Molecular Alterations Associated with Reduced 5-FU Sensitivity in mtDNA-Depleted HGC-27 Cells

Mitochondrial DNA (mtDNA) depletion has been linked to cancer progression and chemoresistance. However, its involvement in the response of Gastric Cancer (GC) to 5-Fluorouracil (5-FU) is unknown. This study sought to investigate the effects of mtDNA depletion on cellular behavior and drug sensitivity of HGC-27 gastric cancer cells.

mtDNA-depleted cells were established, and the effect of 5-FU on viability of the cells, mitochondrial activity, gene and protein expression, and biochemical and functional parameters was subsequently determined. Pathway involvement was further evaluated using pharmacological inhibition analyses.

mtDNA-depleted HGC-27 cells exhibited decreased sensitivity to 5-FU compared to parental cells. Various cancer-related pathways, including HIF-1, TNF, MAPK signaling pathways, and PI3K-Akt, as well as Rap1 signaling, were found to be affected. Metabolic analysis demonstrated a switch towards glycolysis and the pentose phosphate pathway. mtDNA-depleted cells were primarily arrested at G0/G1 phase. These cells displayed an increased clonogenic potential following 5-FU treatment and increased migratory ability. In addition, VEGF-A, IL-6, p21, HIF-1α levels, and the pERK/ERK ratio were significantly elevated in mtDNA-depleted cells. Pharmacological inhibition of HIF-1α and MAPK/ERK signaling reduced cell survival following 5-FU treatment, supporting the potential involvement of these pathways in the adaptive response associated with mtDNA depletion. Raman spectroscopy further indicated that the biochemical response to 5-FU was markedly attenuated in mtDNA-depleted cells.

The findings suggest that mtDNA depletion is associated with a metabolically reprogrammed, stress-adapted phenotype in HGC-27 cells. Mitochondrial genome loss was associated with alterations in multiple pathways, including HIF-1α signaling and the MAPK pathway, particularly its ERK-associated branch, which may contribute to the reduced sensitivity to 5-FU observed in these cells.

These findings suggest a potential link between mitochondrial dysfunction and reduced sensitivity to 5-FU in HGC-27 gastric cancer cells. They also imply that targeting mitochondrial stress-associated signaling pathways may represent a potential strategy to overcome chemoresistance.