Phenformin Induces Cell Cycle Change, Apoptosis, and Mesenchymal-Epithelial Transition and Regulates the AMPK/mTOR/p70s6k and MAPK/ERK Pathways in Breast Cancer Cells

Abstract

Breast cancer remains a world-wide challenge, and additional anti-cancer therapies are still urgently needed. Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin. However, little attention has been given to the role of phenformin in breast cancer. In this study, we reveal the role of phenformin in cell death of the MCF7, ZR-75-1, MDA-MB-231 and SUM1315 breast cancer cell lines. The respective IC50 values of phenformin in MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were 1.184±0.045 mM, 0.665±0.007 mM, 2.347±0.010 mM and 1.885±0.015 mM (mean± standard error). Phenformin induced cell cycle change and apoptosis in breast cancer cells via the AMPK/mTOR/p70s6k and MAPK/ERK pathways. Interestingly, phenformin induced MET (mesenchymal-epithelial transition) and decreased the migration rate in breast cancer cell lines. Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice. Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer.

Fig 1. Phenformin induces cell death of breast cancer cell.

Cells were grown for 24 hours in the presence of the indicated concentration of phenformin. CCK-8 assays were performed to evaluate cell viability. The absorbance was measured at 450 nm using an automated microplate reader. The percent growth inhibition was calculated using the following formula: (OD of the control-OD of the experimental sample)/OD of the control×100%.

Fig 2. Phenformin induces an increase in the percentage of cells in G1 in breast cancer cells.

(A) MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were treated with 1.184 mM, 0.665 mM, 2.347 mM, and 1.885 mM phenformin (the IC₅₀ of each cell lines) respectively for 24 hours. Control cells were treated with solvent (42.9% DMSO in DMEM). The cells were fixed with ethanol, stained with PI and analyzed by flow cytometry. (B) The percentages of cells with a DNA content consistent with each phase of the cell cycle were plotted. The data are presented as the mean±SEM of three replicates per group. Asterisks indicate significant differences at p<0.05 by Student’s t test. Phenformin treated cells were labeled as P and control cells were labeled as C.

Fig 3. Phenformin downregulates cyclin D1 expression and inactivates the MAPK/ERK pathway.

(A) MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were treated with or without phenformin for 24 hours. Cell extracts were analyzed by western blotting to detect the expression of cyclin D1, p-ERK, ERK and GAPDH. (B) Western blotting ratio analysis of p-ERK to ERK, (C) Cyclin D1 to GAPDH. The data are presented as the mean±SEM of three replicates per group. Asterisks indicate significant differences at p<0.05 by Student’s t test. Phenformin-treated cells were labeled as P and control cells were labeled as C.

Fig 4. Phenformin promotes breast cancer apoptosis.

(A) MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells treated with or without phenformin were double-stained with Annexin V and PI, and then analyzed by flow cytometry. (B) The percentages of Annexin V⁺/PI^- cells(early apoptosis) were plotted. (C) Cell extracts were analyzed by western blotting to detect the expression of cleaved caspase 3 and GAPDH. (D) Expression ratio of cleaved caspase 3 to GAPDH. The data are presented as the mean±SEM of three replicates per group. Asterisks indicate significant differences at p<0.05 by Student’s t test. Phenformin-treated cells were labeled as P and control cells were labeled as C.

Fig 5. Activation of AMPK/mTOR/p70s6k signaling by phenformin.

(A) MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were treated with or without phenformin for 24 hours. Cell extracts were analyzed by western blotting to detect the expression of p-AMPK, AMPK, p-mTOR, mTOR, p-p70s6k, p70s6k and GAPDH. (B) Expression ratios analysis of p-AMPK to AMPK, (C) p-mTOR to mTOR, (D) p-p70s6k to p70s6k. The data are presented as mean±SEM of three replicates per group. Asterisks indicate significant differences at p<0.05 by Student’s t test. Phenformin-treated cells were labeled as P and control cells were labeled as C.

Fig 6. Phenformin induces epithelial features in breast cancer cells.

(A) MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were treated with or without phenformin for 24 hours. Cell extracts were analyzed by western blotting to detect the expression of E-cadherin, vimentin and GAPDH. (B) Expression ratios of E-cadherin to GAPDH, (C) vimentin to GAPDH. The data are presented as the mean±SEM of three replicates per group. Asterisks indicate significant differences at p<0.05 by Student’s t test. Phenformin-treated cells were labeled as P and control cells were labeled as C.

Fig 7. Phenformin inhibits MDA-MB-231 cells migration.

(A) After incubation with phenformin for 24 hours, MDA-MB-231 cells (25,000 cells per chamber) were seeded in the upper chamber in serum free medium. The lower chamber contained medium with 10% FBS. After incubation for 16 hours, the cells were removed from the upper surface of the chamber membrane, and the cells on the lower surface of the chamber were stained with crystal violet and counted using a microscope(100X). (B) The number of cells/five fields was plotted. The data are presented as the mean±SEM of three replicates per group. Asterisks indicate significant differences at p<0.05 by Student’s t test.

Fig 8. Phenformin inhibits the metastasis of MDA-MB-231 cells in nude mice.

(A) The luciferase-tagged MDA-MB-231 cells were inoculated intracardially into female nude mice. After the injections, the mice were separated into the control and phenformin groups (10 mice per group). The control group’s drinking water was replaced with 5% sucrose. The phenformin group’s drinking water was replaced with 5% sucrose containing phenformin (300 mgkg^-1). The development of metastasis was monitored using whole mouse fluorescence and bioluminescence imaging(negative control: mice did not receive an intracardiac injection of luciferase-expressing cells). (B)Four weeks after the intracardiac injections, the total flux signals in the control group were significantly higher than those in the phenformin group (p = 0.0065). The data are presented as the mean±SEM and the Wilcoxon rank sum test was used to identify significant differences in total flux between the control and phenformin- treated animals.