| Objective: It is the major purpose in this study to investigate the inhibition of glioma cells affected by tamoxifen combined with (60)~Co γ -rays as well as the radiosensitizing effect of tamoxifen on SHG-44 cells. The study also included research on the effect of apoptosis and cell cycle progress of glioma cells induced by tamoxifen and (60)~Co γ-rays, the effect on the level of cytosolic free calcium affected by tamoxifen was analysed as well. All these researches are to investigate the mechanism of proliferation inhibition and radisensitizing effect of tamoxifen on SHG-44 cells.Methods: 3~H incorporation assay was used to investigate the inhibition effect on DNA synthesis in SHG-44 cells treated with tamoxifen alone or combined with (60)~Co γ -rays, the radiosensitizing effect of tamoxifen on SHG44 cells was also oberseved.Flow cytometry was used to investigate the cell cycle progress and apoptosis treated with tamoxifen alone or combined with (60)Co γ -rays . Morphology feature of apoptosis was observed by laser confocal microscope in SHG-44 cells stained with AO/EB. The cells were immunohistochemically assessed for estrogen receptor expression, using a estrogen-receptor-positive clinical breast cancer specimen as a positive control.Calcium ionophore-induced apoptosis affected by tamoxifen was observed by laser confocal microscope in cells stained with Hochst. Cultured SHG-44 cells were loaded with calcium fluorescent probe Fluo-3/AM , and the level of cytosolic free calcium was determined by laser confocal microscope.Results: Tamoxifen could significantly inhibit DNA synthesis in SHG-44 cells, and it blocked DNA synthesis in a concentration-dependent manner , the combination of tamoxifen and (60)~Co γ -rays showed a synergistic action on it. Tamoxifen shifted the radiation dose -response curve of SHG-44 to theleft, with the decrease of Do values.Tamoxifen could change cell-cycle of SHG-44 cells in a dose-dependent manner, Go/G, and G2/M phase cells increase and S phase cells decrease. Tamoxifen could dose-dependently induce the apoptosis of SHG-44 cells, and karyopyknosis, nuclear fragmentation and apoptotic body in the cells were observed, synergitical effect was observed when associated with ""Co y -rays. Immunohistochemistry of the estrogen receptor revealed intense brown staining of the nuclei of the breast cancer cells, whereas those of the glioma cells remained essentially unstained. These findings suggested that tamoxifen inhibited DNA synthesis in SHG-44 cells and sensitized SHG-44 cells to gama radiation in our model, which may not result from the estrogen receptor-mediated process but from apoptosis pathway and changing cell cycle.Tamoxifen could accelerate calcium ionophore-induced apoptosis . In the presence of extracellular calcium, tamoxifen can dose-dependently and promptly increase cytosolic calcium,which remained at a high level;in the absence , it can also but slowly increase cytosolic calcium , the range and the rate of calcium elevation were lower than that of in the presence.Conlusion: Tamoxifen can sensitize SHG44 cells to gama radiation, the radiation dose -response curve of SHG-44 was changed and shifted to the left, with the decrease of Do values , the model was changed from multi-hit or multi-taget to single-hit or single-taget.Tamoxifen could inhibit DNA synthesis of SHG-44 cells. Tamoxifen enhance radiosensitizing effect of SHG-44 cells to 60Co y -rays through inducing apoptosis and arresting cell in G0/Gi and G2/M.Tamoxifen could increase cytosolic calcium by both releasing intercellular calcium and causing extracellular calcium influx. Tamoxifen could induce apoptosis and arrest the SHG--44 glioma cells in G0/Gi and G2/M , which was related with increament of cytosolic calcium. |
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