Research On The Pyroptosis Induced By Atmospheric Pressure Cold Plasma In Tumor Cells

Plasma,the fourth state of matter,consists of positive and negative ions,electrons,neutral particles,ultraviolet,visible light,etc.Cold atmospheric plasma(CAP),produced at normal atmosphere,has strong biological effects which have proved in the previous studies,and CAP temperature is close to room temperature.In the past decade,CAP has been widely used in the application and studies of sterilization,wound healing,skin and oral disease treatment,especially cancer treatment.The most notable advantage is that CAP can"selectively"kill tumor cells with less damage to normal tissues.Previous studies have shown that CAP caused significant increase of reactive oxidative species(ROS),results in DNA damage and mitochondria damage,then initiates apoptosis.GSDME(Gasdermin E),as one of the potential tumor suppressor genes,belongs to the gasdermin superfamily.Previous reports have shown that the expression of GSDME in some tumor tissues is lower than that in normal tissues due to epigenetic silencing through promoter methylation.GSDME can be cleaved by Caspase-3 to generate a necrotic GSDME-N fragment,targeting to plasma membrane and permeabilizing it by forming GSDME pores,then mediated pyroptosis caused by chemical or biological factors.Upregulation of GSDME expression increases the chemosensitivity of tumors.Furthermore,GSDME also cooperates with p53 to participate in DNA damage stress in tumor cells.Pyroptosis,a form of programmed cell death(PCD),is discovered in recent years and is different from apoptosis in morphology and mechanism.Although we know that CAP kill tumor cells mainly via inducing apoptosis,whether CAP can also induce pyroptosis has not been reported.In this thesis,tumor cell pyroptosis-induced by CAP was investigated.The relationship between the basal level of GSDME protein in various tumor cell lines(lung,gastric and liver cancers)and the CAP sensitivity was explored,and then the pattern and possible mechanism of CAP inducing pyroptosis was studied.The dielectric barrier discharge device(DBD)was performed to generate CAP and the working gas was helium.The main research results consists the following three parts:Part I.the relationship between GSDME expression and the sensitivity of tumor cells to CAP was studied.The results showed that the basal protein levels ofGSDME were significantly different in the adopted tumor cell lines derived from three types of cancer.A dose dependent manner of cell viability after CAP treatment was observed based on the experiments performed with representive cell lines with relative high or low expression GSDME.Moreover,compared to the cell lines with low GSDME expression,the cells lines with relative high GSDME expression are more sensitive to CAP than those with low expression,and these results indicated that the basal levels of GSDME protein in tumor cells have tight relationship with CAP sensitivity.Part ?.The pattern of CAP-induced pyroptosis in tumor cells was explored.The detection of pyroptosis,marked by morphology,GSDME cleavage,lactate dehydrogenase release,apoptotic/necrotic cell ratio,indicated that CAP treatment effectively induced pyroptosis in a dose-dependent manner.Furthermore,the cell lines with relative high GSDME level were observed prone to occur pyrotosis after the same dose CAP treatment,comparing with those with low GSDME level.These results suggested that the pyroptosis occurance-induced by CAP associated tightly with the basal protein level of GADME in tumor cells.Part III.The possible mechanism of CAP-induced pyroptosis was explored.Knocking down GSDME significantly decreased pyroptosis level even GSDMD was found to express in low level.Inhibiting Caspase-3 activity with Caspase inhibitor blocked the cleavage of GSDME and decreased the level of CAP-induced pyroptosis effectively.Then the activation of Caspase-9,the upstream of Caspase-3,was also observed.Scavenge of ROS also attenuated the pyroptosis,GSDME cleavage and Caspase-3 activation distinctly.These results suggested an axis of ROS-Caspase-3-GSDME-pyroptosis in tumor cells after CAP treatment.CAP,as one physical factors but not chemical or biological ones,inducing GSDME mediated pryoptosis and possible mechanism was firstly revealed in this study.Tumor cell pyroptosis consistent to the basal level of GSMDE give a hint,targeting GSDME to enhace curative effect,in cancer CAP therapy in the future.