The Role And Mechanism Of Pyroptosis In X-ray And Anlotinib Therapy For Esophageal Squamous Cell Carcinoma

Background:Esophageal carcinoma(EC)is one of the most common malignant tumors worldwide and ranks seventh and sixth in morbidity and mortality,respectively.Esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC)are the two most common histological types of esophageal cancer,with ESCC being the main type in China.At present,X-ray radiotherapy is still the main treatment method of ESCC,and has achieved remarkable therapeutic effect.However,some patients have low reactivity to radiotherapy,leading to treatment failure.Eventually,local recurrence and distant metastasis of the tumor occurred.Therefore,on the basis of in-depth exploration of molecular mechanisms such as therapeutic efficacy and radioresistance,actively seeking potential sensitization targets and radiotherapy synergies are hot and difficult issues on domestic and foreign research.Pyroptosis is a programmed cell death mode that depends on Caspase and Gasdermin family proteins.It is characterized by cells that expand until their membranes burst,activating the immune system by releasing cytokines.Pyroptosis can lead to immune inflammatory reaction and participate in the genesis and development of tumor.The classical pyroptosis pathway is mediated by inflammasomes with Gasdermin D(GSDMD)cleavage and IL-1βand IL-18 release.In the non classical pyroptosis pathway,the absence of upstream sensory complexes of Caspase-4/5/11 enables these Caspases to be activated by direct binding to intracellular lipopolysaccharides(LPS).Cleaved-Caspase-4/5/11 can also cleave GSDMD,then oligomerize and transfer GSDMD-N to the cell membrane,ultimately forming a plasma membrane pore.In addition to the above two pyroptosis mechanisms,a new GSDME-mediated pyroptosis pathway has been discovered recently:The stimulation leads to cleaved-Caspase-3,which is previously known as a substrate for apoptosis.Caspase-3 induces the cleavage of Gasdermin E(GSDME),and the active GSDME-N terminal is also involved in the formation of plasma membrane pores,which can also lead to pyroptosis.X-ray radiotherapy can cause DNA double-strand breaks and lead to cell death,including apoptosis,necrosis,autophagy,etc.However,the role of pyroptosis in ESCC X-ray radiotherapy has not been reported,which is one of the important directions.Anlotinib is a novel small-molecule multi-target tyrosine kinase inhibitor(TKI)developed in China independently.It can inhibit the receptor of VEGF,PDGF,FGF,c-Kit and other targets,and has the dual effect of anti-tumor angiogenesis and inhibiting tumor growth.Anlotinib monotherapy maintained Class II 2A recommendation,in second-line and above ESCC treatment.Meanwhile,data from a single-arm,multicenter exploratory clinical study(ALTER-E002)showed that patients with advanced ESCC benefit from anlotinib in combination with cisplatin and paclitaxel.However,there are few studies on anlotinib combined with radiotherapy in ESCC treatment.Based on the theory that antiangiogenic drugs can normalize tumor blood vessels,improve the hypoxic microenvironment after radiotherapy and increase the death of tumor cells.Pyroptosis is a mode of immunogenic cell death.Tumor cells with pyroptosis can promote the recruitment of CD8+T cells.By analyzing the RNA-seq data of ESCC in the TCGA database,we found the expression of VEGF,FGF and PDGF was negatively correlated with the degree of infiltration of CD8+T cells.We hypothesized that blocking the above growth factor signals might affect the infiltration level of CD8+T cells by regulating pyroptosis.Anlotinib is a multi-target inhibitor of VEGFR,FGFR and PDGFR,which may enhance the therapeutic effect of ESCC by blocking the above pathways and increasing pyroptosis.Based on the above theories,we speculate that anlotinib combined with X-ray may also have a synergistic effect on ESCC,or reduce the dose of X-ray through the combination of drugs,thereby reducing radiation resistance and side effects.This study focused on exploring the role and mechanism of pyroptosis mediated X-ray or anlotinib in ESCC treatment,as well as the synergic effect of anlotinib in ESCC treatment by enhancing pyroptosis,and achieved expected research results.Methods:1.To explore the relationship and the mechanism between X-ray and pyroptosis in ESCC.1.1 Detection of pyroptosis caused by X-ray:The effects of X-ray treatment on pyroptosis were evaluated by optical microscopy under time gradient and dose gradient,and verified by flow cytometry and lactate dehydrogenase(LDH)detection.The perforation of cell membrane after radiation treatment was detected by transmission electron microscopy.1.2 Detection of basic expression of GSDMs and activation after radiotherapy:The Cancer Genome Atlas(TCGA)database was used to search the expression of Gasdermin family proteins in esophageal cancer,and Western blot was used to verify the expression of Gasdermin family proteins in ESCC cell lines.Western blot was used to detect the activation of GSDMD and GSDME with X-ray irradiation after 96 h.1.3 To verify the effect of GSDME on pyroptosis of ESCC cells after radiotherapy:GSDME overexpression and knockdown cell lines were constructed,and the effect of GSDME expression level on pyroptosis of ESCC cells was detected by Western blot and optical microscope after 10 Gy X-ray irradiation.The