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。这项研究项目的长期目标是开发一种安全有效的策略，使用一种新的阿育吠陀药物成分-Withaferin-A(WA)预防女性乳腺癌。这项临床前研究项目源于我们已发表和未发表的初步研究结果，该结果表明，WA处理通过引起凋亡而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物种(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的诱导有关。值得注意的是，Wa给药显著抑制了人乳腺癌MDA-MB-231裸鼠移植瘤的生长，且未引起任何不良副作用。然而，尽管有这些可喜的结果，我们对水杨酸诱导细胞凋亡的机制(S)的理解仍存在重大差距。例如，Wa介导的ROS产生的确切机制仍然难以捉摸。同样，在Wa诱导的细胞凋亡过程中，ROS产生和JNK1/2激活的下游信号通路也不清楚。这项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，而且还将在转基因小鼠模型中确定WA预防乳腺癌的体内有效性。根据我们的初步研究结果，我们假设WA治疗选择性地导致ROS/JNK介导的乳腺癌细胞凋亡，从而化学预防乳腺癌的发生。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细胞为模型，确定WA介导的ROS产生的机制；(2)以上述细胞株为模型，确定WA介导的JNK1/2激活的机制；(3)利用上述细胞系，确定在WA诱导的细胞凋亡过程中，ROS产生和JNK1/2激活的下游信号通路；MCF-10A细胞系被包括在特定的AIMS 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的机制；(4)确定饮食WA对使用MMTV-neu小鼠预防乳腺癌形成的有效性；以及(5)使用来自对照和WA处理的MMTV-neu小鼠的乳腺肿瘤组织，确定饮食WA预防MMTV-neu小鼠乳腺癌发展的机制。拟议研究的意义：拟议的临床前体内疗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。明确水杨酸抗癌作用机制的价值可以通过多种途径实现，包括寻找水杨酸反应的生物标记物(S)，在未来的临床试验中可能有用，以及优化水杨酸为基础的乳腺癌化学预防方案。

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 This preclinical research project stems from our published and preliminary unpublished results demonstrating that WA treatment decreases viability of cultured breast cancer cells by causing apoptosis and the proapoptotic effect of WA correlates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.