| Objective Oleanolic acid (OA) has traditionally been used in clinical medicine asanti-cancer, anti-inflammatory and hepatoprotection agent. The aim of our study was toresearch whether OA might increase the radiosensivities of tumor cells or not, and thenthe relative mechanism was also investigated.Methods In our study, the rat glioma C6cell line and human lung cancer A549cell linewere used as experimental models and were subsequently assigned into aerobic groupand hypoxic group. The experimental concentration of OA was selected by MTT assay.Clonogenic assay was used to observe the radiosensitivity of C6and A549cells bydifferent treatment, subsequently the survival fractions of irradiated cells calculated toform the fitting curve of doses-effect by the single-hit multi-target model. Meanwhile,D0and Dqvalues of the fitting curve were obtained to calculate the sensitiveenhancement ratios (SERs) of different concentrations of OA. After cytochalasin B (CB)treatment, the alteration of intracellular DNA damage was further measured usingmicronucleus (MN) assay. In order to observe the mechanism of OA radiosensitizingtumor cells, the contents of reduced glutathione (GSH) in irradiated cells with variouspretreatments were determined using glutathione reductase/5,5’dithiobis-(2-nitrob-enzoic acid)(DTNB) recycling assay. At the same condition, the activities ofγ-glutamylcysteine synthetase (γ-GCS) and GSH synthase (GSS)，both key enzyme forGSH synthesis, were detected with the experiment kits. To the hypoxic tumor cell, thechange of hypoxia-inducible factor1-α (HIF-1α) level by OA pretreatment was detectedby Western blot method.Results The results of MTT assay showed that the half inhibition concentration (IC50) of OA on aerobic and hypoxic C6cell was35and80μg/ml respectively,27and81μg/mlon aerobic and hypoxic A549cell. Therefore,20%IC50and30%IC50OA were utilizedin the following experiments. The combined treatment of radiation with OA couldsignificantly decrease the survival fractions of tumor cells. When the concentrations ofOA were20%IC50, the SERs of aerobic, hypoxic C6and A549cells was1.81,1.42,1.26and1.25respectively. After30%IC50OA treated, the SERs of aerobic, hypoxic C6and A549cells was2.26,2.09,1.65and1.71each. The results from MN assaydisplayed that the number of MN in irradiated cells pretreated by diverse concentrationsof OA were significantly enhanced compared with the irradiated cells without OAtreatment. It is therefore indicated that OA could increase the irradiation damage ofDNA. The experimental results from GSH assay and the determination of γ-GCSactivity showed that the synthesis of cellular GSH was significantly reduced by OAprecondition concomitant with the down-regulation of γ-GCS activity; however thereduction of intracellular GSS activity wasn’t found under both aerobic and hypoxicconditions. It therefore indicated that OA could inhibit the GSH synthesis viadecreasingγ-GCS activity, followed by elevating the death of aerobic and hypoxictumor cells exposure to radiation. Additionally, we also obtained that OA pretreatmentcould obviously down-regulate HIF-1α levels after irradiation. Thereby, theradiosensitizing effect of OA on hypoxic cells is involving in the attenuation of GSHcontents and the down-regulation of HIF-1α.Conclusion The results from our research displayed that OA had the radiosensitizingcapacities in C6and A549cells. The mechanism of this sensitization may involve theinhibition of reduced GSH synthesis via the down-regulation of γ-GCS activity, andthen attenuate the antioxidation efficience of tumor cells. To hypoxic tumor cells, theregulation of OA to the radiosensitivity is correlation with both pathways of the reduction of GSH and the decrease of HIF-1α. |
PDF Full Text Request