Effects Of CREB On Proliferation And Apoptosis Of Prostate Cancer Cells

cAMP-response element binding protein(CREB) is a member of family of the basic leucine-zipper transcriptional factors. Direct activation of gene expression by CREB requires phosphorylation by the cAMP-dependent PKA to serine 133 and the target genes of CREB involves the survival, proliferation, metabolism of cell. It has been reported that the higher expression and activation of CREB could be detected in acute lymphoblastic leukemia, breast cancer and non-small cell lung cancer and contribute to the tumor progression. In contrast, inhibition of CREB can restrain proliferation and induce apoptosis of different cancer cells. However, the effects of CREB on proliferation and apoptosis of prostate cancer cells has never been studied.Protein kinase A(PKA) bound to cAMP activates CREB by phosphorylating a specific residue,Ser 133, in the CREB activation domain, which in turn leads to the recruitment of the transcriptional coactivators. The phosphorylation and activation of CREB need extracellular signal, which contains hormones, synaptic activity, growth factors, stress and inflammatory cytokines. In the microenvironment of cancer, tumor associated macrophage(TAM) among the inflammation relates closely with the tumor progression and has been paid more attention in recent years. TAM involves the tumor progression by promoting angiogenesis, lymphangiogenesis, tumor cell activation, immunosuppression, tumor invasion and metastasis. However, the mechanisms are not clear and whether TAM could trigger CREB pathway to affect prostate tumor progression as extracellular signal has never been reported.In view of the roles of CREB in tumor and the importance of TAM in tumor progression, the present research aims to study the expression of CREB in different prostate tissue and their relationship, the roles of CREB in proliferation and apoptosis of prostate cancer cells, and whether TAM could affect prostate tumor progression by inducing CREB expression. This study is composed of the following three parts. Experiment 1: The expression of CREB and p-CREB in different prostate tissue and prostate cell lines and their potential functionIn the first part, the immunohistochemistry of tissue microarray was applied to detect the expression of CREB and p-CREB in normal prostate tissue, hyperplasia of prostate tissue and different grades of prostate cancer of human. The expression of CREB and p-CREB in human prostate cancer tissue was higher than that in normal prostate tissue and hyperplasia of prostate tissue. The result demonstrated that the positive rate and intensity of CREB and p-CREB positively correlated the malignant degree of prostate cancer, indicating that the higher expression of CREB and p-CREB positively correlated the higher proliferation or apoptosis-resistance of prostatic cancer cells.In addition, immunocytochemistry and Western blotting were performed to examine the expression of CREB and p-CREB in myofibroblast stromal cell line WPMY-1, prostate cancer cell lines PC3 and LNCap cells. The protein levels of CREB and p-CREB were increased in PC3 and LNCap cells compared to WPMY-1 cell. The data further supported the result that the expression of CREB and p-CREB positively correlated the proliferation of prostatic cancer cells. Experiment 2: The effects of CREB on proliferation and apoptosis of prostate cancer cellsThe primer for CREB was designed by Vector NTI 11.0 software through searching the mRNA sequence of CREB in NCBI. The coding sequence of CREB was cloned in the pcDNA3.1 vector. The recombinant pcDNA3.1-CREB plasmid was constructed and transfected into human prostate cancer cell lines PC3 and LNCap cells to observe its effects. Transformation was verified by Western blotting analysis of the elevated expression of CREB and p-CREB. The data showed that CREB induced the expression of proliferating cell nuclear antigen(PCNA), a proliferation-related marker, in PC3 and LNCap cells, and CREB-transfected PC3 and LNCap cells showed higher activity of proliferation by CCK-8 assay in vitro. Moreover, CREB can induce the expression of survivin, which serves as an apoptosis-resistant protein, and resistance to apoptosis was observed in cells transfected with pcDNA3.1-CREB. In contrast, knocking down CREB expression by siRNA can down-regulate proliferation and enhance apoptosis of cancer cells. Experiment 3: The role of CREB in TAM related growth and progress of prostate cancer cellsA human acute monocytic leukemia cell line THP-1 cells were induced to macrophage by addition of Phorbol-12-myristate-13-acetate(PMA). Then macrophages were polarized to M1 and M2 phenotypes by stimulation with LPS + IFN-γ, IL-4 respectively. The conditioned medium from M1 and M2 macrophages were collected and added to stimulate prostatic cancer cells to observe its effects on growth of prostatic cancer cells and the possible role of CREB. Compared to unobvious effect of conditioned medium from M1 macrophages, the data of western blotting and CCK-8 assay revealed that conditioned medium from M2 macrophages could enhance the growth of cancer cells, in pace with the induction of CREB. In contrast, knocking down the expression of CREB could inhibit the growth promotion of M2-CM. These data indicated that CREB involves the effect of M2 macrophages on growth of cancer cell. The mechanism of induction of CREB by conditioned medium from M2 macrophages remains to be explored.