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FTY720 inhibits proliferation and epithelial-mesenchymal transition in cholangiocarcinoma by inactivating STAT3 signaling
- Zhaoyang Lu†1,
- Jiabei Wang†1,
- Tongsen Zheng†1,
- Yingjian Liang1,
- Dalong Yin1,
- Ruipeng Song1,
- Tiemin Pei1,
- Shangha Pan1,
- Hongchi Jiang1 and
- Lianxin Liu1, 2Email author
© Lu et al.; licensee BioMed Central Ltd. 2014
Received: 23 June 2014
Accepted: 17 October 2014
Published: 25 October 2014
Interleukin 6 (IL-6)-mediated signal transducers and activators of transcription 3 (STAT-3) phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced myeloid cell leukemia 1 (Mcl-1) expression and resistance to apoptosis. FTY720, a new immunosuppressant, derived from ISP-1, has been studied for its putative anti-cancer properties. This study aimed to elucidate the mechanism by which FTY720 mediates antitumor effects in cholangiocarcinoma (CC) cells.
Three CC cell lines were examined, QBC939, TFK-1, and HuCCT1. The therapeutic effects of FTY720 were evaluated in vitro and in vivo. Cell proliferation, apoptosis, cell cycle, invasive potential, and epithelial- mesenchy-mal transition (EMT) were examined.
FTY720 greatly inhibited CC cells proliferation and EMT in vitro and in vivo, and this effect was associated with dephosphorylation of STAT3tyr705. FTY720 induced apoptosis and G1 phase arrest in CC cells, and inhibited invasion of CC cells. Western blot analysis showed that FTY720 induced cleavage of caspases 3, 8 and 9, and of PARP, in a dose-dependent manner, consistent with a substantial decrease in p-STAT3, Bcl-xL, Bcl-2, survivin, cyclin D1, cyclin E, N-cadherin, vimentin, VEGF and TWIST1. In vivo studies showed that tumor growth and metastasis were significantly suppressed after FTY720 treatment.
These results suggest that FTY720 induces a significant decrease in p-STAT3, which inhibits proliferation and EMT of CC cells, and then induces G1 phase arrest and apoptosis. We have characterized a novel immunosuppressant, which shows potential anti-tumor effects on CC via p-STAT3 inhibition. FTY720 merits further investigation and warrants clinical evaluation.
Human cholangiocarcinoma (CC) arises from the epithelium of the biliary tree. CC encompasses adenocarcinomas arising in the intra or extrahepatic biliary tree and in the gall bladder. CC is a relatively uncommon malignancy in western countries , but has a high incidence in Asia and Latin America [2, 3]. CC is characterized by poor prognosis and a 5-year survival rate less than 5% . Currently, conventional chemotherapy and radiotherapy have not been reported to be effective in improving long-term survival , the only curative treatment for CC is surgical resection. However, the majority of CC patients shows advanced liver involvement and metastasis, and this precludes the use of curative surgical resection. Therefore, there is an urgent need to define the molecular mechanisms underlying CC proliferation and metastasis in order to develop novel therapeutic strategies.
One promising candidate for CC targeted therapy is signal transducer and activator of transcription 3 (STAT3). STAT3 is a transcription factor that is constitutively activated in many types of cancer, contributing to tumor progression via several mechanisms. [6–9] When phosphorylated at tyrosine705, STAT3 undergoes translocation from the cytosol to the nucleus, where it functions as a pivotal transcription factor upregulating gene transcription [10–12]. IL-6 secretion can further increase STAT3 activation levels within tumor cells via an autocrine feedback loop . IL-6–activated STAT3 is crucial for survival of several types of cancer cell, including multiple myeloma, a plasmacytic B-cell malignancy [6, 13]. Studies suggest that IL-6/STAT3 signaling is aberrant in human CC cells and CC tissues with prolonged and sustained STAT-3 phosphorylation [14, 15]. The mechanisms responsible for this atypical IL-6 signaling response are unclear but of pathophysiological importance.
