INCREASE OF GAMMA RADIATION-INDUCED DNA DAMAGE BY MAGNETIC NANOPARTICLES IN HUMAN MELANOMA CELLS

IBAÑEZ, IRENE L.; NEGRIN, LARA M.; MUNICOY, SOFÍA; BELLINO, MARTÍN G.; MOLINARI, BEATRIZ; DURÁN, HEBE

Congreso; XII Congreso Argentino de Física Médica, I Congreso de Física Médica de las Américas y I Congreso de Medicina Nuclear y Diagnóstico por Imágenes de la AATMN; 2014

Sociedad Argentina de Física Médica (SAFIM), Asociación Argentina de Técnicos en Medicina Nuclear y American Association of Physicist in Medicine

I.L.I y L.M.N. han contribuido de manera equitativa a este trabajo. Introduction: The incidence of melanoma has substantially increased worldwide over the last years and it is responsible of the 80% of deaths from skin cancer. The poor outcome of this disease is associated with the resistance of melanoma to conventional cancer therapies such as radio- and chemotherapy. In recent years, the evolution in the field of nanomaterials science has allowed to generate, characterize and manipulate particles at the nanometric scale (1-100 nm). One type of nanoparticle that has attracted significant interest is the Fe3O4 magnetic nanoparticle which can be manipulated under the influence of an external magnetic field. These nanoparticles have high biocompatibility, chemical stability in physiological media and are relatively easy to produce. Moreover, magnetic nanoparticles can increase the intracellular levels of reactive oxygen species (ROS). The additional oxidative stress induced by exogenous agents, such as ionizing radiation, can saturate the intracellular antioxidant capacity and impair the redox balance of cancer cells, leading to selective tumor cytotoxicity, causing damage, in particular at the DNA level. Objectives: The aim of this study was to evaluate the effects of different doses of gamma radiation on the induction and rejoining of DNA double-strand breaks (DSBs) in human melanoma cells treated with Fe3O4 magnetic nanoparticles. Material and Methods: In order to evaluate the cytotoxicity of different concentrations of Fe3O4 magnetic nanoparticles (10-800 μg/ml) in A-375 human melanoma cells, cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) growth assay. ROS induction capacity of Fe3O4 magnetic nanoparticles was assessed by the 2?,7?-dichlorodihydro-fluorescein diacetate (DCFH-DA) assay. Intracellular levels of ROS in cells treated with the lower concentration of nanoparticles that did not induce cytotoxicity were evaluated. The formation of DNA DSBs generated by the combined treatment of gamma radiation (0.5-2 Gy) and magnetic nanoparticles in melanoma cells were determined by the immunocytochemical detection of γ-H2AX foci 30 min post-irradiation. The local formation of γ-H2AX allows microscopic detection of distinct foci that most likely represent a single DSB. The rejoining of DNA DSBs ability was evaluated 24 h post-irradiation. Results: A significant decrease in proliferation rate was observed in A-375 cells treated with low concentrations of magnetic nanoparticles (10 and 20 μg/ml), whereas a significant increase in cell proliferation rate was observed after treatment with high concentration of nanoparticles (200 and 800 μg/ml). Taking into account these results, a 40 μg/ml concentration of magnetic nanoparticles was selected for use in the following assays, considering that this dose did not induce differences in cell growth as compared with non-treated control cells. However, the treatment of melanoma cells for 24 h with this concentration of magnetic nanoparticles increased intracellular levels of ROS as compared with non-treated cells. DNA DSBs analysis exhibited a significant increase in the number of γ-H2AX foci 30 min post-irradiation when cells were treated with the combination of both treatments (gamma radiation and magnetic nanoparticles) as compared with cells exposed to gamma radiation alone. The diminution in the number of γ-H2AX foci 24 h post-irradiation was observed in cells treated with gamma radiation alone for all doses. On the other hand, cells treated with the combination of 1 Gy of gamma radiation and magnetic nanoparticles showed non-significant differences in the number of γ-H2AX foci between 30 min and 24 h post-irradiation. Besides, an increase in the area of γ-H2AX foci from 0.44 to 0.97 μm2 was observed 24 h post-irradiation, showing non-significant differences between treatments at the same time. Conclusions: These results allow us to perform a first evaluation on the use of the combined therapy with gamma radiation and magnetic nanoparticles for human melanoma, showing a promising improvement in the radiation treatment by the addition of nanoparticles. Taking into account the magnetic properties, the biocompatibility and the ability to increase intracellular ROS levels that Fe3O4 nanoparticles show, further studies are required to evaluate the capability of these particles as radiosensitizer agents.