Sensitivity Of Murine Melanoma Cells B16 To Plasma Needle Treatment

Abstract

Melanoma is the most dangerous form of skin cancer originated from the pigment-producing melanocytes in the basal layer of the epidermis. Its capability to produce metastasis, along with the development of quimio or radioresistance makes this kind of cancer very dangerous and hard to heal, so is important an early detection as well as the search of new treatments. B16 murine cell line has been used extensively as a model for the study of this type of cancer. Non thermal plasma needle has been effectively used in the inactivation of microorganisms, in the disinfection of several types of tissues and to accelerate the wound healing processes. It also has been tested as an inductor of apoptosis in HepG2 cancer cell line. The observed effects of plasma upon cells has been attributed to the generation of reactive oxygen/nitrogen species (RONS), which can disrupt the plasmatic membrane and react with several biomolecules including DNA. The aim of the present work is to evaluate the sensitivity of the murine melanoma cell line B16 to a helium-generated plasma needle exposure.

B16 cells were grown at 37°C and 5% CO2 atmosphere, in minimal essential medium with 10% FBS.

Cells were harvested by tripsinization, washed twice with Hanks balanced saline solution and further incubated for at least one hour in MEM at 37°C to recover. Blood samples were obtained from healthy donors by venopunction and lymphocytes were separated by using the Ficoll Hypaque technique. Nucleated cells were collected, washed twice with HBSS, resuspended in RPMI-1640 with 10% FBS and kept at 37°C for at least one hour. Both cell types were resuspended in HBSS and then 200 µl aliquots were distributed in a microwell plate and exposed to plasma generated by a flow of 0.7 LPM of helium through a 13.56 MHz radiofrequency generator at a power of 5 W. The needle was kept at a distance of 2 mm. Cell death was evaluated by the fluorescein diacetate technique. After treatments, cells were stained with a 1:1 fluorescein diacetate (80 µg/ml) and ethidium bromide (50 µg/ml) solution and observed with an epifluorescence microscope. Living cells are stained in green while dead cells are stained in red. Genotoxicity was assessed by means of the comet assay. After treatments, cells were mixed with an equal volume of 1% low melting point agarose, poured on top of fully frosted microscope slides and immerse in cold lysis solution for an hour. Slides were then transferred to an electrophoresis cell, covered with the electrophoresis solution (0.3M NaHO, 0.1 mM Na2EDTA) for 20 minutes to allow DNA unwinding and then a current was applied (20 V, 300 mA, 20 minutes). Slides were stained with 60 µl of a 20 µg/ml ethidium bromide solution and observed under an epifluorescence microscope. Comets were scored by means of the Comet Assay IV Analyzer (Perceptive Instruments Inc.). The results show that the exposure to plasma produced cytotoxicity and genotoxicity on B16 cells in a dose-dependent manner. It was observed that the effect of non-thermal plasma was higher in cells of melanoma of mouse comparing to the effect observed in the lymphocytes.