Role of Proteinase-3 in Apoptosis and Drug Resistance

Proteinase-3 在细胞凋亡和耐药性中的作用

• 批准号: 7226259
• 负责人: Ahmad R. Safa
• 金额: $ 28.58万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7226259
• 关键词: Acetylcysteine; Antineoplastic Agents; Antioxidants; Apoptosis; Apoptotic; Breast Cancer Cell; Cancer cell line; Catalytic Domain; Cell Line; Cell-Mediated Cytolysis; Cells; Ceramides; Cytoplasm; DNA Fragmentation; Data; Daunorubicin; Development; Doxorubicin; Drug resistance; Drug-sensitive; Gene Transfer; Generations; HL-60 Cells; HL60; Human; JUN gene; Knowledge; Laboratories; MCF7 cell; Malignant Neoplasms; Measures; Mediating; Mitochondria; Molecular; Myeloid Leukemia; Numbers; Oligonucleotides; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Principal Investigator; Process; Production; Proteinase 3; Proteins; Reactive Oxygen Species; Resistance; Role; Serine Protease; Site-Directed Mutagenesis; Sphingomyelinase; Tertiary Protein Structure; Therapeutic; Transcription Factor AP-1; Treatment Protocols; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Variant; Vesicle; cancer cell; caspase-3; caspase-8; cytotoxic; cytotoxicity; design; drug efficacy; expression vector; gene therapy; human TNF protein; inhibitor/antagonist; mutant; novel; prevent; programs; research study; resistance mechanism; transcription factor

DESCRIPTION (provided by applicant): We have recently discovered that decreased expression of proteinase-3 (PR3), a serine protease, is associated with drug resistance. Inhibition of cellular PR3 expression by an antisense PR3 oligodeoxynucleotide resulted in a significant reduction fin reactive oxygen species (ROS) generation and drug-induced apoptosis, and increased cellular resistance to doxorubicin (DOX) or daunorubicin (DNR). In this application, we propose to explore the molecular mechanism(s) by which PR3 mediates drug-induced apoptosis and increases the efficacy of anticancer agents, and how its loss causes drug resistance. Our long-term objective is to apply the knowledge obtained from these studies to identify and/or develop novel and more effective cancer therapeutic regimens. The Specific Aims are to (1) determine whether increased PR3 expression by gene transfer experiments enhances the cytotoxic effects of DOX and DNR, and (2) investigate the role of the PR3 in mediating increased reactive oxygen species (ROS) and delineate the molecular mechanism(s) of PR3-mediated drug-induced apoptosis, and (3) determine whether the PR3-mediated drug-induced apoptosis is dependent on the proteolytic activity of PR3 by identifying the domain of PR3 with apoptotic function. Specific Aim 1 will (a) evaluate whether the increase in PR3 enhances DOX- or DNR-induced apoptosis in drug sensitive and resistant cells, (b) determine the role of PR3 in drug-induced ROS production and apoptosis, (c) examine the subcellular localization of PR3, and (d) ascertain biochemically how PR3 expression modulates the effect of DOX on triggering apoptosis. Specific Aim 2 will (a) determine whether the PR3-mediated increase in ROS occurs through a mitochondrion-dependent mechanism, and by increased activation of cellular tumor necrosis factor-a (TNFct), (b) investigate whether inhibition of PR3 expression blocks neutral sphingomyelinase (N-SMase) activation and ceramide generation, and whether increased expression of PR3 activates these processes, and (c) assess whether the PR3-mediated drug induced increase in ROS production induces ceramide generation, and activates c-Jun-Terminal Kinase (JNK) and AP1 transcription factor. In Specific Aim 3, we will (a) construct FLAG-tagged expression vectors containing regions of PR3 that, upon expression in cells, produce PR3 fragments which either contain or lack the full components of catalytic domain of PR3, (b) transfect cells lacking PR3 with these expression vectors, and identify a fragment of PR3 which enhances drug-induced apoptosis, (c) assess the effect of this PR3 fragment in modulating drug-induced apoptosis in the drug sensitive and resistant cell lines, (d) generate deletion mutants as well as perform site-directed mutagenesis studies to refine the domain of PR3 with apoptotic function, and (e) design peptides from this domain and examine whether they are able to modulate the cytotoxicity of DOX and DNR. These studies will aid in understanding how PR3 is capable of mediating drug-induced apoptosis, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.

