The Therapeutic Effect And Mechanism Of Irreversible Electroporation(IRE) Combined With 5-Fu In Esophageal Cancer

Backgrounds:Esophageal cancer is a common malignant tumor of digestive tract and a large number of people around the world die because of it every year.At present,surgery is still the main form of treatment.For unresectable esophageal cancer,the treatment is combined with radiotherapy and chemotherapy,but the effect is not satisfactory.Other local treatments use ablation to relieve symptoms or to shrink the tumor,to achieve the purpose of reoperation,such as radiofrequency ablation,cryoablation and microwave ablation.However,these methods act mainly by thermal effect and lack of selective to tissues.Irreversible electroporation(IRE)technology is also known as nanoknife,It is a new technique that can effectively ablate tumor.IRE is mainly through the release of high voltage direct current,the use of electrodes to transfer electrical pulses,generate a strong electric field,change the cell membrane potential,the phospholipid bilayer rearrangement,so as to produce permanent nanopores on the cell membrane,affect cell homeostasis,and lead to cell apoptosis.Compared with common ablation procedures,IRE relies on transient high-voltage electric field instead of thermal effect,so it is not affected by heat sinks of large blood vessels and has good tissue selectivity to preserve important structures in the ablated area,such as nerves and large blood vessels.There have been extensive studies on the application of IRE in the treatment of solid tumors such as liver cancer,pancreatic cancer,kidney cancer and prostate cancer,while few studies have been conducted a hollow organ on esophageal cancer.Although IRE has a good effect on many tumors,due to the different distribution of IRE electric field,the field intensity in the center of the electrode is high and the surrounding area is low,which makes it difficult to completely ablate the edge of large tumor tissues.As a result,research into combined IRE and other treatments has increased in recent years.Studies have reported that the combined application of IRE and chemotherapy drugs in the treatment of pancreatic cancer and liver cancer has a much better effect than the use of IRE or chemotherapy drugs alone.However,it is not known whether IRE combined with chemotherapy drugs has a better effect on esophageal cancer.Objects:To verify the effect and safety of IRE for esophageal tissue ablation;to evaluate the killing effect of IRE on esophageal cancer cells and observe the effects of different parameters;to explore the specific mechanism of IRE ablation of esophageal cancer cells;to verify the therapeutic effect of IRE combined with chemotherapy drug 5-Fu on esophageal cancer,and to preliminarily explore its mechanism.Methods:1.The abdominal esophagus of New Zealand white rabbits were treated with IRE by plate electrode,and then samples were collected and paraffin sections were removed for HE staining and Masson staining.The pathological changes of esophageal tissues were observed under microscope.2.The effect of IRE on survival rate of esophageal cancer cells EC109 and KYSE30 under different electric field intensity was detected by MTT assay;the changes of cells after IRE treatment were observed under light microscope;cell apoptosis was detected by Western blot and Annexin V-FITC/PI;the effect of IRE on the proliferation of esophageal cancer cells was evaluated by crystal violet staining;the subcutaneous grafts of esophageal cancer cells EC109 were established in nude mice,and the effect of IRE on esophageal cancer was detected in vivo.3.After mitochondrial staining,the morphological changes of mitochondria in esophageal cancer cells treated with IRE were observed by laser confocal microscope;mitochondrial membrane potential and ROS production were detected by flow cytometry;the effects of IRE on mitochondrial cytochrome c and mitochondrial homeostasis related proteins OPA1,TFAM and Nrf2 were detected by Western blot.4.MTT assay and Annexin V-FITC/PI were used to detect the effect of IRE combined with 5-FU on esophageal cancer cells;the subcutaneous grafts of esophageal cancer cells EC 109 were established in nude mice;the therapeutic effects of IRE combined with 5-Fu were detected in vivo,and the side effects of the grafts on other organs were observed by HE staining.5.Western blot and immunofluorescence were used to detect the protein expressions of LC3 and p62 in esophageal cancer cells after 5-FU treatment alone,IRE treatment alone and IRE combined with 5-FU treatment.Results:1.After IRE treatment,a large number of parenchymal cells died in the esophageal tissue of experimental rabbits,while the collagen fibers were well preserved,and compared with the control group,IRE did not cause esophageal fistula and other adverse events.2.IRE treatment can reduce the survival rate of EC109 and KYSE30 cells,and the degree is proportional to the electric field intensity;after IRE treatment,the morphology of esophageal cancer cells changed,and even fragments were produced;IRE treatment induced apoptosis of esophageal cancer cells and inhibited their proliferation.3.After IRE treatment,mitochondrial morphology of esophageal cancer cells changed,diameter and circumference became shorter,and decreased mitochondrial membrane potential,and the increase in ROS production indicated mitochondrial damage,the release of mitochondrial cytochrome C increased,while the expression of mitochondrial homeostasis related proteins OPA1,TFAM and Nrf2 decreased.4.The apoptosis of EC109 and KYSE30 cells was significantly increased after the application of IRE combined with 5-FU;in vivo results showed that the application of IRE combined with 5-Fu reduced tumor volume and weight in nude mice,and had no adverse effects on other organs and tissues.5.After 5-FU treatment of EC109 and KYSE30,the expression of LC3 increased,while the expression of p62 decreased,inducing protective autophagy.However,IRE can reduce the level of autophagy induced by 5-Fu in esophageal cancer cells.Conclusion:The experimental results in this experiment prove that IRE is safe and effective in the application of esophageal tissue of rabbit,it has a lethal effect on esophageal cancer cells,possibly it affects mitochondrial homeostasis and leads to mitochondrial damage,thus promoting the apoptosis of esophageal cancer cells;moreover,the combination of IRE and chemotherapy drug 5-Fu can exert better therapeutic effect,which may be related to the reduction of autophagy in esophageal cancer cells by IRE.