| Objective To observe the radiosensitizing effects of GXHSWAQ-1 (an Anthraquinone compoune extracted from Guangxi Polygonum multiflorum )on human nasopharyngeal carcinoma in vitro and in vivo, and investigate the mechanism of radiosensitivity.Methods (1) CNE-1 cells were incubated with GXHSWAQ-1 at various concentrations, then to determine the No-cytotoxic concentrations. (2) Cloning survival assay was used for studing the radiosensitizing effect of GXHSWAQ-1 monomer after treatment of CNE-1 cells in the hypoxic environment. (3) The expression of genes that related hypoxia, DNA break repair and apoptosis between the experimental groups and the control group under hypoxic condition was detected by the real-time fluorescence quantitative PCR.(4) Changes of cell cycle, apoptosis and contents of ROS between the experimental groups and the control group were analyzed with flowcyometry(FCM). (5) Tumour -bearing model was established by injecting nasopharyngeal carcinoma cells to Nu/Nu nude mice. Mice bearing nasopharyngeal carcinoma were randomized into seven groups: control group, solvent group, the group with GXHSWAQ-1 alone, which had high and low two doses, the group with radiation alone, the group with both GXHSWAQ-1 and radiation,which also had two doses. Each group consisted of 5 or 6 mice. The longest diameters of the tumors were measured to calculate the tumor Absolute growth delay(AGD), Normalized growth delay(NGD) and Enhancement factor(EF).Observed the pathological changes by HE staining and calculated the mitotic index.Results 1.GXHSWAQ-1 had the minor proliferation inhibitory action to the CNE-1 cells. The value of IC50 was 465μg/ml. When the concentration of GXHSWAQ-1 was lower than 62.5μg/ml, there was no cytotoxicity for CNE-1cells (Inhibitory ration is lower than 10% ).2.There were effects radiosensitivity of CNE-1 cells exposed to GXHSWAQ-1 in the no-cytotoxic concentrations(3.9μg/ml and 7.8μg/ml), the sensitization enhancement ration (SER) were 1.436 and 1.832. 3.Expression of HIF-1αwas significantly increased under hypoxia condition. HIF-1αhad no change after treatment with GXHSWAQ-1 alone. Compared with control group, the expression level of KU70 /80, Survivin and hTERT were induced by radiation. Compared with radiation alone group, radiation combined hypoxia group obviously enhanced the expression of KU70/80, Survivin and hTERT. KU70/80, Survivin and hTERT mRNA expression significantly reduced after radiation combined with GXHSWAQ-1 under hypoxic condition.4. The cell percentage (%) of CNE-1 cell distributing among the cell cycle (G0/G1 and G2/M) were 49.03±1.63 and 14.27±0.57 in GXHSWAQ-1 combined radiation group, 53.89±1.12 and 8.0±0.18 in radiation alone group, 72.19±3.736 and 5.81±0.15 in GXHSWAQ-1 group, 66.13±4.02 and 3.57±0.77 in control group, respectively. In GXHSWAQ-1 combined radiation group, there was significant G2/M-phase arrest. The apoptosis ratio (%) of CNE-1 cell was 22.78±1.6 in GXHSWAQ-1 combined radiation group, 19.92±2.0 in radiation alone group, 16.58±0.8 in GXHSWAQ-1 group, and 17.79±1.3 in control group. The apoptosis ratio of combined group was significantly higher than that of other groups(P<0.05).Compared with control group, the relative contents of ROS in CNE-1 cell determined at time of 12h, 24h were 0.47±0.13, 0.71±0.08 in hypoxia group; 2.82±0.23, 3.17±0.35 in GXHSWAQ-1 combined hypoxia group; 2.17±0.23, 1.54±0.27 in radiation alone group; 2.78±0.24, 2.30±0.18 in GXHSWAQ-1 combined radiation group respectively. The relatively content of ROS in CNE-1 cell in GXHSWAQ-1 combined hypoxia group was significantly higher than those in hypoxia group at time of 12h and 24h(P<0.05).The relative content of ROS in CNE-1 cell in GXHSWAQ-1 combined radiation group was significantly higher than those in radiation alone group at time of 12h and 24h(P<0.05).5.The tumor diameter doubling time(d) was 17.02±1.1 in control group, 17.14±0.6 in solvent group, 17.2±0.8 in 12mg/kg GXHSWAQ-1 group, 17.4±1.0 in 4mg/kg GXHSWAQ-1 group , 21.3±1.14 in radiation alone group, 24.1±0.7 in 12mg/kg GXHSWAQ-1 combined radiation group, 24.17±0.6 in 4mg/kg GXHSWAQ-1 combined radiation group respectively. There was no significant differences in the control group, solvent group and in the group with GXHSWAQ-1 alone (P>0.05).Tumor growth inhibitions in combination groups and radiation group were higher than those in control group(P<0.05).Compared with control group, the time of tumor growth delay were 4.28 days in radiation group, 6.9 days in 12mg/kg GXHSWAQ-1 combined radiation group, 6.77 days in 4mg/kg GXHSWAQ-1 combined radiation group respectively. The values of EF(enhancement factor) were 1.612 and 1.601 in 12mg/kg GXHSWAQ-1 combined radiation group and 4mg/kg GXHSWAQ-1 combined radiation group. The mitotic index(%) was 6.08±0.8 in control group, 6.59±0.226 in radiation group, 3.80±0.455 in combination group with high dose, 3.43±0.34 in combination group with low dose respectively. There was no significant differences in the control group and radiation alone group (P>0.05). The mitotic index of combined groups was significantly lower than that in control group (P<0.05).Conclusion 1.There is radiosensitizing effect of CNE-1 cells exposed to GXHSWAQ-1 in the no-cytotoxic concentrations. The mechanism may be inhibiting cells repairing in the hypoxic environment, inducing cells apoptosis,causing G2/M block and enhancing the content of ROS.2.GXHSWAQ-1 had shown a radiosensitizing effect on human nasopharyngeal carcinoma in vivo. |
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