| BackgroundProstate cancer(CaP)is a common disease in aged men. In America, CaP is the most common malignant illness in men, and the second leading cause of cancer death. The effect on prostate cancer of lowering circulating androgens by castration or by reducing plasma testosterone levels through interference with the estrogen-driven diencephalic pituitary feedback mechanism was first observed and described by Huggins in 1941. Patients who develop metastatic disease are often initially treated with hormone deprivation by medical castration or surgical castration. Most metastatic prostatic tumors can therefore be treated effectively by hormonal therapies. However, hormone ablation results in only a temporary regression of these tumors. Inevitably, some tumors in the population become androgen-independent prostate cancer(AICP) usually within 6–18 months, At present, Options following initial treatment include secondline hormonal therapy or systemic chemotherapy when metastatic disease develops. Chemotherapy had led to stabilization of disease and improvement of symptoms but did not increase survival in patients with AICP. The molecular mechanisms by which prostate cancer cells become androgen independent are unknown and remain the focus of intensive research. Several factors have been demonstrated to be involved in the development of androgen-independent growth in prostate cancer. For instance, preexisting genetic changes in prostate cancer stem cells, oncogenes, the inhibition of apoptosis, ligand-independent AR activation, AR hypersensitivity, change of AR specificity (AR mutations), gene fusions and Androgen synthesis in AIPC tissues have been observed in androgen-independent prostate cancer. The molecular Pathogenesis of androgen-independent prostate cancer is so complicated that the mechanism of this disease remain uncertain. Reversible Phosphorylation of proteins play s a key role in many cellular Processes including signal transduction, proliferation apoptosis, differentiation, cytoskeletal regulation and so on. It plays an important role in the pathogenesis of prostate cancer to the androgen-independent state.Objective By the way of proteomics and bioinformatics, we can known about global differential phosphoproteins in androgen-independent prostate and reveal the rule of the phosphoproteins, the function and interactions of as many proteins as possible at the global level. It is useful for us to elucidate the molecular mechanisms underlying the formation of AICP and to identify new molecular targets that can be used to develop treatments for the disease.Materials and methods1. We generated an androgen-independent LNCaP-AI prostatic carcinoma cell line from androgen-dependent LNCaP cells by in vitro cell culture medium with 10% fetal bovine serum(FBS) depleted of steroids by charcoal/dextan-treatment(CDS medium. Tumor cell growth was estimated by the CCK-8 assay, PSA protein was measured in cell culture supernatants from the LNCaP and LNCaP-AI cells with medium containing different concentrations of androgen.2. We extracted the total proteins of LNCaP and LNCaP-AI cell, then used immobilized metal affinity chromatography(IMAC) to enrich phosphoproteins from cell proteins. Proteins extracted from LNCaP and LNCaP-AI were separared by two-dimendional gel electophoresis(2-DE). Analytical gels were stained with silver nitate. The stained gels were analyzed with PDQuest software. The protein spots exhibiting statistically alternations between the two groups through computer image analysis. 3 fold differential protein spots were excised from preparative gels and digested into peptides and analyzed by MALDI-TOF-MS. Protein identification using peptide mass fingerprinting(PMF) was performed by the MASCOT search engine against the MSDB(Swissprot or NCBI) protein database.3. Two differential phosphorylated proteins ,Peroxiredoxin-2 and HSP27, from all the identified proteins were chosen to be testified by western blot analysis to substantiate the 2-DE results.4. By using String database and software to analysis the relationships between the up-regulated, down-regulated and all proteins in LNCaP-AI cells identified by proteomics respectively to discover the relationships and set the associations between the relationships and the biological process. Result1. Initially, the proliferation of LNCaP cells were suppressed rapidly in the absence of hormone and laid in autocrine status after about 1 week. 1 months later, the cells gradually adapted to the no hormone circumstance and 6 months later began to proliferate, at this time the cells become androgen independent LNCaP-AI cell line. LNCaP- AI showed to be androgen-independent and grows equally well with or without androgens. PSA level in LNCaP-AI cell culture supernatant increased parallelly with concentrations of androgen. The secretion of PSA is still responsive to androgens, but to a much lesser extent than in the original LNCaP.2. Through the PDQuest software analyzed the stained gels, a total 28 protein spots had been detected in protein profile of LNCaP-AI and LNCaP cells, which exhibiting a consistent, more than threefold phosphrylation level. Among them, 13 exhibiting protein up-regulation and 15 exhibiting down-regulation in LNCaP-AI cells, compared with LNCaP cells.3. Data were then searched with MASCOT, a total of 17 prosteins were identified, these differential phosphoproteins included cell signaling proteins, cytoskeletal proteins, energy metabolism regulators such as Nucleophosmin, Heat shock protein beta-1, Peroxiredoxin-2 , Peroxiredoxin-6, and so on.4. The western blot data showed that total HSP27 protein was found significantly up-regulated in LNCaP-AI cell lines, while total Pereoxiredoxin-2 protein was found significantly down-regulated in LNCaP-AI cell lined. The extents of the changes of these two proteins in LNCaP-AI were similar to that observed in the 2-DE analysis.5. Supported by the STRING database and document analysis, the interaction of P53 and HSP27 and furthermore the MAPK pathway which involved in may play an important role of leading the transition of androgen-dependent prostate cells into androgen-independent prostate cells. Decrease of the function of eliminating peroxides generated during metabolism may be another important mechanism leads to the transition of androgen-dependent prostate cancer into androgen-independent prostate cancer. conclusionLNCaP-AI cell was developed from LNCaP cell under the circumstance of androgen ablation. This kind of CaP cell model could imitate the process from androgen-dependent prostate cancer to androgen-independent prostate cancer during endocrine therapy of CaP. LNCaP-AI cell was homology with LNCaP cell and could be compared with it. The cell model is an ideal model to study the pathogenesis for revealing signaling pathways of importance for transition of androgen dependent prostate cancer into androgen independent prostate cancer. We successfully identified 17 differential phosphoproteins including cell signaling proteins, cytoskeletal proteins, and energy metabolism regulators by IMPC strategy of phosphoprotein enrichment, proteomics technology and bioinformatics technology. Bioinformatics analysis reveals the interaction of P53 and HSP27 and furthermore the MAPK pathway which involved in and the decrease of the function of eliminating peroxides generated during metabolism may play an important role of leading the transition of androgen-dependent prostate cells into androgen-independent prostate cells. Thus, block of the MAPK signaling pathway and enhance the function of eliminating peroxides may be helpful to androgen-independent prostate cancer. |
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