Isoflavone-mediated radioprotection involves regulation of early endothelial cell death and inflammatory signaling in Radiation-Induced lung injury

Abstract

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 10 Gy 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 inflammatory infiltrates and vascular injury in lungs, including vascular endothelial cells, were observed with SIF treatment at early time points post-XRT. XRT-induced expression of proinflammatory adhesion molecule ICAM-1 cells was reduced by SIF in-vitro and in-vivo in endothelial cells. Molecular changes in endothelial cells with SIF treatment in conjunction with XRT included increased DNA damage, reduced cell viability and cyclin B1, and inhibition of nuclear translocation of NF-κB. Analysis of cell death showed that SIF treatment promoted apoptotic endothelial cell death and decreased XRT-induced type III cell death. In-vitro molecular studies indicated that SIF + XRT increased apoptotic caspase-9 activation and production of IFNβ while reducing the release of inflammatory HMGB-1 and IL-1α, the cleavage of pyroptotic gasdermin D, and the release of active IL-1β, which are all events associated with type III cell death.

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. 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 and curtailing the overall inflammatory response to radiation.

Keywords:

• Radiation
• soy isoflavones
• endothelial cells
• lung inflammation

Acknowledgements

We thank Dr Phillip E. Pellett for his continuous support and encouragement. We thank Dr Lisa M. Abernathy for her technical and intellectual help. We thank Dr Michael Joiner, Dr Kang Chen, and Dr Fulvio Lonardo for their input and expertise throughout the development and progression of these studies. We thank Karri Stark, Jessica Back, Eric Van Bueren, Christopher Yunker, Kali Hankerd, Alexa Cannon, David Hoogstra, and Shoshana Rothstein for their excellent assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

These studies were supported by Wayne State University Boost grant and Wayne State University Research Bridge fund awarded to GGH. MDF was supported by the NIH T32 DCTP HL120822 Detroit Cardiovascular Training Program and the Thomas C. Rumble Fellowship provided by the Graduate School at Wayne State University. The Microscopy, Imaging and Cytometry Resources Core is supported, in part, by the NIH Center grant P30 CA022453 to the Karmanos Cancer Institute at Wayne State University, and the Perinatology Research Branch of the National Institutes of Child Health and Development at Wayne State University.

Notes on contributors

Matthew D. Fountain

Matthew D. Fountain completed these studies as a PhD student in the Department of Biochemistry, Microbiology, and Immunology and department of Oncology at Wayne State University and Karmanos Cancer Institute. Matthew is currently a postdoctoral researcher in the department of Obstetrics, Gynecology and Reproductive Biology at Michigan State University College of Human Medicine with interests in translational research for immunological and cancer drug discovery.

Laura A. McLellan

Laura A. McLellan is a master’s student in biomedical sciences in the Department of Biochemistry, Microbiology, and Immunology, Wayne State University and is currently seeking admission to medical programs with interests in family medicine.

Natalie L. Smith

Natalie L. Smith is a post-baccalaureate student in the Department of Biochemistry, Microbiology, and Immunology, Wayne State University School of Medicine and is seeking admission to advance medical degree programs.

Brian F. Loughery

Brian F. Loughery is a physicist, Assistant Professor in the Department of Oncology, Division of Radiation Oncology at Karmanos Cancer Institute and Wayne State University with interests in clinical automation and imaging for radiation treatment.

Joseph T. Rakowski

Joseph T. Rakowski is a physicist, Associate Professor in the Department of Oncology, Division of Radiation Oncology at Karmanos Cancer Institute and Wayne State University School of Medicine with interests in advanced technology and imaging modalities for radiation treatment.

Harley Y. Tse

Harley Y. Tse is a Professor in the Department of Biochemistry, Microbiology, and Immunology, Wayne State University School of Medicine with interests in inflammation, autoimmunity and autoimmune disease.

Gilda G. Hillman

Gilda G. Hillman is a Professor Emeritus in the Department of Biochemistry, Microbiology, and Immunology, Wayne State University School of Medicine and department of Oncology at Karmanos Cancer Institute. Her main interest is radiobiology and radioprotection for cancer.