| ObjectiveTo evaluate the radiosensitizing effects and the mechanism of irisquinone(IR) and sodium glycididazole(CMNa) on the normoxic and hypoxic VX2 tumor cells and C6 tumor cells. 18F-FDG and 18F-FMISO PET/CT were used to evaluate the early radiosensitizing effect of IR and CMNa in order to explore the feasibility of visual evaluation by molecular nuclear medicine imaging.Methods1. In vitro experiment:The inhibitory effects of IR and CMNa on normoxic and hypoxic VX2 and C6 tumor cells were observed by MTT assay. Calculate the half effective inhibition concentration(IC50)of IR and CMNa and select the experiment concentration of each medicine. Clonogenic assay was used to observe the radiosensitizing effect of IR and CMNa on VX2 and C6 tumor cells and the sensitive enhancement ratio(SER)of each medicine was calculated. The change of glutathione(GSH) content in tumor cells was observed during the different treatment. Tumor cell apoptosis rate was detected by flow cytometry. The level of hypoxia inducible factor-1a(HIF-1a), vascular endothelial growth factor( VEGF)and glucose transport protein(GLUT-1)of hypoxic VX2 and C6 tumor cells which treated by different concentration of IR and CMNa were detected by Western blot method. Tumor cells were incubated with 18F-FDG and 18F-FMISO, then the radioactivity was counted. The 18F-FDG and 18F-FMISO uptake rate of hypoxic tumor cells was obtained at different time points. And the 18F-FDG and 18F-FMISOuptake rate of the control group, IR group, CMNa group, radiation group, IR plus radiation group and CMNa plus radiation group were obtained before and after different treatment.2. In vivo experiment:Rabbit VX2 lung transplant tumor model and rat C6 hindlimb muscle transplant tumor model were established, thirty-six for each tumor model. Then evenly and randomly divided into 6 groups: the control group, IR group, CMNa group, radiation group, IR plus radiation group and CMNa plus radiation group, six in each group. 18F-FDG and 18F-FMISO PET/CT images were performed before and 24 hours after treatment. The tumor tissue maximum standardized uptake(SUVmax) and tumor volume(V) ware recorded. Then the SUVmax percentage change(ΔSUVmax) and tumor volume percentage change(ΔV) were calculated. The animal models were killed after PET/CT imaging, the specimen of heart, liver, spleen, lung, kidney, muscle, necrotic of tumor and non-necrotic of tumor were weighted and the radioactivity was counted. The tumor tissue was stained by hematoxylin and eosin. The expression of HIF-1a, VEGF and GLUT-1 were detected by immunohistochemical method. Correlation analysis between SUVmax and HIF-1a, VEGF and GLUT-1 assay rate were analyzed.Results1. In vitro experiment:The results of MTT assay showed that IR and CMNa had no inhibitory effect on normoxic and hypoxic VX2 and C6 tumor cells at low concentration. The IC50 of IR on normoxic and hypoxic VX2 tumor cells was 48.5ug/ml and 70.8ug/ml,41.2ug/ml and 76.3ug/ml on normoxic and hypoxic C6 tumor cells. The IC50 of CMNa on normoxic and hypoxic VX2 tumor cells was 6.6mg/ml and 13.3mg/ml,7.9mg/ml and 15.6mg/ml on normoxic and hypoxic C6 tumor cells. Therefore, 20% and 30%IC50 IRand CMNa were utilized in the following experiments. Clonogenic assay showed both IR and CMNa could improve the radiosensitivity of normoxic and hypoxic C6 tumor cells. When the concentration of IR was 20%, the SER of normoxic and hypoxic VX2 and C6 tumor cells were 1.05, 1.29 and 1.18, 1.33. When the concentration of IR was 30%, the SER of normoxic and hypoxic VX2 and C6 tumor cells were 1.35, 1.71 and 1.47, 1.94. When the concentration of CMNa was 20%, the SER of normoxic and hypoxic VX2 and C6 tumor cells were 1.02, 1.21 and 1.10, 1.36. When the concentration of CMNa was 30%, the SER of normoxic and hypoxic VX2 and C6 tumor cells were 1.39, 1.69 and 1.46, 2.07.The experiment results of GSH assay showed the content