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STAT3 signaling
Pharmacological Reports 67 (2015) 388–393
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Matrine suppresses proliferation and induces apoptosis in human cholangiocarcinoma cells through suppression of JAK2/STAT3 signaling Nanmu Yang a, Feng Han a, Hong Cui a,*, Jinxi Huang a, Tao Wang b, Yi Zhou a, Jinxue Zhou a a b
Department of Hepatopancreatobiliary Surgery, The Affiliated Tumor Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China Department of Anesthesia, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China
A R T I C L E I N F O
Article history: Received 19 August 2014 Received in revised form 21 October 2014 Accepted 21 October 2014 Available online 5 November 2014 Keywords: Apoptosis Mcl-1 Phytochemical STAT3 signaling Therapy
A B S T R A C T
Background: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling contributes to apoptosis resistance in cholangiocarcinoma. The aim of this study is to check whether matrine, an alkaloid isolated from traditional Chinese herb Sophora flavescens ait, can exert cytotoxic effects against cholangiocarcinoma cells via inactivation of STAT3 signaling. Methods: Mz-ChA-1 and KMCH-1 cholangiocarcinoma cells were treated with matrine at 0.25–2.0 g/L for 48 h and cell viability and apoptosis were assessed. Apoptosis-related molecular changes and STAT3 phosphorylation and transcriptional activities were measured after matrine treatment for 48 h. The effect of expression of a constitutively active STAT3 mutant on matrine-induced apoptosis was determined. Results: Matrine significantly inhibited the viability and induced apoptosis in cholangiocarcinoma cells. Matrine treatment caused loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-9 and -3. Matrine-induced apoptosis was inhibited in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Matrine reduced the phosphorylation levels of Janus kinase 2 (JAK2) and STAT3, inhibited STAT3-dependent transcriptional activity, and downregulated STAT3 target gene Mcl-1. Notably, expression of the constitutively active form of STAT3 significantly antagonized matrine-induced apoptosis of cholangiocarcinoma cells. Conclusion: Matrine can trigger mitochondrial apoptotic death of cholangiocarcinoma cells largely through inhibition of JAK2/STAT3 signaling. Therefore, matrine represents a potentially effective anticancer agent for cholangiocarcinoma. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Cholangiocarcinoma is a highly aggressive malignancy that arises in the biliary tract. It represents the second most common primary hepatobiliary cancer, with an extremely poor prognosis [1]. Because of the nonspecific presentation of early disease, cholangiocarcinoma is often diagnosed at an advanced stage. Most
Abbreviations: Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-aldehyde; EGCG, epigallocatechin-3-gallate; IL-6, interleukin-6; JAK, Janus kinase; JC-1, 5,50 ,6,60 tetrachloro-1,10 ,3,30 -tetraethylbenzimi-dazolylcarbocyanine iodide; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; STAT3, signal transducer and activator of transcription 3. * Corresponding author. E-mail address: [email protected] (H. Cui).
of patients with advanced cholangiocarcinoma are ineligible for potentially curative surgery [2]. Systemic chemotherapy (e.g. gemcitabine plus cisplatin) has been shown to offer survival benefits in unresectable cholangiocarcinoma patients [3]. However, the efficacy of current systemic therapies is still unsatisfactory, in part due to low overall response rates, unwanted toxicities, and development of drug resistance [1]. Therefore, new therapeutic agents for advanced cholangiocarcinoma are urgently needed. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that regulates transcription of genes involved in diverse cellular activities such as proliferation, differentiation, apoptosis, and tumorigenesis [4]. Constitutive activation of STAT3 occurs in many cancers and promotes tumor growth, survival and progression [5]. In cholangiocarcinoma cells, STAT3 is activated in response to interleukin-6 (IL-6) and upregulates myeloid cell leukemia-1 (Mcl-1) expression, resulting
http://dx.doi.org/10.1016/j.pharep.2014.10.016 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
N. Yang et al. / Pharmacological Reports 67 (2015) 388–393
in apoptosis resistance [6]. STAT3 is frequently activated by members the Janus kinase (JAK) family of non-receptor tyrosine kinases [7], which phosphorylate STAT3 at a single C-terminal Tyr residue (i.e., Y705). After the phosphorylated activation, STAT3 undergoes homodimerization and nuclear translocation where it governs the expression of a variety of genes [8]. The JAK2/STAT3 pathway is implicated in tumor survival and chemoresistance and represents a potential target for cancer treatment [5]. Matrine is an alkaloid isolated from traditional Chinese herb Sophora flavescens ait. It possesses many pharmacological and biological activities, such as anti-inflammatory [9], antiviral [10], and anticancer [11] activities. Matrine has been shown to induce apoptosis in a variety of cancer cells [12,13]. Generally, there are two main apoptotic pathways (i.e., the death receptor pathway and the mitochondrial pathway), which converge on activation of caspases, a family of aspartate-specific cysteine proteinases [14]. In the mitochondrial apoptotic pathway, there is an increase in mitochondrial membrane permeability and loss of mitochondrial membrane potential (Dcm), which leads to the release of mitochondrial cytochrome c and consequent activation of procaspase-9 and downstream executioner caspases such as caspase-3. A recent study has demonstrated that matrine can induce apoptosis in human acute myeloid leukemia cells via the mitochondrial pathway [12]. Similarly, matrine has been shown to trigger caspase-dependent apoptosis in human osteosarcoma cells [15]. However, the potential therapeutic activities of matrine and associated molecular mechanisms have not yet been explored in cholangiocarcinoma. Matrine has shown inhibitory effects on STAT3 signaling in distinct biological settings [16,17]. These findings led us to hypothesize that matrine might exert cytotoxic effects against cholangiocarcinoma cells via interfering with activation of STAT3 signaling. To this end, here we examined the effects of matrine on human cholangiocarcinoma cell viability and apoptosis and checked the involvement of JAK2/STAT3 signaling in the action of matrine.