描述（由申请人提供）： 我们最近发现蛋白酶 3 (PR3)（一种丝氨酸蛋白酶）的表达降低与耐药性相关。通过反义 PR3 寡脱氧核苷酸抑制细胞 PR3 表达，导致活性氧 (ROS) 生成和药物诱导的细胞凋亡显着减少，并增加细胞对阿霉素 (DOX) 或柔红霉素 (DNR) 的耐药性。在本申请中，我们打算探索PR3介导药物诱导的细胞凋亡并提高抗癌药物疗效的分子机制，以及它的缺失如何导致耐药性。我们的长期目标是应用从这些研究中获得的知识来识别和/或开发新颖且更有效的癌症治疗方案。具体目标是 (1) 确定通过基因转移实验增加 PR3 表达是否会增强 DOX 和 DNR 的细胞毒性作用，(2) 研究 PR3 在介导活性氧 (ROS) 增加中的作用并描绘 PR3 介导的药物诱导细胞凋亡的分子机制，以及 (3) 确定 PR3 介导的药物诱导细胞凋亡是否依赖于 通过鉴定具有凋亡功能的 PR3 结构域来检测 PR3 的蛋白水解活性。具体目标 1 将 (a) 评估 PR3 的增加是否会增强药物敏感和耐药细胞中 DOX 或 DNR 诱导的细胞凋亡，(b) 确定 PR3 在药物诱导的 ROS 产生和细胞凋亡中的作用，(c) 检查 PR3 的亚细胞定位，以及 (d) 从生化角度确定 PR3 表达如何调节 DOX 触发细胞凋亡的作用。具体目标 2 将 (a) 确定 PR3 介导的 ROS 增加是否通过线粒体依赖性机制以及细胞肿瘤坏死因子-a (TNFct) 的激活增加而发生，(b) 研究 PR3 表达的抑制是否会阻止中性鞘磷脂酶 (N-SMase) 激活和神经酰胺生成，以及 PR3 表达增加是否会激活这些过程，以及 (c) 评估 PR3 表达的增加是否会激活这些过程。 PR3 介导的药物诱导 ROS 产生增加，从而诱导神经酰胺生成，并激活 c-Jun 末端激酶 (JNK) 和 AP1 转录因子。在具体目标 3 中，我们将 (a) 构建含有 PR3 区域的 FLAG 标记表达载体，在细胞中表达时，产生包含或缺乏 PR3 催化结构域完整成分的 PR3 片段，(b) 用这些表达载体转染缺乏 PR3 的细胞，并鉴定增强药物诱导细胞凋亡的 PR3 片段，(c) 评估该 PR3 片段在调节药物诱导细胞凋亡中的作用 药物敏感和耐药细胞系中的细胞凋亡，(d) 生成缺失突变体并进行定点诱变研究，以细化具有凋亡功能的 PR3 结构域，以及 (e) 从该结构域设计肽并检查它们是否能够调节 DOX 和 DNR 的细胞毒性。这些研究将有助于了解 PR3 如何能够介导药物诱导的细胞凋亡，并将有助于开发更有效的化疗或潜在的基因治疗策略。

项目成果

Human GM3 Synthase Attenuates Taxol-Triggered Apoptosis Associated with Downregulation of Caspase-3 in Ovarian Cancer Cells.

• DOI: 10.4236/jct.2012.35065
• 发表时间: 2012-10
• 期刊: Journal of cancer therapy
• 作者: Huang S;Bijangi-Vishehsaraei K;Saadatzadeh MR;Safa AR
• 通讯作者: Safa AR

c-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8-, and caspase-9-dependent apoptosis in breast cancer cells.

• DOI: 10.1016/j.bcp.2008.09.007
• 发表时间: 2008-12-15
• 期刊: BIOCHEMICAL PHARMACOLOGY
• 影响因子: 5.8
• 作者: Day, Travis W.;Huang, Su;Safa, Ahmad R.
• 通讯作者: Safa, Ahmad R.

c-FLIP gene silencing eliminates tumor cells in breast cancer xenografts without affecting stromal cells.

• 发表时间: 2009-10
• 期刊: Anticancer research
• 影响因子: 2
• 作者: T. W. Day;A. Sinn;Su Huang;K. Pollok;G. Sandusky;A. Safa

4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells.

• DOI: 10.1007/s11010-010-0477-7
• 发表时间: 2010-09
• 期刊: MOLECULAR AND CELLULAR BIOCHEMISTRY
• 影响因子: 4.3
• 作者: Bijangi-Vishehsaraei, Khadijeh;Saadatzadeh, Mohammad Reza;Huang, Su;Murphy, Michael P.;Safa, Ahmad R.
• 通讯作者: Safa, Ahmad R.

Human beta-galactoside alpha-2,3-sialyltransferase (ST3Gal III) attenuated Taxol-induced apoptosis in ovarian cancer cells by downregulating caspase-8 activity.

• DOI: 10.1007/s11010-009-0147-9
• 发表时间: 2009-11
• 期刊: MOLECULAR AND CELLULAR BIOCHEMISTRY
• 影响因子: 4.3
• 作者: Huang, Su;Day, Travis W.;Choi, Mi-Ran;Safa, Ahmad R.
• 通讯作者: Safa, Ahmad R.