FTY720 is a synthetic sphingosine immunosuppressant, which is currently undergoing clinical trials for the prevention of kidney graft rejection  and the treatment of relapsing multiple sclerosis . Previous studies indicate that the effect of FTY720 on prolonging the survival of allografts is attributable to the ability of its phosphorylated metabolite to inhibit T-lymphocyte infiltration by targeting several of the sphingosine-1-phosphate (S1P) receptors [18, 19]. Recently, FTY720 has been reported to have a strong antitumor effect on breast cancer , bladder cancer  and leukemia . So far, the feasibility of using this drug in CC treatment has not been studied. The precise mechanism of FTY720 action on cancer cells is not completely understood. Therefore, in this study we aimed to investigate the in vitro and in vivo anticancer potential of FTY720 and to ascertain the precise mechanism by which proliferation and metastasis are inhibited in CC cells.
We investigated the effect of FTY720 on the STAT3 cell survival pathway and found that STAT3 dephosphorylation plays a central role in cell growth arrest, apoptosis and metastasis upon administration of FTY720 to CC cell lines. Dephosphorylation of STAT3tyr705 results in G1 arrest and apoptosis possibly by up-regulation of p27, cleavage of caspase-3 and down-regulation of Mcl-1, cyclin D1 and Bcl-xL. It might also inhibit EMT by up-regulation of E-cadherin and down-regulation of Vimentin and N-cadherin, both in vitro and in vivo.
Cell lines and reagents
The human CC cell line QBC939 was a gift from Prof. Shuguang Wang (Third Military Medical University, Chongqing, China). Human CC cell lines TFK-1 and HuCCT1 were kindly provided by the Cancer Cell Repository, Tohoku University, Japan. All cell lines were cultured in Dulbecco’s modified Eagle's medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco BRL), penicillin G (100,000 U/L) and streptomycin (100 mg/L; Gibco BRL) at 37°C in a humidified atmosphere containing 5% CO2. FTY720 was purchased from Selleckchem (Houston, TX, USA).
Cell viability was assessed using the MTT assay. CC cells were seeded at 2 × 104 per well in 96-well flat-bottomed plates and incubated in 10% FBS supplemented DMEM for 24 h. Cells were treated with FTY720 at various concentrations in the same medium. Controls received dimethyl sulfoxide (DMSO) vehicle at a concentration equal to that in drug-treated cells. After 24 and 48 h, the drug-containing medium was replaced with 200 μL of 10% FBS supplemented DMEM containing 0.5 mg/mL MTT, and cells were incubated in the CO2 incubator at 37°C for 4 h. Medium was removed and the reduced MTT solubilized in 100 μL per well of DMSO. Absorbance was then measured at 570 nm. Six replicates were performed for each experiment.
Cell cycle analysis
Cells were treated with FTY720 and then 106 cells were fixed in 80% ethanol at -20°C for 24 h. Fixed cells were stained according to the Cycle TESTTM PLUS DNA Reagent Kit protocol (BD Biosciences, San Jose, CA, USA) and analyzed by flow cytometry (Beckman Coulter FC 500). The experiment was repeated thrice under the same conditions.
FTY720 treated cells were harvested, washed twice with prechilled PBS and resuspended in 1× binding buffer at a concentration of 1 × 106 cells/ml. One hundred microliters of this cell suspension (1 × 105 cells) was mixed with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) (BD Biosciences) according to the manufacturer’s instructions. The mixed solution was gently vortexed and incubated in the dark at room temperature (25°C) for 15 min. Four hundred microliters of 1× dilution buffer were then added to each tube and cell apoptosis analysis was performed by flow cytometry (BD FACS Calibur) within 1 h.
Cell invasion assays
Eight hours after FTY720 treatment, invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences, NJ, USA) with an 8 μm porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix.
