Gamma-tocotrienol Ameliorates Senescent Cells Induced By Radiochemotherapy

Objectives:Radiotherapy and chemotherapy are widely used in cancer treatment.Under chemoradiotherapy,it will inevitably cause damage to the surrounding normal tissues,inducing acute and chronic radiation damage,such as hematopoietic syndrome,gastrointestinal syndrome,chronic diseases,aging related diseases and so on.These will seriously affect the quality of life of cancer patients.Therefore,it is very important to study the protection of chemoradiotherapy-induced damage.The purpose of the present study is to establish cell senescent model by ionizing radiation（IR）and doxorubicin（DOX）,to explore whetherγ-tocotrienol（GT3）pretreatment can reduce cell senescence and tissue damage caused by radiotherapy and chemotherapy,and to explore the protective mechanism of GT3,so as to provide new methods and strategies for the prevention of chronic injuries induced by radiotherapy and chemotherapy.Methods1.Establishment and intervention of cell aging model（1）Dox-induced cell senescent model:The cells were divided into control group（CTL group）and DOX-treated group.The final concentration of DOX was 250 nm.In the CTL group,the same volume of DMSO was added.After 24 hours of culture,the drug was discarded and passaged to 6-well plate at 5×10⁴/ml.The senescent related parameters were detected on days 1,3,5,and 7 after treatment.（2）Ionizing radiation-induced cell senescent model:The cells were divided into CTL group and IR group.The cells in radiation group were irradiated by 10.0 Gy X-ray and passaged at 5×10⁴/ml on the 3^rd day after radiation.The senescent related parameters were detected on the 10^th day after radiation.（3）GT3 intervention:Based on the senescent model,GT3 group and DOX+GT3（or IR+GT3）group were added.The final concentration of GT3 was 10μM.GT3 was administered 4 hours prior to the DOX or IR exposure and continued to treatment until cell collection.The parameters of cell senescence and death were detected.（4）P2X7 inhibitor PPADS intervention:Based on the senescent model,PPADS group and DOX+PPADS（or IR+PPADS）group were added.The final concentration of PPADS was 20μm,which was administered 2 hours before DOX or IR exposure,and continued to treatment until cell collection.The parameters of cell senescence and death were detected.2.q PCR was used to detect the expression of genes related to cell senescence（p16,p21）,Pyroptosis（P2X7,Caspase-1,Gsdmd,IL-18）,cell apoptosis（Puma）and ferroptosis（Gpx4）.3.Cell aging detection:SA-β-gal chemical staining kit and C12FDG probe with flow cytometry were used to detect the level of cell senescence.4.Measuring cell proliferation:Brd U immunofluorescence staining was used to detect the cell proliferation.5.Detecting DNA damage:53BP1 immunofluorescence was used to detect DNA damage.6.ROS detection:The level of ROS was detected by flow cytometry using the probe DCFDA.7.Western blot was used to detect the expression of P2X7 and CASP-1 in senescent cells.Results1.Effect of chemotherapy（DOX）on senescent mouse embryonic fibroblasts（MEFs）:the expression levels of p16 and p21 increased with the prolongation of culture time,and the difference was statistically significant（P<0.05 or P<0.001）.SA-β-gal staining data showed that the proportion of SA-β-gal positive cells in the DOX group was significantly increased（P<0.001）.The results of C12FDG flow cytometry showed that the average fluorescence intensity of C12FDG was increased in the DOX group（P<0.05）.Brd U immunofluorescence results showed that the Brd U positive rate of DOX group was significantly decreased when compared to the CTL group（P<0.01）.The results of 53BP1 immunofluorescence showed that the average positive foci of53BP1 were increased in the DOX group when compared to the CTL group（P<0.01）.2.The effect of radiotherapy（IR）on the senescent MEFs:The expression levels of p16 and p21 significantly increased after IR treatment in MEFs（P<0.05 or P<0.01）.SA-β-gal staining results showed that the proportion of SA-β-gal positive cells in the IR group was significantly increased（P<0.001）.The results of C12FDG flow cytometry showed that the average fluorescence intensity of C12FDG was increased in the IR group（P<0.001）.DCFDA flow cytometry data showed that the average fluorescence intensity of DCFDA increased in the IR group（P<0.01）.Brd U immunofluorescence staining data showed that the positive cell rate of the IR group decreased significantly（P<0.01）.53BP1 immunofluorescence results showed that the level of DNA damage in the IR group was significantly increased（P<0.001）.3.Effects of GT3 on DOX-induced cell senescence:Compared with the DOX group,the expression levels of p16 and p21 in DOX+GT3 group had no significant change while the proportion of SA-β-gal positive cells decreased significantly（P<0.001）.The average fluorescence intensity of C12FDG decreased（P<0.001）.The rate of Brd U positive cells had no significant difference and the average positive foci of 53BP1 had no significant difference.4.The effect of GT3 on IR induced cell senescence:Compared with IR group,the expression levels of p16 and p21 in IR+GT3 group had no significant change.The ratio of SA-β-gal positive cells decreased significantly（P<0.01）.The average fluorescence intensity of C12FDG decreased（P<0.01）.The average fluorescence intensity of DCFDA decreased（P<0.001）.The positive rate of Brd U staining had no significant difference.The average positive foci of 53BP1 had no significant difference.5.The expression of genes related to pyroptosis,apoptosis and ferroptosis in the senescent cells:In DOX-induced MEF cell senescence,the expression levels of P2X7,CASP-1 and Gsdmd genes increased in a time-dependent manner with significant difference（P<0.05 or P<0.01 or P<0.001）.The expression level of IL-18 also increased,but the difference was not statistically significant（P>0.05）.The expression level of apoptosis related gene Puma increased significantly on the 7^th day（P<0.001）.The expression level of ferroptosis related gene Gpx4 increased,but the difference was not statistically significant（P>0.05）.In the senescent MEFs induced by IR,the expression levels of P2X7 and CASP-1 were significantly increased in the IR group（P<0.05 or P<0.001）,while the expression level of IL-18 had no significant change.6.Effects of inhibiting pyroptotic pathway on senescent cells:PPADS could reduce the expression levels of P2X7 and CASP-1 genes in MEFs exposed to DOX and IR,but the difference was not statistically significant（P>0.05）.After PPADS treatment,the SA-β-Gal positive cell rate of MEF cells induced by DOX or IR had no statistical significance.PPADS treatment did not decrease the fluorescence value of C12FDG and increase the numbers of Brd U⁺cells.7.Effects of GT3 on pyroptotic pathway in senescent cells:Compared with DOX group,the expression level of P2X7 gene in DOX+GT3 group had no significant change,while the expression of CASP-1 gene had a downward trend without significant difference（P>0.05）.The protein expression level of P2X7 was significantly deceased in the DOX+GT3 group（P<0.05）.Conclusions1.Dox and IR could induce MEF cell senescence,DNA damage,ROS production and cell proliferation decrease.2.GT3 can ameliorate DOX-and IR-induced MEF senescence via decreasing the levels of reactive oxygen species.3.In the process of DOX-and IR-induced cellular senescence,the expression of pyroptosis-related genes was increased.4.PPADS could not slow down DOX-or IR-induced MEF senescence.5.The reduction of DOX and IR-induced cell senescent by GT3 is not mediated by inhibition of pyroptotic pathway.