Mechanism of Apoptosis and Inhibition of Tumor Progression and Metastasis by Par-

Par-细胞凋亡及抑制肿瘤进展和转移的机制

• 批准号: 7909261
• 负责人: Vivek M Rangnekar
• 金额: $ 31.88万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7909261
• 关键词: Address; Adenocarcinoma; Amino Acids; Animals; Apoptosis; Apoptotic; Binding; Bone Marrow Transplantation; Brefeldin A; Cancer Model; Cell Nucleus; Cell Surface Receptors; Cell Survival; Cell membrane; Cells; Cessation of life; Conditioned Culture Media; Control Animal; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Distant Metastasis; Endoplasmic Reticulum; Exposure to; Frequencies; Funding; Golgi Apparatus; Growth; In Vitro; Induction of Apoptosis; Inhibition of Apoptosis; Lesion; Leucine Zippers; Liver; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Morphologic artifacts; Mus; NF-kappa B; Neoplasm Metastasis; Normal Cell; PAWR gene; PAWR protein; Pathway interactions; Phosphorylation; Plasmids; Prostate; Prostatic; Prostatic Intraepithelial Neoplasias; Prostatic Neoplasms; Proteins; RNA Interference; Receptor Signaling; Recombinants; Research; Science; Signal Pathway; Spleen; Stromal Cells; TNFSF10 gene; Tertiary Protein Structure; Testing; Therapeutic; Transfection; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Vertebrates; Western Blotting; base; cancer cell; clinically relevant; extracellular; glucose-regulated proteins; in vivo; inhibitor/antagonist; insight; intravenous injection; neutralizing antibody; novel; offspring; prevent; public health relevance; tumor; tumor growth; tumor progression

DESCRIPTION (provided by applicant): Prostate apoptosis response-4 protein (Par-4; also known as PAWR) is a leucine zipper domain protein that is conserved in vertebrates. During the previous funding period, we showed the core effector domain of Par-4 (amino acids 137-195, designated the SAC domain) is necessary and sufficient to induce apoptosis. Interestingly, both Par-4 and its SAC domain induce apoptosis selectively in cancer cells, but not in normal cells. Although ectopic Par-4 and the SAC domain have long been thought to effect apoptosis by acting within the cell nucleus to inhibit NF-kappaB, our recent transient transfection studies unexpectedly displayed apoptosis in neighboring cells that did not express the Par-4 or SAC plasmid. Moreover, conditioned medium (CM) from Par-4 transfected cells induced apoptosis when applied to cancer cells, but not to normal cells; this implies over-expression of Par-4 leads to the secretion of a pro-apoptotic activity. Both Par-4 and the SAC protein were detected in the CM by Western blot analysis, and the apoptotic activity in this CM was neutralized by antibodies against Par-4. The apoptotic activity was diminished in the CM from Par-4 transfectants treated with brefeldin A, indicating Par-4 and SAC secretion (or sPar-4 and sSAC, respectively) occurs by the classical pathway (i.e., from the endoplasmic reticulum to the Golgi, and then to the cell membrane). Importantly, CM from normal and cancer cells contains endogenous Par-4, of which the levels increase upon treatment with TRAIL (a short term exposure that is insufficient to produce apoptosis), thereby indicating secretion is physiologically relevant, and not an artifact associated with over-expression of the Par-4 and SAC constructs. Based on these data, we hypothesize endogenous and exogenous sPar-4 and sSAC exert cancer-specific apoptotic effects via both intracellular and extracellular mechanisms. We will address this hypothesis from a mechanistic perspective, characterizing secreted Par-4 and SAC in prostate cancer cells, and clarifying the means by which they selectively initiate apoptosis in cancer cells. Specifically, we will 1) Delineate the mechanism of Par-4/SAC secretion and apoptosis by sPar-4/sSac in prostate cancer cells; 2) Test the growth suppressive effects of sPar-4 and sSAC domain in prostate cancer models; and 3) Study the inter-dependence of extracellular and intracellular Par-4 in inducing apoptosis in cancer cells. As our recent studies indicate Par- 4 and its SAC domain are secreted, and that recombinant (r) Par-4 or rSAC can induce apoptosis when applied exogenously to cells, these data may hold significant clinical relevance. Our results introduce the novel prospect of using sPar-4 or sSAC to induce apoptosis, not only in local or superficially confined tumors into which Par-4 or SAC can be readily delivered intratumorally, but also to distant metastases by intravenous injection or bone-marrow transplantation, which we will explore in this proposal. PUBLIC HEALTH RELEVANCE: Our research focuses on the secreted forms of Par-4 and it core effector domain SAC, both of which selectively induce apoptosis in cancer cells. We will study these two proteins from a mechanistic perspective both in vitro and in vivo, characterizing secreted Par-4 and SAC in prostate cancer cells, and clarifying the means by which they selectively initiate apoptosis in cancer cells. The proposed science will explore the novel prospect of using sPar-4 or sSAC to induce apoptosis, not only in local or superficially confined tumors into which Par-4 or SAC can be readily delivered intratumorally, but also to distant metastases by intravenous injection or bone-marrow transplantation.

