Effects And Mechanism Of Atmospheric Pressure Plasma On Murine Melanoma Cells

Cold atmospheric plasma(CAP)is generated by ionizing neutral gas molecules/atoms at ambient temperature,leading to a highly reactive gas in which excited molecules,reactive species and transient electric fields exist.Because of its potential to interact with tissue or cells without a significant temperature increase,CAP appears as a promising approach for the treatment of various diseases including wound healing,sterilization,blood coagulation,and concer treatment.Although the increasing evidences suggest that CAP induces various cancer cells to die and offers a promising alternative treatment,the mechanism of its therapeutic effect is little understood.Therefore this study will focus on the interaction of CAP with B16 cell line at a molecular level.The major results are obtained as follows:(1)The effect of CAP on the growth of various tumor cell lines was investigated.Microscope was used to observe the effects of the plasma treatment on cell morphology,and MTT assay was used to detect the anti-proliferation effect of CAP treatment on HepG2,B16,Hela,MCF-7 and epidermal cells of mice.The results showed that cells became shrinkage after CAP treatment and CAP could inhibit cell proliferation of these different cell lines.Additionally,the anti-proliferation effect of plasma treatment was much weaker on mouse subcutaneous cells than B16 cell line.(2)The mechanism of CAP-induced apoptosis in B16 cells was studied.The PI staining and AnnexinV/PI binding assay were used to detect apoptosis in B16 cells.Nitric reductase sand DCFH probe was applied to detect the changes of the intracellular nitric oxide(NO)and reactive oxygen species(ROS)level,and JC-1 probe was then used to measure the mitochondrial membrane potential.The cytochrome C level and Bcl-2/Bax evpression in B16 cells were also investigated.The results showed that the CAP treatment could induce B16 cells apoptosis in dose-dependent manner.The mitochondrial apoptotic pathway was activated,resulting in the loss of mitochondria membrane potential,increasement of ROS and NO level,and release of cytochrome C.Furthermore,the CAP treatment also suppressed the expression of Bcl-2 significantly,and increased the expression of Bax at the same time.(3)The mechanism of CAP affecting B16 cell cycle was studied by PI staining and flow cytometry analysis.The expression of genes and proteins related to cell cycle was analyzed by RT-PCR and Western blotting.The results showed that the CAP treatment could induce apoptosis of B16 cells associated with arrest cell cycle in G2/M phase.The G2/M phase arrest was related to down-regulation of cyclin B1 at both mRNA level and protein level.