Stereotactic Body Radiation Oncology Exert Anti-Tumor Effect Via Rip3-Dependent Necroptosis In Non-Small Cell Lung Cancer Cells

Although hypofractionated radiation therapy (HFRT) in the treatment of early-stage non-small cell lung cancer (NSCLC) achieved a remarkable effect, there is still radiation resistance with unknown mechanism. Previous studies demonstrated that cell apoptosis may play an important role in cancer HFRT, but recent studies showed that cellular necrosis is not an accidental event, but is a programmed process regulated by reception interaction protein 1(RIP1)/RIP3 and mixed lineage kinase domain-like protein (MLKL), which was named cellular necroptosis. This study was to investigate the role and molecular mechanism of RIP1/RIP3 induced cancer cell necroptosis after HFRT. The RIP1/RIP3 and MLKL/p-MLKL expressions levels in the NSCLC cell lines and patient histopathology by Western blotting assays and immunohistochemical assay. The NSCLC cell lines and xenografts were exposed to increasing doses of radiation in the presence of necroptosis inhibitor Nec-1 or RIP3 interference, and the xenografts response of xenografts were also evaluated. On planned time, the death type of tumor cells, were determined by immunofluorescence, fluorescence-activated cell sorting (FACS), and electronic microphotography assays in vitro and in vivo. Our results demonstrated that the RIP3 and MLKL expression was evaluated in the NSCLC cell lines including A549, SPC-A1, and LTEP-A2 compared to RIP1. Importantly, Silencing and inhibition of RIP3 expression could enhance HFRT induced necrosis as well as improve cell cloning information and xenografts growth after HFRT in vitro and in vivo, accompanied by significantly evaluated RIP3 and enhancing MLKL phosphorylation. These results demonstrated that RIP3 activation and the resulting p-MLKL formation that is the key event which is responsible for necroptosis of cancer after HFRT, which is an indispensable pathway separately from apoptosis. This study will provide new idea and target for HFRT personalized therapy, with important theoretic contribution and promising future of application.