Abstract
BackgroundNF-E2-related factor 2 (Nrf2) is involved in the radiation resistance of esophageal squamous cell carcinoma (ESCC), but the underlying molecular mechanism is unclear. The purpose of our study was to explore the role of Nrf2 in the radiation resistance of ESCC and the potential molecular mechanism.ResultsNrf2 expression was introduced into Ec109 and KYSE-30 ESCC cells with lentivirus. CCK-8 and colony formation assays were used to evaluate the effect of Nrf2 on radioresistance in culture. The autophagy level was assessed by western blotting, flow cytometry, and confocal fluorescence microscopy. The effect of Nrf2 on the transcription of Ca2 +/calmodulin-dependent protein kinase II α (CaMKIIα) was studied by chromatin immunoprecipitation. We found that the overexpression of Nrf2 increased the radiation resistance of ESCC cells. Mechanistically, Nrf2 triggered the radiation resistance of ESCC cells by targeting CaMKIIα and subsequently activating autophagy. In addition, we found that Nrf2 directly regulated the transcription of CaMKIIα by binding to its promoter region. The effect of Nrf2 on radiation resistance was also explored in both a xenograft mouse model and ESCC patient samples. Consistent with the results of the in vitro study, high Nrf2 expression level resulted in in vivo radioresistance in an Ec109-derived xenograft mouse model. Furthermore, we also demonstrated that upregulations of both Nrf2 and CaMKIIα was closely related to lower survival rates of ESCC patients.ConclusionsOur study reveals that Nrf2 promotes the radiation resistance of ESCC by targeting CaMKIIα and subsequently activating autophagy, which is characterized by the suppression of phosphorylated mTOR and p62, activation of Beclin 1, and transformation of LC3-I to LC3-II.
Highlights
NF-E2-related factor 2 (Nrf2) is involved in the radiation resistance of esophageal squamous cell carci‐ noma (ESCC), but the underlying molecular mechanism is unclear
We found that calmodulin-dependent protein kinase II α (CaMKIIα) was the key molecule involved in the autophagy activation and radiation resistance induced by Nrf2
Nrf2 triggers radiation resistance in esophageal cancer cells To determine whether Nrf2 is involved in the radiation resistance of esophageal cancer cells, we first used a lentivirus transduction method to overexpress Nrf2 in both Ec109 and KYSE-30 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analyses confirmed that Nrf2 expression was elevated by lentivirus transduction in both Ec109 and KYSE-30 cells (Fig. 1)
Summary
NF-E2-related factor 2 (Nrf2) is involved in the radiation resistance of esophageal squamous cell carci‐ noma (ESCC), but the underlying molecular mechanism is unclear. The purpose of our study was to explore the role of Nrf in the radiation resistance of ESCC and the potential molecular mechanism. Dysregulation of Nrf has been identified as one of Autophagy is a biological process that involves the selfdegradation of cytoplasmic macromolecules and organelles under conditions of nutritional stress. Recent studies have shown that autophagy can mediate the radiation resistance of ESCC. Zhu et al [12] reported that autophagy activation played a key role in ESCC radiation resistance induced by eukaryotic extension factor 2 kinase (eEF2K). It was found that increased autophagy was the main mechanism of ESCC radioresistance induced by liver kinase B1 (LKB1) [14]. The mechanism of autophagy-induced radiation resistance, especially whether autophagy is involved in Nrf2mediated radiation resistance, is worthy of further study
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