Radiosensitizing effect of oleanolic acid on tumor cells through the inhibition of GSH synthesis in vitro

Abstract

Oleanolic acid (OA) is a natural pentacyclic triterpenoid that has been used in traditional medicine as an anticancer and anti-inflammatory agent. The aim of our study was to investigate whether or not OA increases the radiosensivity of tumor cells, and the relative mechanism was also investigated. Clonogenic assay was used to observe the radiosensitivity of C6 and A549 cells following different treatments. The alteration of intracellular DNA damage was determined using a micronucleus (MN) assay. In order to identify the mechanism of OA-mediated radiosensitization of tumor cells, the levels of glutathione (GSH) in irradiated cells following various pretreatments were determined using glutathione reductase/5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay. Under the same condition, the activities of γ-glutamylcysteine synthetase (γ-GCS) and GSH synthase (GSS), both key enzymes for GSH synthesis, were detected using appropriate methods. In order to confirm the radiosensitizing effect of OA on cancer cells by attenuating GSH, N-acetylcysteine (NAC) was added to cells in culture for 12h before irradiation. The results showed that the combined treatment of radiation with OA significantly decreased the clonogenic growth of tumor cells and enhanced the numbers of intracellular MN compared to irradiation alone. Furthermore, it was found that the synthesis of cellular GSH was inhibited concomitantly with the downregulation of γ-GCS activity. Therefore, the utilization of OA as a radiosensitizing agent for irradiation-inducing cell death offers a potential therapeutic approach to treat cancer.