Metformin reduces pancreatic cancer cell proliferation and increases apoptosis through MTOR signaling pathway and its dose-effect relationship

摘要

OBJECTIVE: To study the influences of metformin on the proliferation and apoptosis of pancreatic cancer cells and its dose-effect relationship and crucial molecular mechanism. MATERIALS AND METHODS: With human pancreatic cancer cell line PANC-1 as the study object, different concentrations of metformin were added for intervention. Then, the proliferation of PANC-1 cells was detected via methyl thiazolyl tetrazolium (MTT) assay to determine the dose-effect relationship of metformin in PANC-1 cell proliferation. PANC-1 cells were treated with metformin at three appropriate concentrations as Metformin treatment groups, and an equal amount of dimethyl sulfoxide (DMSO) was added in Control group. Flow cytometry was performed to detect PANC-1 cell cycle and apoptosis, and the apoptosis of PANC-1 cells was also evaluated via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Caspase-3 protein localization and expression in PANC-1 cells were detected using immunofluorescence assay. Besides, the expressions of the apoptosis-associated proteins Caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2 associated X protein (Bax) and phosphatidylinositol 3-hydroxy kinase (PI3K), phosphorylated protein kinase B (p-Akt), and p-mammalian target of rapamycin (mTOR) proteins related to the mTOR pathway were detected using Western blotting. RESULTS: Metformin repressed the proliferation of human pancreatic cancer PANC-1 cells in a concentration-dependent and time-dependent manner. Compared with Control group, Metformin treatment groups (0, 20 and 40 mM) exhibited a higher proportion of PANC-1 cells in G0/G1 phases, and a lower proportion of PANC-1 cells in S phase (p0.05), and the change in the proportion of cells in G2/M phase was not statistically significant (p0.05). Moreover, Metformin treatment groups (0, 20, and 40 mM) had more apoptotic PANC-1 cells, higher expression levels of pro-apoptosis proteins Caspase-3 and Bax and lower expression levels of anti-apoptosis protein Bcl-2 and the mTOR pathway-related proteins PI3K, p-Akt, and p-mTOR in cells than Control group (p0.05). CONCLUSIONS: Metformin modulates the mTOR signaling pathway to reduce the proliferation of pancreatic cancer cell, but increase their apoptosis.