Synergistic anti-tumor effects of liraglutide with metformin on pancreatic cancer cells

Abstract

Either metformin or liraglutide has been reported to have anti-tumor effects on pancreatic cancer cells. However, it is not clear whether their combined treatment has additive or synergistic anti-tumor effects on pancreatic cancer cells. In this study, the human pancreatic cancer cell line MiaPaca-2 was incubated with liraglutide and/or metformin. The cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, and wound-healing and transwell migration assays were used to detect cell viability, clonogenic survival, cell cycle and cell migration, respectively. RT-PCR and western blot analyses were used to determine the mRNA and protein levels of related molecules. Results showed that combination treatment with liraglutide (100 nmol/L) and metformin (0.75 mmol/L) significantly decreased cell viability and colony formation, caused cell cycle arrest, upregulated the level of pro-apoptotic proteins Bax and cleaved caspase-3, and inhibited cell migration in the cells, although their single treatment did not exhibit such effects. Combination index value for cell viability indicated a synergistic interaction of liraglutide and metformin. Moreover, the combined treatment with liraglutide and metformin could activate the phosphorylation of AMP-activated protein kinase (AMPK) more potently than their single treatment in the cells. These results suggest that liraglutide in combination with metformin has a synergistic anti-tumor effect on the pancreatic cancer cells, which may be at least partly due to activation of AMPK signaling. Our study provides new insights into the treatment of patients with type 2 diabetes and pancreatic cancer.

Fig 1. Combined effects of liraglutide and metformin on cell proliferation in human pancreatic cancer cells.

MiaPaca-2 cells were incubated for 48 h with metformin (a) or liraglutide (b) alone or in combination (c) at the specified concentration. Cell viability (top) and cell numbers (middle) for each treatment group were measured using the CCK-8 assay, and the protein level of the cell proliferation marker PCNA was determined by western blot analysis (bottom). Data are shown as means ± SD. n = 4. *P<0.05 vs. control. Lira, liraglutide; Met, metformin; PCNA, proliferating cell nuclear antigen.

Fig 2. Combined effects of liraglutide and metformin on colony formation and cell cycle in human pancreatic cancer cells.

MiaPaca-2 cells were treated with liraglutide (100 nmol/L) and/or metformin (0.75 mmol/L) for 8 days in the colony formation assay (a) or 96 h in cell cycle determination (b). The quantitative histograms of either colony formation or cell cycle distribution are shown in the right. Data are shown are means ± SD. n = 4. *P<0.05 vs. control; ^§P<0.05 vs. liraglutide; ^†P<0.05 vs. metformin. Ctrl, control; Lira, liraglutide; Met, metformin.

Fig 3. Combined effects of liraglutide and metformin on cell apoptosis in human pancreatic cancer cells.

MiaPaca-2 cells were incubated for 48 h with liraglutide (a) or metformin (b) alone or in combination (c) at the specified concentrations. Protein levels of the pro-apoptotic marker cleaved caspase-3 (top) and Bax (bottom) were measured by western blot analysis. Data are shown as means ± SD. n = 4. *P<0.05 vs. control; ^§P<0.05 vs. liraglutide; ^†P<0.05 vs. metformin. Lira, liraglutide; Met, metformin.

Fig 4. Combined effects of liraglutide and metformin on cell migration in human pancreatic cancer cells.

MiaPaca-2 cells were treated with liraglutide (100 nmol/L) and/or metformin (0.75 mmol/L) for 48 h. Wound healing assay (a) and transwell migration assay (b) were performed to analyze cell migration, as shown by either photographs (left) or histograms (right). Data are shown as means ± SD. n = 4. *P<0.05 vs. control; ^§P<0.05 vs. liraglutide; ^†P<0.05 vs. metformin. Ctrl, control; Lira, liraglutide; Met, metformin.

Fig 5. AMPK phosphorylation but not GLP-1 receptor level is upregulated by combination treatment with liraglutide and metformin in human pancreatic cancer cells.

MiaPaca-2 cells were incubated for 48 h with liraglutide (a) or metformin (b) alone or in combination (c) at the specified concentrations. The levels of GLP-1 receptor (GLP-1R) mRNA (top) and protein (middle), and the protein level of p-AMPK (bottom) were measured by RT-PCR and/or western blot analyses. Data are shown as means ± SD. n = 4. *P<0.05 vs. control; ^§P<0.05 vs. liraglutide. Lira, liraglutide; Met, metformin; GLP-1R, glucagon-like peptide-1 receptor; AMPK, AMP-activated protein kinase; p-AMPK, phosphorylated AMPK.