Effects Of ATRA,EGF On Proliferation, Apoptosis And Metastatic Phenotype Of Human Androgen-independent Du145 Cell Line

To investigate Effects of ATRA,EGF on proliferation, apoptosis and metastasis phenotype of human androgen-independent Du145 cell lines, we plan to search for the mechanisms of Du145's proliferation,apoptosis and metastasis in cellular and molecular levels. We hope to find some rationale to employ differentiation induction or apoptosis promotion to treat androgen-independent prostate cancer.This thesis was divided into three parts:Part 1 Effects of ATRA,EGF on proliferation of human androgen-independent Du145 cell lineThis part mainly investigate the ATRA and EGF's effect on growth rate,cell cycle and corresponding molecular mechanism of Du145 cell. And further research the possible signal pathway that EGF promote the proliferation of Dul45 cell. The results were summarized in the following.1. MTT method detection: ATRA decreased the rate of cell growth and EGF increased the rate of cell growth。2. Flow-cytometry detection: ATRA inhibited the cells going from G1 stage to S stage and EGF stimulated the cells going from G1 stage to S stage.3. The expressions of cyclin D1, A and E as well as cyclin-dependent protein kinases (CDKs) CDK2 and CDK4 in the ATRA, EGF treated cells were unchanged comparing to the control.4. Among the inhibitors of cyclin-dependent protein kinases (CDKI), only the expression of p27, but not p16 and p21 was dramatically increased by ATRA and reduced by EGF. The p27 combining with cyclinA/E-CDK2 complex as detected by immuno-precipitation was also obviously increased by ATRA and decreased by EGF, while the mRNA of p27 was not altered.5. The phosphorylated retinoblstoma protein (Rb) was increased, while the unphosphorylated Rb was correspondingly decreased by ATRA while the EGF has the contrary function..6. The inhibitor of PI-3K, LY294002, could not attenuate the difference in the p27 expression and Rb phosphorylation in control and EGF treated cells, but the inhibitors of MEK phosphorylation, PD09059 did, indicating that the EGFregulate the p27 expression by MEK-MAPK pathway but not PI-3K-PKB pathway.It is conceivable that the EGF promotes the signal transduction of growth factor into the cells mainly via MEK/MAPK pathway, results in the inhibition of p27 expression, reducation of p27 inhibitory effect on CDK2 and enhancement of Rb phosphorylation by the activated CDK2, and final stimulation of the separation of phosphorylated Rb from transcription factor E2F. Consequently, E2F translocates into the nuclei to stimulate the synthesis of DNA, leading to the passage of the cells through G1 check-point to the S stage of cell cycle.Part 2 Effects of ATRA,EGF on apoptosis of human androgen-independent Du145 cell lineThis part mainly investigate the ATRA and EGF's effect on apoptosis and corresponding molecular mechanism of Dul45 cell. And further research the effect of ATRA and EGF on skeleton protein F-actin of Du145 cell. The results were summarized in the following:1. By using flow-cytometry, it was found that the susceptibility to cell apoptosis was elevated in ATRA treated cells. This was evidenced by the appearance or significant increase of the apoptotic cell peak (sub-diploid cells peak), and the ratio of apoptotic cells was ATRA group>Control>EGF group.2. Immunoflourescence microscope examination can find obvious apoptotic bodies in ATRA group after AO dying.3. Treated with ATRA,EGF, the expression of T308 phosphorylated PKB and S-136 phosphorylated Bad (a proapoptosis protein in the mitochondria pathway of apoptosis) has obvious deviation, with the order of ATRA group< Control Control > EGF group and the expressions of antiapoptotic Bcl-2 and Bcl-XL were reduced with a reverse order.5. The expressions of anti-oncogene p53, death receptor Fas and the ligand of Fas (FasL) were not altered either in ATRA group or EGF group .6. In EGF group , the caspase-3 exhibited as an un-activated zymogen and was hydrolyzed very little. After the cells were treated with ATRA, the hydrolyzedand activated fragments of caspase-3 was dramatically increased.7. In EGF group, the cell skeleton protein F-actin was arranged in ordered, compact, clear-cut with undisturbed edges. After ATRA treatment, the structure of F-actin was disturbed .The findings in this part suggest that ATRA is a apoptosis stimulating protein. Conversely, EGF is benefit for anti-apoptosis. It is probably that ATRA, as an apoptosis-inducing factor, may down regulate some anti-apoptotic proteins and/or up regulate some proapoptotic proteins. The effects of these decreased anti-apoptotic and increased proapoptotic proteins finally result in the activatiating of caspase -3 which may lead to the destroy of the cell skeleton and the promotion of apoptosis. On the contrary, EGF facilitates the transduction of cell proliferative signals and the cell cycle thus keep the Du145 cell from apoptosis.Part 3 Effects of ATRA,EGF on metastatic phenotype of human androgen-independent Du145 cell lineThis part mainly investigate the ATRA and EGF's effect on metastatic phenotype and corresponding molecular mechanism of Du145 cell. The results were summarized in the following:1. ATRA can restrain the adhension ability of Du145 cell to Fn; EGF canpromote the adhension ability of Du145 cell to Fn2. ATRA can restrain the adhension ability of Du145 cell to HUVEC; EGF can promote the adhension ability of Du145 cell to HUVEC.3. ATRA can restrain the migration ability of Du145 cell; EGF can promote the migration ability of Du145 cell.4. ATRA can restrain the invasion ability of Du145 cell; EGF can promote the invasion ability of Du145 cell.5. EGF can up-regulate the protein expression of β1 subunit of integrin α5β1 in Du145 cell; EGF+PD98059(MAPK signal pathway inhibitor) can down-regulate the protein expression of β1 subunit of integrin α5β1 in Du145 cell;6. EGF can up-regulate the mRNA expression of β1 subunit of integrin α5β1 in Du145 cell; EGF+PD98059 can down-regulate the mRNA expression of β1 subunit of integrin α5β1 in Du145 cell.The findings in this part suggest that ATRA can restrain the adhension,migration and invasion ability of Du145 cell and EGF can promote the adhension,migration and invasion ability of Dul45 cell. Because the receptor of Fn is integrin α5β1, we further investigate the MAPK singal pathway's effect on the expression of α5 and β1 subunit. Flow-cytometr and Western-blot indicate that the expression of subunit α5 were not altered either in EGF group or EGF+PD98059 group but the expression of subunit β1 were changed with the order EGF+PD98059 group < Control < EGF group. RT-PCR indicate EGF+PD98059 can down-regulate the mRNA expression of β1, EGF can up-regulate the mRNA expression of β1. So we can conjecture that the regulation function may in the level of mRNA and the change of the expression of mRNA was concord to the change of the adhension ability to Fn and migration.