Figure 3. Induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells.

Apoptosis was evaluated by FACS analysis, after cell labeling with propidium iodide (PI) and FITC-Annexin V. MCF-7 (a) and MDA-MB-231 (b) cells were both unlabeled and untreated (CTR−), labeled and not treated (CTR + ), treated with DoHuRu/POPC or with DoHuRu/DOTAP for 48 and 72 h using IC₅₀ concentrations, as indicated. The lower left quadrants of each panels show the viable cells, which exclude PI and are negative for FITC-Annexin V binding. The upper left quadrants contain the non-viable, necrotic cells, negative for FITC-Annexin V binding and positive for PI uptake. The lower right quadrants represent the cells in early apoptosis, that are FITC-Annexin V positive and PI negative. The upper right quadrants represent the cells in late apoptosis, positive for both FITC-Annexin V binding and for PI uptake. The experiments were performed at least three times with similar results. Quantitative analysis of viable, non-viable (necrotic), early and late apoptotic MCF-7 (c) and MDA-MB-231 (d) cells after 48 and 72 h of treatments are shown. Data are expressed as percentage of untreated control cells and are reported as mean of four independent experiments ± SEM (n = 24); ***p < 0.001 vs. control (untreated cells). (e) DNA fragmentation assay on MCF-7 and MDA-MB-321 cells treated or not (C) with IC₅₀ concentrations of DoHuRu/POPC (Ru^POPC) and DoHuRu/DOTAP (Ru^DOTAP) for 48 h, and with IC₅₀ doses (17 and 19 μM, respectively) of cisplatin (cDDP) as the positive control for DNA fragmentation. After incubation, the DNA was extracted and visualized on 1.5% agarose gel as detailed in the Methods section. The lane in the middle corresponds to the molecular weight markers. The agarose gel is representative of three independent experiments.