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Rabbit anti-cytochrome c polyclonal antibody (#4272) was obtained from Cell Signaling Technology (Beverly, MA, USA), rabbit anti-phospho-STAT3 (Tyr705) monoclonal antibody (ab76315) and rabbit anti-STAT3 monoclonal antibody (ab32500) from Abcam (Cambridge, MA, USA), goat anti-phospho-JAK2 (Tyr1007/1008) polyclonal antibody (sc-21870), goat anti-JAK2 polyclonal antibody (sc-34479), mouse anti-Mcl-1 monoclonal antibody (sc-56152), and rabbit anti-b-tubulin polyclonal antibody (sc-9104) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), mouse anti-mitochondrial heat shock protein 70 (mtHSP70) monoclonal antibody (MA3-028) from Thermo Scientific (Rockford, IL, USA), and mouse anti-Flag monoclonal antibody from Sigma–Aldrich. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Cell culture and drug treatment Two human cholangiocarcinoma cell lines (KMCH-1 and MzChA-1) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). They were maintained in DMEM supplement with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 8C in a 5% CO2 humidified incubator. For cytotoxic assay, cells at a density of 4 103 cells/well in 96well plates were treated with matrine at 0.25–2.0 g/L for 48 h and cell viability was assessed using the MTT assay. For inhibitor assay, cells were pretreated with Ac-DEVD-CHO (50 mM) for 1 h prior to exposure to matrine. Transfection of constitutively active STAT3 mutant At 70% confluence, cells were transiently transfected with pRC/CMV-STAT3C-Flag plasmid or empty vector (0.1 mg) using Lipofectamine 2000, according to the manufacturer’s instructions. After incubation for 24 h, the transfected cells were treated with matrine for 48 h. The cells were then harvested and subjected to apoptosis analysis.
Materials and methods
Cytotoxic assay
Reagents and antibodies
Cytotoxic assay was performed using the MTT method. Briefly, after drug treatment for 48 h, the MTT solution (5 mg/mL) was added to the cell cultures and incubated at 37 8C for 4 h. After removal of MTT, dimethyl sulfoxide (DMSO) solution was added to dissolve formazan crystals. Absorbance was measured at a wavelength of 570 nm. Cell viability was expressed as a percentage of control without matrine treatment.
Matrine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (AcDEVD-CHO) was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The STAT3 reporter construct (pLuc-TK/STAT3) was prepared as described previously [18], which harbors seven copies of an annealed oligonucleotide corresponding to a STAT3-specific binding site from the human C-reactive protein gene [19]. The Renilla luciferase reporter pRL-TK and the Dual Luciferase Reporter Assay System were purchased from Promega (Madison, WI, USA). The pRC/CMV-STAT3C-Flag plasmid (#8722), which expresses a constitutively activated STAT3 mutant, was obtained from AddGene (Cambridge, MA, USA). Annexin V Apoptosis Kit was purchased from Becton Dickinson Biosciences (San Diego, CA, USA), JC-1 Mitochondrial Membrane Potential Assay Kit from Biotium (Hayward, CA, USA), Mitochondria/Cytosol Fractionation Kit from BioVision (Mountain View, CA, USA), and Enhanced Chemiluminescence Detection Kit from Amersham Biosciences (Piscataway, NJ, USA). Caspase-3 Activity Kit and Caspase-9 Activity Kit were purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China).
Apoptosis analysis Cell apoptosis was detected using the annexin-V/propidium iodide (PI) assay. Briefly, after drug treatment, cells were collected, pelleted, and resuspended in annexin-V binding buffer. The cell suspension was incubated with fluorescein isothiocyanateconjugated annexin-V and PI for 20 min in the dark. The stained cells were analyzed by flow cytometry. Mitochondrial membrane potential assay Mitochondrial membrane potential (Dwm) was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit. The cyanine dye JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) accumulates in the mitochondria in a potential-dependent manner, which is accompanied by the shift from green to red fluorescence emission. After matrine treatment, cells were stained with 10 mM JC-1 at 37 8C for 15 min in the dark. Cells were washed and subjected to flow cytometric analysis. The
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ratio of the red/green fluorescence was calculated and presented in arbitrary units. A decrease in this ratio reflects loss of Dwm. Subcellular fractionation and Western blot analysis To determine the release of cytochrome c from mitochondria, subcellular fractions were extracted using the mitochondrial/ cytosol fractionation kit. Briefly, after matrine treatment, cells were resuspended in the cytosol extraction buffer containing protease inhibitors and homogenized on ice. After centrifugation at 700 g for 10 min, the supernatant was collected and further centrifuged at 10,000 g for 30 min at 4 8C. Then the supernatant was collected as the cytosolic fraction. The pellet was lysed in the mitochondrial extraction buffer mix containing protease inhibitors and kept as the mitochondrial fraction. Changes in mitochondrial and cytosolic cytochrome c levels were examined using Western blot analysis, as described below. Briefly, protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After blocking nonspecific sites, the membrane was incubated overnight at 4 8C with the primary antibodies. After incubation with HRP-conjugated secondary antibodies for 1 h, the immune complexes were detected by the enhanced chemiluminescence detection kit. Densitometric analysis of protein expression was performed using the Quantity One program (Bio-Rad, Hercules, CA, USA). Assessment of caspase activation The activities of caspase-3 and caspase-9 were determined by the colorimetric method. In brief, after treatment, cells were pelleted, washed and incubated in lysis buffer on ice for 10 min. The lysates were then incubated with the reaction buffer containing caspase-3 substrate (acetyl-Asp-Glu-Val-Asp-p-nitroanilide) or caspase-9 substrate (acetyl-Leu-Glu-His-Asp-p-nitroanilide) at 37 8C for 4 h. Caspase activities were measured by spectrofluorometry. Luciferase reporter assay Cells (2 104 cells/well in 24-well plates) were transiently transfected with 0.2 mg pLuc-TK/STAT3 and 0.04 mg pRL-TK as a transfection efficiency control. At 12 h after transfection, cells were treated with matrine for 48 h. Cells were washed and lysed according
Fig. 2. Matrine induces mitochondrial apoptosis in cholangiocarcinoma cells. MzChA-1 and KMCH-1 cells were treated with matrine at the IC50 concentration for 48 h and apoptosis-related cellular and molecular changes were examined. (A) Detection of apoptosis by flow cytometry analysis. Representative dot plots of annexin-V and PI staining are shown. (B) Bar graphs depict mean percentages of annexin-V-positive apoptotic cells from three independent experiments. Error bars represent SD. *p < 0.05 vs. control. (C) Loss of Dwm was determined by flow cytometry using JC-1 staining. The ratio of the red/green fluorescence was calculated and results were expressed as percentage of control values (assigned 100%). *p < 0.05 vs. control. (D) Western blot analysis of cytochrome c (Cyto c) protein levels in cytosol and mitochondrial fractions of cells treated with or without matrine. Representative blots from three independent experiments are shown. b-Tubulin and mtHSP70 were used as cytoplasmic and mitochondrial loading controls, respectively. Fig. 1. Cytotoxic effects of matrine on human cholangiocarcinoma cells. (A) Chemical structure of matrine. (B) KMCH-1 and Mz-ChA-1 cells were treated with different concentrations of matrine for 48 h and cell viability was assessed using the MTT assay. The viability of untreated cells (control) was arbitrarily assigned 100%. *p < 0.05 vs. control.
