DNA-thePKcs Inhibitors NU7441 Promote Gastric Cancer Radiation Tolerate Cells Injury And Apoptosis Research

Research purposes: This study through different doses of high-energy irradiation to 6 groups of gastric cancer cell lines,BGC823 and MGC803 screen radiation tolerate,made clear the phosphorylated proteins from DNA-PKcs and high expression caused radiation resistance molecular mechanism of gastric cancer cells DNA-PKcs inhibitors NU7441 can promote gastric cancer radiation damage tolerate cells apoptosis,reverse radiation resistance;CX-4945 joint CK2 inhibitors to further promote apoptosis,radiation tolerate in order to improve gastric cancer radiation sensitivity to provide a new strategy.Research methods:(1)Traditional culture SGC7901,HGC27,MKN45,MKN28,BGC823 and MGC803,6 strains of different gastric cancer cell line MGC803 altogether,under a microscope to observe the morphology of cell lines,6 cases treated cells in good condition and stable after the wedding,when the nutrient solution by clarifying becoming weak yellow,namely into the logarithmic phase,the strongest cell vitality,suitable for used in experiments,the cells according to 0 gy,2 gy,4 gy,6 gy high-energy rays total radiation dosage,each cell by VARIAN linac downward 6 MV X-ray irradiation,the dose rate 300 cgy/min,distance is 100 cm,radiation field 10 cm * 10 cm,equivalent tissue culture plate underneath fillings.(2)6 groups of cell lines to high-energy radiation exposure in thermostatic cultivation in the box to cells in good condition,stability to extend,through cell cloning experiments culture 12 h,24 h after the draw different time survival bar bar graph of the gastric cancer cell lines.(3)By Western blot technique to detect DNA-PKcs in different cell lines and phosphorylation(NU7441 phosphorylation sites)influence the protein level and GAPDH,comparison of the resistance of two strains of relative differences in gastric cancer cells and other cells,and further detect relative radiation resistance cell line.(4)Using CCK8(determined by MTT)detection of two strains of gastric cancer tolerate cells to DNA-PKcs inhibitors NU7441 IC50(24h)value,formed by cloning experiments mapped to join small dose NU7441 pretreatment time-cell survival curve and blank control group,two strains of gastric cancer tolerate cells by 24 h after irradiation,low-dose NU7441 pretreatment CCK8(determined by MTT),clone formation test cells survive,Western blot detection PARP1,cleaved-caspase3,gamma-H2 AX,GAPDH protein expression,immunofluorescence test damage index.(5)Small doses NU7441 CX-4945 and CK2 inhibitors combined radiation tolerate cells for 24 h after irradiation,CCK8(determined by MTT),clone formation test cells survive,Western blot test cell damage repair protein indicators,immunofluorescence test cell damage indicator.The results of the study:(1)Cell plate cloning experiment 0 gy,2 gy,4 gy,6 gy corresponding doses,12 h of gastric cancer BGC823 cell survival rate was 74%,70%,65%,70%;SGC7901 cells survival rate of 74%,68%,54%,68%;MGC803 cell survival rate was 74%,70%,65%,74%;HGC27 cell lines of survival rate was 53%,57%,50%,43%;MKN45 cells survival rate of 78%,74%,56%,74%;MKN28 cell survival rate was 78%,74%,56%,78%;24 h of gastric cancer BGC823 cell survival rate was 67%,65%,60%,67%;SGC7901 cells survival rate of 64%,65%,58%,65%;MGC803 cell survival rate was 60%,61%,58%,60%;HGC27 cell lines of survival rate was 58%,56%,52%,50%;MKN45 cells survival rate of 53%,47%,45%,47%;MKN28 cell survival rate was 52%,49%,35%,49%.And map out the different gastric cancer cell lines survival bar graph,Western blot method to detect the 4 gy PARP1 protein expression after 6 mv X-ray show BGC823,MGC803 significantly higher than the other four groups of cell lines,cleaved protein-caspase3,gamma H2 AX protein expression BGC823,MGC803 cells significantly lower than the other four groups of cell lines,the difference was statistically significant(P<0.05),show the relative radiation tolerate,immunofluorescence method to detect gamma H2 AX,DAPI fluorescence degree significantly weaker than the other four groups of cell lines,the difference was statistically significant(P<0.05).(2)By Western blot method to detect 6 groups the DNA after 4 gy 6 mv X-ray-PKcs and phosphorylation(NU7441 phosphorylation sites)influence the protein level and GAPDH,p-the grey value of DNA-PKcs and DNA-PKcs significantly higher than the other four groups of cell lines,the difference was statistically significant(p < 0.05).(3)By CCK experiment of different concentrations of NU7441 BGC823,MGC803 cell survival after radiotherapy and draw out the survival curve,BGC823 IC50 alue concentration of 3.724 umol/L,MGC803 IC50 alue concentration of 4.704 umol/L,as NU7441 concentration increased,cell survival rate decreased significantly,the dose of radiation tolerate cell lines BGC823 NU7441 and blank control group,MGC803 0 h,12 h,24 h,48 h of cell survival curve can be obtained,blank control group in cell survival rate was significantly higher in small doses NU7441 group,by Western blot method to detect BGC823,MGC803 cell lines blank control group,and join NU7441 gamma-H2 AX grey value,found to join NU7441 cell lines gamma-H2 AX grey value is obvious rise,immunofluorescence show to join NU7441 gamma-H2 AX fluorescence intensity increases,the difference was statistically significant(P < 0.05).(4)Using DNA CX-4945 joint NU7441 PKcs inhibitors and CK2 inhibitors,compared to only NU7441 controls are used to show time-survival curves suggest combination can further promote apoptosis,by Western blot,combination gamma-H2 AX grey value increase,immunofluorescence show to join NU7441 gamma-H2 AX fluorescence intensity increases,the difference was statistically significant(P < 0.05).Research conclusions:(1)This research through the six groups of different cell lines after radiation exposure BGC823 selection,relative radiation resistance cell line MGC803 gastric cancer cell line.(2)The phosphorylated proteins from DNA-PKcs and high expression is closely related to the radiation resistance of gastric cancer cells,may be related to cellular repair mechanisms.(3)Has been clear about the DNA-PKcs inhibitors NU7441 can contribute to the damage of gastric cancer radiation tolerate cells apoptosis.(4)NU7441 joint CX-4945 can further promote the apoptosis of gastric cancer radiotherapy tolerate,improve efficacy of radiotherapy.