FTY720 inhibits proliferation and epithelial-mesenchymal transition in cholangiocarcinoma by inactivating STAT3 signaling

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

FTY720 is cytotoxic to CC cells in a dose- and time-dependent manner. (A) MTT assay showing percentage of viable CC cells treated with 0, 5, 10, 15 and 20 μmol/L of FTY720 for 24 h. Data are presented as mean ± SD from three independent experiments. (B) CC cells were treated with FTY720 or vehicle for 72 hr, and proliferation measured using MTT assays. (C) Flow cytometry results of annexin V-PI stained CC cells after exposure to FTY720 (0 or 10 μmol/L) for 24 h. An increase in apoptotic cells following treatment with FTY720 is shown. Data are presented as the mean ± SD from three independent experiments. (D) A representative example of apoptosis of QBC939 cells treated with 10 μmol/L of FTY720 for 24 h.

Figure 2

FTY720 induces cell death in a caspase-dependent manner. (A) Caspase activation and PARP cleavage following treatment with FTY720. Lysates from CC cells treated with 0, 5 and 10 μmol/L FTY720 for 24 h were probed for cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and cleaved-PARP by western blotting. (B) Caspase inhibition protects against FTY720-induced cell death. CC cells were incubated in 0.1% DMSO, 5 μmol/L FTY720 or a combination of Q-VD-OPH (20 μmol/L) and 5 μmol/L FTY720, followed by annexin V-PI staining 24 h later. Data are presented as the mean ± SD from three independent experiments. (C) Lysates from treated cells were probed for cleaved-caspase-3 or cleaved-PARP by western blotting. The western blot is representative of three independent experiments. β-Actin was used as the internal control.

Figure 3

FTY720 reduces constitutive and inducible p-STAT3 in CC cells, and downregulates the expression of anti- or proapoptotic proteins. (A) CC cells were treated for 24 h with or without FTY720 and analyzed for the indicated protein by western blotting. (B) FTY720 reduced IL-6 induced p-STAT3 expression in CC cells as shown in the western blot. β-Actin was used as the internal control. All assays were performed in triplicate.

Figure 4

Effect of FTY720 on cell cycle proteins and cell cycle progression. (A) FTY720 induces expression of p16 and p27 and reduces expression of cyclin D1, CDK4, cyclin E and CDK2. CC cells were treated with FTY720 at the indicated concentrations for 24 h. Lysates were then prepared immediately and analyzed by western blotting for cyclin D1, CDK4, cyclin E, CDK2, p16 and p27. β-Actin was used as the internal control. All assays were done in triplicate. (B) Cell cycle analysis of FTY720-treated CC cells showing arrest in G1 phase. CC cells were incubated with FTY720 for 24 h. The percentage of cells in each phase of the cell cycle is presented as the mean ± SD from three independent experiments. Following treatment with FTY720 for 24 h, there was a significant increase in the percentage of cells in G0/G1 relative to the control group. (C) A representative example of cell cycle arrest in QBC939 cells treated with FTY720 for 24 h.

Figure 5

FTY720 inhibits the invasive potential of CC cells in vitro . (A) FTY720 significantly inhibited the invasive capacity of QBC939, TFK-1 and HuCCT1 cells. ***P <0.0001. The results are presented as the mean ± SD of experiments performed in triplicate. (B) Western blot showing that FTY720 increased the expression of E-cadherin and decreased the expression of N-cadherin, vimentin, VEGF and TWIST1 in CC cells. β-Actin was used as the internal control. All assays were performed in triplicate. (C) Single and merged images show immunofluorescence staining of N-cadherin (green) and vimentin (red). The cell nucleus is stained blue by DAPI.

Figure 6

FTY720 inhibits proliferation and metastasis of CC in vivo . (A) Photomicrographs of xenograft tumors in nude mice. Representative images of a mouse in each group are presented. Tumor volumes in FTY720-treated mice were smaller than those of control mice. (B) The multiple tumor masses formed by the HuCCT1 cells in the FTY720-treated group were much smaller than those formed by HuCCT1 cells in the control group. (C) Tumors from different groups were immunostained for indicated molecules. CD31-stained microvessels were counted to record microvessel density. Apoptotic cells were counted to give the apoptosis index and cells expressing Ki-67 were counted to calculate the proliferation index. Pictures are representative of three independent experiments. (D) Western blot detection of the indicated molecules in tumor samples. β-Actin was used as the internal control. All assays were performed in triplicate. The results are expressed as the mean ± SD of three independent experiments; ***P <0.0001.