to the dual luciferase assay protocol. The luciferase activity in the cytosolic supernatant was measured on a luminometer. Relative luciferase activity was determined by normalization of Firefly luciferase activity to Renilla luciferase activity.
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Statistical analysis Data are presented as means standard deviation (SD). The difference among the means of multiple groups was analyzed by one-way analysis of variance (ANOVA) followed by the Tukey test. A p value of <0.05 was considered as statistically significant. Results Cytotoxic effects of matrine on human cholangiocarcinoma cells
Fig. 3. Matrine induces caspase-3-dependent apoptosis in cholangiocarcinoma cells. (A) Cells were treated with or without matrine at the IC50 concentration for 48 h and the activities of caspase-9 and -3 were measured. The relative caspase activity is expressed as fold change compared to untreated cells (assigned 1). * p < 0.05 vs. untreated cells. (B) Cells were pretreated with Ac-DEVD-CHO (50 mM) for 1 h prior to matrine treatment. After incubation for 48 h, cell apoptosis was measured using the annexin V/PI staining assay. *p < 0.05 vs. control; #p < 0.05 vs. matrine treatment.
KMCH-1 and Mz-ChA-1 cells were exposed to matrine at different concentrations and the cytotoxicity of matrine was evaluated after 48 h. Compared to control cells, matrine treatment caused a significant (p < 0.05) and dose-dependent reduction in cell viability (Fig. 1B). The IC50 (concentration required to reduce viability by 50%) after exposure to matrine for 48 h was approximately 1.0 and 0.5 g/L in Mz-ChA-1 and KMCH-1 cells, respectively. In the following experiments, cholangiocarcinoma cells were treated with the IC50 concentration of matrine. Matrine induces apoptosis of cholangiocarcinoma cells via the mitochondrial death pathway Next, we examined the effect of matrine on apoptosis of cholangiocarcinoma cells. As shown in Fig. 2A and B, matrine treatment caused significant apoptosis in both Mz-ChA-1 and KMCH-1 cells, with a 5- and 8-fold increase in apoptosis vs. corresponding control, respectively. The induction of apoptotic
Fig. 4. Matrine suppresses JAK2/STAT3 signaling in cholangiocarcinoma cells. (A) Western blot analysis of total and phosphorylated JAK2 and STAT3 in cholangiocarcinoma cells treated with or without matrine. Representative blots are shown in left panels. Bar graphs (right panels) represent densitometric analysis of three independent experiments. (B) Cells were transfected with pLuc-TK/STAT3 and pRL-TK 12 h before the treatment with matrine for additional 48 h. Dual luciferase assays were performed. The results were expressed as relative luciferase activity compared to control cells arbitrarily assigned as 1. (C) Western blot analysis of Mcl-1 protein in cholangiocarcinoma cells treated with or without matrine. Representative blots are shown in left panels. Bar graphs (right panels) represent densitometric analysis of three independent experiments. *p < 0.05 vs. untreated cells.
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cells, the expression vector encoding a constitutively activated mutant of STAT3 or empty control vector was transfected into cholangiocarcinoma cells. Transfected cells were confirmed by Western blot analysis (Fig. 5A). Expression of constitutively active STAT3 had no significant effect on cholangiocarcinoma cell survival (Fig. 5B). Notably, matrine-induced apoptosis of cholangiocarcinoma cells was significantly antagonized by the delivery of constitutively active STAT3, compared to transfection with empty vector (Fig. 5B). Discussion
Fig. 5. Restoration of STAT3 signaling impairs the proapoptotic activity of matrine in cholangiocarcinoma cells. (A) Cells were transfected with constitutively active STAT3 plasmid or empty vector and Western blot analysis of STAT3-Flag protein with antiFlag antibody. Representative blots of three independent experiments with similar results are shown. (B) Annexin-V/PI staining analysis of apoptotic death. Cells were transfected with constitutively active STAT3 plasmid or empty vector with or without subsequent matrine treatment. *p < 0.05 vs. untreated control; #p < 0.05 vs. transfection with STAT3-expressing plasmid before matrine treatment.
