Phenformin induces epithelial features in breast cancer cells.

<p>(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.</p

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

<p>(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.</p

Phenformin inhibits MDA-MB-231 cells migration.

<p>(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.</p

Activation of AMPK/mTOR/p70s6k signaling by phenformin.

<p>(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.</p

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

<p>(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.</p

Phenformin induces cell death of breast cancer cell.

<p>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%.</p

Phenformin promotes breast cancer apoptosis.

<p>(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.</p

Inhibitors of Mutant Isocitrate Dehydrogenases 1 and 2 (mIDH1/2): An Update and Perspective

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are homodimeric enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) in the tricarboxylic acid cycle. However, mutant IDH1/2 (mIDH1/2) reduces α-KG to the oncometabolite 2-hydroxyglutarate (2-HG). High levels of 2-HG competitively inhibit the α-KG-dependent dioxygenases involved in histone and DNA demethylation, thereby impairing normal cellular differentiation and promoting tumor development. Thus, small molecules that inhibit these mutant enzymes may be therapeutically beneficial. Recently, an increasing number of mIDH1/2 inhibitors have been reported. In this review, we summarize the molecular basis of mIDH1/2 and the activity, binding modes, and progress in clinical application of mIDH1/2 inhibitors. We note important future research directions for mIDH1/2 inhibitors and discuss potential therapeutic strategies for the development of mIDH1/2 inhibitors to treat IDH1/2 mutated tumors

Multivariate analysis of CIA.

<p>OR, odds ratio; CI, confidence interval.</p

Univariate analysis using Cox proportional risk modeling in patients with IDC.

<p>HR: hazard ratio; 95%CI: 95% confidence interval; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2.</p