of GSH in hypoxic tumor cells was higher than in normoxic tumor cells. The synthesis of cellular GSH was reduced by IR and CMNa. And the longer time of IR and CMNa action, the lower GSH in tumor cells. The content of GSH in normoxic and hypoxic VX2 and C6 tumor cells were increased after 2Gy radiation. The content of GSH in tumor cells significantly decreased in IR or CMNa plus radiation group. Flow cytometry showed the tumor cell apoptosis rates in IR or CMNa plus radiation group were higher than radiation group. The radiosensitizing effect of 30%IC50 concentration was more powerful than 20%IC50 concentration. Western blot showed the level of HIF-1a, VEGF, GLUT in IR or CMNa plus radiation group were lower than radiation group. The radiosensitizing effect of 30%IC50 concentration was more powerful than 20%IC50 concentration. 18F-FDG and 18F-FMISO uptake experiment showed rabbit VX2 lung transplant tumor model had low 18F-FMISO uptake. The uptake of 18F-FDG and 18F-FMISO was increased in hypoxic state compared with normoxic tumor cells and influenced by hypoxic level and lasting time. The 18F-FDG and 18F-FMISO uptake level of IR and CMNa plus radiation group significantly decreased which was lower than radiation group, and the difference between groups was statistically significant(P<0.05).2. In vivo experiment:18F-FDG and 18F-FMISO PET/CT images were performed on VX2 tumor-bearing rabbits and C6 tumor-bearing rats. Rabbit VX2 lung transplant tumor had positive uptake of 18F-FDG and negative uptake of 18F-FMISO. Rat C6 hindlimb muscle transplant tumor had positive uptake of both 18F-FDG and 18F-FMISO. The SUVmax of 18F-FMISO uptake was lower than 18F-FDG uptake in the same tumor model.24 hours after treatment, the SUVmax of tumors in the control group, IR group and CMNa group increased, the difference between before and 24 hours after treatment was statistically significant(P<0.05) and the difference between groups was no significant(P>0.05). The SUVmax of tumors in the radiation group, IR plus radiation group and CMNa plus radiation group reduced, the difference between before and 24 hours after treatment was statistically significant(P<0.05). The SUVmax of tumors in the medicine plus radiation group was lower than radiation group(P<0.05), and the difference between IR and CMNa was no significant(P>0.05).24 hours after treatment, the tumor volume of rabbits grew up in six groups, the tumor volume percentage change(ΔV) of radiation group, IR plus radiation group and CMNa plus radiation group were lower than the rest three groups. The tumor volume of rats grew up in the control group, IR group and CMNa group(P<0.05), while the tumor volume of rats in the rest three groups were similarly to the beginning(P>0.05).18F-FDG and 18F-FMISO were excreted by kidney. The radioactivity of non-necrosis was nearly ten times of necrosis. Immunohistochemistry revealed that tumor area of 18F-FDG and 18F-FMISO accumulation showed high expression of HIF-1a, VEGF and GLUT-1. The SUVmax of 18F-FDG and 18F-FMISO uptake was significant positivecorrelation with HIF-1a, VEGF and GLUT-1 expression(P all<0.05). There was the highest correlation coefficient between the SUVmax of 18F-FDG and GLUT-1. There was the highest correlation coefficient between the SUVmax of 18F-FMISO and HIF-1a.ConclusionThe results from our research displayed that IR and CMNa had the radiosensitizing effect on normoxic and hypoxic VX2 and C6 tumor cells. The mechanism of the sensitization was the inhibition of GSH synthesis and damage repair. IR or CMNa plus radiation could reduce the level of HIF-1a, VEGF and GLUT-1 expression which were consistent with the 18F-FDG and 18F-FMISO PET/CT imaging results. The results indicated that 18F-FDG and 18F-FMISO PET/CT images could evaluate the early reaction of radiation sensitizers before morphology change. |
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