Abstract
Micronucleus assay is a test used to evaluate genotoxic damage in cells, which can be caused by various factors, like ionizing radiation. Interactions between radiation energies and DNA can cause breakage, leading to use chromosomal mutations or loss of genetic material, important events that could be induced in solid tumors to mitigate its expansion within human body. Melanoma has been described as a tumor with increased radio resistance. This work evaluated micronuclei percentages (%MN) in human melanoma cells (SK-MEL-37), irradiated by gamma radiation, with doses between 0 and 16Gy. Cell suspensions were irradiated in PBS by a 60Co source in doses between 0 and 16Gy, and incubated by 48h. Then cell membranes were lysed in the presence of SYTOX Green and EMA dyes, preserving nuclear membranes. Using this method, EMA-stained nuclei could be discriminated as those derived from dead cells, and SYTOX nuclei and micronuclei could be quantified. Micronuclei percentages were found to be proportional to dose, (R2 = 0.997). Only the highest dose (16Gy) could induce statistically significant increase of MN (p<0.0001), although cultures irradiated by 4, 8 and 16Gy showed significant increase of dead cell fractions. Calculation of the nuclei-to-beads ratio showed that 8 and 16Gy could reduce melanoma cell proliferation. Results showed that although cell death and loss of proliferative capacity could be observed on cultures irradiated at lower doses, genotoxic damage could be induced only on a higher dose. Resistance to radiation-induced genotoxicity could explain a relatively high radio resistance of melanoma tumors.
Highlights
INTRODUCTIONMelanoma is classically described as a tumor that exhibits relative resistance to the destructive effects of ionizing radiation both in vitro [9] and in observations from clinical studies [10]
The National Cancer Institute (INCA) estimates about 600,000 new cases of cancer in Brazil in 2016/2017, including those characterized as non-melanoma skin [1], that have high prevalence, but low mortality that represent an object of little relevant study
There is a chance of underestimation of mortality by relativizing its incidence to the total number of cancer in Brazil, but the mortality calculated by the INCA in 2013 approached 25% of the cases of melanoma, an index considered high
Summary
Melanoma is classically described as a tumor that exhibits relative resistance to the destructive effects of ionizing radiation both in vitro [9] and in observations from clinical studies [10] In this way, radiotherapy has lost its importance in the treatment of this type of tumor, a notable resurgence of the defense of the technique has appeared in recent years [11,12,13]. The technique is based on the observation of accumulation of cytoplasmic micronuclei by blocking cytokinesis (CBMN) but without inhibiting karyokinesis of the cell nucleus after DNA duplication In this way, cells treated by a given compound would exhibit two nuclear bodies, being called binucleate cells. The test protocol was useful to detect (radiation-induced) cytotoxicity and changes in cell division, as detected by preferential staining with EMA and by calculating the nuclei-to-bead ratios and proven to be suitable to study clastogenic effects of radiation on in vitro human melanoma model
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