The Research Of Ectopic Expressed Neurotrophic Peptide On Proliferation Of Human Prostate Cancer Cells By Up-Regulating Expression And Function Of The Androgen Receptor

Prosaposin is a highly conserved glycoprotein (65-72kDa, 527 amino acids), the gene was localized to the long arm of chromosome 10 (q21-22) and encompasses 17 kb of genomic sequence with 14 exons. Prosaposin is the precursor of four small heat-stable sphingolipid activator proteins (saposins A, B, C and D, which are required for the enzymatic hydrolysis of sphingolipids in lysosomes). In addition to its intracellular presence and function, Prosaposin is also expressed as a secretory protein and also a well-known neurotrophic factor in vivo and in vitro. As a secretory protein, Prosaposin could promote survival, prevent apoptosis and stimulate synapse growth of neuroglial-derived cells. Prosaposin functional sequence is localized to a 12-amiono acid stretch at NH₂-terminal end of the saposin C domain (LIDNNRTEELLY). Several synthetic peptides (14-22 residues, e.g. TX14A) derived from this region are equally as bioactive as Prosaposin. Prosaposin, saposin C, and prosaptides exert their neurotrophic effects by binding to a putative high affinity G protein-coupled receptor (GPCR).Some reports demonstrated that inactivation of prosaposin gene affected the development of the prostate gland. Koochekpour S. et al affirmed that prosaposin gene amplified and overexpressed in prostate cancer cells, and also demonstrated that exogenous saposin C and Prosaptide TX14A stimulated growth, metastasis and invasion via activated MAPK, PI₃K/AKT et al singling pathways in prostate cancer.Prostate cancer is the highly strong lethal disease, once relapsing most of the patients will develop into hormone-resistant or hormone-independent prostrate cancer. At present, this is no effective treatment options for the patients. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor (AR) has been' shown to play a critical role because AR was the only overexpressed persistently molecular during the development and progression of the AIPC. The AR is a ligand-dependent transcription factor of the nuclear steroid hormone receptor superfamily. Binding androgen, AR can be activated by phosphorylation. The active AR enters into the nuclear and binds to the androgen-responsive element (ARE) of the target DNA in order to activate the target gene transcription and promote cells growth. During the development of AIPC the singling pathways AR mediating involved in AR gene mutation, AR gene amplification, the aberrant regulation of AR by growth factors and cytokines, and AR cofactors change. Evidences have been shown that ligand-independent activation of AR is concerned with the development of AIPC.The present study is aimed to determine effect of the function domain of saposin C (neurotrophic peptide, NP) on androgen receptor (AR) expression and transcriptional activity and cells proliferation in prostate cancer. We constructed DNA vectors that can express NP or a chimeric peptide of a viral TAT transduction domain and NP by gene cloning technology. The effect of ectopic expression of NP with or without the TAT transduction domain on cell growth was examined by MTT assay and flow cytometry. Then reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and transient transfection experiments were used to determine the effect of NP on AR expression and activation.The Results showed that the eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed and confirmed by sequencing, and they can normally express in prostate cancer cells. MTT and flow cytometry analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation, and promoted the cells to enter S and G₂/M phase. The RT-PCR and Western blot analyses displayed that TAT-NP and NP induced AR gene expression. Dual-luciferace experiment results demonstrated that TAT-NP and NP activated transactivation of the AR.Collectively, our study showed that NP, as a growth stimulating factor to PCa cells, exerted the activity by increasing the expression and activation of the AR in a ligand-independent manner. Further investigation focused on how NP regulates the AR will give more insight into its role in PCa progression as well as a new cancer therapeutic target.