Vanillic acid inhibits TGF-β type I receptor to protect bone marrow mesenchymal stem cells from radiation-induced bystander effects.

Abstract

Background: Radiotherapy is a major treatment option for non-small cell lung cancer (NSCLC); however, irradiated tumor cells can damage non-irradiated cells through radiation-induced bystander effects (RIBE), which can affect the therapeutic efficacy. The study aimed to investigate the mechanism underlying RIBE and the protective effects of vanillic acid (VA) on human bone marrow mesenchymal stem cells (BMSCs).

Methods: We established two irradiation models to investigate RIBE. First, we established the A549 cell irradiation model alone, and tested the expression of cathepsin B (CTSB) and transforming growth factor-beta 1 (TGF-β1) by western blot and immunofluorescence staining. Next, we established a co-culture model of A549 cells and BMSCs. After 2 Gy X-rays irradiation of A549 cells, BMSCs cell viability was detected using Cell Counting Kit-8 (CCK-8), reactive oxygen species (ROS) level was detected using flow cytometry, and CTSB, TGF-β type I receptor (TGFβRI), p62 (sequestosome 1), BECLIN1, microtubule-associated protein light chain 3 (LC3), etc., were detected using western blot. Phosphorylated histone H2AX (γH2AX), CTSB, lysosomal-associated membrane protein 1 (LAMP1), and TGFβRI expression levels were detected by immunofluorescence staining. Molecular docking and molecular dynamics simulation, and a CCK-8 assay were used to screen for molecules from Astragalus membranceus that inhibited TGFβRI activity, to protect BMSCs from RIBE. Lastly, we validated VA activity in vivo.

Results: In this study, 2 Gy X-rays radiation on A549 cells was found to result in an increase in CTSB and TGF-β1, while CTSB inhibitor CA074Me reduced the radiation-induced TGF-β1 increase. In the co-culture model of A549 cells and BMSCs, 2 Gy X-rays radiation on A549 cells resulted in increase of TGFβRI expression in BMSCs, which led to an increase in ROS, and resulted in DNA damage and the inhibition of BMSCs proliferation. The small molecule VA from Astragalus membranaceus inhibited TGFβRI activity and restored the proliferation of BMSCs.

Conclusions: Our findings reveal that radiation causes CTSB overexpression in A549 cells, which further promotes TGF-β1 expression. TGF-β1 activates its receptors on BMSCs to increase ROS levels in BMSCs, while reducing lysosomal double-chain CTSB (dc-CTSB), which results in decreased BMSCs autophagy and an inability to clear ROS, and thus inhibits proliferation. VA inhibits TGFβRI to restore the proliferation of BMSCs, and in vivo, VA can enhance the killing effect of radiation on tumors.