Tumor xenografts in nude mice
In these studies, tumor xenografts were established by standard techniques in 8-week-old nude mice (BALBc nu/nu) . In brief, each mouse was injected subcutaneously with 3 × 106 QBC939 cells and 3 × 106 HuCCT1 cells suspended in PBS. Tumor size was measured by Vernier calipers, and tumor volume was calculated as described previously . Once the tumors reached an average of 90 mm3, the treatment began. For the treatment group, FTY720 was administered by daily i.p. injection of 10 mg/kg/day for 20 days. After treatment, mice in both the treatment and control groups (n = 10 in each group) were sacrificed. Tumor tissues were collected, snap-frozen and embedded in paraffin for further analysis.
This study does not involve human subjects, human material, or human data. All nude mice were treated and all procedures were conducted in accordance with the guidelines for experimental animals approved by the Animal Care and Use Committee of Harbin Medical University, Harbin, China.
In vivoinvasive assay
HuCCT1 cells (3 × 106 cells in 200 μL) and QBC939 (3 × 106 cells in 200 μL) were injected into the intraperitoneal cavity as previously described . Animals were randomized to receive either FTY720 (10 mg/kg/d, i.p.) or vehicle at 1 week after injection. The mice were sacrificed at 4 weeks after tumor cell injection.
Western blot analysis
Protein isolation was performed as described previously , and western blot analysis was achieved via established protocols . The primary antibodies used were against N-cadherin, E-cadherin, p16 and vimentin (Abcam, Cambridge, MA, USA); p27, STST3, p-STAT3, cleaved PARP, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bcl-xL, and Bcl-2 (Cell Signaling Technology, Danvers, MA, USA); cyclin D1, VEGF, TWIST1, Bax, survivin, cyclin E, CDK2, CDK4 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Briefly, cells seeded on coverslips were fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich) for 10 min and permeabilized with 0.1% (v/v) Triton X-100 for 5 min at room temperature. The cells were then incubated overnight with primary antibodies at 4°C, followed by incubation with fluorescent secondary antibody for 1 h at room temperature. After final washes with PBS, coverslips were mounted using an anti-fade mounting solution containing 4',6-diamidino-2-phenylindole (DAPI; Vector Lab) and images were examined and captured.
Immunohistochemistry was performed as described previously  using Ki-67, CD31 and cleaved caspase-3 antibodies (Cell Signaling Technology).
All data are expressed as mean values ± standard deviation (SD). Comparisons among multiple groups were made with a one-way analysis of variance followed by Dunnett's t-test. A value of “p <0.05” was considered to be statistically significant.
FTY720 is a potent anti-CC agent and induces apoptosis in CC cells
FTY720 induces cell death in a caspase-dependent manner by cleavage of caspases 3, 8 and 9
FTY720 inhibits constitutive and inducible STAT3 phosphorylation in CC cells, and affects the expression of anti- or proapoptotic proteins
FTY720 downregulates cyclin D1 and cyclin E, increases p27 and p16 expression and induces G1 cell cycle arrest in CC cells
FTY720 inhibits the invasive potential of CC cells in vitro
FTY720 inhibits tumor growth and metastasis of CCin vivo
Cholangiocarcinoma is an aggressive disease, with a poor response to the treatments that are currently available, including the standard gemcitabine [30, 31]. To this end, we examined a new agent for the treatment of CC. FTY720 is a chemical substance derived by modifying an immunosuppressive metabolite and has been shown to possess anti-cancer properties in various types of cancer . However, the efficacy of FTY720 against CC has not been previously assessed. Herein, we have demonstrated that FTY720 induces apoptosis and cell cycle arrest, inhibits EMT of CC cells and in vivo tumor growth in a nude mouse model without notable toxic side-effects.
