Exenatide modulates tumor-endothelial cell interactions in human ovarian cancer cells

Abstract

Diabetes and cancer are prevalent diseases whose incidence is increasing globally. Diabetic women have a moderate risk increase in ovarian cancer, suggested to be due to an interaction between these two disorders. Furthermore, patients manifesting both diseases have associated worse prognosis, reduced survival and shorter relapse-free survival. According to current recommendations, incretin drugs such as Exenatide, a synthetic analog of Exendin-4, and Liraglutide are used as therapy for the type 2 diabetes (T2D). We studied the effects of GLP-1 and Exendin-4 on migration, apoptosis and metalloproteinase production in two human ovarian cancer cells (SKOV-3 and CAOV-3). Exendin-4 inhibited migration and promoted apoptosis through caspase 3/7 activation. Exendin-4 also modulated the expression of key metalloproteinases (MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2). Vascular endothelial cells, which contribute to the formation and progression of metastasis, were also analyzed. TNF-α-stimulated endothelial cells from iliac artery after Exendin-4 treatment showed reduced production of adhesion molecules (ICAM-1 and VCAM-1). Additionally, incretin treatment inhibited activation of apoptosis in TNF-α-stimulated endothelial cells. In the same experiment, MMPs (MMP-1 and MMP-9), which are relevant for tumor development, were also reduced. Our study demonstrated that incretin drugs may reduce cancer cell proliferation and dissemination potential, hence limiting the risk of metastasis in epithelial ovarian cancer.

Keywords: Exenatide; MMPs; apoptosis; endothelial cells; ovarian cancer.

Figure 1

GLP-1R Western blot and transwell-based migration assay. Representative Western blot image of protein extracts from SKOV-3, CAOV-3 and patient biopsies (A). Ovarian cancer cell lines were stimulated for 24 h with Exendin-4 (50 nM), Exendin-4 (50 nM) pre-treatment for 24 h and GLP-1 Antagonist 9–36 (50 nM) in both SKOV-3 (B) and CAOV-3 (C). Relative fluorescence units (RFU). Mean values ± s.e.m. are shown. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 Control vs different conditions. One-way ANOVA followed with Dunnett’s post hoc.

Figure 2

Exendin-4 effects on apoptosis, cell viability and signaling pathway. Ovarian cancer cell lines were stimulated for 24 h with Exendin-4 (50 nM), Exendin-4 (50 nM) pre-treatment for 24 h and Camptothecin (CPT, 100 nM) in both SKOV-3 (A, B, C) and CAOV-3 (D, E, F). Caspase 3/7 activation in SKOV-3 (A) and CAOV-3 (D), cell viability in SKOV-3 (B) and CAOV-3 (E) and phosphoIκB in SKOV-3 (C) and CAOV-3 (F) was analyzed. Relative fluorescence units (RFU). Mean values ± s.e.m. are shown. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 Control vs different conditions. One-way ANOVA followed with Dunnett’s post hoc.

Figure 3

TNF-α-mediated metalloproteinase profile after Exendin-4 treatment in SKOV-3 cancer cells. Protein levels of MMP-1 (A), MMP-2 (B), MMP-7 (C), MMP-9 (D), MMP-10 (E) and MMP-13 (F) were analyzed by multiplex analysis after 24 h incubation with TNF-α (10 ng/mL), Exendin-4 (50 nM) and GLP-1 agonist (100 nM). Mean values ± s.e.m. are shown. n = 3–6 per group. Gray bars, TNF-α-stimulated. White bars, non-TNF-α-stimulated. *P < 0.05, **P < 0.01, ***P < 0.001 TNF-α vs different conditions. ^#P < 0.05 Unstimulated vs Exendin-4 or GLP-1 group. One-way ANOVA followed with Dunnett’s post hoc.

Figure 4

VEGF, TNF-α concentration in ovarian cancer cells and Caspase 3/7 activation in endothelium. VEGF protein levels in SKOV-3 (A) and CAOV-3 (D) and TNF-α protein levels in SKOV-3 (B) and CAOV-3 (E) after 24 h incubation with Camptothecin (100 nM), Exendin-4 (50 nM) and GLP-1 (100 nM). Representative Western blot image of GLP-1R expression in human Aortic Endothelial Cells (AoEC), Iliac Artery Endothelial Cells (IAEC) and Coronary Artery Endothelial Cells (CAEC) (C). Caspase 3/7 activation in TNF-α (10 ng/mL) stimulated IAEC after incubation with Exendin-4 (1 nM and 10 nM), and GLP-1 (10 nM and 100 nM) (F). Mean values ± s.e.m. are shown. n = 3–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001 TNF-α vs different conditions. One-way ANOVA followed with Dunnett’s post hoc.

Figure 5

Exendin-4 modulates endothelial-cell response after TNF-α stimulation. Iliac Artery Endothelial Cells (IAEC) were stimulated for 24 h with TNF-α (10 ng/mL), Exendin-4 (1 nM and 10 nM) and GLP-1 (10 nM and 100 nM). Protein concentration of VCAM-1 (A), ICAM-1 (B), MMP-1 (C), MMP-9 (D), TIMP1 (E) and TIMP-2 (F). Mean values ± s.e.m. are shown. n = 6 per group. *P < 0.05, **P < 0.01 TNF-α vs different conditions. One-way ANOVA followed with Dunnett’s post hoc.