Abstract P3-03-03: Phenolic compounds from pimenta dioica berries (allspice) inhibit breast cancer cell proliferation by autophagy induction

Abstract

Abstract Background and methods:Recent studies have shown that plant-derived dietary compounds have potential as chemopreventive 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).An aqueous extract of Certified-organic Allspice (AAE) was prepared and tested on several BrCa cell lines for potential antitumor activities by techniques as western blotting, clonogenic survival, ELISA, flow cytometry, quantitative-RT- PCR, promoter reporter assay and microscopy. Results: AAE reduced the viability and clonogenic growth of BrCa cells (IC50∼100μg/ml) with significantly reduced cytotoxicity against 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, including high vacuolation, increase in levels of GFP-LC3-positive puncta, and LC3-II protein levels. Silencing the ATG gene expression in both ER+ and ER- cells by siRNA prevented AAE-induced cell death. In ERα+ cell line MCF-7, ATG7 silencing abrogated AAE cytotoxicity by 80% and in ERα- MDA-MB231 cells, 50% cell death was rescued. Inhibiting ATG5 gene yielded similar yet less pronouced results. Autophagy was induced by Endoplasmic reticulum (ER) stress, confirmed by increase in ER stress marker GRP78, but no change in ATP levels. Further, AAE caused inhibition of mTOR signaling and showed enhanced cytotoxicity when combined with an mTOR inhibitor, rapamycin. AAE reduced ERα protein as well ERα-mRNA levels in MCF-7 and T47D cells. Incubation with AAE (100μg/ml) caused 50% decrease in ERα protein level while its mRNA decreased by 25%. Furthermore, AAE induced down regulation of ERα was partially rescued by co-incubation with the 26S-proteasome inhibitor MG132. AAE also exhibited anti-metastatic activities against MDA-MB231 cells where it decreased invasive potential and caused decreases in mRNA levels of EMT markers N-cadherin, vimentin, slug and increase in Twist. Activity based purification combined with chemical identification strategies resulted in identification of several novel candidate compounds with characteristics of polyphenols. In conclusion, we determined the anti-cancer properties of an aqueous extract of Allspice on BrCa cells, which principally affects cell survival by inducing cell death by autophagy and ERα down regulation. It reduced EMT in metastatic BrCa cells thus enhancing the clinical potential of AAE as an adjuvant for BrCa treatment or dietary intervention. [Grant Support: Sheila and David Fuente Cancer Biology program of Sylvester Cancer Center (LZ) and PHS Grant No. R01 CA156776 (BLL)]. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-03.