Beneficial Effects Of Ionizing Radiation To Enhance Anti-Cancer Effects Of RIG-Like Receptor Stimulus
Authors: Yoshino Hironori , Kashiwakura Ikuo 
Published: October 2019

Introduction
The immune system is composed of innate and adaptive immunity. Antigen presenting cells (APCs), such as macrophages and dendritic cells, serve as a link between innate and adaptive immunity. Furthermore, APCs express pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns. Retinoic acid-inducible gene-I (RIG-I)-like receptors [RLRs; RIG-I and melanoma differentiation-associated gene 5 (MDA5)] are a type of PRRs and sense virus-derived RNA or a synthetic analog of dsRNA polyinosinic-polycytidylic acid [poly(I:C)]. Although macrophages are resistant to ionizing radiation, it remains unclear whether radiation affects RLR expression in the macrophages. Therefore, the effects of ionizing radiation on the RLR expression in macrophages and the response against poly(I:C) were herein investigated. Additionally, the anti-cancer effects of poly(I:C) and the combination treatment of poly(I:C) with ionizing radiation was examined in human lung cancer A549 cells.

Methods
For preparation of human macrophage-like cells, the human acute monocytic leukemia THP1 cells were treated with phorbol 12-myristate 13-acetate and then differentiated into macrophage-like cells. To stimulate RLRs, poly(I:C)/LyoVecTM (InvivoGen), which is a complex between poly(I:C) and the transfection reagent LyoVecTM, was used on macrophage-like differentiated THP-1 cells. X-irradiation was performed with an X-ray generator at a dose rate of 102.0–104.0 cGy/min. The viable cells were counted by trypan blue exclusion assay. The expression of RLRs was analyzed by reverse transcription polymerase chain reaction (RT-PCR) or western blotting. The interferon (IFN)-β expression and tumor necrosis factor (TNF)-α concentration present in culture supernatants were analyzed by RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The cell death analysis was performed by fluorescein isothiocyanate labeled annexin V and propidium iodide (PI) staining and analyzed by flow cytometry.

Results
The effects of ionizing radiation on RLR expression were first investigated. Both non-irradiated and X-irradiated (1–10 Gy) macrophage-like cells expressed RIG-I and MDA-5, with no significant difference in expression levels. Next the response of macrophage-like cells to poly(I:C)/LyoVecTM (500 ng/ml) was examined. Although the expression of IFN-β was not observed in non-stimulated macrophage-like cells, the poly(I:C)/LyoVecTM-stimulated macrophage-like cells expressed IFN-β. In X-irradiated macrophage-like cells, IFN-β expression after poly(I:C)/LyoVecTM stimulation was comparable with that of non-irradiated cells. Similar to the IFN-β expression, no significant difference in concentration of TNF-α were observed after poly(I:C)/LyoVecTMstimulation in non-irradiated and irradiated cells. These results suggest that ionizing radiation did not affect RLR expression or the response against poly(I:C) in macrophages. We next investigated the anti-cancer effects of poly(I:C)/LyoVecTM against human lung cancer A549 cells. Treatment with poly(I:C)/LyoVecTM (500 and 1000 ng/ml) suppressed the A549 cell growth (approximately 70% and 80% inhibition, respectively). Furthermore, the treatment with poly(I:C)/LyoVecTM induced both annexin V(+)/PI(−) (early apoptotic cells) and annexin V(+)/PI(+) cells (late apoptotic/necrotic cells). Finally, the combination treatment of poly(I:C) with 2 Gy was tested. The cell growth suppressive effects of 2 Gy resulted in 30% inhibition, whereas the combination of 2 Gy with poly(I:C)/LyoVecTM (500 and 1000 ng/ml) resulted in 85% and 90% inhibition, respectively. Correspondingly, the proportion of early apoptotic cells and late apoptotic/necrotic cells were higher in the combination of 2 Gy with poly(I:C)/LyoVecTM compared with 2 Gy alone or poly(I:C)/LyoVecTMalone. These results suggest that poly(I:C) and ionizing radiation synergistically exhibit anti-cancer effects against A549 cells.

Conclusion
This study demonstrated that ionizing radiation synergistically acted with RLR stimulation in suppressing the growth of human lung cancer cells without affecting the expression of RLRs in macrophages. Therefore, the combination of radiation therapy with RLR stimulus is expected to be an effective cancer therapy.

Disclosures:
No relevant conflicts of interest to declare.