Figure 1.
Metformin promoted antiproliferative activity in MCF-7 cells.
(A) MCF-7 cells were treated with metformin (2.5, 5, 10, 20 mM) for 24, 48 and 72 hours and cell proliferation was analyzed using the MTT assay. Data was expressed as the number of viable cells compared with the control. (B) MCF-7 cells were treated with 10 mM metformin for 24, 48 and 72 hours and cell proliferation was analyzed by Trypan blue exclusion assay. Data was expressed as the number of viable cells compared with the control. (C) MCF-7 cells were treated with 10 mM metformin for 48 hours. Cell proliferation was determined by measuring BrdU incorporation using flow cytometry. (D) MCF-7 cells were treated with 10 mM metformin for 24, 48 and 72 hours. DNA cell cycle was analyzed by propidium iodide staining and measured by flow cytometry. Sub G1 phase, G0 – G1 phase, S phase, and G2-M phase were analyzed using flow cytometry. Results were reported in Table 1. * p<0.05 vs. control, ** p<0.001 vs. control, *** p<0.0001 vs. control.
Table 1.
Metformin promoted cell cycle arrest and apoptosis.
Figure 2.
Metformin increased cell death and caspase activity in MCF-7 cells.
(A–C) MCF-7 cells exposed to 10 mM metformin for 48 and 72 hours were analyzed with flow cytometry. Results were expressed as percentage of PI positive cells. (D–F) Apoptosis was determined by measuring active caspase-3 and -7 positive MCF-7 cells after 48 and 72 hours of treatment using flow cytometry. Results were expressed as mean fluorescence arbitrary units (AU). (G–H) MCF-7 cells were treated with 10 mM metformin for 24 and 48 hours. Apoptosis was determined by Annexin V propidium iodide staining and measured by flow cytometry. Apoptotic cells were measured as percentage of AV+/PI- stained cells. ** p<0.001 vs. control, *** p<0.0001 vs. control.
Figure 3.
Metformin decreased the activation of insulin signaling pathway.
(A) A western blot of IRβ, p-IRβ (Tyr 1162/1163), Akt, p-Akt1/2/3 (Ser 413), ERK1/2 and p-ERK1/2 (Thr 202/Tyr 204) with β-actin as the control band. (B) Western blot ratio analysis of p-IRβ (Tyr 1162/1163) and IRβ, (C) p-Akt1/2/3 (Ser 413) and Akt and (D) p-ERK1/2 (Thr 202/Tyr 204)and ERK1/2 in MCF-7 cells after treatment with metformin (10 mM) for 24, 48 and 72 hours. * p<0.05 vs. control; ** p<0.001 vs. control.
Figure 4.
Metformin increased AMPK activity in MCF-7 cells.
(A) A western blot of AMPKα, p-AMPKα (Thr 172), p70S6K and p-p70S6K (Thr 389) with β-actin as the control band. (B) Western blot ratio analysis of AMPK and p-AMPK (Thr 172), (C) p70S6K and p-p70S6K (Thr 389) in MCF-7 cells after treatment with metformin (10 mM) for 24, 48 and 72 hours. * p<0.05 vs. control; ** p<0.001 vs. control and *** p<0.0001 vs. control.
Figure 5.
Metformin induced cell apoptosis associated with FOXO3a.
(A) Real Time PCR ratio analysis of mRNA levels of FOXO3a after treatment with metformin (10 mM) for 48 and 72 hours in MCF-7 cells. (B) Western blot of p-FOXO3a (Ser 253), p-FOXO3a (Ser 413), FOXO3a and β-actin from MCF-7 cells after treatment with metformin (10 mM) for 24, 48 and 72 hours. (C) Western blot ratio analysis of p-FOXO3a (Ser 253) and FOXO3a, (D) p-FOXO3a (Ser 413) and FOXO3a, and (E) FOXO3a and β-actin. (F) Real Time PCR analysis of mRNA levels of cyclin D1 (CCND1 gene), and (G) p27 (CDKN1B gene), after treatment with metformin (10 mM) for 48 and 72 hours. * p<0.05 vs. control; ** p<0.001 vs. control and *** p<0.0001 vs. control.
