Molecular Imaging And Biological Evaluation Of The Radiosensitive Effect Of Oleanolic Acid

Objective The aim of this study was to elucidate the radiosensitizing effect and mechanism of oleanolic acid（OA）which is a kind of natural drug extracted from Chinese herbal medicine.¹⁸F-FluoromisonidazolPositronEmissionTomography/Computed Tomography(¹⁸F-FMISO PET/CT)was used to evaluate the changes of tumor hypoxia and the radiosensitizing effect of OA on C6 glioma tumor model.The biological changes of tumors were also detected via histopathological and immunohistochemical studies in order to investigate the mechanism of OA and evaluate the feasibility and superiority of¹⁸F-FMISO PET/CT in assessing tumor hypoxia and radiosentization.Methods1.In vitro experiments:The radiosensitizing effect and mechanism of OA on C6 glioma cells were detected in vitro by MTT,Clonogenic survival assay,Real-time PCR and Western blot.MTT assay:Hypoxic C6 cells model was established as described in the literature using 100 umol/l CoCl₂.Normoxic and hypoxic C6 tumor cells were treated by gradient concentrations of OA for 12 h,24 h,48 h.The absorbance of cells was measured at 570 nm using microplate reader.The inhibition ratios were calculated and tumor cells survival curves were plotted.Then the half effective inhibition concentration(IC₅₀)of OA on normoxic and hypoxic C6 cells at 24 h time-point were obtained by software.Clonogenic survival assay:The radiosensitizing effect of OA on normoxic and hypoxic C6 tumor cells were observed by Clonogenic survival assay.Normoxic and hypoxic C6 tumor cells were treated with 20%IC₅₀0 or 30%IC₅₀0 concentration OA for 24 h,then irradiation with gradient doses from 0 to 8 Gy were followed immediately.Tumor colony forming efficiency of each group was obtained two weeks later.Oxygen enhancement ratio（OER）and sensitive enhancement ratio（SER）were calculated.Real-time PCR and Western blot:The effect of 20%IC₅₀0 or 30%IC₅₀0 concentration OA with or without radiation on the variations of hypoxia inducible factor-1a（HIF-1α）expression at RNA and protein levels were observed by Real-time PCR and Western blot,respectively.2.Animal experiments:The radiosensitizing effect of OA on C6 glioma tumor model was observed in vivo based on tumor volume and survival time of tumor-bearing rats.Rat hindlimb transplant C6 tumor model was established and divided into four groups,the control group,OA group,radiation group and OA combined with radiation group,six tumor-bearing rats for each group.Tumor volume of each rat was calculated weekly and the growth curves were plotted.Tumor inhibition rates were calculated based on the average tumor volume in each group after treatment.Survival analysis was performed on the death time of each tumor-bearing rats.3.¹⁸F-FMISO PET/CT imaging For the hypoxic imaging experiment,C6 tumor bearing rats were established and divided into four groups,the control group,OA group,radiation group and OA combined with radiation group,six tumor-bearing rats for each group.¹⁸F-FMISO PET/CT imaging was performed before and 24 h after treatment.Visual analysis combined with semi-quantitative analysis were used for PET/CT image interpretation.Select the brightest image plane and outline the region of interest for PET/CT image data analysis.Then the maximal standard uptake value of tumor（SUVmax-tumor）and the maximal standard uptake value of contralateral limb muscle（SUVmax-muscle）were acquired automatically.Tumor muscle ratio of SUVmax（TMR）was used as the index for statistical analysis.4.Pathological experiments Tumor bearing rats were euthanized for pathological experiments,include gross observation of pathology,hematoxylin and esosin staining and immunohistochemical staining.The changes of tumor biological indicators like HIF-1α,Glut-1,Ki67,P53 and micro-vessel density（MVD）were calculated.5.Statistical analysis Results of in vitro