Effect of Apatinib Combined with Interferon-γ Mediated Ferroptosis of Gastric Cancer Cells to Activate cGAS-STING Path-way to Promote Anti-Tumor Immune Response

ZHAO Yanzheng; GUO Qing; JI Yanxia; SU Ying; WANG Congmin

doi:10.13730/j.issn.2097-2148.2025.08.003

Abstract：Objective To explore the therapeutic effect of Apatinib combined with interferon-γ on gastric cancer and its possible mechanism.Methods Taking the human gastric cancer cell line MGC803 as the research objects,the ob-jects were randomly divided into the control group[only given the same amount of 1％dimethyl sulfoxide(DMSO)],Ap-atinib group(intervened with Apatinib at 8 μmol/L),interferon-γ group(intervened with interferon-γ at 100 U/ml),Ap-atinib+interferon-γ group(after pretreatment with interferon-γ at 100 U/ml for 24 h,8 μmol/L Apatinib was added)and Apatinib+interferon-γ+ferrostatin-1(Fer-1)group(after pretreatment with interferon-γ 100 U/ml for 24 h,Ap-atinib 8 μmol/L and Fer-1 1 μmol/L was added),subsequent experiments were performed 72 h later.Cell migration and invasion were detected by the Transwell assay,the amount of lactatedehydrogenase(LDH)released from cells,malondi-aldehyde and reduced glutathione(GSH)contents were detected by kits,intracellular Fe2+content was detected by Ferro Orange probe;mitochondrial structure was detected by transmission electron microscopy;mitochondrial membrane poten-tial was detected by JC-1 staining;the levels of mitochondrial DNA(mtDNA)and cytoplasmic dsDNA(cDNA)in cells were detected by reverse transcription polymerase chain reaction(RT-PCR).A tumor-bearing mouse model of gastric cancer was constructed,and they were randomly divided into control group,interferon-γ group[interferon-γ 1.5 U/(kg·d)intraperitoneally inj ected],Apatinib group[Apatinib 200 mg/(kg·d)by gavage]and Apatinib+interferon-γ group[interferon-γ 1.5 U/(kg·d)intraperitoneally injected and Apatinib 200 mg/(kg·d)by gavage simultaneously],all groups were treated for 20 d,with 5 mice in each.Immunofluorescence staining was used to detect the expression of Mi-toSOX-Red in cancer cells and the stimulator of interferon genes(STING)and golgi matrix protein 130(GM130)co-lo-calization in cancer tissues;protein expressions of human prostaglandin-endoperoxide synthase 2(PTGS2),solute carrier family 7 member 1 1(SLC7A11)and glutathione peroxidase4(GPX4)and cancer tissues p-STING,STING,phosphoryla-ted-TANK-binding kinase 1(p-TBK1)and phosphorylated-interferon regulatory factor 3(p-IRF3)were detected by Western blot;the levels of reactive oxygen species and lipid reactive oxygen species in cells and the proportion of immune cells were detected by flow cytometry.Results Compared with Apatinib group,the cell survival rate of Apatinib+in-terferon-γ group reduced,when the concentration of Apatinib was 8 μmol/L,the combined treatment with interferon-γachieved an effective inhibitory concentration.Compared with the MGC803 cells in the control group,the leakage of LDH in the Apatinib group increased,the number of invasive cells and migratory cells decreased,the levels of Fe2+,reactive oxygen species,lipid reactive oxygen species and malondialdehyde and the expression of PTGS2 increased,the content of GSH and the expressions of SLC7A11 and GPX4 decreased,and mitochondrial breaks and crists were absent,the fluores-cence intensity ratio of J-aggregate/Monomers and mtDNA level decreased,the relative fluorescence intensity and cDNA level of MitoSOX-Red increased,and the mtDNA level decreased(all P＜0.05).Compared with the Apatinib group,the leakage of LDH in MGC803 cells in the Apatinib+interferon-γ group increased,the number of invasive cells and migra-tory cells decreased,the levels of Fe2+,reactive oxygen species,lipid reactive oxygen species and malondialdehyde and the expression of PTGS2 increased,the content of GSH and the expressions of SLC7A11 and GPX4 decreased,and mito-chondrial breaks and crists were absent,the fluorescence intensity ratio of J-aggregate/Monomers and the level of mtDNA also decreased,and the relative fluorescence intensity of MitoSOX-Red and cDNA level also increased(all P＜0.05).A tumor-bearing mouse model of gastric cancer was constructed,compared with the control group,the tumor volume and weight of mice in the Apatinib group decreased,the co-localization fluorescence intensity of STING and cis-GM130 in tumor tissues and the expressions of p-STING,p-TBK1 and p-IRF3 increased,the proportions of DC,CD8+T cells and central memory T cells increased,the proportion of exhausted T cells decreased(all P＜0.05);compared with the Apa-tinib group,the tumor volume and weight of mice in the Apatinib+interferon-γ group both decreased,the co-localization fluorescence intensity of STING and cis-GM130 in tumor tissues and the expressions of p-STING,p-TBK1 and p-IRF3 in-creased,the proportions of DC,CD8+T cells and central memory T cells increased,the proportion of exhausted T cells decreased(all P＜0.05).Conclusion Interferon-γ combined with Apatinib treatment may promote mtDNA release in-duced by ferroptosis-related mitochondrial stress and initiate the cyclic guanosine monophosphate adenylate synthetase(cGAS)-STING pathway,improve the tumor microenvironment,promote anti-tumor immune responses and enhance the sensitivity of gastric cancer cells to Apatinib.