| Objective To invstigate effects of low-dose radiation on the expression of immunogenic membrane molecules calreticulin (CRT) and MHC-I/II on the surface of human renal clear cell carcinoma 786-0 cells, to explore a better play of radiotherapy for the cellular immunotherapy and lead to a synergistic anti-tumor effect, to provide basic experimental data for further improved efficacy of cancer treatmentMethods The inhibitory activity of low-dose radiation on cell line 786-0 was tested by the CCK-8 assay. The expression of CRT,MHC-I and MHC-II, on the membrane of the 786-0 cell line after radiation, was measured by flow cytometry, and CRT was also visual displayed by immunofluorescence photography.Results 1 The results of CCK-8 assay: The inhibition rates on the proliferation abilities of four 786-0 cell line groups was increased with the increasing radiation dose as 0Gy,6Gy, 12Gy and 24Gy, and the median inhibition dose (IC50) after 24h was 12Gy. 2 Impacts on the expression of membrane molecule CRT in the four 786-0 groups irradiated by 0Gy, 6Gy, 12Gy and 24Gy respectively: The mean fluorescence intensity of CRT determined by flow cytometry as follows : In the control group, CRT(0Gy) 13.32%±2.01%; In the experimental groups irradiated by 6Gy, 12Gy, and 24Gy, the CRT mean fluorescence intensities were 78.25%±5.83%, 202.24%±16.47% and 83.51%±8.26% respectively. The CRT exposure of each experimental group was significantly higher than that of the control group(P<0.001).Among them, the strongest effect on CRT expression was induced by 12Gy radiation, followed by 24Gy radiation, and the weakest effect was induced by 6Gy radiation. Along with the dose increase of irradiation, the average calreticulin fluorescence intensities were increased gradually at first, then shown a downward trend. As compared 12Gy radiation group with 24Gy and 6Gy radiation groups, the differences appeared significantly (P values <0.05).3 Impacts on the expression of membrane molecule MHC-I in the four 786-0 groups irradiated by 0Gy, 6Gy, 12Gy and 24Gy respectively: The mean fluorescence intensity of MHC-I determined by flow cytometry as follows : In the control group, the mean fluorescence intensity of MHC-I(0Gy) was 8.24%±1.25%; In the experimental groups irradiated by 6Gy, 12Gy, and 24Gy, the mean fluorescence intensities of MHC-I were 16.26%±1.81%, 27.39%±2.43% and 48.72%±3.85% respectively. The MHC-I exposure of each experimental group was significantly higher than that of the control group(P values <0.05). As the irradiation dose increases, the average MHC-I fluorescence intensities were increased gradually, such upregulation was shown to be dose dependent, with higher radiation doses resulting in higher MHC-I expression. 4 Impacts on the expression of membrane molecule MHC-II in the four 786-0 groups irradiated by 0Gy, 6Gy, 12Gy and 24Gy respectively: The mean fluorescence intensity of MHC-II determined by flow cytometry as follows : In the control group, the mean fluorescence intensity of MHC-II(0Gy) was 9.07%±1.43%; In the experimental groups irradiated by 6Gy, 6Gy, and 12Gy, the mean fluorescence intensities of MHC-II were 11.46%±1.55%, 23.82%±2.61% and 31.52%±2.63% respectively. The MHC-II exposure of each experimental group was significantly higher than that of the control group(P values <0.05). As the irradiation dose increases, the average MHC-II fluorescence intensities were increased gradually, such upregulation was also shown to be dose dependent, with higher radiation doses resulting in higher MHC-II expression.Conclusions 1 After irradiation 24h, the expressions of CRT, MHC-I and MHC-II, the immunogenicity associated molecules on 786-0 cell surface, upregulated along with dose increase, indicating that in addition to the direct killing effect, radiotherapy also can enhance the immunogenicity of tumor cells, which might improve the ability of body's immune cells to recognize tumor antigens and leads to stronger antitumor effects.2 Although a certain relevance were shown between radiation dose and the expression of CRT, MHC-I and MHC-II on the surface of 786-0 cells, while lower dose of radiation can achieve this effect, indicating that low-dose radiation might be able enough to upregulate the immuneogenicity of 786-0 cells, the result suggests that low-dose radiation can induce immune response against tumor, thus avoiding severe immune suppression as well as damages on normal organs or tissue caused by large dose of radiation. These results suggest that low-dose radiotherapy could upregulate CRT and MHC class I/II molecules which related to the immunogenicity of the tumor cells. In light of these findings, radiotherapy combined immunotherapy might be considered in relapsing patients with renal clear cell carcinoma after receiving the standard treatment. |
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