cell death was associated with loss of Dcm (Fig. 2C) and release of cytochrome c from mitochondria to the cytosol (Fig. 2D). Moreover, caspase-9 and -3 activities were significantly elevated in matrine-treated KMCH-1 and Mz-ChA-1 cells, as compared to corresponding control (Fig. 3A). To confirm the requirement of caspase-3 activation for matrine-induced apoptosis, cells were pretreated with Ac-DEVD-CHO before matrine exposure. Apoptosis analysis revealed that Ac-DEVD-CHO pretreatment significantly impaired apoptotic cell death induced by matrine (Fig. 3B). Matrine inhibits JAK2/STAT3 signaling in cholangiocarcinoma cells Given that STAT3 signaling is constitutively activated in cholangiocarcinoma and contributes to cell survival [6], we next checked whether matrine-induced cell death of cholangiocarcinoma cells was associated with STAT3 inactivation. As shown in Fig. 4A, matrine treatment caused a significant reduction in the level of phosphorylated STAT3 (Tyr705) relative to control cells. Moreover, the expression level of phosphorylated JAK2 (Tyr1007/ 1008) was significantly lowered by matrine treatment (Fig. 4A). Luciferase reporter assay demonstrated that constitutive STAT3 transcription activity was blocked by matrine-treated cholangiocarcinoma cells (Fig. 4B). Consistent with inactivation of STAT3, the expression of Mcl-1, an anti-apoptotic target gene of STAT3, was decreased by matrine treatment (Fig. 4C). Restoration of STAT3 signaling antagonizes the proapoptotic activity of matrine in cholangiocarcinoma cells To further explore whether STAT3 activity directly influences the proapoptotic activity of matrine in cholangiocarcinoma
Naturally occurring phytochemicals are an important source of anticancer agents [20]. The dietary phytochemicals quercetin and epigallocatechin-3-gallate (EGCG) have been shown to inhibit the growth of cholangiocarcinoma cells [21]. Matrine shows anticancer activities in a number of malignancies such as melanoma [11] and pancreatic cancer [13]. Our data revealed that matrine treatment for 48 h caused a dose-dependent inhibition of the viability of cholangiocarcinoma cells. Annexin-V staining analysis further demonstrated that matrine significantly induced apoptotic death in cholangiocarcinoma cells. During apoptosis the plasma membrane remains intact, which prevents the release of cellular contents and establishment of cancer-prone inflammation niche [22]. Therefore, induction of apoptosis is a desired response to anticancer treatment. In agreement with our findings, matrine also triggers apoptotic death in hepatocellular carcinoma cells [23]. To the best of our knowledge, this is the first report showing that matrine exerts anticancer effects against cholangiocarcinoma cells via induction of apoptosis. Our data further showed that matrine treatment resulted in loss of Dcm, release of mitochondrial cytochrome c, as well as activation of both caspase-9 and -3 in cholangiocarcinoma cells. These findings suggest that the proapoptotic activity of matrine in cholangiocarcinoma is mediated by activation of the mitochondrial death cascade. Caspase-3 is a key effector caspase in the apoptosis pathway [24]. Pharmacological inhibition of caspase-3 activity was found to block the apoptosis induction by matrine, confirming that matrine induces caspase-3-dependent apoptosis in cholangiocarcinoma cells. The activation of caspase-3-dependent mitochondrial death pathway by matrine has also been described in several other types of cancers [12,25]. JAK/STAT3 signaling is constitutively activated in cholangiocarcinoma and represents an important target for treatment of this disease [6]. Inhibition of STAT3 signaling by sorafenib, a multikinase inhibitor was found to sensitize cholangiocarcinoma cells to tumor necrosis factor-related apoptosis-inducing ligandmediated apoptosis and suppress cholangiocarcinoma tumor growth [26]. Inactivation of JAK/STAT3 signaling was shown to mediate the chemopreventive effects of quercetin and EGCG in cholangiocarcinoma cells [21]. Similarly, we found that matrine significantly decreased the phosphorylation levels of STAT3 and JAK2 and STAT3-dependent transcriptional activity in cholangiocarcinoma cells. Matrine-mediated inactivation of JAK2/STAT3 provides a mechanistic explanation for its cytotoxicity in cholangiocarcinoma cells. To confirm the mediating role of STAT3 signaling, we transfected the constitutively active STAT3 mutant into cholangiocarcinoma cells before exposure to matrine. The results showed that restoration of STAT3 activation antagonizes the proapoptotic activity of matrine in cholangiocarcinoma cells. Mcl-1 is a well-established antiapoptotic target gene of STAT3 [6]. We found that matrine treatment significantly reduced the expression of Mcl-1 in cholangiocarcinoma cells. Mcl-1 reduction has been found to sensitize cholangiocarcinoma cells to apoptotic stimuli [27]. It has been documented that short hairpin RNA-mediated depletion of Mcl-1 enhanced the induction of
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cholangiocarcinoma cell apoptosis by the multikinase inhibitor BAY 43-9006 [27]. Triptolide, a bioactive ingredient extracted from the Chinese herb Tripterygium wolfordii Hook F, has been shown to cause apoptotic cell death of human cholangiocarcinoma cells through inhibition of Mcl-1 [28]. These studies indicate the important role for Mcl-1 in apoptosis resistance of cholangiocarcinoma cells. Our results collectively suggest that matrine triggers apoptotic death in cholangiocarcinoma cells via suppression of JAK2/STAT3 signaling and downregulation of Mcl-1. In conclusion, we provide first evidence that matrine is capable of inducing mitochondrial apoptotic death of cholangiocarcinoma cells, most likely through inactivation of the JAK2/STAT3 pathway and downregulation of the antiapoptotic protein Mcl-1. Therefore, matrine may represent a novel anticancer agent for cholangiocarcinoma, especially with constitutive activation of JAK2/STAT3 signaling. Conflict of interest The authors declared no conflict of interest. Funding None. References [1] Ramı´rez-Merino N, Aix SP, Corte´s-Funes H. Chemotherapy for cholangiocarcinoma: an update. World J Gastrointest Oncol 2013;5(7):171–6. [2] Brown KM, Parmar AD, Geller DA. Intrahepatic cholangiocarcinoma. Surg Oncol Clin N Am 2014;23(2):231–46. [3] Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med 2010;362(14):1273–81. [4] Junk DJ, Bryson BL, Jackson MW. HiJAK’d signaling; the STAT3 paradox in senescence and cancer progression. Cancers (Basel) 2014;6(2):741–55. [5] Xiong A, Yang Z, Shen Y, Zhou J, Shen Q. Transcription factor STAT3 as a novel molecular target for cancer prevention. Cancers (Basel) 2014;6(2):926–57. [6] Isomoto H, Kobayashi S, Werneburg NW, Bronk SF, Guicciardi ME, Frank DA, et al. Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells. Hepatology 2005;42(6): 1329–38. [7] Bournazou E, Bromberg J. Targeting the tumor microenvironment: JAK-STAT3 signaling. JAKSTAT 2013;2(2):e23828. [8] Darnell Jr JE, Kerr IM, Stark GR. JAK-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264(5164):1415–21. [9] Shi D, Zhang J, Qiu L, Li J, Hu Z, Zhang J. Matrine inhibits infiltration of the inflammatory Gr1(hi) monocyte subset in injured mouse liver through inhibition of monocyte chemoattractant protein-1. Evid Based Complement Alternat Med 2013;2013:580673.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Matrine suppresses proliferation and induces apoptosis in human cholangiocarcinoma cells through suppression of JAK2/STAT3 signaling Nanmu Yang a, Feng Han a, Hong Cui a,*, Jinxi Huang a, Tao Wang b, Yi Zhou a, Jinxue Zhou a a b
