Abstract B30: 15-Methylene-Eburnamonine: A preventative agent against breast to brain metastases by targeting circulating tumor cells and cancer stem cells through induction of oxidative stress

Abstract

One in eight women in the United States will develop invasive breast cancer during their lifetime. Of these women, a significant portion (10-16%) will develop metastases to the brain, resulting in significant morbidity and poor prognosis. Symptoms of brain metastasis are devastating and include dizziness, nausea, vertigo, headaches, impaired vision and cognitive functions. Brain metastases arise from highly aggressive triple negative (estrogen, progesterone, and Her2 receptor negative) or Her2 positive (estrogen and progesterone receptor negative, and Her2 positive) mammary tumors and often afflict young, premenopausal women. Less than 2% of women with such lesions survive past 2 years. Since there are currently no effective therapies against brain metastases, the best approach is to prevent their formation by targeting circulating tumor cells before they engraft at distant sites. It has been suggested that cancer stem cells (CSCs) contribute to the metastatic spread in many solid tumors, including those of the breast. A successful preventative agent must kill circulating tumor cells and CSCs before they have a chance to colonize a secondary site while having minimal toxicity against normal tissue. Eburnamonine (EBN) is a pharmacologically active compound derived from the flowering plant Periwinkle with vasoregulatory and anti-hypoxic properties. It has been found to cross the blood-brain-barrier and has been used as a treatment for a number of cerebrovascular disorders, including ischemia and anoxia. EBN has no known neurotoxicity, in fact it has been found to be neuroprotective. A derivative of EBN, 15-methylene-(-)-eburnamonine (15-M-EBN), with increased cytotoxity against cancer cells has been synthesized by our group. We found that EBN is cytotoxic against triple negative human breast cancer cells, MDA-MB-231 (LC50=42.0 μM). Our derivative, 15-M-EBN, is even more effective against triple negative human breast cancer cells, MDA-MB-231 (LC50=14.0 μM), as well as against triple negative breast to brain metastatic cells, MDA-MB-231BR (LC50=26.4 μM); and Her2 positive breast to brain metastatic cells, MDA-MB-231BR-Her2 (LC50=27.2 μM). Mammosphere assay indicated that 15-M-EBN kills CSCs found in MDA-MB-231, triple negative breast cancer cells (LC50=10.3 μM), as well as those found in triple negative breast to brain metastatic cells, MDA-MB-231BR, (LC50=26.2 μM) and Her2 positive breast to brain metastatic cells, MDA-MB-231BR-Her2 (LC50=19.8 μM). Also, by using 3D cultures and coatings of various extracellular matrix component proteins, we established that 15-M-EBN is active under the conditions of environmental-mediated drug-resistance. Staining with Annexin V showed that 15-M-EBN induces apoptosis that we demonstrate to be mediated through the activation of caspases 3, 6, and 7 and cleavage of cytokeratin 18. Addition of reducing agents (N-Acetyl-L-cysteine, glutathione, or DTT) to 15-M-EBN treated cultures blocked its cytotoxic effects, demonstrating that 15-M-EBN acts through the induction of oxidative stress. Furthermore, to establish that the methylene group on the fifteenth carbon in the structure of 15-M-EBN is the reactive site, our group synthesized a compound with a methyl group in place of the methylene (15-methyl-EBN). It was tested in cultures of breast cancer cells and showed that replacing the methylene with the methyl group abrogates the cytotoxic effects of this compound. Taken together our data show that 15-M-EBN kills triple negative and Her2 positive breast cancer cells and CSCs derived from these tumors through an induction of oxidative stress, overcomes the pro-tumorigenic effects of the tumor microenvironment while exhibiting minimal toxicity against normal tissue. These findings suggest that 15-M-EBN has a high potential to become a preventative agent against breast to brain metastases. Citation Format: Mary Minyi Zheng, Britney Harris, James Woods, David Colby, Julia Kirshner. 15-Methylene-Eburnamonine: A preventative agent against breast to brain metastases by targeting circulating tumor cells and cancer stem cells through induction of oxidative stress. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B30.