activation of GSDME under time gradient and dose gradient was further detected by Western blot.1.4 Detection of the influence of GSDME expression level on radiosensitivity of ESCC:Based on colony formation assay under dose gradient,survival curve was drawn by clicking multi-target model to detect the influence of GSDME on radiosensitivity.The subcutaneous transplanted tumor model of Ctrl-Eca109 or shGSDME-Eca109 nude mice was constructed using BALB/c nude mice.X-ray treatment was performed to calculate the tumor volume and plot the growth curve to study the effect of GSDME on the growth of transplanted tumors.Ki-67 proliferation and DNA damage were detected by IHC to verify the effect of GSDME on radiosensitivity in vivo.1.5 Clinical data and specimen collection:Biopsy pathological specimens and clinical case data of advanced ESCC patients who could not tolerate chemotherapy and chose radiotherapy alone before treatment were collected.The expression of GSDME was evaluated by immunohistochemistry(IHC).Chi-square test was used to determine the correlation between radiotherapy effect and GSDME expression in ESCC patients after 3 months of treatment.Kaplan-Meier curve evaluated the effects of GSDME on Overall Survival(OS)and Progression-free survival(PFS).1.6 Identification of upstream Caspase of GSDME:After the application of Caspase-3 specific inhibitor Z-DEVD-FMK,the influence on pyroptosis of cells was detected,and the activation of Caspase-1,Caspase-4,Caspase-5,Caspase-7 and Caspase-9 was further detected by Western blot.According to the activation of Caspase protein,Caspase-3 knock-down cell line was constructed.After X-ray treatment,Western blot and LDH were used to further verify the upstream regulatory gene of GSDME.2.To explore the role of pyroptosis in anlotinib combination with radiotherapy for ESCC.2.1 Analysis of target-related factors of anlotinib therapy in TCGA database:RNA-seq data of ESCC were obtained from TCGA data,and a single correlation chart was realized by R software package ggstatsplot,and Spearman’s correlation analysis was used to describe the correlation.2.2 Detection of the effect of anlotinib on the biological function of ESCC cells:CCK-8 cell proliferation curve and EdU assay were used to detect the effect of anlotinib on the proliferation of ESCC cells.Transwell assay and Western blot assay were used to detect the expression of EMT-related molecules to evaluate the effect on invasion and metastasis of ESCC.2.3 The synergistic effect of anlotinib on ESCC radiotherapy in vitro:ESCC cells were divided into four groups:control group,anlotinib treatment group,X-ray treatment group and anlotinib combined X-ray treatment group.After different treatments,colony formation assay used to detect cell proliferation,and DNA damage indicators were detected by immunofluorescence and Western blot.Based on the results of cell proliferation inhibition,Bliss independent model was constructed to evaluate the synergistic effect of anlotinib which was combined with radiotherapy in vitro.2.4 Establishment of subcutaneous transplanted tumor model of ESCC nude mice:X-ray and anlotinib were treated in different mode,tumor volume was calculated and growth curve was drawn to study the effect of anlotinib which was combined with radiotherapy on the growth of transplanted tumor.IHC verified Ki-67 proliferation and DNA damage proteins.Bliss model was constructed based on the results of tumor growth inhibition to verify the synergistic effect of anlotinib which was combined with radiotherapy in vivo.2.5 Detection of ESCC cell pyroptosis caused by anlotinib:The bubble blowing of ESCC cells after anlotinib treatment were observed and recorded by optical microscope,and further verified by flow cytometry and transmission electron microscopy.The activation of pyroptosis related proteins was detected by Western blot.2.6 Detection of the effect of anlotinib combined with X-ray treatment on pyroptosis of ESCC cells:Optical microscopy and flow cytometry were used to detect pyroptosis of ESCC cells after combined treatment,and Western blot was used to further detect the activation of pyroptosis related proteins GSDME and Caspase-3.2.7 Clinical data and specimen collection:Primary tumor tissues of surgically resected ESCC patients were collected and GSDME expression was evaluated by Western blot and IHC.Chi-square test was used to detect the relationship between GSDME expression and clinical features.Kaplan-Meier method was used to analyze and draw the survival curve to evaluate the effect of GSDME expression on OS.Univariate and multivariate analyses based on Cox proportional risk models were used to evaluate whether GSDME could be used as a prognostic predictor in patients with ESCC.Results:1.The effect and mechanism of X-ray on ESCC pyroptosis.1.1 X-ray could induce pyroptosis of ESCC cells:After 96 hours of 10 Gy X-ray irradiation,the ESCC cells were observed under the optical microscope with obvious bubble blowing,increased LDH release,and the cells with propidium iodide(PI)and Annexin V positive gradually increased.Transmission electron microscopy showed that the cells were enlarged and the integrity of the cell membrane was destroyed.It was confirmed that X-ray can induce ESCC pyroptosis in a time-dependent and dose-dependent manner to a certain extent,and the pyroptosis induced by large dose segmentation was more obvious.1.2 