Department of Hepatopancreatobiliary Surgery, The Affiliated Tumor Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China Department of Anesthesia, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China
A R T I C L E I N F O
Article history: Received 19 August 2014 Received in revised form 21 October 2014 Accepted 21 October 2014 Available online 5 November 2014 Keywords: Apoptosis Mcl-1 Phytochemical STAT3 signaling Therapy
A B S T R A C T
Background: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling contributes to apoptosis resistance in cholangiocarcinoma. The aim of this study is to check whether matrine, an alkaloid isolated from traditional Chinese herb Sophora flavescens ait, can exert cytotoxic effects against cholangiocarcinoma cells via inactivation of STAT3 signaling. Methods: Mz-ChA-1 and KMCH-1 cholangiocarcinoma cells were treated with matrine at 0.25–2.0 g/L for 48 h and cell viability and apoptosis were assessed. Apoptosis-related molecular changes and STAT3 phosphorylation and transcriptional activities were measured after matrine treatment for 48 h. The effect of expression of a constitutively active STAT3 mutant on matrine-induced apoptosis was determined. Results: Matrine significantly inhibited the viability and induced apoptosis in cholangiocarcinoma cells. Matrine treatment caused loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-9 and -3. Matrine-induced apoptosis was inhibited in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Matrine reduced the phosphorylation levels of Janus kinase 2 (JAK2) and STAT3, inhibited STAT3-dependent transcriptional activity, and downregulated STAT3 target gene Mcl-1. Notably, expression of the constitutively active form of STAT3 significantly antagonized matrine-induced apoptosis of cholangiocarcinoma cells. Conclusion: Matrine can trigger mitochondrial apoptotic death of cholangiocarcinoma cells largely through inhibition of JAK2/STAT3 signaling. Therefore, matrine represents a potentially effective anticancer agent for cholangiocarcinoma. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Cholangiocarcinoma is a highly aggressive malignancy that arises in the biliary tract. It represents the second most common primary hepatobiliary cancer, with an extremely poor prognosis [1]. Because of the nonspecific presentation of early disease, cholangiocarcinoma is often diagnosed at an advanced stage. Most
Abbreviations: Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-aldehyde; EGCG, epigallocatechin-3-gallate; IL-6, interleukin-6; JAK, Janus kinase; JC-1, 5,50 ,6,60 tetrachloro-1,10 ,3,30 -tetraethylbenzimi-dazolylcarbocyanine iodide; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; STAT3, signal transducer and activator of transcription 3. * Corresponding author. E-mail address: [email protected] (H. Cui).
of patients with advanced cholangiocarcinoma are ineligible for potentially curative surgery [2]. Systemic chemotherapy (e.g. gemcitabine plus cisplatin) has been shown to offer survival benefits in unresectable cholangiocarcinoma patients [3]. However, the efficacy of current systemic therapies is still unsatisfactory, in part due to low overall response rates, unwanted toxicities, and development of drug resistance [1]. Therefore, new therapeutic agents for advanced cholangiocarcinoma are urgently needed. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that regulates transcription of genes involved in diverse cellular activities such as proliferation, differentiation, apoptosis, and tumorigenesis [4]. Constitutive activation of STAT3 occurs in many cancers and promotes tumor growth, survival and progression [5]. In cholangiocarcinoma cells, STAT3 is activated in response to interleukin-6 (IL-6) and upregulates myeloid cell leukemia-1 (Mcl-1) expression, resulting
http://dx.doi.org/10.1016/j.pharep.2014.10.016 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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in apoptosis resistance [6]. STAT3 is frequently activated by members the Janus kinase (JAK) family of non-receptor tyrosine kinases [7], which phosphorylate STAT3 at a single C-terminal Tyr residue (i.e., Y705). After the phosphorylated activation, STAT3 undergoes homodimerization and nuclear translocation where it governs the expression of a variety of genes [8]. The JAK2/STAT3 pathway is implicated in tumor survival and chemoresistance and represents a potential target for cancer treatment [5]. Matrine is an alkaloid isolated from traditional Chinese herb Sophora flavescens ait. It possesses many pharmacological and biological activities, such as anti-inflammatory [9], antiviral [10], and anticancer [11] activities. Matrine has been shown to induce apoptosis in a variety of cancer cells [12,13]. Generally, there are two main apoptotic pathways (i.e., the death receptor pathway and the mitochondrial pathway), which converge on activation of caspases, a family of aspartate-specific cysteine proteinases [14]. In the mitochondrial apoptotic pathway, there is an increase in mitochondrial membrane permeability and loss of mitochondrial membrane potential (Dcm), which leads to the release of mitochondrial cytochrome c and consequent activation of procaspase-9 and downstream executioner caspases such as caspase-3. A recent study has demonstrated that matrine can induce apoptosis in human acute myeloid leukemia cells via the mitochondrial pathway [12]. Similarly, matrine has been shown to trigger caspase-dependent apoptosis in human osteosarcoma cells [15]. However, the potential therapeutic activities of matrine and associated molecular mechanisms have not yet been explored in cholangiocarcinoma. Matrine has shown inhibitory effects on STAT3 signaling in distinct biological settings [16,17]. These findings led us to hypothesize that matrine might exert cytotoxic effects against cholangiocarcinoma cells via interfering with activation of STAT3 signaling. To this end, here we examined the effects of matrine on human cholangiocarcinoma cell viability and apoptosis and checked the involvement of JAK2/STAT3 signaling in the action of matrine.
