The Research On The Role Of PI3K/Akt Signal Transduction Pathway In Radiosensitization Of Tumor Cell Lines

Objective: To explore the molecular mechanism of increasing radiosensitization effect on tumor cell by inhibiting PI3K/Akt signal transduction pathway; To explore whether the use of LY294002 which inhibits PDK/Akt signal transduction pathway could increase the radiosensitization effect of docetaxel, cisplatin, celecoxib on tumor cells.Methods: Hela cells and MCF-7cells were cultured in vitro. The subjects were divided into 9 groups: control group, radiation group, LY294002+ radiation group, docetaxel+ radiation group, cisplatin+ radiation group, celecoxib+ radiation group, LY294002+ docetaxel+ radiation group, LY294002+ cisplatin+ radiation group, LY294002+celecoxib+ radiation group. The subjects were disposed with proper drugs for 24h, and then were treated with X ray. The Bad, COX-2, Ku-70, Ku80, NF-κB mRNA were detected by RT-PCR method, and the Bad, pBad, Akt, pAkt, COX-2 protein were examined by Western blot; the apoptosis ratio of each group cells were detected by flow cytometer; The damage of cell DNA was detected by neutral comet electrophoresis; The radiosensitization effect of HeLa was detected by cell colony formation methods. The Dq, D₀, SF₂ and SER of each group were calculated, and the cell survival curve was gained by single-hit multi-target model.Results: On Hela cells: (1) According to the control group, the expressive level of Bad mRNA of LY94002 + docetaxel/cisplatin/celecoxib+ radiation group are higher, and COX-2, Ku-70, NF-κB mRNA expression are lower, Ku80 mRNA expression is not changed significantly; (2) The expressive level of Bad protein of LY94002+ docetaxel/cisplatin/celecoxib+ radiation group are higher than control group, COX-2, pAkt protein are lower; and the difference of Akt protein expression is not significant; (3)The celecoxib inhibits the COX-2, NF-κB mRNA expression; and inhibits COX-2, pAkt protein expression; (4) The apoptosis ratio of LY94002+ docetaxel/cisplatin/celecoxib+ radiation group is higher than only drug + radiation group; (5) The Dq, D₀, SF₂ of LY94002 + drug+ radiation groups are lower than drug+ radiation groups significantly, the SER of LY294002+docetaxel+ radiation group is 1.35 times of docetaxel+ radiation group, the SER of LY294002+cisplatin+ radiation group is 1.26 times of cisplatin+ radiation group, the SER of LY294002+celecoxib+ radiation group is 1.22 times of celecoxib+ radiation group; (6) The tail distance of HeLa cell comet electrophoresis of LY294002 + drug+ radiation groups is longer than drug+ radiation groups. On MCF-7 cells: (1) The Dq, D₀, SF₂ of LY94002 + drug+ radiation group are lower than drug+ radiation group significantly, the SER of LY294002+docetaxel+ radiation group is 1.47 times of docetaxel+ radiation group, the SER of LY294002+cisplatin+ radiation group is 1.03 times of cisplatin+ radiation group, the SER of LY294002+celecoxib+ radiation group is 1.22 times of celecoxib+ radiation group; (2) The expression of pAkt protein of LY294002 + drug+ radiation group is lower than control, and the expression of Akt protein has no difference between LY294002 + drug+ radiation group and control. (3) The apoptosis ratio of LY94002+ docetaxel/cisplatin/celecoxib+ radiation group is higher than only drug + radiation group.Conclusions: (1) The PI3K/Akt signal transduction pathway was actived after the radiation of X ray; the radiosensitization of docetaxel and cisplatin could active the pathway obviously, it was a early reaction. (2) The radiosensitization of celecoxib suppressed the activation of the pathway. (3) Inhibiting PI3K/Akt signal transduction pathway could increase the radiosensitization effect of docetaxel, cisplatin, celecoxib on HeLa and MCF-7 cell. (4) Inhibiting PI3K/Akt signal transduction pathway may increase radiosensitization of tumor cell in three aspects: â‘  Inhibiting Ku70 to suppress the DNA recovery after damaging; â‘¡ Inhibiting the expression of COX-2 and NF-κB to suppress the cell division growth; â‘¢ Enhancing the expression of Bad to promote cell apoptosis. Part I The role of PI3K/Akt signal transduction pathway in drug radiosensitization of human cervical carcinoma HeLa cell lineObjective: To explore the role of PI3K/Akt signal transduction pathway in drugs (docetaxel, cisplatin, celecoxib) radiosensitization of human cervical carcinoma HeLa cell line.Methods: Hela cells were cultured in vitro, the IC₅₀ of docetaxe, cisplatin or celecoxib on HeLa cells for 24h. The subjects were divided into 5 groups: control group, radiation group, docetaxel+ radiation group, cisplatin+ radiation group, celecoxib+ radiation group; the subjects were disposed with proper drugs for 24h, then were treated with 6 Gy X ray. After 1h, the pAkt protein was detected by western blot and immumofluorescence methods.Results: (1) IC₅₀ of docetaxel, cisplatin or celecoxib on HeLa cells for 24h were 8.82±2.36μg/mL, 12.04±2.58μg/mL, 185.74±46.62μmol/L respetively; (2) The expressive level of pAkt protein: control group 0.1553±0.0019, radiation group 0.3630±0.0037, docetaxel+ radiation group 0.5121±0.0113, cisplatin+ radiation 0.4909±0.0052, celecoxib+ radiation 0.2076±0.0029, the difference among each group is significant (F=2168.521, P<0.05);Conclusions: The PI3K/Akt signal transduction pathway was actived after the radiation of X ray; the radiosensitization of docetaxel and cisplatin could actived the pathway obviously; the radiosensitization of celecoxib suppressed the activation of the pathway. Part II The study