Breast Cancer Prevention by Ayurvedic Medicine Constituents

描述（由申请人提供）： 乳腺癌仍然是美国女性癌症相关死亡的主要原因。该研究项目的长期目标是开发一种安全有效的策略，利用新型阿育吠陀药物成分——Withaferin-A (WA) 来预防女性乳腺癌。该临床前研究项目源于我们已发表和初步未发表的结果，表明 WA 治疗通过引起细胞凋亡来降低培养的乳腺癌细胞的活力，并且 WA 的促凋亡作用与活性氧 (ROS) 的产生、c-Jun-N 末端激酶 (JNK1/2) 的激活以及促凋亡蛋白 Bax、Bak 和 Bim 的诱导相关。值得注意的是，WA 给药显着延缓了雌性裸鼠中 MDA-MB-231 人乳腺癌异种移植物的生长，且不会引起任何有害的副作用。然而，尽管取得了这些有希望的结果，但我们对 WA 诱导细胞凋亡机制的理解仍存在重大差距。例如，WA 介导的 ROS 产生的精确机制仍然难以捉摸。同样，在 WA 诱导的细胞凋亡执行过程中 ROS 产生和 JNK1/2 激活的下游信号通路尚不清楚。本申请中提出的研究不仅将填补我们知识中的这些机制空白，还将确定 WA 在转基因小鼠模型中预防乳腺癌的体内功效。根据我们的初步研究结果，我们假设 WA 治疗选择性地引起乳腺癌细胞中 ROS/JNK 介导的细胞凋亡，从而对乳腺癌发生进行化学预防。具体目标：本项目的具体目标是：（1）以MDA-MB-231、MCF-7、BRI-JM04和MCF-10A细胞系为模型，确定WA介导的ROS产生机制； (2) 以上述细胞系为模型，确定WA介导的JNK1/2激活机制； (3) 使用上述细胞系确定 WA 诱导的细胞凋亡执行过程中 ROS 产生和 JNK1/2 激活的下游信号通路； MCF-10A细胞系包含在具体目标1-3中，以深入了解正常乳腺上皮细胞对WA诱导的细胞凋亡的相对抵抗机制； (4) 使用MMTV-neu小鼠确定膳食WA给药预防乳腺癌的功效； (5) 使用来自对照和 WA 治疗的 MMTV-neu 小鼠的乳腺肿瘤组织，确定饮食 WA 可以预防 MMTV-neu 小鼠乳腺癌发展的机制。拟议研究的意义：拟议的临床前体内功效研究的积极结果将为临床试验提供动力，以确定 WA 对人类乳腺癌的化学预防效果。定义 WA 抗癌作用机制的价值可以通过多种方式实现，包括鉴定 WA 反应的生物标志物，这些生物标志物可能在未来的临床试验中有用，以及优化基于 WA 的乳腺癌化学预防方案。 公共卫生相关性： 该研究项目的长期目标是开发一种安全且非内分泌的策略，使用阿育吠陀药物成分房颤素-A (WA) 预防女性乳腺癌。本申请中提出的研究将： (a) 确定 WA 导致乳腺癌细胞凋亡的机制；定义 WA 诱导细胞凋亡机制的内在未来价值在于优化基于 WA 的化学预防方案，并合理设计使用 WA 和其他机制不同的药物的协同组合，以实现更大的化学预防功效； (b) 使用转基因小鼠模型 (MMTV-neu) 确定 WA 预防乳腺癌发生的体内功效；体内功效的证明是启动临床试验以确定 WA 对抗人类乳腺癌活性的先决条件。总之，本申请中提出的假设驱动和以机制为重点的研究将为 WA 作为人类乳腺癌化学预防剂的临床开发提供必要的临床前数据。

DESCRIPTION (provided by applicant): Breast cancer continues to be a leading cause of cancer related deaths among women in the United States. The long-term goal of this research project is to develop a safe and effective strategy for prevention of breast cancer in women using a novel Ayurvedic medicine constituent- Withaferin-A (WA). This preclinical research project stems from our published and preliminary unpublished results demonstrating that WA treatment decreases viability of cultured breast cancer cells by causing apoptosis and the proapoptotic effect of WA correlates with generation of reactive oxygen species (ROS), activation of c-Jun- N-terminal kinases (JNK1/2), and induction of proapoptotic proteins Bax, Bak, and Bim. Noticeably, WA administration significantly retards growth of MDA-MB-231 human breast cancer xenografts in female nude mice without causing any harmful side effects. Despite these promising results, however, significant gaps exist in our understanding of the mechanism(s) of WA-induced apoptosis. For example, the precise mechanism of WA-mediated ROS production remains elusive. Likewise, the signaling pathways downstream of ROS production and JNK1/2 activation in execution of WA-induced apoptosis are unclear. Studies proposed in this application will not only fill these mechanistic gaps in our knowledge but also determine in vivo efficacy of WA for prevention of breast cancer in a transgenic mouse model. Based on the results of our preliminary studies, we hypothesize that WA treatment selectively causes ROS/JNK-mediated apoptosis in breast cancer cells leading to chemoprevention of mammary carcinogenesis. Specific Aims: The specific aims of this project are to: (1) Determine the mechanism of WA-mediated ROS production using MDA-MB-231, MCF-7, BRI-JM04, and MCF-10A cell lines as a model; (2) Determine the mechanism of WA-mediated activation of JNK1/2 using the above mentioned cell lines as a model; (3) Determine the signaling pathways downstream of ROS production and JNK1/2 activation in execution of WA- induced apoptosis using above cell lines; the MCF-10A cell line is included in specific aims 1-3 to gain insight into the mechanism of relative resistance of normal mammary epithelial cells towards WA-induced apoptosis; (4) Determine the efficacy of dietary WA administration for prevention of mammary carcinogenesis using MMTV-neu mice; and (5) Determine the mechanism by which dietary WA may prevent breast cancer development in MMTV-neu mice using mammary tumor tissues from control and WA-treated MMTV-neu mice. Significance of the Proposed Research: Positive outcome of the proposed preclinical in vivo efficacy study will provide impetus for clinical trials to determine chemopreventive effectiveness of WA against human breast cancer. The value of defining the mechanism of anticancer effect of WA may be realized in a variety of ways including identification of biomarker(s) of WA response potentially useful in future clinical trials and optimization of WA-based chemopreventive regimens against breast cancer. PUBLIC HEALTH RELEVANCE: The long-term objective of this research project is to develop a safe and non-endocrine strategy for prevention of breast cancer in women using Ayurvedic medicine constituent withaferin-A (WA). Studies proposed in this application will: (a) define the mechanism by which WA causes apoptotic cell death in breast cancer cells; the inherent future value of defining the mechanism of WA-induced apoptosis resides in optimization of WA-based chemopreventive regimens and rational design of synergistic combinations using WA and other mechanistically distinct agents to achieve even greater chemopreventive efficacy; and (b) determine in vivo efficacy of WA for prevention of breast carcinogenesis using a transgenic mouse model (MMTV-neu); demonstration of in vivo efficacy is a prerequisite for initiation of clinical trials to determine activity of WA against human breast cancer. In summary, the hypothesis-driven and mechanistically-focused studies proposed in this application will provide preclinical data necessary for clinical development of WA as a chemopreventive agent against breast cancer in humans.