Methylene blue and photodynamic therapy for melanomas: Inducing different rates of cell death (necrosis and apoptosis) in B16-F10 melanoma cells according to methylene blue concentration and energy dose

Abstract

Background: Photodynamic therapy (PDT) is a cancer treatment based on the interaction between the photosensitizing agent methylene blue (MB), light, and molecular oxygen. MB has antibacterial properties and can to bind to melanin. Here, we investigated whether MB based PDT (MB-PDT) could decrease viability and induce death of murine melanoma B16-F10 cells.

Methods: B16-F10 cells were incubated with different concentrations of MB (0, 1, or 2 µg/mL) and exposed to a diode red laser with a wavelength of 660 nm and power output of 100 mW/cm². The energy dose and density varied from 0 J and 0 J/cm² to 100.8 J and 720 J/cm². Cell viability was measured using the trypan blue exclusion assay of cell viability and confirmed by performing an MTT assay. The morphological type and cell death rates were determined using fluorescence microscopy with acridine orange and ethidium bromide. The presence and rate of apoptosis were evaluated via Annexin V-Alexa Fluor/propidium iodide staining and flow cytometry analysis.

Results: MB-PDT decreased cell viability and increased cell death (necrosis and apoptosis) in a drug- and light-dose dependent manner. Morphological characteristics of necrosis were observed immediately after treatment, and apoptotic characteristics were observed after 3 h. The apoptosis and necrosis rates varied with the drug and light doses, with 2 µg/mL MB and a 100.8 J energy dose inducing the highest rates.

Conclusions: We demonstrated that MB-PDT reduced murine melanoma B16-F10 cell viability and induced cell death in a drug- and light-dose dependent manner.

Keywords: Apoptosis; Cancer; Melanoma B16-F10; Methylene blue; PDT , MB.; Photodynamic therapy.