Publication Date

2015-07-24

Availability

Embargoed

Embargo Period

2017-07-23

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2015-05-29

First Committee Member

Bal L. Lokeshwar

Second Committee Member

Silvina Levis-Dusseau

Third Committee Member

Ralf Landgraf

Fourth Committee Member

Samita Andreansky

Fifth Committee Member

Zhibin Chen

Abstract

Plant-derived dietary compounds have shown potential as chemopreventive and anticancer agents to prolong survival and reduce breast cancer deaths. Lack of specific mechanism of action is the leading concern for adopting these edible compounds as chemopreventive or adjuvant therapeutics against breast and other cancers. The unripe berries of Pimenta Dioica, also called Allspice, are widely used in cuisines worldwide, contain a cornucopia of bioactive compounds with potential therapeutic properties. Limited studies on the potential anticancer compounds present in Allspice prompted us to hypothesize and test potential antiproliferative agents in an aqueous extract of Allspice (AAE). The potential anti-tumor activity of an Aqueous Allspice Extract (AAE) was tested on Breast Cancer cells. AAE reduced the viability and clonogenic growth of BrCa cells (IC50≈100μg/ml) but had limited toxicity in non-tumorigenic, quiescent cells (IC50 >200μg/ml). AAE induced antiproliferative activity was inconsistent with cell cycle arrest, apoptosis or necrosis, but was strongly associated with characteristics of autophagy. The characteristics included high vacuolation, increase in levels of autophagy marker protein LC3 and showed LC3 positive puncta. Silencing the autophagy related gene (ATG) expression in both estrogen receptor+ (ER+) and ER- cells by siRNA prevented AAE-induced cell death. In an ERα+ cell line MCF-7, ATG7 silencing abrogated AAE cytotoxicity by 80% and in the ERα- MDA-MB231 cells, 50% cell death was rescued. Inhibiting ATG5 gene yielded similar yet less pronouced results. Further, AAE down-regulated the Akt/mTOR signaling, which potentially led to autophagy induction. AAE showed enhanced cytotoxicity in BrCa cells when combined with an mTOR inhibitor, rapamycin. AAE also exhibited anti-metastatic activities against MDA-MB231 cells where it decreased invasive potential and caused decrease in mRNA levels of EMT markers N-cadherin, Vimentin, Slug. AAE was also found to significantly decrease Estrogen Receptor α (ERα) protein level in MCF7 and T47D cells, through both proteasome-mediated degradation and transcriptional down-regulation mechanisms. Oral administration of AAE showed anti-tumor activity in vivo on MB231 xenografts in athymic mice. Tumor growth rate was reduced by 17% in gavaged mice. Further, tumor growth latency delay in palpable tumor growth was significantly increased in mice pre-treated with AAE for two weeks (38%) and in those animals injected with MB231 cells pre-incubated with low concentration of AAE for 2 days. These results demonstrate antitumor and chemopreventive potential of AAE against breast cancer.

Keywords

Allspice; autophagy; breast cancer; chemoprevention; mTOR signaling

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