ANTIPROGESTINS AND ACTIVE CELL DEATH IN BREAST CANCER

抗孕激素与乳腺癌中的活性细胞死亡

• 批准号: 2611391
• 负责人: PATRICIA V SCHOENLEIN
• 金额: $ 21.23万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2611391
• 关键词: MCF7 cell; apoptosis; athymic mouse; breast neoplasms; cell growth regulation; cell line; chemoprevention; combination chemotherapy; disease /disorder model; enzyme linked immunosorbent assay; estrogen receptors; gene expression; hormone regulation /control mechanism; immunocytochemistry; mifepristone; neoplasm /cancer chemotherapy; northern blottings; progesterone receptors; protein kinase A; protein kinase C; pulsed field gel electrophoresis; tamoxifen; transforming growth factors; western blottings

DESCRIPTION: (adapted from the applicants abstract) Antiprogestins are a promising new class of mammary tumor inhibitors with a unique mechanism of action which does not seem to involve a classical progesterone antagonism. The objective of this application is to confirm an additive antitumor activity and to clarify the intracellular mechanism of action of the antiprogestin mifepristone, and the antiestrogen tamoxifen, in inhibition of growth of established tumors (chemotherapy) and in prevention or delay of tumor development (chemoprevention/adjuvant setting) in clinically relevant breast cancer models. The focus of the mechanistic studies is to explore the role of (and the specific pathways for) induction of apoptosis in breast cancer cells as a treatment response. The importance of (and the effect of treatments on) estrogen (ER) and progesterone (PR) receptors as well as the potential additive or synergistic interaction between mifepristone and tamoxifen will also be explored. The research design includes the use of the ER(+) and PR(+) MCF-7 and the ER(-) and PR(-) MDA-231 human breast cancer cell lines grown in culture (in vitro studies) or inoculated into female nude mice supplemented with 17BETA-estradiol (in vivo studies). Companion experiments utilizing the MXT [hormone-dependent ER(+) and PR(+)] and MXT-OVEX [hormone-independent ER(-) and PR(-)] homograft mammary carcinoma models of the mouse are also included to compare the antitumor effects in a normal (nonimmunologically suppressed) animal with those induced in nude mice. The following cellular parameters and their time-dependent sequence will be assessed: morphological features (e.g., mitotic and apoptotic indices), in vitro cell viability (cytotoxicity), Cdk1 protein concentration, DNA fragmentation, PKA and PKC isoform activity and subcellular distribution, as well as the changes in the expression of bax, bcl2 and TGFBETA isoforms. The expression of other known "cell death genes" or "cell cycle determinants" will also be included whenever they seem appropriate. These various outcome measures will be evaluated using immunocytochemistry, pulsed field gel electrophoresis, Western blots, Northern analysis, specific ELISA, enzyme kinetics and saturation lignad binding assays. The results generated from the described studies will confirm an additive antitumor activity of mifepristone and tamoxifen. This activity is mainly the result of a steroid receptor-mediated interaction with the cell suicide mechanism which involves distinct changes in gene expression (an increase in the bax/bcl2 ratio) in distinct subpopulations (different receptor status) of breast cancer cells. These changes initiate a direct and/or an indirect cell death command via distinct PKC or PKA second messenger signaling pathways and/or via expression or specific TGF-b isoforms. Induction of apoptosis in two distinct subpopulations of breast cancer cells and/or through multiple reinforcing molecular biochemical mechanisms would provide an excellent rationale for a combination therapy. The expected additive effect may delay malignant progression, reduce metastatic spread and increase survival of breast cancer patients. The studies could also impact the future clinical development of "antiprogestins" for this indication.

描述：（改编自申请人摘要）抗植物是一个 有希望的新型乳腺肿瘤抑制剂，具有独特的机制 似乎不涉及古典孕酮拮抗作用。 该应用的目的是确认添加剂抗肿瘤 活动并阐明细胞内作用机理 抗植物米非生酯和抗雌激素他莫昔芬，以抑制 已建立肿瘤（化学疗法）的生长以及预防或延迟 临床相关的肿瘤发育（化学预防/辅助设置） 乳腺癌模型。 机械研究的重点是探索 （以及特定途径）凋亡在乳房中的作用 癌细胞作为治疗反应。 重要性（以及 雌激素（ER）和孕酮（PR）受体的处理以及 Mifepristone和 他莫昔芬也将被探索。 研究设计包括使用 ER（+）和PR（+）MCF-7以及ER（ - ）和PR（ - ）MDA-231人乳房 在培养（体外研究）中生长的癌细胞系或接种 雌性裸鼠补充了17beta-雌二醇（体内研究）。 使用MXT [激素依赖性ER（+）和PR（+）]的伴侣实验 和mxt-overx [激素独立的ER（ - ）和PR（ - ）]同源乳腺 还包括小鼠癌模型以比较抗肿瘤 在正常（非绝种抑制）动物中的影响 在裸鼠中诱导。 以下蜂窝参数及其 将评估时间依赖性序列：形态学特征（例如， 有丝分裂和凋亡指数），体外细胞活力（细胞毒性），CDK1 蛋白质浓度，DNA片段化，PKA和PKC同工型活性以及 亚细胞分布以及Bax表达的变化， Bcl2和TGFBETA同工型。 其他已知的“细胞死亡基因”的表达 或“单元循环决定因素”，每当它们看起来 合适的。 这些各种结果指标将使用 免疫细胞化学，脉冲场凝胶电泳，蛋白质印迹， 北方分析，特定的ELISA，酶动力学和饱和木质 绑定测定。 从描述的研究产生的结果将 确认米非司酮和他莫昔芬的添加抗肿瘤活性。 这 活性主要是类固醇受体介导的相互作用的结果 具有涉及基因明显变化的细胞自杀机制 在不同亚群中的表达（BAX/BCL2比率增加） （不同的受体状态）乳腺癌细胞。 这些变化启动 通过不同的PKC或PKA直接和/或间接的细胞死亡命令 第二使者信号通路和/或通过表达或特定的TGF-B 同工型。 在两个不同的乳房亚群中诱导凋亡 癌细胞和/或通过多种增强分子生化 机制将为联合疗法提供一个很好的理由。 预期的添加剂效应可能会延迟恶性进展，减少 转移扩散并增加乳腺癌患者的存活率。 这 研究还可能影响未来的临床发展 此指示的“抗叶膜蛋白”。