The Action Mechanism Of EGCG Induced Apoptosis Of Human Lung Adenocarcinoma Cells By Regulating The Expression Of Ku70and Its Acetylation State

Chapter One EGCG induces A549cells to apoptosisObjective To investigate the apoptosis-inducing effect and the cytostasis effect of EGCG in the human lung adenocarcinoma A549cells. Methods1. A549cell lines were cultured with RPMI1640medium, then were treated with different concentrations of EGCG2. Cytostasis effect was studied using MTT, apoptosis was analysis by flow cytometry and microscope observe the change in form.3. Established the transplanted tumor model of human lung adenocarcinoma, A549cells were implanted into the right axillary fossa’s subcutaneous tissue of nude mice. After the model of lung carcinoma was established, the animals were randomized into3groups:the control group、EGCG group and the cisplatin group.4. Tumor growth and animal weight were observed. After these were administered, all mice were put to death. The weight of the transplantation tumor was observed. Results1. Treated with EGCG, the survival ratio decreased, and EGCG induced apoptosis. The treated A549cells showed turning smaller and round, chromosome condensed, pyknosis, forming quite a few apoptosis bodies.2. In EGCG group the volume of tumor tissues were smaller than the NS group, with the inhibition rate of37%, while the cisplatin group was50%. However, the animal weight in EGCG group was heavier than cispaltin group. Conclusion Both in vitro and in vivo, EGCG can induce apoptosis of human lung adenocarcinoma A549cells, and its toxicity effect is less than cisplatin in vivo.Chapter Two EGCG induces A549cells to apoptosis through Bcl-2family membersObjective To investigate whether the Bcl-2family members contribute to the apoptosis-inducing effect of EGCG in the human lung adenocarcinoma A549cells. Methods Different proteins were detected by Western Blot method, and different mRNA were detected by RT-PCR or real-time PCR. Results1. Treated with EGCG, the expression of protein and mRNA of Bax increased, while Bcl-xl decreased, and the expression of protein of caspase-3(17KDa) increased in vitro.2. In vivo, in EGCG group, the expression of protein and mRNA of Bax was more than NS group, less than cisplatin group, but the Bcl-xl was opposite. Conclusion In the human lung adenocarcinoma A549cells, EGCG can upregulate the expressions of Bax and Caspase-3(17KDa), and downregulate the expression of Bcl-xl, both in a time-dependent manner and in a dose-dependent manner.Chapter Three EGCG induces A549cells to apoptosis through Ku70Objective To investigate whether the Ku70contribute to the apoptosis-inducing effect of EGCG in the human lung adenocarcinoma A549cells. Methods Different proteins were detected by Western Blot method, different mRNA were detected by RT-PCR or real-time PCR, and identification of interaction between Bax and Ku70by co-immunoprecipitation.2. To confirm the role of Ku70in EGCG-induced apoptosis in A549cell lines, cells were transfected with pSliencer4.1-CMV-Ku70, then treated with different concentrations of EGCQ finally, different proteins were detected by Western Blot method, and apoptosis was analysis by flow cytometry. Results1. Treated with EGCQ the expression of protein and mRNA of Ku70decreased, and the interaction between Bax and Ku70was disturbance in vitro.2. In vivo, in EGCG group, the expression of protein and mRNA of Ku70was less than NS group, more than cisplatin group, and the interaction between Bax and Ku70was weaken in EGCG group as compared with NS group, while stronger than cisplatin group.3. When Ku70is inhibited, A549cells become sensitive to EGCQ while there is no effect on the expression of Bax. Conclusion1. Constructing RNA interfering vector targeting Ku70was succeeded.2. In the human lung adenocarcinoma A549cells, EGCG can downregulate the expression of Ku70and interfere the interaction between Bax and Ku70both in a time-dependent manner and in a dose-dependent manner.3. EGCG induces A549cells to apoptosis through downregulating Ku70. Chapter Four EGCG induces A549cells to apoptosis by regulating the acetylation state of Ku70Objective To investigate whether the acetylation state of Ku70contribute to the apoptosis-inducing effect of EGCG in the human lung adenocarcinoma A549cells. Methods1. Identification of the level of the acetylation state of Ku70by co-immunoprecipitation.2. To confirm the role of the acetylation state of Ku70in EGCG-induced apoptosis in A549cell lines, cells were transfected with pCDNA3.1(+)-Ku70, which was higher expression level of Ku70, through site mutation directed by PCR, we cloned the pCDNA3.1(+)-Ku70539/542R, and cells also were transfected with pCDNA3.1(+)-Ku70539/542R, which could not be acetylated in site K539or K542.Then different group cells were treated with different concentrations of EGCQ and different proteins were detected by Western blot method, apoptosis was analysis by flow cytometry, and identification of the level of the acetylation state of Ku70by co-immunoprecipitation. Results1. Treated with EGCG, the level of the acetylation state of Ku70increased in vitro.2. In vivo, in EGCG group, the level of the acetylation state of Ku70was more than cisplatin group, while less than cisplatin group.3. When the site of K539and K542is mutated, A549cells become resistant to EGCQ while there is no effect on the expression of Bax, however, the interaction between Bax and Ku70turned strong. Conclusion1. Constructing over-expression and mutated Ku70was succeeded.2. In the human lung adenocarcinoma A549cells, EGCG can upregulate the level of the acetylation state of Ku70, both in a time-dependent manner and in a dose-dependent manner.3. EGCG induces A549cells to apoptosis through upregulating the level of the acetylation state of Ku70.