Guiqi Baizhu decoction enhances radiosensitivity in non-small cell lung cancer by inhibiting the HIF-1α/DNA-PKcs axis-mediated DNA repair

Abstract

Background

Radiotherapy is one of the main treatments for non-small cell lung cancer (NSCLC), and radiosensitivity is a determinant of its efficacy. Therefore, enhancing the radiosensitivity is of great significance to improve the clinical efficacy of non-small cell lung cancer (NSCLC).

Purpose

This study intended to investigate the radiosensitisation effect and mechanism of Guiqi Baizhu decoction (GQBZD) on non-small cell lung cancer (NSCLC) and the role of hypoxia-inducible factor-1 alpha (HIF-1α)/DNA-dependent protein kinase catalytic subunit (DNA-PKcs) axis-mediated DNA non-homologous end joining (NHEJ) repair in NSCLC radiotherapy.

Study design

In vivo experimental model was Lewis subcutaneous transplantation tumor model of in C57 black 6 (C57BL/6) mice, and in vitro experimental models were A549, H1299 and H460 cells.

Methods

In vivo experimental model was Lewis subcutaneous transplantation tumor model of in C57 black 6 (C57BL/6) mice. After the model was successfully established, the tumor was irradiated locally with 4 Gy X-ray, and 10.465 g/kg Guiqi Baizhu Decoction (GQBZD) was administered by gavage on the second day after irradiation for a total of 10 days. The morphological changes in tumour tissues were observed by HE staining, Ki67 levels in tumour tissues were detected by immunohistochemistry, the apoptosis in tumour cells were detected by Tunel staining. In vitro experimental models were different NSCLC cells (A549, H1299 and H460), irradiated by 2 Gy X-rays and then intervened with 5%, 10% and 20% Guiqi Baizhu Decoction (GQBZD)-containing serum for 24 h. A549 stably-transformed cell lines knocking down and overexpressing HIF-1α were also constructed by lentiviral transfection. The cell proliferation was detected by CCK-8 and clone formation, the apoptosis and cell cycle was detected by flow cytometry. Network pharmacology and transcriptomics to investigate key targets and pathways of GQBZD effects on NSCLC irradiation, further validated by immunofluorescence and Western blot.

Results

In vivo experiments confirmed that GQBZD combined with irradiation could inhibit the growth of Lewis subcutaneous transplantation tumor, reduce the expression of Ki67 and promote the apoptosis of tumour cells. In vitro experiments confirmed that GQBZD combined with irradiation inhibited the proliferation of different NSCLC cells, promoted NSCLC cell apoptosis and G2/M-phase arrest, and induced the expression of phosphorylated histone H2AX (γ-H2AX) in NSCLC cells, which showed a good radiosensitisation effect. Mechanistically, GQBZD exerts its radiosensitisation effect on NSCLC mainly through the HIF-1α signalling pathway. Meanwhile, under irradiation conditions, the expression of HIF-1α and DNA-PKcs were positively correlated, and HIF-1α had a regulatory effect on DNA-PKcs, promoting DNA-PKcs-dependent non-homologous end joining (NHEJ) repair. In addition, GQBZD combined irradiation down-regulated the expression of HIF-1α, DNA-PKcs, and NHEJ repair-related proteins in NSCLC cells, while reversing the expression of HIF-1α, DNA-PKcs, and NHEJ repair-related proteins in overexpressing HIF-1α A549 cell, thereby enhancing radiosensitivity in NSCLC.

Conclusion

This study provides an in-depth exploration of the radiosensitisation effect of GQBZD and provides an important experimental basis for the study of Chinese medicine in the field of cancer radiosensitisation, and further extends the extensibility of GQBZD on the basis of the previous study.