Study Of Apoptosis Induced By Nonsecond Steep Pulsed Fileds Through Extrinsic Apoptosis Pathway In SKOV3Cells And The Safety Of Treatment Suboutaneous Transplantation Tumor

Following the reversible electroporation are adjuvant to chemotherapy drug therapy for cancer and energy controllable steep pulse witch is base on irreversible electroporation directly kill cancer cell, nonsecond plused electric filed (nsPEF) witch is base on intracellular electromanipulation (IEM) becomes gradually well know.It is not damaging cell membranes but to activate the cell membrane signal transduction pathway or to change the structure or function of subcellular organelles, inducing of apoptosis, so as to achieve the goal of eradication of tumor cells.A large number of studies confirm that nsPEF could induce cell apoptosis.Apoptosis is the cell under certain physiological or pathological conditions,follow the established procedures of programmed cell death process. Classic apoptosis pathway inducing endogenous(mitochondrial) apoptosis pathway and extrinsic apoptosis pathway.Fas-mediated apoptosis pathway plays an important role in the endogenous(mitochondrial) apoptosis pathway.For the nsPEF induced apoptosis mainly in the study of mitochondrial apoptosis pathway in vitro and effectiveness of the treatment in vivo.Our group test confirmed nsPEF able to activate the apoptotic pathway of ovarian cancer SKOV3cells endogenous (mitochondrial) apoptotic pathway and endoplasmic reticulum.Therefore, this research is to study the follow two aspects:1、 Induction of apoptosis of ovarian cancer cells and influence on Fas-mediated apoptosis pathway by nanosecond pulsed electric field;2、 Safety evaluation of nonsecond plused electric filed in treatment of subcutaneously transplanted tumor.PartⅠ Induction of apoptosis of ovarian cancer cells and influence on Fas-mediated apoptosis pathway by nanosecond pulsed electric fieldObjective:To investigate the ovarian cancer SKOV3cell apoptosis effect induced by nonsecond plused electric fileds (nsPEF) and the influence of Fas-mediated apoptosis.Methods:SKOV3cell were exposed to the45KV/cm,50ns、100ns、200ns,30pulses. Flow cytometry were used to assay apoptosis. Agarose gel electrophoresis was used to detect DNA ladder.Real time PCR(RT-PCR) and Western blot analysis was used to measure the level of Fas、FasL、Caspase-8and Bid expression.Results:Compared with the control group, Flow cytometry results revealed that the late apoptosis rates and (or) necrosis were significantly higher than those in control group (3.03%±0.57%)(p<0.05), with apoptosis rates and (or) necrosis being (18.31%±0.65%)、(45.55%±3.71%)、(47.47%±7.01%) in the groups of50ns、100ns、200ns, respectively. A typical DNA ladder pattern of internucleosomal fragmentation was observed in the groups of50ns、100ns, but200ns not clear. RT-PCR revealed that the mRNA expression of Fas、FasL、 Caspase-8and Bid were significantly increased in groups of50ns、100ns, but significantly decreased in group of200ns (p<0.05). Meanwhile,Western blot analysis demonstrated that the Fas、FasL、 Caspase-8and Bid expression were significantly higher in groups of50ns、100ns,but significantly decreased in group of200ns (p<0.05).Conclusion:45KV/cm,50ns、100ns nonsecond plused electric fileds can induce apoptosis in ovarian cancer SKOV3cells and Fas-mediated apoptosis pathway is activated. Part Ⅱ Safety evaluation of nonsecond plused electric filed in treatment of subcutaneously transplanted tumorObjective:To evaluate the safety of nonsecond plused electric filed (nsPEF) in the treatment of subcutaneously transplanted tumor.Methods:The models of human melanoma A375cell were established in32BALB/c female nude mice,and all animals were divided into two groups randomly:the nsPEF group(n=10)and the control group(n=10).nsPEF group tumors were exposed to the40KV/cm,200ns,1Hz,1000pulses.general condition and survival time after nsPEFs treatment were monitored.Measurement of the tumor temperature instantly change before and after nsPEF treatment. Comet assay was used to detect the normal tissues surrounding the tumor.The Pathological of the liver and kidney were detected by HE staining.Results:Compared with the control group, nude mice generally in good condition,processing at the untreated skin and the formation of ulcers can heal itself, The mean survival time was significantly longer in nsPEF group(60.6±6.9d) than in control group(17.7±5.4d)(P<0.01). The increase ratio of life span reached up to242%. We directly measured this temperature increase a very small by thermocouple.No comet tailing was observed,indicating that no DNA lesions after nsPEF treatment.The Pathological of the liver and kidney were no significant change.Conclusion:The nsPEF is verified to be safe.