| Background and purpose of studyThe glioma is one with highest morbidity in the intracranial malignant primary tumors.Although the clinical treatments for patients with glioma include surgery, radiotherapy and chemotherapy, presently, prognosis is very poor for those patients with malignant gliomas.The median survival time in patients with glioblastoma presents not more than 15 months.According to the World Health Organization(WHO), gliomas can be graded as I-IV, and gliomas with grade III and IV are highly malignant and aggressive ones. The failure of current glioma radiotherapy might be resulted from local tumor recurrence, in which radiation resistance of tumor may be one major reason in radiotherapy. A line of growing evidences have indicated that existence of glioma stem cells or tumor stem cells, glioma micro-environment, and DNA damage/repair mechanism might contribute the generation of radiation resistance. It is essential to study radiation biology of glioma stem cells and related regulation signaling pathway underling radiation resistance, which may also be one candidate way to establish a new radiotherapy strategy by targeting glioma stem cells.Evidence has shown that Wnt signaling is a critical regulatory factor in stem cell proliferation and differentiation. Some studies also indicated activation of Wnt/?-catenin signaling in glioma cells, suggesting its involvement in human glioma occurrence.However, previous data had mainly focused on killing effects of high-dose radiation on glioma cells, it is still lack of studies on its role in low-dose radiation on glioma cells or tumor stem-like cells. By combining previous literatures and our pilot observation on stimulation effect of low-dose radiation on neural stem cell, we thus hypothesize that low-dose radiation may stimulate proliferation and growth of glioma cells or glioma stem cell through induction of Wnt signaling activation, which merits further investigation.Therefore, U251 human glioma cell line was applied in this study, by using method of dispersing and cloning culture, low-dose radiation cell models and transplanted glioma animal models, and administration of selective Wnt singling inhibitor. Major objectives are focused on effect of low-dose radiation stimulating viability, proliferation, growth of glioma cells or tumor stem-like cells, and possible Wnt/?-catenin signaling mechanism involving in this biological process.Methods1. Cell culture in vitro method: The dispersing and cloning culture were applied to U251 cells by in serum-containing medium and serum-free medium respectively.2. Transplanted glioma in vivo method:The U251 glioma cells or glioma stem-like cells were injected subcutaneously into nude mice for the purpose to observe formation,growth and proliferation of transplanted tumors.3. Cell model with low-dose radiation: X-ray low-dose radiation(0.3Gy, 0.6Gy) was applied to observe effect on proliferation and growth of glioma cells.4. Administration of Wnt signal inhibitor: Wnt signal inhibitors(IWR-1) was used to observe role of Wnt signaling pathway activation in proliferation of glioma cells and low-dose radiation stimulation effect.5. Cell viability: MTT assay and clone formation were performed to compare cell viability and proliferation of glioma cells or glioma stem-like cells among distinct groups.6. Immunocytochemistry: Immunoflurescent staining was carried out to observe cell localization of proliferation and Wnt signaling molecules in glioma cells.7. Western blot method: Western blotting was performed to detect expression levels and changes of Wnt signal-related proteins in cultured cell and transplanted tumor tissue.Results1. The proliferation of U251 human glioma cells, clone growth and cellular location of Wnt/?-catenin signaling pathway-related molecules By establishment of dispersing culture for U251 cells and cloning culture for tumor stem cells, characteristics of glioma cells or stem-like cells were observed by immunofluorescence to CD133(tumor stem cell marker), GFAP(astrocytic cell marker), Ki-67(cell proliferation marker), Nestin(neural stem cell marker), BrdU(nuclear incorporation for cell division) and Wnt signaling molecules. It revealed that most cultured U251 cells in fusiform or elliptic were in active proliferation and growth, showed strong CD133 immunoreactivty and weak GFAP positivity. The clones or neurospheres were generated after 5-7d culture with serum-free culture medium, and clone cells showed strong expression of Ki-67 or nestin proteins.Cellular location of Wnt1, Wnt3 a, Wnt5 a and ?-catenin was observed in almost all glioma cells. Data thus indicated that U251 glioma cells might exhibit tumor stem cells marker,active proliferation and abundant expression of Wnt signaling pathway-related proteins.2. The promoting effect of low-dose radiation on stimulating proliferation of glioma cells clone formation and intervention of Wnt signaling pathway Cell viability and proliferation of glioma cells or stem-like cells were observed by using MTT assay,clone formation, migration test, and immunostaining detection of Ki-67 and proliferative cell nuclear antigen(PCNA). The result showed that low-dose radiation stimulated cell proliferation of glioma cells, promoted clone formation, and increased expression level of Ki-67 and PCNA compared to that of control. Wnt signaling inhibitor IWR-1 could significantly inhibit cell proliferation and migration of glioma cells, clone formation stimulated by low-dose radiation. Data indicated that low-dose radiation could stimulate proliferation and migration of glioma cells, in which Wnt signaling activation might beinvolved in mediation biological effect of low-dose radiation.3. The stimulating effect of low-dose radiation promoting formation and growth of transplanted tumor and intervention of Wnt signaling pathway By establishment of transplanted tumor model with injection of U251 cells or glioma stem-like cells subcutaneously into nude mice, the tumor formation, growth, morphology and Ki-67 immunostaining of transplanted glioma tumors were observed at d4, 7, 10, 13, 16, 19 and22 after cell injection. Results showed that transplanted tumors were obviously formed during period of d13 to d22. The low-dose radiation significantly increase size, volume and weight of transplanted tumors, and Ki-67-potive cells compared with that of control.Administration of Wnt signaling inhibitor IWR-1 could block stimulating effect of low-dose radiation on tumor growth. Data indicated that low-dose radiation could promote formation and growth of transplanted glioma tumors, in which activation of Wnt signaling pathway might be involved in this process.4. Analysis of intracellular signaling pathways underling glioma cell proliferation and tumor growth induced by low-dose radiation By using 24h/48 h cultured glioma cells and d22 transplanted tumor samples and Western blotting, expression and phosphorylation levels of Wnt AKT, ERK1/2 and JNK signaling pathway were observed for analysis of their functional states or activation. It revealed that low-dose radiation increased expression of Wnt1,Wnt2,Wnt3 a,Wnt5a and ?-catenin proteins and decreased expression of p-?-catenin compared with control. Besides, expression of AKT,p-AKT,ERK1/2 and p-ERK1/2 was up-regulated while JNK and p-JNK was down-regulated in low-dose radiation group compared with control. Wnt inhibitor IWR-1 treatment could decrease or turnover stimulating effect induced by low-dose radiation. Data further confirmed that activation or involvement of Wnt signaling pathway during proliferation of glioma cells and growth of tumors stimulated by low-dose radiation. It also suggested enrollment of AKT and ERK1/2 signaling activation in the biological process induced by low-dose radiation. However, precise relationship of Wnt signaling and AKT or ERK1/2signaling activation and pathological significance need further investigation.ConclusionBy in vitro and in vivo experiments, it revealed that the low-dose ionizing radiation could stimulate proliferation of human glioma cells(or tumor stem-like cells) and formation of transplanted tumors, in which abnormal activation of Wnt/?-catenin signaling pathways might be involved in this low-dose radiation stimulation process. Data of this study will be beneficial for us to understand pathogenesis of human glioma, and it may also provide valuable evidence for establishing new radiotherapy of human glioma by targeting on glioma stem cells. |
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