of increasing the radiosensitization effect of HeLa cell by inhibiting PI3K/Akt signal transduction pathwayObjective: To explore whether the use of LY294002 which inhibits PI3K/Akt signal transduction pathway could increase the radiosensitization effect of docetaxel, cisplatin, celecoxib on HeLa cells and raise the DNA impair of HeLa cell.Methods: The subjects were divided into 9 groups: control group, radiation group, LY294002+ radiation group, docetaxel+ radiation group, cisplatin+ radiation group, celecoxib+ radiation group, LY294002+ docetaxel+ radiation group, LY294002+ cisplatin+ radiation group, LY294002+celecoxib+ radiation group. The subjects were disposed with proper drugs for 24h, and then were treated with X ray. The radiosensitization effect of HeLa was detected by cell colony formation methods. The Dq, D₀, SF₂ and SER of each group were calculated, and the cell survival curve was gained by single-hit multi-target model; the damage of cell DNA was detected by neutral comet electrophoresis.Results: (1)The Dq, D₀, SF₂ of LY94002 + drug+ radiation groups are lower than drug+ radiation groups significantly, the SER of LY294002+docetaxel+ radiation group is 1.35 times of docetaxel+ radiation group, the SER of LY294002+cisplatin+ radiation group is 1.26 times of cisplatin+ radiation group, the SER of LY294002+celecoxib+ radiation group is 1.22 times of celecoxib+ radiation group; (2) The tail distance of HeLa cell comet electrophoresis of LY294002 + drug+ radiation groups is longer than drug+ radiation groups, the difference is significant (P<0.05).Conclusions: Inhibiting PI3K/Akt signal transduction pathway could increase the radiosensitization effect of docetaxel, cisplatin, celecoxib on HeLa, and rise the impair DNA of the cell. PartIII The mechanism of increasing radiosensitization effect on HeLa cell by inhibiting PI3K/Akt signal transduction pathwayObjective: To explore the molecular mechanism of increasing radiosensitization effect on HeLa cell by inhibiting PI3K/Akt signal transduction pathway.Methods: The subjects were divided into 9 groups: control group, radiation group, LY294002+ radiation group, docetaxel+ radiation group, cisplatin+ radiation group, celecoxib+ radiation group, LY294002+ docetaxel+ radiation group, LY294002+ cisplatin+ radiation group, LY294002+celecoxib+ radiation group. The subjects were disposed with proper drugs for 24h, and then were treated with X ray. The Bad, COX-2, Ku-70, Ku80, NF-κB mRNA were detected by RT-PCR method, and the Bad, pBad, Akt, pAkt, COX-2 protein were examined by Western blot; the apoptosis ratio of each group cells were detected by flow cytometer.Results: (1) According to the control group, the expressive level of Bad mRNA of LY94002 + docetaxel/cisplatin/celecoxib+ radiation group are higher, and COX-2, Ku-70, NF-κB mRNA expression are lower, Ku80 mRNA expression is not changed significantly; (2) The expressive level of Bad protein of LY94002+ docetaxel/cisplatin/celecoxib+ radiation group are higher than control group, COX-2, pAkt protein are lower; and the difference of Akt protein expression is not significant; (3) The celecoxib inhibits the COX-2, NF-κB mRNA expression; and inhibits COX-2, pAkt protein expression; (4) The apoptosis ratio of LY94002+ docetaxel/cisplatin/ceiecoxib+ radiation group is higher than only drug + radiation group.Conclusions: Inhibiting PI3K/Akt signal transduction pathway may increase radiosensitization of HeLa in three aspects: (1) Inhibiting Ku70 to suppress the DNA recovery after damaging; (2) Inhibiting the expression of COX-2 and NF-κB to suppress the cell division growth; (3) Enhancing the expression of Bad to promote cell apoptosis. Part IV The study of increasing the radiosensitization effect of MCF-7 cell by inhibiting PI3K/Akt signal transduction pathwayObjective: To explore whether the use of LY294002 which inhibits PI3K/Akt signal transduction pathway could increase the radiosensitization effect of docetaxel, cisplatin, celecoxib on human breast cancer cell MCF-7.Methods: The subjects were divided into 9 groups: control group, radiation group, LY294002+ radiation group, docetaxel+ radiation group, cisplatin+ radiation group, celecoxib+ radiation group, LY294002+ docetaxel+ radiation group, LY294002+ cisplatin+ radiation group, LY294002+celecoxib+ radiation group. The subjects were disposed with proper drugs of for 24h, and then were treated with X ray. The radiosensitization effect of MCF-7 was detected by cell colony formation methods. The Dq, D₀, SF₂ and SER of each group were calculated, and the cell survival curve was gained by single-hit multi-target model; the damage of cell DNA was detected by neutral comet electrophoresis.Results: (1) The Dq, DO, SF2 of LY94002 + drug+ radiation group are lower than drug+ radiation group significantly, the SER of LY294002+docetaxel+ radiation group is 1.47 times of docetaxel+ radiation group, the SER of LY294002+cisplatin+ radiation group is 1.03 times of cisplatin+ radiation group, the SER of LY294002+celecoxib+ radiation group is 1.22 times of celecoxib+ radiation group; (2) The expression of pAkt protein of LY294002 + drug+ radiation group is lower than control, and the expression of Akt protein has no difference between LY294002 + drug+ radiation group and control; (3) The apoptosis ratio of LY94002+ docetaxel/cisplatin/celecoxib+ radiation group is higher than only drug + radiation group.Conclusions: Inhibiting PI3K/Akt signal transduction pathway could increase the radiosensitization effect of docetaxel, cisplatin, celecoxib on MCF-7 cell.