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 This preclinical research project stems from our published and preliminary unpublished results demonstrating that WA treatment decreases viability of cultured breast cancer cells by causing apoptosis and the proapoptotic effect of WA correlates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

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA142604-05
Application #
8590113
Study Section
Chemo/Dietary Prevention Study Section (CDP)
Program Officer
Seifried, Harold E
Project Start
2009-12-04
Project End
2014-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
5
Fiscal Year
2014
Total Cost
$274,439
Indirect Cost
$93,291
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Hahm, Eun-Ryeong; Lee, Joomin; Singh, Shivendra V (2014) Role of mitogen-activated protein kinases and Mcl-1 in apoptosis induction by withaferin A in human breast cancer cells. Mol Carcinog 53:907-16
Kim, Su-Hyeong; Singh, Shivendra V (2014) Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells. Cancer Prev Res (Phila) 7:738-47
Antony, Marie L; Lee, Joomin; Hahm, Eun-Ryeong et al. (2014) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ?-tubulin. J Biol Chem 289:1852-65
Nagalingam, Arumugam; Kuppusamy, Panjamurthy; Singh, Shivendra V et al. (2014) Mechanistic elucidation of the antitumor properties of withaferin a in breast cancer. Cancer Res 74:2617-29
Sehrawat, Anuradha; Sakao, Kozue; Singh, Shivendra V (2014) Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells. Breast Cancer Res Treat 146:543-55
Vyas, Avani R; Singh, Shivendra V (2014) Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone. AAPS J 16:1-10
Hahm, Eun-Ryeong; Singh, Shivendra V (2013) Autophagy fails to alter withaferin A-mediated lethality in human breast cancer cells. Curr Cancer Drug Targets 13:640-50
Hahm, Eun-Ryeong; Lee, Joomin; Kim, Su-Hyeong et al. (2013) Metabolic alterations in mammary cancer prevention by withaferin A in a clinically relevant mouse model. J Natl Cancer Inst 105:1111-22
Hahm, Eun-Ryeong; Moura, Michelle B; Kelley, Eric E et al. (2011) Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species. PLoS One 6:e23354
Hahm, Eun-Ryeong; Lee, Joomin; Huang, Yi et al. (2011) Withaferin a suppresses estrogen receptor-? expression in human breast cancer cells. Mol Carcinog 50:614-24

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