to control group. To the best of our knowledge, no prior studies with ASC has been reported in tumor bearing mouse models so far, and our overall findings suggest that ASC has an enormous potential for the treatment of HCC through the modulation of STAT3 signaling cascade. Thus, overall, our in vitro and in vivo experimental findings clearly provide novel mechanistic insights into the molecular actions of ASC and provide a strong rationale to pursue the use of this isoprenoid antibiotic to improve the treatment outcome of HCC patients.
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This research work was supported by grants from Singapore Ministry of Health's National Medical Research Council to GS under its Individual research grants funding scheme. KMH was supported by grant from the National Medical Research Council of Singapore, Biomedical Research Council of Singapore, and the Singapore Millennium Foundation. KSA was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean Ministry of Education, Science and Technology (MoEST) (No. 2011‐0006220). This work was also supported by grants from the Singapore Ministry of Education Tier 2 [MOE2012‐T2‐2‐139], NUHS Bench‐to‐Bedside‐To‐Product [R‐184‐000‐243‐515] and Cancer Science Institute of Singapore, Experimental Therapeutics I Program [R‐713‐001‐011‐271] to APK.
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2014.12.008.
Dai Xiaoyun, Ahn Kwang Seok, Kim Chulwon, Siveen Kodappully Sivaraman, Ong Tina H., Shanmugam Muthu K., Li Feng, Shi Jizhong, Kumar Alan Prem, Wang Ling Zhi, Goh Boon Cher, Magae Junji, Hui Kam M., Sethi Gautam, (2015), Ascochlorin, an isoprenoid antibiotic inhibits growth and invasion of hepatocellular carcinoma by targeting STAT3 signaling cascade through the induction of PIAS3, Molecular Oncology 9, doi: 10.1016/j.molonc.2014.12.008.