Objective To investigate the effect of copper homeostasis imbalance on radiation-induced mitochondrial dysfunction and epithelial-mesenchymal transition (EMT) in lung epithelial cells.
Methods The human non-small cell lung cancer cell line A549 (with the characteristics of alveolar type Ⅱ epithelial cells) and human normal lung epithelial cell line BEAS-2B were used as research objects. These cell lines were divided into control, irradiation, and irradiation + ammonium tetrathiomolybdate (TTM) groups. The irradiation and irradiation + TTM groups received 10 Gy X-ray irradiation at a dose rate of 1.02 Gy/min, and the irradiation + TTM group was treated with 15 μmol/L TTM, a copper ion-specific chelator, immediately after irradiation. Copper ion content was detected through the copper ion colorimetric method. Mitochondrial membrane potential was detected via 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide staining. Adenosine triphosphate (ATP) level was quantified through the luciferase method. Mitochondrial reactive oxygen species (ROS) level was detected with MitoSOX fluorescent probes. The expression levels of neural cadherin (N-cadherin), epithelial cadherin (E-cadherin), vimentin, copper uptake protein 1 (CTR1), and ATPase copper transporting alpha (ATP7A) proteins were detected through western blot analysis. The relative expression levels of the cadherin genes (CDH)1, CDH2, vimentin gene (VIM), and solute carrier family 31 member 1 gene (SLC31A1), and ATP7A mRNA were detected through quantitative real-time polymerase chain reaction. The intergroup comparison of measurement data was performed via independent samples t-test.
Results In the two cell lines, compared with the control group, the irradiation group showed significantly increased copper ion content, increased CTR1 expression level, decreased ATP7A expression level, increased relative expression level of SLC31A1 mRNA, decreased relative expression level of ATP7A mRNA, decreased ATP level, decreased mitochondrial membrane potential, increased mitochondrial ROS level, decreased relative expression levels of E-cadherin and CDH1 mRNA, and increased relative expression levels of N-cadherin, vimentin and CDH2, VIM mRNA (t=4.333–12.942, all P<0.05). Compared with the irradiation group, the changes of the above indicators in the irradiation+TTM group were reversed: copper ion content decreased, CTR1 expression level decreased, ATP7A expression level increased, relative expression level of SLC31A1 mRNA decreased, relative expression level of ATP7A mRNA increased, ATP level recovered, mitochondrial membrane potential recovered, mitochondrial ROS level decreased, relative expression levels of E-cadherin and CDH1 mRNA recovered, and relative expression levels of N-cadherin, vimentin and CDH2, VIM mRNA decreased (t=3.223–11.732, all P<0.05).
Conclusions Radiation can induce the abnormal expression of copper transporters in lung epithelial cells, leading to copper homeostasis imbalance and mitochondrial dysfunction, thereby promoting the occurrence of EMT. TTM can effectively reverse this process.
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