GSDME mediated X-ray induced ESCC pyroptosis:According to the TCGA database and Western blot detection of Gasdermin family proteins,GSDMD and GSDME were expressed in esophageal cancer cells.GSDME was obviously activated with 10 Gy X-ray irradiation after 96 h.GSDME overexpression and knockdown cell lines were constructed,and the cell morphology and Western blot results showed that GSDME expression was positively correlated with the degree of pyroptosis of ESCC cells.1.3 The expression of GSDME was correlated with radiosensitivity of ESCC:The colony formation assay based on gradient dose X-ray irradiation using click-multi-target model to plot survival curve showed that the expression level of GSDME was positively correlated with radiosensitivity.The growth rate and size of the transplanted tumor after radiotherapy in the GSDME knockout group were faster,and the proliferation of Ki-67 was increased,and the DNA damage protein was reduced compared wiht control.The radiosensitivity of transplanted tumor in nude mice was reduced by GSDME knockout.1.4 GSDME affected radiotherapy reactivity and prognosis of ESCC patients:Efficacy evaluation and survival analysis showed that the expression of GSDME affected the efficacy and survival of ESCC patients treated with radiotherapy alone.1.5 X-ray mediated ESCC pyroptosis by Caspase-3/GSDME:The application of Caspase-3 specific inhibitor Z-DEVD-FMK showed that the activation of GSDME was inhibited,suggesting that the activation of GSDME may be regulated by upstream Caspase-3.Western blot results showed that Caspase-3 was activated after radiotherapy.X-ray irradiation was performed after Caspase-3 knockdown,and the results showed that X-ray may mediate pyroptosis of ESCC cells by activating Caspase-3 to cleave GSDME.2.Pyroptosis mediated the synergic effect of X-ray and anlotinib on ESCC.2.1 Analysis of target-related factors of anlotinib therapy in TCGA database:TCGA RNA-seq data analysis showed that the expressions of anlotinib target-related factors,VEGF,FGF and PDGF,were negatively correlated with CD8+T cell infiltration.2.2 Anlotinib inhibited ESCC malignant phenotype:CCK-8 and EdU experiments showed that anlotinib could inhibit ESCC cell proliferation;Transwell assay and Western blot assay showed that the invasion and metastasis ability of ESCC cells was inhibited after anlotinib treatment.2.3 Anlotinib had synergistic effect on ESCC radiotherapy in vitro:48 h after 1 μM anlotinib treatment combined with 6 Gy X-ray irradiation,the typical features of pyroptosis could be observed.Colony formation assay results showed that the combination of anlotinib and radiotherapy increased the proliferation inhibition of ESCC cells compared with the anlotinib and radiotherapy alone groups.Bliss model results showed that anlotinib had a synergistic effect on ESCC.Immunofluorescence and Western blot analysis of DNA damage and repair related proteins showed that the combination of anlotinib and radiotherapy increased DNA damage and decreased DNA repair.These results suggest that anlotinib can enhance the therapeutic effect of ESCC with radiotherapy in vitro.2.4 Anlotinib had synergistic effect on ESCC radiotherapy in vivo:The results of subcutaneous tumor formation experiment in BALB/c nude mice showed that anlotinib can improve radiotherapy reactivity in vivo,slow down the growth rate and reduce the volume of transplanted tumors.The IHC results showed that Ki-67 positive cells decreased and DNA damage protein expression increased.Bliss independent model results showed that anlotinib had a synergistic effect on ESCC in vivo.2.5 Anlotinib could induce pyroptosis of ESCC cells:The results of optical microscope,flow cytometry and transmission electron microscopy showed that the occurrence of pyroptosis could be observed 48 hours after the application of 1 μM anlotinib.Western blot analysis showed that 1 μM anlotinib induced the activation of GSDME and ESCC pyroptosis.2.6 Anlotinib could enhance ESCC radiosensitivity through GSDME-mediated pyroptosis:cell morphological changes and flow cytometry results showed that pyroptosis significantly increased in the anlotinib combined with X-ray treatment group.Western blot analysis showed that anlotinib could enhance ESCC radiosensitivity through GSDME-mediated pyroptosis2.7 The expression level of GSDME was correlated with the prognosis of ESCC patients:IHC staining results of tumor tissue slices of ESCC patients showed differences in GSDME expression among patients.And 68 cases were in the high expression group and 47 cases were in the low expression group.Kaplan-Meier curve showed that patients with high GSDME expression had better OS,and univariate and multivariate analyses showed that GSDME could be used as a prognostic predictor in ESCC patients.Conclusion:1.Pyroptosis was an important common mechanism that mediated X-ray,anlotinib,or the synergistic effect of both.2.X-ray mediated ESCC pyroptosis by Caspase-3/GSDME.3.GSDME expression affected the radiosensitivity of ESCC and is positively correlated with the prognosis of ESCC patients receiving radiotherapy alone.4.Anlotinib inhibited ESCC malignant phenotype and has a synergistic effect on ESCC radiotherapy.5.Anlotinib could induce pyroptosis and might enhance the radiosensitivity of ESCC through GSDME-mediated pyroptosis.6.Low GSDME expression is associated with poor prognosis in ESCC patients.