Study On The Effect And Mechanism Of Tamoxifen On The Radiosensitivity In Glioma Cell SHG44

Objective: The aim of this study is to investigate the effects of tamoxifen combined with ⁶⁰Coγ-rays on DNA damage and DNA repair in SHG-44 cells. The study also examined the activity of Caspase 9, the expression of PKCα,Cyclin D1and Bcl-2,the effects on PI3K/Akt pathway caused by tamoxifen combined with ⁶⁰Coγ-rays was also analysed .All these researches are to investigate the effects and mechanism of tamoxifen on the radiosensitivity in glioma cell SHG44.Methods: The DNA damage and DNA repair were measured by alkaline single cell gelelectrophoresis assay;the activity of Caspase 9 was determined by spectrophotometric methods; Changes of the expression of protein (PKCα,Cyclin D1,Bcl-2,Akt,p-Akt,p-c-Raf,p-PTEN,p-PDK1,p-GSK-3β) caused by tamoxifen combined withγ-ray irradiation was investigated by Western-blot analysis.Results: 1,The alkaline single cell gelelectrophoresis assay showed that:the initial DNA damage caused byγ-ray irradiation was aggravated by tamoxifen,the OTM value of the combined groups increased 89.5% when compared with the radiation group immediately after treatments (p <0.05);The DNA repair was time dependent,the combined treatment delayd the DNA repair,the DNA strain breaks of other groups were more efficiently rapaired,while there was still significant residual DNA strain break in the combined groups 2 hours after treatment. 2,The spectrophotometric methods showed that:compared with the control group the activity of Caspase 9 of other groups increased ,the activity was significantly increased 48 hours after treatments compared with 24 hours(p<0.05);The activity of the combined groups was significantly increased at the same time compared with other groups(p<0.05);Compared with 10μmol/L+2Gy groups ,the activity of 10μmol/L+4Gy groups increased 21.9% and 73.4% after 24 hours and 48 hours respectively(p<0.05).3,Western blot analysis showed that:The expressions of PKCαand Bcl-2 were decreased after irradiation,and tamoxifen enhanced the down-regulation of the expression of PKCαand Bcl-2 induced by irradiation. The expressions of Cyclin D1 was increased after irradiation,but tamoxifen can reverse the increase of Cyclin D1 induced by radiation.4,Western blot analysis showed that:The combination of tamoxifen andγ-ray irradiation has no significant effects on the expression of Akt,p-c-Raf,p-PDK1. The expression of protein p-Akt was increased after irradiation,but tamoxifen can reverse the increase of p-Akt induced by radiation. The expression of protein p-PTEN was increased after irradiation,tamoxifen enhanced the up-regulation of the expression of protein p-PTEN induced by irradiation.The combination of tamoxifen andγ-ray irradiation suppress the expression of protein p-GSK-3β.Conclusion: 1,Tamoxifen increases the irradiation-induced initial DNA damage and delays the DNA repair at the concentration of 10μM. 2,10μM tamoxifen enhances the radiosensitivity of SHG44 cells by raising the level of Caspase 9 activity time-dependently through the intrinsic mitochondrial pathway.3,Tamoxifen of 10μM decreases the expressions of PKCα,Cyclin D1,Bcl-2,p-Akt and p-GSK-3βand increases the expression of p-PTEN. It shows no effects on the expression of p-c-Raf-1,Akt and p-PDK1.The tamoxifen-induced decreased expression of PKCαacts as a trigger to enhance the radiosensitivity of SHG44 cells through suppressing the PI3K/Akt signaling pathway.