The IL-6/STAT3 pathway plays an important role in human cancers. STAT proteins comprise a seven member family of latent cytoplasmic transcription factors [10, 33]. Accumulating data suggest that aberrant STAT signaling, and in particular STAT3 initiated cascades, participate in the development and progression of human cancers [10, 11]. Numerous studies have shown that STAT3 inhibitors have tumor suppressive effects on various tumors. AG490, the most popular STAT3 inhibitor, can induce CC cell apoptosis and inhibit CC/mycosis fungoides tumor cell proliferation [14, 15, 34]. New STAT3 inhibitors also can inhibit tumor proliferation [35, 36], chemo-therapy resistance  and metastasis . We therefore presumed that STAT3 would be a good target for CC treatment, and our results indeed show that FTY720 inhibited proliferation and EMT in CC mainly through the IL-6/STAT3 pathway.
FTY720 has been demonstrated to inhibit proliferation of various tumors [20, 39, 40]. So we tried to test whether FTY720 could inhibit CC proliferation. Cell viability analysis (MTT assays) showed that FTY720 could induce a dramatic reduction in cell viability in all three CC cell lines tested. After cells were treated with FTY720 for 24 h, we observed a significant decrease in the S-phase population, and induction of G1 arrest. FTY720 induced significant expression of the cyclin-dependent kinase (CDK) inhibitors p16 and p27. Both p16 and p27 block the formation of cyclin-CDK complexes, allowing Rb to become activated and to halt the cell cycle. In addition to inducing p16 and p27, FTY720 also downregulated cyclin D1 and cyclin E in CC cells contributing to arrest in the G1 phase.
FACS analysis also showed that the inhibitory effect on CC cell growth by FTY720 was also related to induction of apoptosis. Our results show that FTY720-induced apoptosis is associated with cleavage of caspases 8, 9 and 3, and PARP, suggesting that the drug activates both the extrinsic and intrinsic apoptotic pathways. Further, FTY720-induced apoptosis is in large part dependent on caspase activation. In CC cells, FTY720 also modulates the expression of the antiapoptotic proteins. Of the Bcl-2 family members, the expression of Bcl-xL, Bcl-2 and survivin was significantly reduced, while Bax expression was increased. In additional to inhibition of tumor proliferation, numerous studies demonstrated that FTY720 could inhibit tumor metastasis [20, 41, 42]. And our study also demonstrate that incubation of CC cells with FTY720 leads to the loss of N-cadherin and vimentin and to the accumulation of E-cadherin. Furthermore, FTY720 significantly inhibited the invasive capacity of CC cells. We also examined the ability of FTY720 to suppress the growth and metastasis of human CC cancer cell xenografts in nude mice. We found a significant reduction in relative tumor size and metastatic nodules in FTY720-treated animals compared with untreated controls. In addition, the suppression of proliferation by FTY720 was confirmed by decreased Ki-67 expression. Increased numbers of apoptotic cells and activated protein levels of apoptosis–related proteins, such as cleaved-PARP, cleaved-caspase-9, cleaved-caspase-8 and cleaved-caspase-3, were accompanied by decreased p-STAT3 expression in the FTY720-treated animals.
IL-6 plays an important role in the growth and survival of CC cells [43, 44]. Our results indicate that FTY720 exerts an important inhibitory effect on the IL-6 signal transduction pathway by inhibiting constitutive and inducible STAT3 phosphorylation. STAT3 directly and indirectly upregulates the expression of genes that are required for uncontrolled proliferation and invasion of tumor cells [45, 46]. In our study, the FTY720-induced reduction of Bcl-xL, Bcl-2, N-cadherin, vimentin, cyclin E and cyclin D1 may result, at least in part, through an inhibitory effect on the STAT3 pathway. Importantly, FTY720 overcomes the activation of p-STST3 which was induced by IL-6.
In conclusion, our results show that the novel synthetic sphingosine immunosuppressant, FTY720, has potent activity against CC in vitro and in vivo. Its ability to target mainly the IL-6/STAT3 pathway and downstream anti-apoptotic, EMT and cell cycle proteins, suggest its viability as part of the therapeutic armamentarium for CC. Our results provide preclinical rationale for clinical development of FTY720 for the treatment of CC.
This study was supported by Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1122), the National Natural Science Foundation of China (Grant No. 81302060, No. 81301807, No. 81272705 and No. 81201878) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20102307120008). These funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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