描述（由申请人提供）：前列腺凋亡反应-4蛋白（PAR-4；也称为PAWR）是一种亮氨酸拉链结构域蛋白，在脊椎动物中保守。在上一个资金期间，我们展示了Par-4的核心效应域（氨基酸137-195，指定为SAC域）是必要的，足以诱导凋亡。有趣的是，Par-4及其SAC结构域都在癌细胞中有选择地诱导细胞凋亡，但在正常细胞中却不会。尽管长期以来人们认为异位PAR-4和SAC结构域通过在细胞核内作用以抑制NF-kappab来影响凋亡，但我们最近的瞬时转染研究意外地表现出邻近细胞中未表达PAR-4或SAC质粒的凋亡。此外，当par-4转染的细胞中的条件培养基（CM）诱导凋亡时，将凋亡应用于癌细胞，但不适用于正常细胞。这意味着Par-4的过表达导致促凋亡活性的分泌。通过Western印迹分析在CM中检测到PAR-4和SAC蛋白，并且该CM中的凋亡活性被针对PAR-4的抗体中和。从用Brefeldin A处理的PAR-4转染物中的CM中，凋亡活性降低了，表明PAR-4和SAC分泌（或SPAR-4和SSAC）（分别是经典途径）（即，从内质网向戈尔基氏菌到戈尔基氏菌）发生，然后到达细胞膜）。重要的是，来自正常和癌细胞的CM包含内源性PAR-4，其中水平在治疗时的水平增加（短期暴露不足以产生细胞凋亡），从而表明分泌在生理上是相关的，而不是与过度相关的文物 - PAR-4和SAC构建体的表达。基于这些数据，我们假设内源性和外源性SPAR-4和SSAC通过细胞内和细胞外机制发挥癌症特异性凋亡作用。我们将从机械的角度来解决这一假设，以前列腺癌细胞中的分泌Par-4和SAC为特征，并阐明它们有选择地启动癌细胞凋亡的手段。具体而言，我们将1）描述前列腺癌细胞中SPAR-4/SSAC的PAR-4/SAC分泌的机理； 2）测试前列腺癌模型中SPAR-4和SSAC结构域的生长抑制作用； 3）研究细胞外和细胞内PAR-4在癌细胞诱导凋亡中的相互依赖性。正如我们最近的研究表明，第4杆及其SAC结构域是分泌的，重组（R）Par-4或RSAC可能会诱导外源性应用于细胞时凋亡，这些数据可能具有显着的临床相关性。我们的结果介绍了使用SPAR-4或SSAC诱导细胞凋亡的新型前景，不仅在局部或表面限制的肿瘤中，可以在肿瘤内易于肿瘤，还可以在肿瘤内输送PAR-4或SAC，还可以通过静脉注射或骨row来延伸转移移植，我们将在本提案中探索。公共卫生相关性：我们的研究重点是Par-4和IT核心效应域SAC的分泌形式，这两者都选择性地诱导癌细胞中的凋亡。我们将从体外和体内从机械角度研究这两种蛋白质，以前列腺癌细胞中分泌的Par-4和SAC为特征，并阐明它们有选择地启动癌细胞凋亡的手段。拟议的科学将探索使用SPAR-4或SSAC诱导细胞凋亡的新型前景，不仅在局部或表面上受限制的肿瘤中，可以在肿瘤内容易地输送PAR-4或SAC，还可以通过静脉注射或骨骼注射或骨头转移。 - arrow移植。