Circulating cell-free mitochondrial DNA as a redox-associated marker of mitochondrial homeostasis disruption during...

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an emerging non-invasive marker across cancers. Yet, in gastric cancer (GC), its relationship to tissue mtDNA content, oxidative remodeling and somatic mtDNA variants, and thus its basis in mitochondrial homeostasis, remains poorly defined. We analyzed 169 individuals: 70 GC patients, 29 with precancerous gastric lesions and 70 healthy controls. MtDNA copy number was measured by quantitative PCR, and plasma biomarkers of oxidative damage (8-hydroxy-2'-deoxyguanosine, 8OH-dG; 4-hydroxynonenal, 4HNE) and of antioxidant capacity (glutathione peroxidase-1, GPX-1) were measured by ELISA. MtDNA variants were identified by next-generation sequencing. In MKN-28 GC cells, mitochondrial transcription factor A (TFAM) was manipulated with lentiviral vectors to alter mtDNA content. The results showed that GC progression was accompanied by higher ccf-mtDNA, shifts in plasma oxidative damage and antioxidant markers, and accumulation of mtDNA variants, alongside lower mtDNA content in cancers than in adjacent tissues. In GC cells, lowering mtDNA content by TFAM silencing promoted the malignant phenotype and increased intracellular superoxide-related fluorescence. By multiplex immunohistochemistry, tissue markers of mitochondrial maintenance and mtDNA release declined without a rise in caspase-3. D-loop variants already present at the precancerous stage suggest early mitochondrial changes, whereas predicted deleterious coding variants affecting respiratory chain components were observed in GC tissues. These findings support a redox-associated model of mitochondrial homeostasis that links impaired mitochondrial maintenance, mtDNA instability, and ccf-mtDNA accumulation, and provide a tissue-anchored mechanistic basis for the circulating mtDNA changes detected by plasma-based approaches in GC.