Objective To investigate the effects of radiation-induced copper homeostasis imbalance on mitochondrial dysfunction and subsequent epithelial-mesenchymal transition.

Methods Human lung epithelial cells A549 and bronchial epithelial cells BEAS-2B were exposed to a single dose of 10 Gy (dose rate 1.02 Gy/min) of X-rays, with the addition of 10 μM of the specific copper ion chelator tetrathiomolybdate (TTM) . Intracellular copper ion concentrations were measured using a copper (Cu²⁺) colorimetric assay kit; intracellular ATP levels were detected using a luciferase assay; JC-1 staining was used to detect mitochondrial membrane potential; the mitochondrial reactive oxygen species (ROS) fluorescent probe MitoSOX was used to detect mitochondrial ROS; Western Blot analysis was used to detect the protein levels of E-cadherin, vimentin, N-cadherin, and copper ion transport proteins (CTR1 and ATP7A); Detect mRNA expression levels of epithelial-mesenchymal transition-related genes (CDH1, CDH2, VIM) and copper ion transport protein-encoding genes (SLC31A1 and ATP7A) using qRT-PCR. Intergroup comparisons were analyzed using the independent samples t-test method.

Results Copper colorimetric assay results showed that copper ion concentrations in both types of lung epithelial cells significantly increased after irradiation (P<0.05), but the addition of TTM could inhibit the radiation-induced increase in copper ion concentrations within lung epithelial cells (P<0.05) ; Western Blot results showed that the expression of the copper import protein CTR1 increased, while the expression of the copper export protein ATP7A decreased after irradiation, but the expression changes were reversed after TTM administration; qRT-PCR results showed that the expression of SLC31A1 (the gene name of CTR1) significantly increased (P<0.01) after irradiation, ATP7A gene expression was significantly decreased (P<0.05), and this phenomenon was reversed after TTM administration (P<0.01); luciferase assay results showed that ATP levels in both types of lung epithelial cells were significantly decreased after irradiation (P<0.05), and ATP levels in cells were significantly restored after TTM administration (P<0.05) ; JC-1 staining results showed that mitochondrial membrane potential significantly decreased after irradiation, and mitochondrial membrane potential levels improved after TTM administration; MitoSOX fluorescent probe results showed that mitochondrial ROS levels significantly increased in the irradiation group, and TTM administration reduced mitochondrial ROS levels. Finally, changes in EMT markers were detected. Western Blot results showed that after irradiation, the expression of the epithelial marker E-cadherin protein decreased, while the expression of the mesenchymal markers N-cadherin and Vimentin proteins increased. In the irradiation plus TTM group, the changes were the opposite. qRT-PCR results showed that the expression of the epithelial marker CDH1 was significantly reduced (P<0.01) after irradiation, while the expression levels of the mesenchymal markers CDH2 and VIM were significantly increased (P<0.01). The changes in the irradiation plus TTM group were the opposite.

Conclusion In radiation-induced damage to lung epithelial cells, abnormal expression of copper transport proteins leads to copper homeostasis imbalance, causing mitochondrial dysfunction, which in turn triggers EMT. Treatment with the copper ion chelator TTM can reverse this phenomenon.