Oncolytic adenovirus type 11-induced ferroptosis of esophageal squamous cell carcinoma cells involves in mitochondrial impairment and the mTOR pathway

BACKGROUND: Human adenovirus type 11 (HAdV-11) has several advantages compared with human adenovirus type 5 (HAdV-5), including improved infectivity of tumor cells and the potential for intravenous delivery. Our previous study demonstrated that HAdV-11 efficiently infected various human cancer cell lines including esophageal squamous cell carcinoma (ESCC) cell lines, however the cytotoxicity was not effective in some cell lines. The underlying functional mechanisms for this discrepancy are not clear. METHODS: Cell viability was assessed using an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay, and viral replication was quantified by tissue culture infectious dose 50 (TCID50) and quantitative polymerase chain reaction (qPCR). Flow cytometry was employed to analyze apoptosis, mitochondrial reactive oxygen species, lipid oxidation, mitochondrial mass, mitochondrial membrane potential, and intracellular Fe²⁺ levels. Transmission electron microscopy was used for ultrastructural analysis. Protein expression and mitochondrial localization were evaluated by Western blotting and immunofluorescence staining, while reduced glutathione levels were measured using a commercial assay kit. RESULTS: We report here that HAdV-11 exerts its anti-ESCC effects via two pathways. Firstly, via the mitochondrial pathway, HAdV-11 promotes mitochondrial fission, thereby enhancing mitophagy, suppressing glutathione peroxidase 4 expression, and increasing lipid peroxidation, which ultimately induces ferroptosis; interestingly, HAdV-11-induced mitophagy attenuates ferroptosis in this context. Secondly, HAdV-11 triggers both mitophagy and ferroptosis through the mammalian target of rapamycin (mTOR) pathway. Our findings indicate that mitochondrial fission and mTOR signaling are closely associated with HAdV-11-induced cytotoxicity in ESCC cells. CONCLUSIONS: HAdV-11-induced ferroptosis in ESCC cells involves mitochondrial impairment and the mTOR pathway. These findings provide insights into HAdV-11’s oncolytic mechanisms and suggest strategies for enhancing its therapeutic efficacy in ESCC.