The Effects Of Incubation Temperature On Tumor Cell Survival After Nanosecond Pulsed Electric Fields Treatment And The Mechanism Study

OBJECTIVE:As a major public health problem around the world, part of cancer can be effectively cured by traditional treatment, such as surgery, chemotherapy or radiotherapy in early stage. It is essential to find new therapeutic method to improve survival of those cancer patients. Recent years, a new technology using high pulsed electric field has emerged into biology and medicine by applying nanosecond pulse electric fields (nsPEF) to treat cancer. This study confirmed that the changes of the tumor microenvironment caused by nsPEF treatment contribute to the ablation of tumor. Different incubation temperature were chosen for cells exposed to nsPEF treatment. The relative survival rate, apoptosis rate, generation of reactive oxygen species, changes of mitochondrial membrane potential were deteced in order to investigate the effect of outer temperature on tumor cell survival after nsPEF and the molecular mechanisms. MATERIALS AND METHODS:Experiments in vivo included the observation of infiltration of inflammatory cell insides the tumor on the hepatocellular carcinoma cell line HCCLM3tumor-bearing nude mice and the coagulation embolism of the nsPEF-treated rat liver by HE Staining. In vitro, melanoma B16f10, hepatocellular carcinoma cell line HCCLM3, hepatocellular carcinoma cell line7721, pancreatic cancer cell line Panc-1were incubated in different temperature after nsPEF treatment to investigate the effect of outer temperature on tumor cell survival. After cells were treated by nsPEF and incubated at different outer temperature (ice,4C,25C,37C), the apoptosis rate and24h proliferation rate were detected by flow cytometry and CCK-8assay respectively. The generation of reactive oxygen species and the changes of mitochondrial membrane potential were detected by flow cytometry and microscopy with DCFH-DA and JC-1respectively in further research. Different concentration of sucrose or pre-incubation with NAC were also tested to investigate the effect of osmotic pressure of incubation solution on tumor cell survival.RESULTS:In vivo, there was wide coagulation embolism found in the rat liver after nsPEF treatment.The tumor volume gradually decreased into scar shape(p<0.05),with diffused necrosis of tumor cells and the infiltration of inflammatory cells inside the tumor. In vitro, different incubation temperature had different effects on the relative survival rate. The lower the incubation temperature was, the more sensitive cells were to the nsPEF treatment. The lower the incubation temperature was, the higher the percent of apoptosis cells was. Low temperature nsPEF increased generation of reactive oxygen species and caused decrease in the mitochondrial membrane potential. B16f10cells were the most sensitive cells to the change of incubation temperature,while panc-1cells were the least sensitive cells to the change of incubation temperature. Hyperosmotic medium with sucrose could protect cells treated with nanosecond pulsed electric fields and incubated on the ice by reducing the ROS generation, so did the treatment of pre-incubation with NAC for1h.CONCLUSION:There are three major tumor microenvironment changes that enhance nsPEF ablation effects:the early apoptosis and necrosis of tumor cells, the secondary necrosis of tumor caused by the vascular embolism, the self-destruction induced by the enriched inflammatory cells inside the tumor. Incubation temperature affect tumor cell survival after nanosecond pulsed electric fields through the change of ROS generation. The sensitiveness to the change of incubation temperature depends on the cell type. Hyperosmotic medium with sucrose and the treatment of pre-incubation with NAC could protect cells exposed to nsPEF and incubated on the ice by reducing the ROS generation.