Soy Isoflavones Mediate Radioprotection By Regulating Early Endothelial Cell Death And Inflammatory Signaling In Radiation-Induced Lung Injury

Matthew Donovan Fountain, Wayne State University


Purpose: Vascular damage and inflammation are limiting toxic effects of lung cancer radiotherapy, which lead to pneumonitis and pulmonary fibrosis. We have demonstrated that soy isoflavones (SIF) mitigate these toxic effects at late time points after radiation. However, the process by which SIF impacts the onset of radiation-induced inflammation remains to be elucidated. We have now investigated early events of radiation-induced inflammation and identified cellular and molecular signaling patterns by endothelial cells that could be modified by SIF to control vascular damage and the initiation of lung inflammation.

Materials and Methods: Histopathological, cellular and molecular studies were performed on mouse lungs from C57Bl/6 mice treated with 10Gy of thoracic radiation (XRT) in conjunction with daily oral SIF treatment given prior and after radiation. Parallel studies were performed in-vitro using EA.hy926 endothelial cell line with SIF and radiation. Immunohistochemistry, western blots analysis, and flow cytometry were performed on lung tissue or EA.hy926 cells to analyze endothelial cells, their patterns of cell death or survival, and signaling molecules involved in inflammatory events.

Results: Histopathological differences in vascular injury and inflammatory infiltrates in lungs were observed with SIF treatment at early time points post-radiation. In conjunction, SIF+XRT reduced ICAM-1 in-vitro and in-vivo in lung endothelial cells at 2 to 60 days post-XRT. SIF increased XRT-induced DNA damage to endothelial cells and inhibited cell cycle signaling and transcription factors involved in cell growth. Although no differences were observed in the frequency of senescent cells after XRT and SIF+XRT, SIF+XRT caused decreased ICAM-1 expression in senescent cells both in-vitro and in-vivo studies. Furthermore, SIF treatment promoted apoptotic endothelial cell death and decreased XRT-induced type III cell death. In-vitro molecular studies showed that SIF+XRT increased apoptotic caspase-9 activation and release of IFN as well as reduced the release of inflammatory HMGB-1 and IL-1, the cleavage of pyroptotic gasdermin D, and the release of active IL-1, which are all events associated with type III cell death. Further in-vitro experiments identified that caspase-9 was necessary but not sufficient for SIF-induced changes to patterns of XRT-induced cell death, and various features of mitochondrial homeostasis were modulated by SIF treatment.

Conclusions: SIF+XRT caused changes in patterns of endothelial cell death and survival, proinflammatory molecule release, and adhesion molecule expression at early time points post-XRT associated with early reduction of immune cell recruitment. Caspase-9 was necessary for the observed changes in SIF-mediated XRT-induced cell death and correlated with SIF-mediated changes to mitochondrial homeostasis, suggesting that SIF serves as a molecule to prime cells before XRT. These findings suggest that SIF could mediate its radioprotective effects in irradiated lungs by limiting excessive immune cell homing via vascular endothelium into damaged lung tissue via changes to patterns in cell death and curtail the overall inflammatory response to radiation.