This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Prosaposin, a promoter of prostate carcinogenesis and a novel therapeutic target: We have previously demonstrated overexpression and/or genomic amplification of prosaposin in human prostate cancer cells and tissues. So far our published data showed that as a soluble pluripotent growth factor, prosaposin or one of its active domains (i.e., saposin C) increases prostate cancer cells growth, motility and invasion, production of matrix-degrading prtoteolytic enzymes (e.g., MMPs, uPA), and function as a cell survival and anti-apoptotic factor. TX14A, a synthetic peptide, derived from the trophic amino acid sequence of prosaposin has shown biological activities similar to prosaposin in prostate cancer cells. To evaluate the potential of prosaposin as a molecular target, we designed a chemical conjugate of TX14A and plant toxin saporin (TX14A-SAP) and by using in vitro cytotoxicity assays, we tested its effect on prostate cancer cell lines (PC-3, DU-145, LNCaP). In all three prostate cancer cell lines, we found a potent cell-killing effect. The conclusion from this study was that the TX14A-trophic peptide of the toxin-chimera could be used to deliver the plant toxin (Saporin) to the cells. While this study was performed as a proof of principle, due to the potential hepatocellular toxicity associated with the saporin itself, we decided to employ other strategies. 1. Conjugation of TX14A peptide to chemotherapy drugs. Our search for the most commonly used therapeutic medicine in clinical oncology dealing with advanced metastatic or hormone refractory prostate cancer led to the followings: Mitoxantrone, Docetaxel, and DoxorubicinWe decided to conjugate the TX14A peptide to these chemotherapy agents. Following communication and consultation with 2 independent chemists, it became clear that covalent conjugation between the peptide and the drugs is technically possible. However, because of the unique structure of the peptide and its active group and also the biologically cyto-active chemical group in the drugs, due to chemical alteration in the structure either the peptide or the drug will be inactivated. 2. Antisense-TX14A peptide strategy to target prostate cancer cells. In this method, our goal was to design and synthesize short oligopeptides that could structurally complement the trophic-peptide sequence of prosaposin. Therefore, the free ligand concentration will be reduced and consequently its biological activity will be prevented. We used the hydropathic score and 3-5 or 5-3 reading direction of nucleotide sequence of TX14A (Fig. 1). We designed and synthesized a number of anti-sense peptides (with 8 to 10 amino acids) to complement the biologically active sequence of prosaposin or saposin C (i.e., TX14A-peptide). These peptides are synthesized to e95% purity with minor structural modification to stabilize them and to resist enzymatic degradation by exo- and endo-peptidases. We evaluated found that one of these peptides (LL8) has the ability to compete with the native ligand (PSAP) or its active molecular derivatives (saposin C or TX14A) and to prevent their growth stimulatory effect in competitive inhibition assaysas compared to control inactive (scrambled) peptide-NN8. LL-8 peptide did not show any inhibition for PC-3 cells migration and invasion in vitro. By using a sensitive proliferation/cytotoxicity assay, we tested LL8-peptide on other PCa cells (e.g., DU-145, LNCaP), we did not find any significant differences between the treatment and control groups or between the LL8-peptide and the scrambled peptide-NN8. After this observation, we concentrated our effort to characterize the effect of other anti-sense peptides on a set of androgen-sensitive and androgen-independent metastatic PCa cell lines (Fig. 2). 3. Evaluation of RNA-interference as a targeting strategy for PSAP in prostate cancer. Previously, we have reported PSAP-overexpression and genomic amplification in PC-3 cell line. Since our goal was to interfere with the biological activities of the soluble PSAP, we decided to inhibit or decrease the amount of PSAP-production (secreted PSAP level). As an alternative strategy, we designed and constructed Project 1 continued . . . PSAP-siRNA mammalian expression vectors and established stable transfectants of PSAP-siRNA PC-3 cells (Fig. 3). In addition, since LNCaP cells have a very low level of PSAP expression, we have also established PSAP-overexpressing stable transfectants in these cells. Currently, we are examining the differences among the parental and stable transfectants of PC-3 and LNCaP cells for basal cell proliferation, migration and invasion, and the production of matrix-degrading proteolytic enzymes (MMP-9 and uPA). This study will establish a link between PSAP expression level and prostate cancer cells proliferative and invasive characteristics.
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