Fig 1.
Effect of glucose availability and medium renewal on proliferation of MDA-MB-231 cells.
(A) MDA-MB-231 cells were grown for 72 hours in high-glucose (4.5 g/L), low-glucose (1 g/L) and glucose-free RPMI-1640 (with 10% FBS). Medium was not renewed for 72 hours. Glucose concentrations in media were determined every 24 hours. Results are means±SEM (n = 3). *P≤0.05 vs. 0 hours. (B) MDA-MB-231 cells were grown for 72 hours in high-glucose, low-glucose and glucose-free RPMI-1640 (with 10% FBS). Medium was renewed every 24 hours. Control experiment was carried out in serum-free low-glucose RPMI-1640. Cell number was determined by Hoechst staining. Results are means±SEM (n = 4). *P≤0.05 vs. 0 hours. (C) MDA-MB-231 cells were grown for 72 hours in high-glucose, low-glucose and glucose-free RPMI-1640 (with 10% FBS). Medium was not renewed for 72 hours. Cell number was determined by Hoechst staining. Results are means±SEM (n = 3). *P≤0.05 vs. 0 hours.
Fig 2.
Medium renewal blocks anti-proliferative effects of metformin in cultured MDA-MB-231 cells.
MDA-MB-231 cells were treated with metformin in (A) high-glucose (4.5 g/L), (B) low-glucose (1 g/L) or (C) glucose-free RPMI-1640 (with 10% FBS) for 48 hours. Medium was renewed after 24 hours. Viability was determined by MTS assay. Results are means±SEM (n = 3). *P≤0.05. (D) MDA-MB-231 cells were treated with metformin in low-glucose RPMI-1640 (with 10% FBS) for 24 hours. Proliferation was determined by BrdU assay. Results are means±SEM (one experiment, 8 replicates). *P≤0.05. MDA-MB-231 cells were treated with metformin in (E) low-glucose or (F) high-glucose RPMI-1640 (serum-free) for 48 hours. Medium was renewed after 24 hours. Viability was determined by MTS assay. Results are means±SEM (n = 2). *P≤0.05. (G) MDA-MB-231 cells were treated with 5 mM metfromin for 72 hours in low-glucose RPMI-1640 (with 10% FBS) without medium renewal. Glucose concentrations in media were determined every 24 hours. Results are means±SEM (n = 3). *P≤0.05 vs. 0 hours. (H, I) Different numbers of MDA-MB-231 cells were treated with 5 mM metformin in low-glucose RPMI-1640 (with 10% FBS) for 72 hours with (black) or without (white) medium renewal. Medium was renewed every 24 hours. (H) MTS assay and (I) Hoechst staining were used to estimate viability and cell number. Results are means±SEM (n = 3). *P≤0.05.
Fig 3.
Low concentrations of 2-deoxyglucose modulate metformin-stimulated AMPK activation in MDA-MB-231 cells.
MDA-MB-231 cells were treated with metformin and 600 μM 2-DG for 24 hours in high-glucose (4.5 g/L), low-glucose (1 g/L) and glucose-free RPMI-1640 (with 10% FBS). Western blot was used to measure (A) phosphorylation of AMPK (Thr172) and (B) phosphorylation of ACC (Ser79). Results are means±SEM (n = 4). *P≤0.05.
Fig 4.
Co-treatment with metformin and 2-DG suppresses proliferation of MDA-MB-231 cells with or without medium renewal.
(A) MDA-MB-231 cells were treated with 5 mM metformin and 600 μM 2-DG in low-glucose (1 g/L) RPMI-1640 (with 10% FBS). The experiment was performed with (+) or without (-) medium renewal. Relative number of cells at each time point was determined by Hoechst staining. Results are means±SEM (n = 3). *P≤0.05.
Fig 5.
Low concentrations of 2-DG and metformin reduce viability of MDA-MB-231 cells synergistically.
(A) MDA-MB-231 cells were treated with metformin and 600 μM 2-DG for 72 hours in low-glucose (1 g/L) RPMI-1640. Medium was renewed daily. Viability was determined by MTS assay. Results are means±SEM (n = 3–4). *P≤0.05. (B) MDA-MB-231 cells were treated with metformin and 600 μM 2-DG for 72 hours in low-glucose RPMI-1640. Medium was renewed daily. Cell number was determined by Hoechst assay. Results are means±SEM (n = 3–4). *P≤0.05. (C) MDA-MB-231 cells were treated metformin and 600 μM 2-DG in low-glucose RPMI-1640 for 24 hours. Proliferation was determined by BrdU assay. Results are means±SEM (n = 3). *P≤0.05.