LSUHSC COBRE: PROJ 1: PRESPOSIN, A PROMOTER OF PROSTATE CARCINOGENESIS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 前列腺激活蛋白原，前列腺癌发生的促进剂和新的治疗靶点： 我们先前已经证明了在人前列腺癌细胞和组织中鞘脂激活蛋白原的过表达和/或基因组扩增。到目前为止，我们发表的数据表明，作为可溶性多能生长因子，鞘脂激活蛋白原或其活性结构域之一（即，鞘脂激活蛋白C）增加前列腺癌细胞的生长、运动性和侵袭，基质降解蛋白分解酶（例如，MMPs，uPA），并且作为细胞存活和抗凋亡因子起作用。TX 14 A是一种合成肽，来源于saposin原的营养氨基酸序列，在前列腺癌细胞中显示出与saposin原相似的生物活性。为了评估saposin作为分子靶点的潜力，我们设计了TX 14 A和植物毒素saporin的化学缀合物（TX 14 A-SAP），并通过使用体外细胞毒性测定，我们测试了其对前列腺癌细胞系（PC-3，DU-145，LNCaP）的作用。在所有三种前列腺癌细胞系中，我们发现了一种有效的细胞杀伤作用。本研究的结论是，毒素嵌合体的TX 14 A营养肽可用于将植物毒素（Saporin）递送至细胞。虽然本研究是作为原则证明进行的，但由于与皂草素本身相关的潜在肝细胞毒性，我们决定采用其他策略。 1. TX 14 A肽与化疗药物的缀合。 我们在临床肿瘤学中寻找最常用的治疗药物来治疗晚期转移性或激素难治性前列腺癌，结果如下：米托蒽醌、多西他赛和阿霉素 我们决定将TX 14 A肽与这些化疗剂缀合。在与2名独立化学家进行沟通和咨询后，很明显肽和药物之间的共价缀合在技术上是可能的。然而，由于肽及其活性基团的独特结构以及药物中的生物细胞活性化学基团，由于结构的化学改变，肽或药物将失活。 2.靶向前列腺癌细胞的反义TX 14 A肽策略。在这种方法中，我们的目标是设计和合成短的寡肽，可以在结构上互补的saposin原的营养肽序列。因此，游离配体浓度将降低，从而其生物活性将被阻止。我们使用水疗评分和3-5 或5-3 TX 14 A核苷酸序列的阅读方向（图1）。我们设计并合成了许多反义肽（具有8至10个氨基酸）以补充saposin或saposin C的生物活性序列（即，TX 14 A-肽）。这些肽的合成纯度为95%，具有微小的结构修饰，以稳定它们并抵抗外肽酶和内肽酶的酶促降解。 我们评估发现，这些肽之一（LL 8）具有与天然配体（PSAP）或其活性分子衍生物（saposin C或TX 14 A）竞争的能力，并且在竞争性抑制测定中阻止它们的生长刺激作用 与对照无活性（乱序）肽-NN 8相比。LL-8肽在体外对PC-3细胞的迁移和侵袭无抑制作用。通过使用灵敏的增殖/细胞毒性测定，我们在其他PCa细胞（例如，DU-145，LNCaP），我们没有发现处理组和对照组之间或LL 8-肽和乱序肽-NN 8之间的任何显著差异。 在此观察之后，我们集中精力来表征其他反义肽对一组雄激素敏感性和雄激素非依赖性转移性PCa细胞系的作用（图2）。 3. RNA干扰作为前列腺癌PSAP靶向策略的评估。在此之前，我们已经报道了在PC-3细胞系中PSAP过表达和基因组扩增。 由于我们的目标是干扰可溶性PSAP的生物活性，因此我们决定抑制或减少可溶性PSAP的量。 PSAP产生（分泌的PSAP水平）。作为替代策略，我们设计并构建了 项目1继续进行。. . PSAP-siRNA哺乳动物表达载体和建立的PSAP-siRNA PC-3细胞的稳定转染子（图3）。此外，由于LNCaP细胞具有非常低水平的PSAP表达，我们还在这些细胞中建立了PSAP过表达的稳定转染子。目前，我们正在研究PC-3和LNCaP细胞的亲本和稳定转染子之间的差异，用于基底细胞增殖、迁移和侵袭，以及基质降解蛋白水解酶（MMP-9和uPA）的产生。本研究将建立PSAP表达水平与前列腺癌细胞增殖和侵袭特性之间的联系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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 Doxorubicin We 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 assays as 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.