Metformin transiently inhibits colorectal cancer cell proliferation as a result of either AMPK activation or increased ROS production

Abstract

Metformin is a widely used and well-tolerated anti-diabetic drug that can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear. We studied the anti-cancer activity of metformin on colorectal cancer (CRC) by using the drug to treat HT29, HCT116 and HCT116 p53-/- CRC cells. Metformin reduced cell proliferation and migration by inducing cell cycle arrest in the G0/G1 phase. This was accompanied by a sharp decrease in the expression of c-Myc and down-regulation of IGF1R. The anti-proliferative action of metformin was mediated by two different mechanisms: AMPK activation and increase in the production of reactive oxygen species, which suppressed the mTOR pathway and its downstream targets S6 and 4EBP1. A reduction in CD44 and LGR5 expression suggested that the drug had an effect on tumour cells with stem characteristics. However, a colony formation assay showed that metformin slowed the cells' ability to form colonies without arresting cell growth, as confirmed by absence of apoptosis, autophagy or senescence. Our finding that metformin only transiently arrests CRC cell growth suggests that efforts should be made to identify compounds that combined with the biguanide can act synergistically to induce cell death.

Figure 1

Metformin (Met) decreased the proliferation, migration, and invasion of HT29, HCT116 and HCT116 p53−/− cells. (a) Cell proliferation was evaluated in vitro by means of BrdU incorporation in the absence (Ctrl) or presence of 5 mM Met after 24, 48 and 72 hours’ treatment. The results are shown as mean values ± SD compared with the control group (**P < 0.01, ****P < 0.0001). (b) The wound healing assay was conducted after Met treatment (0.6 mM for HT29 and HCT116 p53−/−; 1.25 mM for HCT116) for 90 hours (HT29), 38 hours (HCT116) or 40 hours (HCT116 p53−/−). (c) The chamber invasion assay was performed after treatment with 0.6 mM or 1.25 mM Met for 96 hours (HT29) or 72 hours (HCT116 and HCT116 p53−/−).

Figure 2

Metformin (Met) increases the percentage of cells in the G0/G1 phase, and affects the expression of various cell cycle regulatory proteins in HT29, HCT116 and HCT116 p53−/− cells. (a) Flow cytometric analysis of proliferating cells 72 hours after the addition of 5 mM Met. The results are representative of three independent experiments. (b) Immunoblots of p-Rb, cycD1, cycE and c-Myc in cells treated for 72 hours with 5 mM Met or left untreated. β-actin was used as the loading control. Full-size blots are shown in Supplementary Fig. S10. (c) Immunohistochemistry of c-Myc.

Figure 3

Metformin (Met) does not induce apoptosis, autophagy and senescence in HT29, HCT116 and HCT116 p53−/− cells. (a) Graphic representation of the results obtained from Annexin V assay (upper panel) and western blot analysis of Caspase-3 and PARP activation after treatment with 5 mM Met for 72 h (lower panel). Vinculin was used as the loading control. (b) Immunoblotting determination of LC3B and BECN1 protein after treatment with 5 mM Met for 72 hours; Vinculin was used as the loading control. Full-size blots are shown in Supplementary Fig. S11. (c) β-galactosidase staining of untreated cells (−) and cells treated with Met for 72 hours (+). The images were acquired at 20x magnification. At the bottom, human primary thyrocytes carrying the ER:RAS vector were left untreated or treated with 200 nM 4-hydroxytamoxifen (4OHT) for seven days, and used as positive controls (magnification 40x). The data are representative of at least three independent experiments.

Figure 4

Metformin (Met) reversibly inhibits colony formation. (a) One hundred cells were grown for 12 days in 6-well plates with or without Met (5 mM). The “rescued” colonies (R) are cells treated with Met for 12 days and grown for a further 12 days in fresh complete medium without Met. (b) Graphic representation of the results. The bars indicate the mean value ± SD of three independent experiments (**P < 0.01, ***P < 0.001). (c) One hundred “rescued” cells (R) were grown for 12 days in 6-well plates in presence or absence of Met. (d) Graphic representation of the results. The bars indicate the mean value ± SD of three independent experiments (*P < 0.05, ***P < 0.001).

Figure 5

Metformin (Met) causes oxidative stress and induces mitochondrial depolarization. The cells were treated with 5 mM Met for 72 hours and a significant increase in ROS production was measured in HCT116 and HCT116 p53−/− cell lines. The bars represent the mean value ± SD of three independent experiments (***P < 0.001) (a). In line with ROS increase, HCT116 and HCT116 p53−/− also showed a stronger mitochondrial depolarisation respect to HT29 (b). Mitochondrial depolarization was revealed by decrease of JC-1 PE and increase of JC-1 Alexa-488 signal intensities measured in the related cells. Before harvesting the cells, 1 μl of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added as a positive control.

Figure 6

Metformin (Met) reversibly inhibits the mTOR pathway and IGF1R protein. (a) Immunoblots of cells treated with 5 mM Met for 72 hours, or left untreated. The “rescued” cells (R) were grown for 72 hours with Met and for a further 72 hours in fresh complete medium without Met. Vinculin was used as the loading control (in HCT116 and HCT116 p53−/− cells vinculin is the same for AMPK, S6 and 4EBP1 since they were analysed on the same blots). (b) Tables showing densitometric quantification of the western blot bands normalized to vinculin. The ratio of each phosphorylated protein to the corresponding total protein band was calculated and quantified with respect to the untreated sample (−) set to 1.0. The results are representative of three independent experiments. Full-size blots are shown in Supplementary Figs S12–S14.

Figure 7

Metformin (Met) affects the expression of the CRC stemness markers LGR5 and CD44. (a) qRT-PCR of CD44 and LGR5 in HT29, HCT116 and HCT116 p53−/− cells. (b) In situ hybridisation of LGR5 in HT29 cells. Hematoxylin and eosin (H&E) staining is the same as in Fig. 2.