and in vivo experiments were analyzed via statistical software SPSS17.0 in order to study the radiosensitization and mechanism of OA.The correlation and regression analysis between TMR and tumor biological indicators HIF-1α,Glut-1,Ki67,P53 and MVD were tested respectively.Results1.In vitro experiments MTT assay indicated that OA inhibited the normoxic and hypoxic C6 tumor cells activity and induced cell death in a dose-dependent and time-dependent manner.The inhibitory effect of OA on normoxic cells was stronger than that of hypoxic cells.At 24 h time point,IC₅₀0 of OA for normoxic C6 tumor cells was 42μg/mL and it was 68μg/mL for hypoxic C6 tumor cells.Clonogenic survival assay showed that the survival rate of hypoxic C6 tumor cells was higher than normoxic C6 tumor cells when exposed to the same dose of radiation,and the oxygen enhancement ratio（OER）was 1.15.Both 20%IC₅₀0 and 30%IC₅₀concentration of OA had radiosensitive effect on normoxic and hypoxic C6 tumor cells.The sensitive enhancement ratio（SER）of OA at 20%IC₅₀0 concentration was 1.51 for normaxic C6 tumor cells and 1.64 for hypoxic C6 tumor cells.Meanwhile,SER of OA at 30%IC₅₀0 concentration was 1.72 for normaxic C6 tumor cells and 1.87 for hypoxic C6 tumor cells.Real-time PCR and Western blot reflected that OA at concentration of 20%IC₅₀0 and30%IC₅₀0 combined with radiation could down-regulate HIF-1αmRNA and protein levels of C6 tumor cells significantly.HIF-1αmRNA level of hypoxic C6 tumor cells treated with 20%IC₅₀0 or 30%IC₅₀0 concentration OA combined with radiation was lower than radiation group（t=3.35,5.58,P<0.05）.HIF-1αprotein level of hypoxic C6 tumor cells treated with 20%IC₅₀0 or 30%IC₅₀0 concentration OA combined with radiation was lower than radiation group（t=7.98,13.62,P<0.01）.Compared with the control group,OA at the concentration of 30%IC₅₀0 down-regulated the HIF-1αmRNA level of hypoxic C6 tumor cells（t=3.21,P<0.05）.There was no significantly differences of HIF-1αprotein level between the OA group and control group.2.Animal experiments Before and 24 h after treatment,tumor volumes in control group were（0.62±0.05,1.65±0.09）cm³,tumor volumes in OA group were（0.65±0.04,1.69±0.11）cm³,tumor volumes in radiation group were（0.62±0.04,0.78±0.19）cm³,tumor volumes in OA combined with radiation group were（0.63±0.08,0.81±0.26）cm³.There was no significantly difference between control group and OA group.Tumor inhibition rates of radiation and OA combined with radiation group were 52.73%and 51.91%,respectively.There was no significantly difference of inhibitory effect between radiation group and OA combined with radiation group.Survival time of tumor-bearing rats in control group,OA group,radiation group and OA combined with radiation group were 43.67±3.3（33⁵2）d,44.62±3.1（35⁵4）d,53.3±2.2（43⁶1）d and 56.8±2.6（44⁶5）d,respectively.Sixty days after tumor cells inoculated into rats,survival rates of tumor-bearing rats in control group,OA group,radiation group and OA combined with radiation group were 0,0,16.7%and 33.3%,respectively.The survival differences between each groups were statistically significant via Kaplan-Meier analysis（χ²=12.5,P<0.01）.3.¹⁸F-FMISO PET/CT imaging¹⁸F-FMISO PET/CT imaging was performed of each tumor-baring rat before and 24 h after treatment.Before treatment,TMR of tumor-bearing rats was（1.29±0.12）.The criterion of tumor hypoxia based on TMR was set at 1.2.TMR>1.2 was defined as the hypoxic tumor.The proportion of hypoxic tumor before treatment was91.7%（22/24）.24 h after treatment,the proportions of hypoxic tumor in control group,OA group,radiation group and OA combined with radiation group were 100%（6/6）,100%（6/6）,66.7%（4/6）and 33.3%（2/6）,respectively.TMRs of each group before and 24 h after treatment were compared statistically.TMRs of control group before and after treatment were（1.28±0.08,2.78±0.16,t=22.89,P<0.01）.TMRs of OA group before and