Cerium Oxide Nanoparticles Modulate Cellular Health and Oxidative Stress in Breast Carcinoma Cells

3005 Background: One of the unique strengths of radiation medicine is its ability to induce cytotoxicity, while an associated challenge is its unintended bystander effects. These paradoxical effects are brought about by the radiation interacting directly with the cellular targets or by free-radical mediated oxidative damage. Studies of nanoceria in cells have shown wide ranging effects from radioprotection in lymphocytes to radiotoxicity in leukemia cells. We investigated the effects of cerium oxide nanoparticles on cellular health in the context of its ability to modulate apoptosis and reactive oxygen species in breast carcinoma cells. Purpose: To demonstrate that unmodified cerium oxide nanoparticles modulates apoptosis and reactive oxygen species in breast cancer cells. Methods: MDA MB231 cells were cultured in 24 well plates at a density of 0.1 x 106 cells/well. The cells were treated with 0, 25, 50, 100, or 200 μg/mL cerium oxide nanoparticles, respectively, for 72 hours. The treated samples were labeled with either Annexin V FITC (AV) and Propidium Iodide (PI), or with dihydrorhodamine 123 (DHR123). Samples labeled with AV/PI were imaged under a confocal microscope. The percentage of viable (AV-/PI-), early apoptotic (AV+/PI-), late apoptotic (AV+/PI+), and necrotic cells (AV-/PI+) were assessed by measuring the fluorescence intensities (green BL1-H and red YL2-H) using flow cytometry. Cells treated with 100 μM Cisplatin acted as positive control for apoptosis. DHR123 treated samples were assessed for green fluorescence by flow-cytometry at the FL1-H channel using an excitation wavelength of 488 nm and an emission of 530nm after correcting for autofluorescence. Results: Our studies showed that: 1) There was normal or increased Annexin V green fluorescence in cells not treated or treated respectively with cisplatin while decreased or absent Annexin V was noted on the cell surfaces of samples treated with nanoparticles (Fig. 1). 2) The number of healthy cells increased proportionately along with a decrease in viable, early apoptotic, late apoptotic, and necrotic cells in samples treated with increasing concentration of cerium oxide nanoparticles, in comparison to the untreated and the cisplatin treated samples (Fig. 2). 3) The amount of cells exhibiting the oxidation product rhodamine 123 decreased proportionately in samples treated with increasing concentration of cerium oxide nanoparticles, in comparison to the untreated samples (Fig. 3). Conclusions: Cerium oxide nanoparticles modulated apoptosis and reactive oxygen species in a dose dependent manner in breast carcinoma cells. Fig. 1. Reduced or absent Annexin V is demonstrated on the surface of MDA-MB231 cells treated with cerium oxide nanoparticles. Representative images of untreated cells (A) and Cisplatin treated cells (B) analyzed by confocal live imaging showed increased annexin V on the cell surfaces (bottom panel), while the cells treated with 25, 50, 100, and 200 μg/mL cerium oxide nanoparticles (C-F) showed reduced or absent Annexin V on the cell surface. Top panel shows representative images of Propidium iodide labeled primary and secondary necrotic cells. Fig. 2. Intracellular CeO2 decreased the amount of early apoptotic, late apoptotic, and necrotic cells. Untreated (A) and breast cancer cells treated with cisplatin (B) or 25, 50, 100, and 200 μg/ml nanoceria (C-F) were analyzed with quadrant gating for green fluorescence (Annexin V, FL1-A) and red fluorescence (Propidium Iodide, FL2-A). The percentage of cells exhibiting healthy, early apoptotic, late apoptotic, and necrotic cells decreased in a dose dependent manner in comparison to untreated or cisplatin treated cells. Fig. 3. Intracellular CeO2 decreased the amount of reactive oxygen species in a dose dependent manner. Cells not treated (A) or treated with 25, 50, 100, and 200 μg/ml nanoceria (B-E), along with 10 μM DHR123 was assessed using flow cytometry.