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Int. J. Mol. Sci. 2018, 19, 1107 9 of 17 human alveolar basal epithelial cells to PDT-induced apoptosis. Inter alia the viability assay was used, in contrast to our result MB alone had little effect on cell viability, MB treatment followed by PDT significantly decreased cell viability [57]. According to their results, it was reported that MB was more toxic to leukemia cells then to normal peripheral blood mononuclear cells. This indicates MB to be more toxic in cancer cells than in normal cells [58]. The cytotoxic effect of MB and its derivatives was also proven on murine mammary tumor cell lines, showing a dark toxicity of 7.9% for MB with a concentration of 18.7 μM/L [59]. A cytotoxic effect of MB could also be found on human brain tumor cells suggesting that the treatment of MB may be useful in the therapeutic applications of human brain tumors [60]. In conclusion, our study suggests a significant potential of combined MB-660 nm laser therapy in HNSCC, but MB alone has a remarkable toxicity in HNSCC tumor cell lines. First, the maximal photodynamic effect was assessed. Therefore the best concentration of MB, exposure time of MB, and time of laser exposure were researched. It was considered that the minimal MB exposure time with minimal light exposure with minimal toxicity for tumor cells trough MB alone and the maximal toxicity for tumor cells in combination of MB and light exposure had to be detected to find a reasonable adaptability. Toxicity of MB The American National Institutes of Health and Public Health Service published in 2008 toxicology and carcinogenesis studies of methylene blue trihydrate. Groups of 50 rats received 5, 25, or 50 milligrams of methylene blue trihydrate per kilogram body weight five days per week for two years. Another group of 50 mice received 2.5, 12.5, or 25 milligrams of methylene blue per kilogram of body weight for the same duration. Animals receiving methylcellulose alone served as controls. Exposure to methylene blue caused pancreatic islet tumors in male rats and small intestine tumors in male mice. Malignant lymphomas in male and female mice were possibly associated with methylene blue trihydrate exposure. Methylene blue trihydrate caused blood abnormalities and anemia in male and female rats and mice [61]. Chang and coworker [62] investigated the cytotoxicity of various dyes in corneal endothelial cells in a rabbit model. Structural changes in corneal endothelial cells after dye exposure were evaluated by light microscopy and transmission electron microscopy. MB 0.20% did not induce significant damage to corneal endothelial cells. Significant cytotoxicity was observed with higher dye concentrations, postoperative corneal edema due to endothelial toxicity was caused [62]. Kirszberg and coworker [58] measured the cytotoxicity of MB by MTT assay in erythroleukemic and melanoma lineages, comparing it with normal cells, lymphocytes, and melanocytes. The authors observed that MB was more toxic on erythroleukemic cells than to normal peripheral blood mononuclear cells. The group found that MB was able to inhibit in vitro growth of erythroleukemic cells. The study group concluded MB to be more cytotoxic for tumoral cells and suggests MB to be used as a chemotherapeutic agent [58]. MB exposure at concentration of 160 μM for 6–8 min caused up to 75% loss of cells compared to control (sham, scattered light in neighboring wells). Exposure to a diode laser at 660 nm at a fixed area dose of 95 J/cm2 for 2–8 min resulted in a phototoxic effect on top of MB-toxicity (p < 0.001). Therefore, the best specific effects were achieved with high concentration of MB, and as short treatment time as possible, and with the maximal laser exposure time. Taken the cell viability results together: since MB alone has an intensive, up to 75% toxicity without light exposure, the MB treatment time was considered to keep as short as possible by high MB concentrations, and the maximal light-dependent cell toxicity was chosen, which was by 8 min laser exposure. The final preferred conditions: 4 min MB exposure with the concentration of 160 μM and 8 min laser exposure, allowed up to 95% reduction of the cell viability in comparison to the control, and 41% of this cell loss was due to light specific effects and not to MB only. These optimized conditions were used in clonogenic survival assays resulting in significant reduction of growing clones in both SCC-25 and Detroit 562 cells if MB treatment was combined with 8 min 660 nm diode laser exposure. MB treatment alone has shown a visible reduction of the number of colonies, but it was notPDF Image | Photodynamic Low Level Laser Squamous Cell Carcinoma
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