after treatment were（1.29±0.16,2.61±0.22,t=17.7,P<0.01）.TMRs of radiation group before and after treatment were（1.28±0.06,1.24±0.32,t=1.09,P>0.05）.TMRs of OA combined with radiation group before and after treatment were（1.30±0.07,1.15±0.13,t=3.26,P<0.05）.TMR growth rates of control and OA group were 117.18%and 102.32%.TMR reduced rates of radiation group and OA combined with radiation group were 3.12%and 11.54%.4.Pathological experiments Gross observation showed that C6 tumors were round and nodular with clear boundaries.After incision,the margin of tumor was like reddish fish and the central region was like grayish white surimi.HE staining indicated that the property of C6 tumor cells was obvious heterogeneity,large nuclei,dense and disordered arrangement.Meanwhile,the necrotic area was red stained.Irreversible damage of tumor cells were found in radiation group and OA combined with radiation group such as nuclear pyknosis,nucleolysis,nuclear rupture and so on.Immunohistochemical analysis was carried out to assess tumor biological indicators like HIF-1α,Glut-1,Ki67,P53 and MVD.The percentage of HIF-1αpositive cells in control group,OA group,radiation group and OA combined with radiation group were（84.53±4.91,81.82±6.45,52.53±6.87,38.87±6.32）%,respectively.The percentage of Glut-1 positive cells in control group,OA group,radiation group and OA combined with radiation group were（89.24±5.88,89.87±3.94,67.25±9.76,45.09±9.61）%,respectively.The percentage of Ki67 positive cells in control group,OA group,radiation group and OA combined with radiation group were（35.57±4.39,36.52±4.62,19.53±6.78,15.85±4.83）%,respectively.The percentage of P53 positive cells in control group,OA group,radiation group and OA combined with radiation group were（53.06±10.41,52.84±11.93,27.79±12.45,30.38±13.02）%,respectively.MVD of tumor in control group,OA group,radiation group and OA combined with radiation group were 19.01±5.34,18.37±4.98,9.06±3.35,9.67±3.68,respectively.Significant differences of these tumor biological indicators were observed between groups（F=78.83,45.94,25.43,7.98,9.25,P<0.01,respectively）.Pairwise compare was acquired via LSD statistical analysis.There was no significant differences of HIF-1α,Glut-1,Ki67,P53 and MVD between control group and OA group.The percentage of HIF-1αand Glut-1 positive cells in OA combined with radiation group were lower than radiation group（P<0.05）.However,there was no significant difference of Ki67,P53 and MVD between OA combined with radiation group and radiation group.5.Statistical analysis As an index for¹⁸F-FMISO uptake,TMR showed positive correlation with tumor biological indicators like HIF-1α,Glut-1,Ki67,P53 and MVD（r=0.92,0.86,0.89,0.70,0.81,P<0.01）.TMR was used as dependent variable,HIF-1α,Glut-1,Ki67,P53 and MVD were used simultaneously as independent variables for multivariate linear regression analysis.The results indicated that the percentage of HIF-1αpositive cells in tumor tissue was an independent factor for TMR value（P<0.01）.Conclusion The present experiments indicated that oleanolic acid which is a kind of natural drug extracted from Chinese herbal medicine increased the radiosensitivity of C6 tumor both in vitro and in vivo.The primary mechanism of this radiosentizing effect involves in the down-regulation of HIF-1αmRNA and protein.Oleanolic acid combined with radiation inhibited the growth rates of tumors and prolonged the survival period of tumor-bearing rats effectively.¹⁸F-FMISO PET/CT was sensitive and noninvasive for monitoring tumor hypoxia and the radiosentizing effect of oleanolic acid.HIF-1α,Glut-1,Ki67,P53 and MVD in tumor were down-regulated by oleanolic acid combined with radiation.As an index for¹⁸F-FMISO uptake,TMR showed positive correlation with tumor biological indicators,in which the percentage of HIF-1αpositive cells in tumor tissue was an independent factor for TMR value.