Background Metformin is a widely prescribed anti-diabetes drug with potential utilities for cancer therapies. Several previous studies have related metformin to the reduced risk of cholangiocarcinoma (CCA), highlighting its potentialities for the treatments of CCA. Methods In this study, cell viability assay and colony formation assay were used to test the inhibition effect of metformin on Mz-ChA-1 cells. The NMR-based metabonomic analysis was conduct to compare the differences between the metformin-treated (Met) and control (Ctrl) groups of the Mz-ChA-1 cells. Significant metabolites were identified from multivariate statistical analysis of 1D 1 H-NMR spectral data, and differential metabolites were identified from the pair-wise t-test of the metabolite levels. Significantly altered metabolic pathways were identified based on characteristic metabolites which were determined by a combination of the significant metabolites and differential metabolites. Results Here, we demonstrated that metformin treatment could inhibit the proliferation of the CCA cell line Mz-ChA-1 in a dose-dependent manner. The NMR-based metabonomic analysis showed a distinct discrimination between the Met and Ctrl groups of the Mz-ChA-1 cells. Moreover, The Met group exhibited promoted glycolysis and suppressed TCA cycle compared with the Ctrl group. While metformin treatment decreased non-essential amino acids, it also increased essential amino acids and UDP-GlcNAc, implying the occurrence of autophagy and cell cycle arrest in metformin-treated CAA cells . Conclusions This work provides a mechanistic understanding of the anticancer effect of metformin on CAA, and is beneficial to the further development of metformin as an anticancer drug
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