Figures & data
Figure 1. γ-irradiation had a cytotoxic effect on LN229 and U251 cells. (A) γ-irradiation had a manner of time-depend and dose-depend effect on LN229 cell proliferation. LN229 cells treated with 6, 9, 12, 15, 18, 21 Gy γ-irradiation were detected to have a reduction on cell proliferation ability at the time point of 24, 48, 72, 96, 120 hours. (B) Compared with LN229, U251 had a resistance to γ-irradiation. Cell proliferation assay showed that U251 treated with γ-irradiation did not have obvious differentiation compared with control group. There existed a clear difference in the sensitivity of LN229 and U251. (C) In apoptosis assay, LN229 had the manner of time-depend and dose-depend to γ-irradiation. LN229 cells with a larger dose of irradiation showed a tendency to early apoptosis at 96 and 120 hours, while this effect could not be detected in U251 cells. Data are mean ± SD of 3 independent experiments. * meant P value was less than 0.05 compared with NC group.
Figure 2. γ-irradiation could induce the Epithelial-to-Mesenchymal Transition. Both LN229 and U251 cells manifested the EMT proteins expression changed. β-catenin and Vimentin were upregulated, while E-cadherin and Claudin were suppressed. GAPDH was used as reference. This phenomenon was more significant at 96 hours than 48 hours. According to these changes, EMT was launched after γ-irradiation. * meant P value was less than 0.05 compared with NC group.
Figure 3. Curcumin could rescue the induction of EMT via suppressing of Hedgehog signaling pathway. With the treatment of 18 Gy γ-irradiation for 96 hours, EMT process of cells was reversed. The expression of β-catenin and Vimentin were restrained. The expression of E-cadherin and Claudin were recovered. The expression of Gli1 and SMO were inhibited, and that of Sufu which acted as an inhibitor of Gli1 was increased. * meant P value was less than 0.05 compared with NC group.
Figure 4. The Immunofluorescence assay showed that the induction of EMT was rescued by Curcumin. Nucleus were stained with DAPI (blue), while E-cadherin, Vimentin and β-catenin were shown in red. The merged pictures were combining with DAPI and the target proteins. γ-IR group showed the induction of EMT in both LN229 and U251, however the CCM+γ-IR group had restrained the process of EMT. All images were taken microscopically (40 ×).
Figure 5. The cell migration and invasion assays revealed that the metastasis ability was restricted in combined therapy group in both LN229 and U251 cells. The γ-irradiation (18Gy) group showed more tendency to metastasize than NC group and CCM (20 μM) group. Adding with Curcumin (20 μM), combined therapy showed the least metastasis ability. * meant P value was less than 0.05 compared with NC group. ** meant P value was less than 0.01 compared with NC group.
Figure 6. In addition with Curcumin (20 μM) and γ-irradiation (18Gy) as combined therapy, Curcumin did not reverse the formation of early apoptosis promoted by γ-irradiation. In LN229, Curcumin could advance the cytotoxic effect of γ-irradiation remarkably, however this phenomenon was slightly in U251. (A) The cell proliferation assay showed the promotion of Curcumin was remarkable in LN229, while that of U251 was slightly. The bar graph merged 2 cell line together for a more intuitive observation. (B) Cell apoptosis assay showed the combined therapy had limited influence on early apoptosis of U251, however it was more effective in LN229. * meant P value was less than 0.05 compared with NC group. (C) A model for Curcumin increasing the efficiency of γ-irradiation by regulating Hedgehog signaling pathway.
Figure 7. The bioluminescence images showed that tumors formed by LN229 were sensitive to γ-irradiation. The size of tumors were detected every 5 d and had decreased gradually in CCM group, γ-IR group, CCM+γ-IR group in 15 d. The tumors formed by U251 were resist to γ-irradiation. The columns on the right represented the fluorescence intensity of tumors. * meant P value was less than 0.05 compared with NC group.
Figure 8. The Immunohistochemistry showed the effect of Curcumin on induction of EMT in vivo. All images was taken microscopically (40 ×). The expression of E-cadherin was upregulated in CCM+γ-IR group and Vimentin was downregulated compared with NC group. Both LN229 and U251 showed the similar results.
