Radiation-induced tumor cell death is widely attributed to multiple distinct mechanisms, mainly including autophagy, apoptosis and necrosis. However, a critical unanswered question is which death pathway a tumor cell chooses in response to various fractionated doses of ionizing radiation (IR). Mitochondrial pathways control both cell apoptosis and necrosis, and play roles in autophagy. The aim of this study was to investigate the role and mechanism of the mitochondrial-dependent cell death regulated by PGAM5/FUNDC1/BCL-xL/DRP1 axis in radiotherapy response of non-small cell lung cancer. Cell death of A549 and Calu-3 NSCLC cell lines induced by various fractionated doses of X-ray radiation (0, 2, 4, 6, 8, 12, 15, 20 Gy/Fraction and 2Gy×9Fraction) was detected by electron microscopy, western blotting, and flow cytometry. Protein interactions were detected by immunofluorescence and immunoprecipitation. Relative low-dose fractionated radiation (LDFRT, <6 Gy /Fraction) were used to induce apoptosis, whereas hypofractionated radiation (HFRT, 6–20 Gy/Fraction) was chosen to induce necrosis in both A549 and Calu-3 cells. Our results demonstrated that LDFRT induced degradation of BCL2-like 1 (BCL2L1), a prominent anti-apoptosis molecule, and released the mitochondrial phosphatase PGAM5 from BCL2L1, which led to its activation and subsequent dephosphorylation of FUN14 domain-containing protein-1 (FUNDC1) at Ser13. The dephosphorylated form of FUNDC1 interacted with light chain 3 (LC3) and induced mitophagy. In addition, peak levels of mitophagy appeared at about 8 hours after 6Gy/Fraction X-ray irradiation in A549, and 4Gy/Fraction X-ray irradiation in Calu-3 cells. Interestingly, the receptor-interacting protein kinase (RIP)1/RIP3 necrosome complex was formed and recruited to the mitochondrial surface at HFRT and then activated PGAM5, dynamin-related protein (Drp1), and mixed lineage kinase domain-like (MLKL) to execute mitochondrial fragmentation and programed cell necrosis. It is worth mentioning that HFRT did not notably induce degradation of BCL2L1 or mitophagy. The PGAM5/FUNDC1/BCL-xL/DRP1 axis might determine mitochondrial fate and the mode of cell death in response to various fractionated doses of X-ray radiation in NSCLC. Targeting these pathways will be useful to understand radiation-induced cell death and may improve the radiotherapeutic effect on human cancers.
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