Figure 6.
Metformin increased cell apoptosis as measured by p27, Bax, Bcl-2 and cleaved caspase-3 in MCF-7 cells.
(A) Western blot of p27 and β-actin from MCF-7 cells that were treated with metformin (10 mM) for 24, 48 and 72 hours. (B) Western blot ratio analysis of p27 and β-actin. (C) Western blot of Bax, Bcl-2 and β-actin from MCF-7 cells that were treated with metformin (10 mM) for 24, 48 and 72 hours. (D) Western blot ratio analysis of Bax and β-actin, (E) Bcl-2 and β-actin, and (F) Bax and Bcl-2 at 24, 48 and 72 hours. (G) Western blot of cleaved caspase-3 and β-actin from MCF-7 cells that were treated with 10 mM metformin for 24, 48, and 72 hours. (H) Western blot ratio analysis of cleaved caspase-3 and β-actin at 24, 48, and 72 hours. * p<0.05 vs. control; ** p<0.001 vs. control.
Figure 7.
Antiproliferative effect of metformin was associated with oxidative stress and apoptosis.
(A–C) MCF-7 cells were treated with metformin (10 mM) for 48 and 72 hours and ROS was determined by the CM-H2DCFDA assay, and measured by flow cytometry. (D) MCF-7 cells were treated with metformin (10 mM) for 48 hours, and then treated with H2O2, (1 mM) for an additional 4 hours and cell viability was analyzed. (E) Production of H2O2 by MCF-7 cells after treatment with metformin for 24 hours (10 mM) or metformin (10 mM) + catalase (20 µg mL−1). * p<0.05 vs. control; *** p<0.0001 vs. control; # p<0.001 vs. H2O2 and & p<0.0001 vs. metformin.
Figure 8.
Antiproliferative effect of metformin was associated with oxidative stress and p-p38 MAPK activation.
(A) MCF-7 cells were treated with metformin (10 mM) or metformin + antioxidant enzymes (apocynin, SOD or catalase) for 48 hours and percentage of dead cells was determined by Trypan blue exclusion assay. (B–C) Western blot and western blot ratio analysis of p38 and p-p38 MAPK (Thr 180/Tyr 182), and (D–G) Catalase, MnSOD and CuZnSOD in MCF-7 cells after treatment with metformin (10 mM) for 24, 48 and 72 hours. * p<0.05 vs. control; ** p<0.001 vs. control; *** p<0.0001 vs. control; # p<0.05 vs. metformin; and ### p<0.001 vs. metformin.
Figure 9.
Metformin demonstrated antiproliferative effect in LLC WRC-256 tumor cells.
LLC WRC-256 cells were treated with metformin (0.5, 1.25, 2.5 and 5 mM) for 24 (A) and 48 (B) hours and cell proliferation was analyzed using the MTT assay. Data was expressed as number of viable cells compared with the control. (C–F) mRNA levels of cyclin D1 (CCND1 gene), pRb (PRB1 gene), p53 (TP53 gene) and p27 (CDKN1B gene), respectively, after treatment with metformin (5 mM) for 48 hours in LLC-WRC-256 breast cancer cells using real time PCR. * p<0.05 vs Control, ** p<0.001 vs Control, *** p<0.0001 vs Control and # p<0.05 vs Metformin 2.5 mM.
Figure 10.
The direct and indirect antiproliferative effects of metformin.
Schematic representation of the effects of metformin on tumor development. The indirect mechanism of action of metformin is mediated by the improvement of insulin sensitivity and decreased insulin levels which consequently decreases tumor growth. The direct mechanism of action of metformin is associated with cell cycle modulation, cell death and up-regulation of tumor suppression genes.