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Rabbit anti-cytochrome c polyclonal antibody (#4272) was obtained from Cell Signaling Technology (Beverly, MA, USA), rabbit anti-phospho-STAT3 (Tyr705) monoclonal antibody (ab76315) and rabbit anti-STAT3 monoclonal antibody (ab32500) from Abcam (Cambridge, MA, USA), goat anti-phospho-JAK2 (Tyr1007/1008) polyclonal antibody (sc-21870), goat anti-JAK2 polyclonal antibody (sc-34479), mouse anti-Mcl-1 monoclonal antibody (sc-56152), and rabbit anti-b-tubulin polyclonal antibody (sc-9104) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), mouse anti-mitochondrial heat shock protein 70 (mtHSP70) monoclonal antibody (MA3-028) from Thermo Scientific (Rockford, IL, USA), and mouse anti-Flag monoclonal antibody from Sigma–Aldrich. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Cell culture and drug treatment Two human cholangiocarcinoma cell lines (KMCH-1 and MzChA-1) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). They were maintained in DMEM supplement with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 8C in a 5% CO2 humidified incubator. For cytotoxic assay, cells at a density of 4 103 cells/well in 96well plates were treated with matrine at 0.25–2.0 g/L for 48 h and cell viability was assessed using the MTT assay. For inhibitor assay, cells were pretreated with Ac-DEVD-CHO (50 mM) for 1 h prior to exposure to matrine. Transfection of constitutively active STAT3 mutant At 70% confluence, cells were transiently transfected with pRC/CMV-STAT3C-Flag plasmid or empty vector (0.1 mg) using Lipofectamine 2000, according to the manufacturer’s instructions. After incubation for 24 h, the transfected cells were treated with matrine for 48 h. The cells were then harvested and subjected to apoptosis analysis.
Materials and methods
Cytotoxic assay
Reagents and antibodies
Cytotoxic assay was performed using the MTT method. Briefly, after drug treatment for 48 h, the MTT solution (5 mg/mL) was added to the cell cultures and incubated at 37 8C for 4 h. After removal of MTT, dimethyl sulfoxide (DMSO) solution was added to dissolve formazan crystals. Absorbance was measured at a wavelength of 570 nm. Cell viability was expressed as a percentage of control without matrine treatment.
Matrine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (AcDEVD-CHO) was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The STAT3 reporter construct (pLuc-TK/STAT3) was prepared as described previously [18], which harbors seven copies of an annealed oligonucleotide corresponding to a STAT3-specific binding site from the human C-reactive protein gene [19]. The Renilla luciferase reporter pRL-TK and the Dual Luciferase Reporter Assay System were purchased from Promega (Madison, WI, USA). The pRC/CMV-STAT3C-Flag plasmid (#8722), which expresses a constitutively activated STAT3 mutant, was obtained from AddGene (Cambridge, MA, USA). Annexin V Apoptosis Kit was purchased from Becton Dickinson Biosciences (San Diego, CA, USA), JC-1 Mitochondrial Membrane Potential Assay Kit from Biotium (Hayward, CA, USA), Mitochondria/Cytosol Fractionation Kit from BioVision (Mountain View, CA, USA), and Enhanced Chemiluminescence Detection Kit from Amersham Biosciences (Piscataway, NJ, USA). Caspase-3 Activity Kit and Caspase-9 Activity Kit were purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China).
Apoptosis analysis Cell apoptosis was detected using the annexin-V/propidium iodide (PI) assay. Briefly, after drug treatment, cells were collected, pelleted, and resuspended in annexin-V binding buffer. The cell suspension was incubated with fluorescein isothiocyanateconjugated annexin-V and PI for 20 min in the dark. The stained cells were analyzed by flow cytometry. Mitochondrial membrane potential assay Mitochondrial membrane potential (Dwm) was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit. The cyanine dye JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) accumulates in the mitochondria in a potential-dependent manner, which is accompanied by the shift from green to red fluorescence emission. After matrine treatment, cells were stained with 10 mM JC-1 at 37 8C for 15 min in the dark. Cells were washed and subjected to flow cytometric analysis. The
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ratio of the red/green fluorescence was calculated and presented in arbitrary units. A decrease in this ratio reflects loss of Dwm. Subcellular fractionation and Western blot analysis To determine the release of cytochrome c from mitochondria, subcellular fractions were extracted using the mitochondrial/ cytosol fractionation kit. Briefly, after matrine treatment, cells were resuspended in the cytosol extraction buffer containing protease inhibitors and homogenized on ice. After centrifugation at 700 g for 10 min, the supernatant was collected and further centrifuged at 10,000 g for 30 min at 4 8C. Then the supernatant was collected as the cytosolic fraction. The pellet was lysed in the mitochondrial extraction buffer mix containing protease inhibitors and kept as the mitochondrial fraction. Changes in mitochondrial and cytosolic cytochrome c levels were examined using Western blot analysis, as described below. Briefly, protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After blocking nonspecific sites, the membrane was incubated overnight at 4 8C with the primary antibodies. After incubation with HRP-conjugated secondary antibodies for 1 h, the immune complexes were detected by the enhanced chemiluminescence detection kit. Densitometric analysis of protein expression was performed using the Quantity One program (Bio-Rad, Hercules, CA, USA). Assessment of caspase activation The activities of caspase-3 and caspase-9 were determined by the colorimetric method. In brief, after treatment, cells were pelleted, washed and incubated in lysis buffer on ice for 10 min. The lysates were then incubated with the reaction buffer containing caspase-3 substrate (acetyl-Asp-Glu-Val-Asp-p-nitroanilide) or caspase-9 substrate (acetyl-Leu-Glu-His-Asp-p-nitroanilide) at 37 8C for 4 h. Caspase activities were measured by spectrofluorometry. Luciferase reporter assay Cells (2 104 cells/well in 24-well plates) were transiently transfected with 0.2 mg pLuc-TK/STAT3 and 0.04 mg pRL-TK as a transfection efficiency control. At 12 h after transfection, cells were treated with matrine for 48 h. Cells were washed and lysed according
Fig. 2. Matrine induces mitochondrial apoptosis in cholangiocarcinoma cells. MzChA-1 and KMCH-1 cells were treated with matrine at the IC50 concentration for 48 h and apoptosis-related cellular and molecular changes were examined. (A) Detection of apoptosis by flow cytometry analysis. Representative dot plots of annexin-V and PI staining are shown. (B) Bar graphs depict mean percentages of annexin-V-positive apoptotic cells from three independent experiments. Error bars represent SD. *p < 0.05 vs. control. (C) Loss of Dwm was determined by flow cytometry using JC-1 staining. The ratio of the red/green fluorescence was calculated and results were expressed as percentage of control values (assigned 100%). *p < 0.05 vs. control. (D) Western blot analysis of cytochrome c (Cyto c) protein levels in cytosol and mitochondrial fractions of cells treated with or without matrine. Representative blots from three independent experiments are shown. b-Tubulin and mtHSP70 were used as cytoplasmic and mitochondrial loading controls, respectively. Fig. 1. Cytotoxic effects of matrine on human cholangiocarcinoma cells. (A) Chemical structure of matrine. (B) KMCH-1 and Mz-ChA-1 cells were treated with different concentrations of matrine for 48 h and cell viability was assessed using the MTT assay. The viability of untreated cells (control) was arbitrarily assigned 100%. *p < 0.05 vs. control.
