Abstract 3555: Inhibition of breast cancer stem cells (CSC) self-renewal and growth by CAPE, a product of propolis

Abstract

Abstract Introduction. Cancer stem cells (CSC) are implicated in tumor metastasis, recurrence, and high patient’ mortality, therefore, substances impairing their activity, e.g., self-renewal, could be invaluable as novel cancer therapeutics. We propose that CAPE (caffeic acid phenethyl ester), a component of propolis produced by honeybees, could be such a novel anti-CSC agent. We previously showed that CAPE, which is innocuous to normal human mammary epithelial cells, inhibits growth of MDA-MB-231 (231) cells, multidrug resistance gene expression, NF-kB transcription factors, EGFR, its phosphorylation, and VEGF formation. These cells are often used as a model of human triple-negative breast cancer, hence, we hypothesized that CAPE could also affect CSC-mediated effects. Methods. We isolated CSC from 231 cells and from nude mouse xenografts induced by 231 cell inoculation, and propagated them in low adherence tissue culture plates in the absence of serum. CSC characteristics, e.g., exclusion of Hoechst 33342 dye, a measure of drug resistance, and the presence of CD44+/CD24−/low/EpCAMlow phenotype, were assessed by flow cytometry, while clonal growth was assayed in soft agar. Results. CSC were grown as mammospheres and, when dissociated into single cells, formed mammospheres again. This process was repeated for many generations and is considered a sign of self-renewal. These cells excluded Hoechst 33342 dye, exhibited characteristic CD44+/CD24−/low/EpCAMlow phenotype, and generated progenitors in presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE inhibited CSC self-renewal, progenitors’ generation, and suppressed growth of CSC-derived tumor cells. When mammosphere-derived single cells were treated with CAPE for 4.5 days, CD44 levels were decreased by ∼95%, while another cell population containing lower CD44 content concurrently increased by ∼10-fold. This decrease in CD44 levels is probably responsible for the following results. An incubation of CSC (as single cells) with different CAPE doses (0-40 microM, one treatment only) for 4 days caused cell growth inhibition in a dose-dependent fashion. After pre-treating cells with CAPE, the same numbers of live cells were taken from each well and plated on soft agar. Clonal growth was also inhibited in a dose-dependent fashion depending on CAPE dose used for the pre-treatment. Conclusions. These results suggest that CAPE causes pronounced changes in CSC characteristics manifested by inhibition of clonal growth in soft agar, a sign of decreased potential of malignancy, and this conclusion is strengthened by the concurrent decrease in CD44 content. Cumulatively, our results strongly suggest that CAPE could be a powerful inhibitor of cancer stem cell growth and functions that are responsible for CSC-mediated recurrence of breast cancer and increased patient mortality. [This work was supported in part by grants BCTR0600476 & ES00260] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3555.