Abstract 3344: CAPE-induced inhibition of breast cancer stem cells (CSC) self-renewal and growth by differentiation to a less malignant phenotype
Authors: Omene Coral O. , Wu Jing , Qu Song , Smith Julia , Frenkel Krystyna 
Published: June 2012

Abstract
Introduction: Cancer stem cells (CSC) are implicated in tumor metastasis, recurrence, and high patient mortality, thus, 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, a honeybee product, could be such an agent. CSC are known for their chemoresistance. We previously showed that CAPE inhibits growth of MDA-231 (231) cells, a model of human triple-negative breast cancer, multidrug resistance gene expression, NF-κB, EGFR, and VEGF. We hypothesized that CAPE could also affect CSC-mediated effects.
Methods: We isolated CSC from 231 cells and mouse xenografts, and propagated them in low adherence tissue culture plates without serum. CSC characteristics, ie Hoechst 33342 dye exclusion and CD44+/CD24-/low/EpCAMlow phenotype, were assessed by flow cytometry, while clonal growth was assayed in soft agar. CSC were treated with CAPE (0-40 μM) for 4 or 7 days. Apoptosis was assessed by Annexin V/propidium iodide staining. Cell cycle analysis was done according to standard protocol and analysis by ModFit software.
Results: 231-derived CSC grow as mammospheres (MMS), and when dissociated into single cells, form MMS 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 growth of CSC-derived tumor cells.
Treatment of CSC with CAPE for 4.5 days decreased CD44 levels by ∼95%, while another cell population containing 10-100-fold lower CD44 content concurrently increased. Incubation of CSC with different CAPE doses for 4 days caused dose-dependent cell growth inhibition. 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, but apoptosis was not induced as determined by Annexin V/PI assay. Instead, CSC were noted to significantly progress from a quiescent cell cycle state in G0/G1 (82%), S phase (12%) to a cycling state with an increase in S phase (41%) and subsequent decrease in G0/G1 (54%).
Conclusions: These results suggest that CAPE causes pronounced changes in CSC characteristics manifested by inhibition of clonal growth in soft agar and concurrent significant decrease in CD44 content, a sign of decreased potential of malignancy. This conclusion is strengthened by the observation that the CSC are induced into a more differentiated state that is less malignant after CAPE treatment.
Our results strongly suggest that CAPE could be an inhibitor of CSC making them more susceptible to chemotherapy. [grants BCTR0600476 & ES00260]
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3344. doi:10.1158/1538-7445.AM2011-3344