Liraglutide exhibits potential anti-tumor effects on the progression of intrahepatic cholangiocarcinoma, in vitro and in vivo

Abstract

Glucagon-like peptide 1 receptor (GLP-1R) agonist is an emerging anti-diabetic medication whose effects on the risk and progression of cholangiocarcinoma (CCA) are controversial. This study aimed to elucidate the roles of GLP-1R and its agonists on intrahepatic CCA (iCCA) progression. Expressions of GLP-1R in iCCA tissues investigated by immunohistochemistry showed that GLP-1R expressions were significantly associated with poor histological grading (P = 0.027). iCCA cell lines, KKU-055 and KKU-213A, were treated with exendin-4 and liraglutide, GLP-1R agonists, and their effects on proliferation and migration were assessed. Exendin-4 and liraglutide did not affect CCA cell proliferation in vitro, but liraglutide significantly suppressed the migration of CCA cells, partly by inhibiting epithelial-mesenchymal transition. In contrast, liraglutide significantly reduced CCA tumor volumes and weights in xenografted mice (P = 0.046). GLP-1R appeared downregulated when CCA cells were treated with liraglutide in vitro and in vivo. In addition, liraglutide treatment significantly suppressed Akt and STAT3 signaling in CCA cells, by reducing their phosphorylation levels. These results suggested that liraglutide potentially slows down CCA progression, and further clinical investigation would benefit the treatment of CCA with diabetes mellitus.

Keywords: Cholangiocarcinoma; Diabetes mellitus; Glucagon-like peptide 1 receptor; Liraglutide.

Figure 1

GLP-1R expression in iCCA tumor tissues. (a) The figures represent the scoring of GLP-1R intensity in iCCA tissues, only the membranous staining pattern was defined as 3 + as it suggests the function of the membrane-bound receptor. (b) GLP-1R expressions in CCA tumor tissues are not different for patients with or without diabetes mellitus (Student’s t-test). (c) The expression of GLP-1R is not associated with the overall survival of patients with iCCA (Log-rank test). Scale bars represent 20 µm.

Figure 2

Effects of GLP-1R agonist on iCCA cell proliferation. (a, b) All examined iCCA cell lines express GLP-1R proteins without a significant difference in levels. (c) Neither exendin-4 nor liraglutide, two different GLP-1R agonists, show any effects on CCA cell proliferation. Western blots are representatives of 3 biological replications, and the graph represents the average band intensities from 3 biological replications. Protein expressions are normalized by GAPDH.

Figure 3

Liraglutide suppresses the migration of CCA cells by suppressing the expression of GLP-1R and epithelial-mesenchymal (EMT) markers. (a, b) Liraglutide significantly suppresses the migration of iCCA cells in both KKU-055 and KKU-213A. (c, d) The expression of GLP-1R and the EMT markers were correspondingly decreased in liraglutide-treated iCCA cells. Western blots are representatives of 3 biological replications, and the graphs represent the average band intensities from 3 biological replications. Protein expressions are normalized by GAPDH, in which the expression in the control groups is assigned as the factor of 1. Scale bars represent 100 µm. (*P < 0.05, Student’s t-test).

Figure 4

Liraglutide inhibits Akt and STAT3 pathways in CCA cells. (a, b) The phosphorylation of Akt and STAT3 is suppressed after both iCCA cells were treated with liraglutide. However, the ERK phosphorylation is slightly inhibited and statistically significant in only KKU-055. Western blots are representatives of 3 biological replications, and the graphs represent the average band intensities from 3 biological replications. Protein expressions are normalized by GAPDH, which the expression in the control groups is assigned as the factor of 1. (*P < 0.05, ***P < 0.001, Student’s t-test).

Figure 5

Liraglutide suppresses the growth of KKU-213A xenografts in vivo. (a) BALB/c Rag-2-/- Jak3-/- (BRJ) immunodeficient mice were subcutaneously inoculated with KKU-213A and then randomized into the control and liraglutide treatment groups (N = 5 mice/group). (b) CCA tumor volumes are significantly reduced in the liraglutide-treated group (*P < 0.001, Two-way ANOVA with Tukey’s multiple comparisons), and (c, d) the tumor weights are accordingly reduced (Student’s t-test). (e) More necrotic areas are observed in the tumors from liraglutide-treated mice. (T tumor cells, N necrotic area, F fibrotic area, Scale bars represent 100 µm).

Figure 6

Liraglutide suppresses the expression of GLP-1R and multiple signaling pathways in vivo. (a, b) Liraglutide suppresses the expression of GLP-1R in KKU-213A xenografted tumors in vivo. It also inhibits the phosphorylation of Akt and STAT3 consistently with the in vitro experiments. The expressions of downstream targeted proteins of Akt and STAT3, for instance, apoptotic protein- caspase-3 is increased, and cell cycle regulatory protein- cyclin D1 is decreased, corresponding with the reduced tumor volumes and tumor weights in mice receiving liraglutide. Each lane of Western blot is from a xenografted tumor in one mouse, where M is the lane loaded with molecular weight marker. (*P < 0.05, ***P < 0.001, Student’s t-test).