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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 前列腺癌促进剂和新的治疗靶点--丙皂苷 我们以前已经证明了在人类前列腺癌细胞和组织中过表达和/或基因组扩增。到目前为止，我们发表的数据表明，作为一种可溶性的多潜能生长因子，丙皂苷或其活性区域之一(即皂苷C)可促进前列腺癌细胞的生长、运动和侵袭，产生降解基质的原分解酶(如MMPs、uPA)，并作为细胞存活和抗凋亡因子发挥作用。TX14A是一种合成肽，来源于丙皂苷的营养氨基酸序列，在前列腺癌细胞中显示出类似于丙皂苷的生物活性。为了评价丙皂苷作为分子靶标的潜力，我们设计了TX14A和植物毒素皂苷的化学偶联物(TX14A-SAP)，并通过体外细胞毒性试验，检测了其对前列腺癌PC-3、DU-145、LNCaP细胞的作用。在所有三种前列腺癌细胞系中，我们都发现了一种强大的细胞杀伤作用。本研究的结论是，毒素嵌合体的TX14A营养多肽可以用来将植物毒素(Saporin)输送到细胞内。虽然这项研究是作为原则的证明，但由于与皂苷本身相关的潜在肝细胞毒性，我们决定采用其他策略。 1.TX14A多肽与化疗药物的偶联我们寻找临床肿瘤学中最常用的治疗晚期转移性或激素耐药前列腺癌的药物，结果如下：米托蒽醌、多西紫杉醇和阿霉素 我们决定将TX14A多肽与这些化疗药物偶联。在与两位独立的化学家沟通和协商后，很明显，多肽和药物之间的共价偶联在技术上是可能的。然而，由于多肽及其活性基团的独特结构，以及药物中具有生物细胞活性的化学基团，由于结构上的化学变化，无论是多肽还是药物都会失活。 2.靶向前列腺癌细胞的反义TX14A多肽策略在这种方法中，我们的目标是设计和合成能够在结构上补充丙皂苷营养肽序列的短寡肽。因此，游离基的浓度会降低，从而阻止其生物活性。我们使用TX14A核苷酸序列的水病理评分和3-5或5-3阅读方向(图1)。我们设计并合成了一系列反义肽(含8~10个氨基酸)来补充丙皂苷或皂苷C的生物活性序列(即TX14A-肽)。这些多肽被合成到E95%的纯度，经过轻微的结构修改以稳定它们，并抵抗外肽酶和内肽酶的酶降解。 我们在竞争抑制实验中发现，这些多肽中的一个(LL8)具有与天然配体(PSAP)或其活性分子衍生物(皂苷C或TX14A)竞争并阻止它们的生长刺激作用的能力 与对照失活(杂乱)多肽-NN8相比。IL-8多肽对PC-3细胞的体外迁移和侵袭无明显抑制作用。通过灵敏的增殖/细胞毒试验，我们测试了LL8多肽对其他前列腺癌细胞(如DU-145，LNCaP)的作用，我们没有发现治疗组和对照组之间以及LL8多肽和杂交肽NN8之间的任何显著差异。 在这一观察之后，我们集中精力研究了其他反义肽对一组雄激素敏感和雄激素非依赖性转移的前列腺癌细胞株的影响(图2)。 3.评价RNA干扰作为前列腺癌PSAP靶向治疗策略的价值。以前，我们已经报道了PSAP在PC-3细胞系中的过表达和基因组扩增。由于我们的目标是干扰可溶性PSAP的生物学活性，我们决定抑制或减少 PSAP-产生(分泌型PSAP水平)。作为一种替代策略，我们设计和构建了 项目1正在继续。。。 构建了PSAP-siRNA哺乳动物表达载体，并建立了稳定的PSAP-siRNA PC-3细胞表达载体(图3)。此外，由于LNCaP细胞中PSAP的表达水平很低，我们还在这些细胞中建立了PSAP过表达的稳定转基因细胞。目前，我们正在研究PC-3和LNCaP细胞亲代和稳定转染体在基底细胞增殖、迁移和侵袭以及产生基质降解蛋白水解酶(MMP9和uPA)方面的差异。这项研究将建立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.