Proteomic and Metabolomic Profiling Reveal Mitochondrial Transplantation-Mediated Reprogramming in Gastric Cancer...

Mitochondria provide multiple functions for cellular physiology. Transplantation of mitochondria isolated from gastric epithelial cells GES-1 reducing the malignancy of gastric cancer cells AGS was previously reported. To elucidate the underlying mechanisms, TMT-based proteomic analysis coupling ingenuity pathway software prediction revealed that 257 upregulated and 34 downregulated proteins were implicated in 14 signaling pathways, including mitochondrial cell dysfunction (data became available from ProteomeXchange with identifier PXD061705). The upregulation of p53, Bax, p-Akt^S473, p-mTOR^S2448 and the downregulation of Sirt 3, p-NRF2^S40, and HO-1 were further verified by western blotting. In the metabolomic analysis, 3 upregulated and 8 downregulated metabolites involved in glycolysis, TCA cycle, pentose phosphate pathway (PPP) and ATP production were identified. Aligning the catalytic step of these metabolites in glycolysis and TCA cycle, the lower fructose 1,6-bisphosphate, 2-phosphoglyceric acid and phosphoenolpyruvate was coupled with higher isocitrate while the reduced α-ketoglutarate, malate, ATP and NADH all implied an accumulation of pyruvate in the cytosol. Western blotting, along with pyruvate and lactate assays, showed decreased extracellular lactate due to upregulated MCT1 (lactate importer) and downregulated MCT4 (lactate exporter). The higher pyruvate was caused by increased LDHB (lactate-to-pyruvate conversion) and a decrease in mitochondrial pyruvate carrier (MPC). With the finding that transplanted GES-1 mitochondria led to an accumulation of pyruvate, the inhibitory effect of elevated pyruvate on migrated AGS cells was observed. In conclusion, combining proteomic and metabolomic analysis revealed the underlying mechanisms for understanding how transplanted GES-1 mitochondria attenuate AGS gastric cancer malignancy.