| Objective: Esophageal cancer is the tenth most common cancer worldwide and the sixth in all cancer-related deaths.China has a high incidence of esophageal cancer,around half of the world’s total cases.More than 85% of Chinese patients are squamous cell carcinoma,which is significantly different from European and American countries in terms of epidemiology and histological types.Radiation therapy plays an important role in the comprehensive treatment of esophageal cancer.Concurrent chemoradiotherapy is the only radical regimen for inoperable locally advanced esophageal cancer,and its effectiveness needs to be improved urgently.Immunotherapy is developing rapidly in advanced esophageal squamous cell carcinoma.There is a synergistic effect between radiotherapy and immunotherapy,and the specific mechanism needs to be further studied.The purpose of this study is: 1.To observe esophageal squamous carcinoma cell pyroptosis and its influencing factors after radiotherapy;2.To explore the underlying mechanism of pyroptosis in esophageal squamous cell carcinoma cells post radiotherapy;3.To evaluate the effect of pyroptosis on radiosensitivity;4.To explore the role of radiotherapy-mediated pyroptosis in anti-tumor immunity.Methods: 1.Observe the pyroptosis phenomenon and influencing factors of esophageal cancer cells after radiotherapy through transmission electron microscope,optical microscope,flow cytometry,LDH release detection,and pyroptotic executive protein activation.2.Through WB(Western Blot)experiments and the effects of silencing and overexpression of the pyroptotic executive protein GSDME on the pyroptotic phenotype after radiotherapy to clarify the executive protein of pyroptosis after radiotherapy;3.Application of caspase-3 inhibitor Z-DVED-FMK and silencing caspase-3,observing the activation of pyroptosis executive protein after application of ROS inhibitor NAC,and exploring the pathway mechanism of pyroptosis induced by radiotherapy in esophageal squamous cell carcinoma cells;4.Observing pyroptosis after silencing GSDME and caspase-3 The effect of pyroptosis on the radiosensitivity of esophageal squamous cell carcinoma cells;5.the pyroptotic KYSE-150 cells after radiotherapy were co-cultured with the macrophages induced by THP-1,and the macrophages were detected by flow cytometry.Changes of cell-related markers to explore the effect of pyroptosis on the polarization of tumor-associated macrophages and anti-tumor immunity.Results: 1.The esophageal squamous cell carcinoma cell line KYSE-150 has a tendency of pyroptosis after radiotherapy observed by transmission electron microscope.2.Observing the morphological changes of cells after radiotherapy under an optical microscope,it was found that the cells of two esophageal cancer cell lines KYSE-150 and TE-1 showed different death patterns after radiotherapy,suggesting that different types of death occurred.3.Transcriptome microarray data of 22 esophageal squamous cell lines indicated that esophageal squamous cell carcinoma mainly expressed GSDME in the Gadermin family,and the expression of other gasdermin family proteins was low,and KYSE-150 cell line highly expressed GSDME,while TE-1 Expression of GSDME is relatively low.Differences in the expression of pyroptosis-related genes between cancer and adjacent tissues were calculated of the 179 patients with esophageal squamous cell carcinoma.GSDME was highly expressed in cancer,and the difference was the most significant among all pyroptosis-related genes.4.The results of WB indicated that radiotherapy can activate GSDME protein in a time-dependent manner,and decompose it into the N-terminal with membrane perforation activity to induce pyroptosis,but cannot activate other proteins of the gasdermin family.5.The KYSE-150 cell line and TE-1 cell line were subjected to flow cytometry after radiotherapy.The KYSE-150 cell line with high GSDME expression had pyroptosis after radiotherapy,while the TE-1 cell line with low GSDME expression had apoptosis.6.After radiotherapy,LDH in the culture medium of KYSE-150 cell line and TE-1 cell line both increased,but KYSE-150 cell line was significantly higher than that of TE-1 cell line;7.Silencing GSDME in KYSE-150 cell line,after radiotherapy The results of flow cytometry showed that necrotic cells decreased,apoptotic cells increased,and the release of LDH decreased compared with the NC group,suggesting that the pyroptotic phenotype transformed into apoptosis;GSDME was overexpressed in the TE-1 cell line,and the results of flow cytometry after radiotherapy showed that Necrotic cells increased,apoptotic cells decreased,and LDH release increased compared with the Vector group,suggesting that the apoptotic phenotype transformed into pyroptosis,and the expression level of GSDME protein regulated the death phenotype of esophageal cancer after radiotherapy.8.The activation of caspase-3increased after radiotherapy,and the application of caspase-3 inhibitor Z-DVED-FMK and silencing of caspase-3 can inhibit the activation of GSDME,which proves that the activation of caspase-3 is the upstream signal of GSDME activation.9.The application of ROS inhibitor NAC can partially rescue the activation of caspase-3 and GSDME after radiotherapy,indicating that ROS is involved in the occurrence of pyroptosis after radiotherapy.10.After silencing GSDME,the colony formation experiment of KYSE-150 cell line after radiotherapy showed no significant change in radiosensitivity.Silencing of GSDME was accompanied by increased caspase-3 activation.11.Considering that silencing GSDME may cause cells to change from pyroptosis to apoptosis,CASP3 was further silenced,and no significant change in radiosensitivity was found.Flow cytometry demonstraed that the percentage of apoptotic cells decreased,and necrotic cells increased.12.THP-1 cells were used to induce differentiation into macrophages,and the KYSE-150 cell line after radiotherapy was used for co-culture.Flow cytometry showed that the positive ratio of M1 polarization marker CD86 was lower than that without radiotherapy and silence GSDME group added.Conclusion: 1.Cells showed different death patterns after radiotherapy,suggesting that different types of death occurred.2.Radiation therapy induced GSDME-mediated pyroptosis in esophageal squamous cell carcinoma cell line KYSE-150.3.Radiotherapy induced pyroptosis in high expression GSDME cells and apoptosis in low expression GSDME cells.4.The level of GSDME mediates the transformation of pyroptosis and apoptosis in esophageal squamous cell carcinoma cells after radiotherapy.5.Radiotherapy mediates pyroptosis through the ROS-CASP3-GSDME pathway.6.There was no significant change in the radiosensitivity of esophageal squamous cell carcinoma cells after inhibiting the pyroptosis pathway.7.Pyroptosis in esophageal squamous cell carcinoma cells after radiotherapy may regulate anti-tumor immunity by inducing TAM cells to polarize to M1... |
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