The radiosensitizing effect of Paeonol on lung adenocarcinoma by augmentation of radiation-induced apoptosis and inhibition of the PI3K/Akt pathway

Abstract

Purpose: To investigate the radiosensitizing effect and mechanism of action by the natural product Paeonol on lung adenocarcinoma both in vitro and in vivo.

Materials and methods: Two lung adenocarcinoma cell lines (human lung adenocarcinoma cell line A549 and mouse Lewis lung carcinoma (LLC) cell line) were chosen for this research. In order to select the experimental concentrations of Paeonol, cytotoxicity was determined using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) assay. A clonogenic assay was performed to measure the radiosensitizing effects. Apoptosis was determined by the Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) assay and flow cytometry. Protein expression was analyzed by Western blotting. To test the radiosensitizing effect in vivo, a transplanted tumor model was established.

Results: The MTT assay showed that Paeonol inhibited proliferation of cells. Paeonol concentration ranged from an IC₅ (5% inhibiting concentration) to an IC₂₀ and was used at non-toxic concentrations for subsequent experiments. The clonogenic assay showed that Paeonol enhanced the radiosensitivity of cells. Data from the Tunel assay and flow cytometry verified that Paeonol enhanced radiation-induced apoptosis. Paeonol inhibited the activation of the PI3K/AKT (Phosphatidylinositol 3-kinase/ Protein Kinase B) pathway and down-regulated the expression of COX-2 (Cyclooxygenase-2) and Survivin. Paeonol (1718 mg/kg) combined with 10 Gy irradiation inhibited the growth of a transplanted tumor model in vivo, resulting in the longest tumor growth time, tumor growth delay and the highest inhibition ratio when compared with the radiotherapy alone group.

Conclusions: It is reported for the first time that Paeonol has a radiosensitizing effect on lung adenocarcinoma both in vitro and in vivo. This effect could be related to the augmentation of radiation-induced apoptosis and the inhibition of the PI3K/Akt signalling pathway and its downstream proteins: COX-2 and Survivin.

Keywords::

• Radiosensitization
• Paeonol
• lung cancer
• apoptosis
• PI3K/Akt pathway

Acknowledgements

The authors would like to thank the Teaching and Research Section of Nuclear Medicine and Institute of Clinical Pharmacology of Anhui Medical University for advice on experimental conditions and helpful comments and discussions.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This research was supported in part by the Natural Science Foundation of China (No. 81071986 and No. 81272739). It was also supported by the Radiotherapy Department of the first affiliated hospital of Anhui Medical University for irradiation and the Central Laboratory for flow cytometry.