to the dual luciferase assay protocol. The luciferase activity in the cytosolic supernatant was measured on a luminometer. Relative luciferase activity was determined by normalization of Firefly luciferase activity to Renilla luciferase activity.
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Statistical analysis Data are presented as means standard deviation (SD). The difference among the means of multiple groups was analyzed by one-way analysis of variance (ANOVA) followed by the Tukey test. A p value of <0.05 was considered as statistically significant. Results Cytotoxic effects of matrine on human cholangiocarcinoma cells
Fig. 3. Matrine induces caspase-3-dependent apoptosis in cholangiocarcinoma cells. (A) Cells were treated with or without matrine at the IC50 concentration for 48 h and the activities of caspase-9 and -3 were measured. The relative caspase activity is expressed as fold change compared to untreated cells (assigned 1). * p < 0.05 vs. untreated cells. (B) Cells were pretreated with Ac-DEVD-CHO (50 mM) for 1 h prior to matrine treatment. After incubation for 48 h, cell apoptosis was measured using the annexin V/PI staining assay. *p < 0.05 vs. control; #p < 0.05 vs. matrine treatment.
KMCH-1 and Mz-ChA-1 cells were exposed to matrine at different concentrations and the cytotoxicity of matrine was evaluated after 48 h. Compared to control cells, matrine treatment caused a significant (p < 0.05) and dose-dependent reduction in cell viability (Fig. 1B). The IC50 (concentration required to reduce viability by 50%) after exposure to matrine for 48 h was approximately 1.0 and 0.5 g/L in Mz-ChA-1 and KMCH-1 cells, respectively. In the following experiments, cholangiocarcinoma cells were treated with the IC50 concentration of matrine. Matrine induces apoptosis of cholangiocarcinoma cells via the mitochondrial death pathway Next, we examined the effect of matrine on apoptosis of cholangiocarcinoma cells. As shown in Fig. 2A and B, matrine treatment caused significant apoptosis in both Mz-ChA-1 and KMCH-1 cells, with a 5- and 8-fold increase in apoptosis vs. corresponding control, respectively. The induction of apoptotic
Fig. 4. Matrine suppresses JAK2/STAT3 signaling in cholangiocarcinoma cells. (A) Western blot analysis of total and phosphorylated JAK2 and STAT3 in cholangiocarcinoma cells treated with or without matrine. Representative blots are shown in left panels. Bar graphs (right panels) represent densitometric analysis of three independent experiments. (B) Cells were transfected with pLuc-TK/STAT3 and pRL-TK 12 h before the treatment with matrine for additional 48 h. Dual luciferase assays were performed. The results were expressed as relative luciferase activity compared to control cells arbitrarily assigned as 1. (C) Western blot analysis of Mcl-1 protein in cholangiocarcinoma cells treated with or without matrine. Representative blots are shown in left panels. Bar graphs (right panels) represent densitometric analysis of three independent experiments. *p < 0.05 vs. untreated cells.
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cells, the expression vector encoding a constitutively activated mutant of STAT3 or empty control vector was transfected into cholangiocarcinoma cells. Transfected cells were confirmed by Western blot analysis (Fig. 5A). Expression of constitutively active STAT3 had no significant effect on cholangiocarcinoma cell survival (Fig. 5B). Notably, matrine-induced apoptosis of cholangiocarcinoma cells was significantly antagonized by the delivery of constitutively active STAT3, compared to transfection with empty vector (Fig. 5B). Discussion
Fig. 5. Restoration of STAT3 signaling impairs the proapoptotic activity of matrine in cholangiocarcinoma cells. (A) Cells were transfected with constitutively active STAT3 plasmid or empty vector and Western blot analysis of STAT3-Flag protein with antiFlag antibody. Representative blots of three independent experiments with similar results are shown. (B) Annexin-V/PI staining analysis of apoptotic death. Cells were transfected with constitutively active STAT3 plasmid or empty vector with or without subsequent matrine treatment. *p < 0.05 vs. untreated control; #p < 0.05 vs. transfection with STAT3-expressing plasmid before matrine treatment.
