An efficient method for the α-methylenation of carbonyl groups is reported, and this transformation is accomplished by a facile elimination of trifluoroacetate during the formation of the olefin. This method represents an improvement beyond existing protocol in cases of steric hindrance, and we have demonstrated the utility of the process across a series of ketones, lactams, and lactones. Additionally, we have applied this method to produce semisynthetic derivatives of the natural products (+)-sclareolide and (-)-eburnamonine, in which the carbonyl group is proximal to bulky functional groups. Mechanistic insight is also provided from a time course of (19)F NMR. Biological evaluation of the natural-product-derived enones led to the identification of a derivative of (-)-eburnamonine with significant cytotoxicity (LC(50) = 14.12 μM) in drug-resistant MDA-MB-231 breast cancer cells.
Multiple myeloma (MM) is an incurable bone marrow malignancy of the B cell lineage. Utilizing multiplex Luminex technology we measured levels of 25 cytokines in the plasma of normal donors (n = 177), those with monoclonal gammopathy of undetermined significance (n = 8), and MM patients (n = 55) with either active disease, on treatment, or in remission. The cytokine levels were compared between normal donors and MM patients as well as between various phases of MM, and discriminant analysis was used to create a predictive classification model based on the differentially expressed cytokines. Evaluating age- and gender-dependence of cytokine expression, we determined that with age there is a shift toward a pro-inflammatory environment. Moreover, we observed a strong gender bias in cytokine expression. However, the profile of differentially expressed cytokines was heavily skewed toward an anti-inflammatory, pro-tumorigenic response in patients with MM. Significantly, our predictive model placed all patients in remission in the same category as those with active disease. Thus, our study demonstrates that the homeostasis of systemic cytokines is not restored when MM patients enter remission, suggesting that once an individual has cancer, the microenvironment is permanently altered and the system is primed for a relapse.
The biological role of installing a critical exocyclic enone into the structure of the alkaloid, (−)-eburnamonine, and characterization of the new chemical reactivity by quantitative NMR without using deuterated solvents are described. This selective modification to a natural product imparts potent anticancer activity as well as bestows chemical reactivity toward nucleophilic thiols, which was measured by quantitative NMR. The synthetic strategy provides an overall conversion of 40%. In the key synthetic step, a modified Peterson olefination was accomplished through the facile release of trifluoroacetate to create the requisite enone in the presence of substantial steric hindrance.
Recent studies suggest that leukemia stem cells (LSCs) play a critical role in initiation, propagation, and relapse of leukemia. Herein we show that (−)-15-methylene-eburnamonine, a derivative of the alkaloid, (−)-eburnamonine, is cytotoxic against acute and chronic lymphocytic leukemias (ALL and CLL) and acute myelogenous leukemia (AML). The agent also reduces primary LSC frequency in vitro. The cytotoxic effects appear to be mediated via the oxidative stress pathways. Furthermore, we show that the compound kills AML, ALL, and CLL stem cells, and using a novel humanized bone marrow murine model of leukemia (huBM/NSG), it reduces progenitor cell engraftment.
BACKGROUND: While most studies investigate the biology of the active disease, the focus of our study was on the state of clinical remission, during which patients are considered healthy and tumor free. We demonstrate that the ‘disease-promoting latency’ factor(s) are sustained post therapy and likely contribute to tumor re-growth and drug-resistance that accompanies the relapse. We propose that therapies aimed at shrinking the tumor do not restore the homeostasis within the tissue; thus, tumor-associated stroma remains post therapy sustaining the production of pro-tumorigenic factors that ultimately induce a relapse. Multiple myeloma (MM) is an incurable bone marrow malignancy of B cell lineage that accounts for 20% of deaths from hematologic malignancies. Despite the development of potent new regimens, nearly all MM patients relapse and become refractory to treatment; thus the median survival rate remains 3-5 years. The goal of this study was to determine whether the balance of the systemic cytokines returns to normal during complete remission and whether we can identify a cytokine signature specific for various phases of the disease. METHODS: Utilizing multiplex technology of Luminex we measured the plasma levels of 25 cytokines in normal donors (n=177) and MM patients (n=54) either at diagnosis/relapse, treatment, or remission phases of MM. The cytokine levels were compared between normal donors and MM patients as well as between various phases of the MM, and discriminant analysis was used to create predictive classification of disease phase based on the levels of differentially expressed cytokines. RESULTS: Based on fold-change analysis, we identified 15 cytokines that were differentially expressed between normal donors and MM patients, whose profile was heavily skewed toward a pro-tumorigenic Th2 response. Moreover, chemokines (IL-8, eotaxin, MCP1) were also upregulated in plasma of patients. Significantly, the expression of cytokines in patients in clinical remission was maintained at the same levels as in patients with active disease and our predictive model placed all patients in remission in the same category as those at diagnosis/relapse. CONCLUSIONS: Our study demonstrates that remission is not a state of health, but tumor latency, and that once an organism has cancer, the system is primed for relapse and emergence of secondary neoplasms. Thus, the cytokines maintained in circulation will induce proliferation and confer drug resistance of the minimal residual disease, and chemokines will stimulate systemic dissemination of tumor cells leading to the colonization of secondary sites. We believe that such systemic alteration of tissue homeostasis is consistent for all cancers; thus our findings suggest the need to shift the paradigm of clinical intervention to include homeostasis-stabilizing interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-499. doi:1538-7445.AM2012-LB-499
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.
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