Metformin induces apoptosis of pancreatic cancer cells

Abstract

Aim: To assess the role and mechanism of metformin in inducing apoptosis of pancreatic cancer cells.

Methods: The human pancreatic cancer cell lines ASPC-1, BxPc-3, PANC-1 and SW1990 were exposed to metformin. The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of metformin was tested.

Results: In each pancreatic cancer cell line tested, metformin inhibited cell proliferation in a dose dependent manner in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays). Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction. Enzymelinked immunosorbent assay (ELISA) showed that metformin induced apoptosis in all pancreatic cancer cell lines. In Western blot studies, metformin induced poly-ADP-ribose polymerase (PARP) cleavage (an indicator of caspase activation) in all pancreatic cancer cell lines. The general caspase inhibitor (VAD-fmk) completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1, the caspase-8 specific inhibitor (IETD-fmk) and the caspase-9 specific inhibitor (LEHD-fmk) only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells. We also observed that metformin treatment dramatically reduced epidermal growth factor receptor (EGFR) and phosphorylated mitogen activated protein kinase (P-MAPK) in both a time- and dose-dependent manner in all cell lines tested.

Conclusion: Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines. And the metformin-induced apoptosis is associated with PARP cleavage, activation of caspase-3, -8, and -9 in a time- and dose-dependent manner. Hence, both caspase-8 and -9-initiated apoptotic signaling pathways contribute to metformin-induced apoptosis in pancreatic cell lines.

Figures

Figure 1

Metformin inhibits proliferation/survival in the basal-like subtype of pancreatic cancer cells. A: ASPC-1, BxPc-3, PANC-1 and SW1990 were plated onto 96-well plates with either complete medium or medium containing a series doses of metformin; B: ASPC-1, BxPc-3, PANC-1 and SW1990 were grown in triplicates in the absence or presence of metformin at different concentrations for 2-3 wk.

Figure 2

Metformin induces apoptosis and blocks cell cycle progression in the pancreatic cancer cells.

Figure 3

Metformin increases PARP cleavage, reduces levels of pro-caspase-8, -9, -3, and induces apoptosis in pancreatic cancer cells.

Figure 4

Activation of both caspase-8 and caspase-9 contributes to metformin-induced apoptosis in pancreatic cancer cells. A: ASPC-1; B: BxPc-3; C: PANC-1.

Figure 5

Metformin lowers EGFR expression levels and inhibits downstream signaling in pancreatic cancer cells. A: ASPC-1; B: BxPc-3; C: PANC-1.