Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells.

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration-dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role

Metformin promoted antiproliferative activity in MCF-7 cells.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) MCF-7 cells were treated with 10 mM metformin for 48 hours. Cell proliferation was determined by measuring BrdU incorporation using flow cytometry. (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098207#pone-0098207-t001" target="_blank">Table 1</a>. * p<0.05 vs. control, ** p<0.001 vs. control, *** p<0.0001 vs. control.</p

Antiproliferative effect of metformin was associated with oxidative stress and p-p38 MAPK activation.

<p>(<b>A</b>) 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>B–C</b>) Western blot and western blot ratio analysis of p38 and p-p38 MAPK (Thr 180/Tyr 182), and (<b>D–G</b>) 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.</p

The direct and indirect antiproliferative effects of metformin.

<p>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.</p

Metformin increased AMPK activity in MCF-7 cells.

<p>(<b>A</b>) A western blot of AMPKα, p-AMPKα (Thr 172), p70S6K and p-p70S6K (Thr 389) with β-actin as the control band. (<b>B</b>) Western blot ratio analysis of AMPK and p-AMPK (Thr 172), (<b>C</b>) 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.</p

Metformin increased cell death and caspase activity in MCF-7 cells.

<p>(<b>A–C</b>) 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. (<b>D–F</b>) 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). (<b>G–H</b>) 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.</p

Metformin demonstrated antiproliferative effect in LLC WRC-256 tumor cells.

<p>LLC WRC-256 cells were treated with metformin (0.5, 1.25, 2.5 and 5 mM) for 24 (<b>A</b>) and 48 (<b>B</b>) hours and cell proliferation was analyzed using the MTT assay. Data was expressed as number of viable cells compared with the control. (<b>C–F</b>) 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.</p

Metformin promoted cell cycle arrest and apoptosis.

<p>MCF-7 cells were treated with metformin (10 mM for 24, 48 and 72 hours). DNA cell cycle was analyzed by propidium iodide staining and measured by flow cytometry. Values expressed in percent.</p><p>*p<0.05 vs. control;</p><p>**p<0.001 vs. control;</p><p>***p<0.0001 vs. control.</p

Antiproliferative effect of metformin was associated with oxidative stress and apoptosis.

<p>(<b>A–C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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.</p