cell death was associated with loss of Dcm (Fig. 2C) and release of cytochrome c from mitochondria to the cytosol (Fig. 2D). Moreover, caspase-9 and -3 activities were significantly elevated in matrine-treated KMCH-1 and Mz-ChA-1 cells, as compared to corresponding control (Fig. 3A). To confirm the requirement of caspase-3 activation for matrine-induced apoptosis, cells were pretreated with Ac-DEVD-CHO before matrine exposure. Apoptosis analysis revealed that Ac-DEVD-CHO pretreatment significantly impaired apoptotic cell death induced by matrine (Fig. 3B). Matrine inhibits JAK2/STAT3 signaling in cholangiocarcinoma cells Given that STAT3 signaling is constitutively activated in cholangiocarcinoma and contributes to cell survival [6], we next checked whether matrine-induced cell death of cholangiocarcinoma cells was associated with STAT3 inactivation. As shown in Fig. 4A, matrine treatment caused a significant reduction in the level of phosphorylated STAT3 (Tyr705) relative to control cells. Moreover, the expression level of phosphorylated JAK2 (Tyr1007/ 1008) was significantly lowered by matrine treatment (Fig. 4A). Luciferase reporter assay demonstrated that constitutive STAT3 transcription activity was blocked by matrine-treated cholangiocarcinoma cells (Fig. 4B). Consistent with inactivation of STAT3, the expression of Mcl-1, an anti-apoptotic target gene of STAT3, was decreased by matrine treatment (Fig. 4C). Restoration of STAT3 signaling antagonizes the proapoptotic activity of matrine in cholangiocarcinoma cells To further explore whether STAT3 activity directly influences the proapoptotic activity of matrine in cholangiocarcinoma
Naturally occurring phytochemicals are an important source of anticancer agents [20]. The dietary phytochemicals quercetin and epigallocatechin-3-gallate (EGCG) have been shown to inhibit the growth of cholangiocarcinoma cells [21]. Matrine shows anticancer activities in a number of malignancies such as melanoma [11] and pancreatic cancer [13]. Our data revealed that matrine treatment for 48 h caused a dose-dependent inhibition of the viability of cholangiocarcinoma cells. Annexin-V staining analysis further demonstrated that matrine significantly induced apoptotic death in cholangiocarcinoma cells. During apoptosis the plasma membrane remains intact, which prevents the release of cellular contents and establishment of cancer-prone inflammation niche [22]. Therefore, induction of apoptosis is a desired response to anticancer treatment. In agreement with our findings, matrine also triggers apoptotic death in hepatocellular carcinoma cells [23]. To the best of our knowledge, this is the first report showing that matrine exerts anticancer effects against cholangiocarcinoma cells via induction of apoptosis. Our data further showed that matrine treatment resulted in loss of Dcm, release of mitochondrial cytochrome c, as well as activation of both caspase-9 and -3 in cholangiocarcinoma cells. These findings suggest that the proapoptotic activity of matrine in cholangiocarcinoma is mediated by activation of the mitochondrial death cascade. Caspase-3 is a key effector caspase in the apoptosis pathway [24]. Pharmacological inhibition of caspase-3 activity was found to block the apoptosis induction by matrine, confirming that matrine induces caspase-3-dependent apoptosis in cholangiocarcinoma cells. The activation of caspase-3-dependent mitochondrial death pathway by matrine has also been described in several other types of cancers [12,25]. JAK/STAT3 signaling is constitutively activated in cholangiocarcinoma and represents an important target for treatment of this disease [6]. Inhibition of STAT3 signaling by sorafenib, a multikinase inhibitor was found to sensitize cholangiocarcinoma cells to tumor necrosis factor-related apoptosis-inducing ligandmediated apoptosis and suppress cholangiocarcinoma tumor growth [26]. Inactivation of JAK/STAT3 signaling was shown to mediate the chemopreventive effects of quercetin and EGCG in cholangiocarcinoma cells [21]. Similarly, we found that matrine significantly decreased the phosphorylation levels of STAT3 and JAK2 and STAT3-dependent transcriptional activity in cholangiocarcinoma cells. Matrine-mediated inactivation of JAK2/STAT3 provides a mechanistic explanation for its cytotoxicity in cholangiocarcinoma cells. To confirm the mediating role of STAT3 signaling, we transfected the constitutively active STAT3 mutant into cholangiocarcinoma cells before exposure to matrine. The results showed that restoration of STAT3 activation antagonizes the proapoptotic activity of matrine in cholangiocarcinoma cells. Mcl-1 is a well-established antiapoptotic target gene of STAT3 [6]. We found that matrine treatment significantly reduced the expression of Mcl-1 in cholangiocarcinoma cells. Mcl-1 reduction has been found to sensitize cholangiocarcinoma cells to apoptotic stimuli [27]. It has been documented that short hairpin RNA-mediated depletion of Mcl-1 enhanced the induction of
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cholangiocarcinoma cell apoptosis by the multikinase inhibitor BAY 43-9006 [27]. Triptolide, a bioactive ingredient extracted from the Chinese herb Tripterygium wolfordii Hook F, has been shown to cause apoptotic cell death of human cholangiocarcinoma cells through inhibition of Mcl-1 [28]. These studies indicate the important role for Mcl-1 in apoptosis resistance of cholangiocarcinoma cells. Our results collectively suggest that matrine triggers apoptotic death in cholangiocarcinoma cells via suppression of JAK2/STAT3 signaling and downregulation of Mcl-1. In conclusion, we provide first evidence that matrine is capable of inducing mitochondrial apoptotic death of cholangiocarcinoma cells, most likely through inactivation of the JAK2/STAT3 pathway and downregulation of the antiapoptotic protein Mcl-1. Therefore, matrine may represent a novel anticancer agent for cholangiocarcinoma, especially with constitutive activation of JAK2/STAT3 signaling. Conflict of interest The authors declared no conflict of interest. Funding None. References [1] Ramı´rez-Merino N, Aix SP, Corte´s-Funes H. Chemotherapy for cholangiocarcinoma: an update. World J Gastrointest Oncol 2013;5(7):171–6. [2] Brown KM, Parmar AD, Geller DA. Intrahepatic cholangiocarcinoma. Surg Oncol Clin N Am 2014;23(2):231–46. [3] Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med 2010;362(14):1273–81. [4] Junk DJ, Bryson BL, Jackson MW. HiJAK’d signaling; the STAT3 paradox in senescence and cancer progression. Cancers (Basel) 2014;6(2):741–55. [5] Xiong A, Yang Z, Shen Y, Zhou J, Shen Q. Transcription factor STAT3 as a novel molecular target for cancer prevention. Cancers (Basel) 2014;6(2):926–57. [6] Isomoto H, Kobayashi S, Werneburg NW, Bronk SF, Guicciardi ME, Frank DA, et al. Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells. Hepatology 2005;42(6): 1329–38. [7] Bournazou E, Bromberg J. Targeting the tumor microenvironment: JAK-STAT3 signaling. JAKSTAT 2013;2(2):e23828. [8] Darnell Jr JE, Kerr IM, Stark GR. JAK-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264(5164):1415–21. [9] Shi D, Zhang J, Qiu L, Li J, Hu Z, Zhang J. Matrine inhibits infiltration of the inflammatory Gr1(hi) monocyte subset in injured mouse liver through inhibition of monocyte chemoattractant protein-1. Evid Based Complement Alternat Med 2013;2013:580673.
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