Cell death plays a central role in normal physiology and in disease. Common to apoptotic and necrotic cell death is the eventual loss of plasma membrane integrity. We have produced a small organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, that rapidly accumulates in the cytosol of dying cells coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90 kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was noninvasively imaged by SPECT/CT using an (111)In-tagged compound. This versatile compound should enable the imaging of cell death in most experimental settings.

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Introduction: MsDef1, a 45-amino acid cysteine-rich peptide from the seed of Medicago sativa is an antifungal defensin small protein. It exhibits broad-spectrum antifungal activity against fungal pathogens of plants at low micromolar concentrations. The common vulnerability of fungal and cancer cells determines the utility of MsDef1 as a potential anti-tumor agent. Results: The solution dynamics of 15N-labeled MsDef1, 15N longitudinal relaxation (T1) and 15N-1H Nuclear Overhauser Effect (NOE) shows that GlcCer binds at two sites on the peptide molecule, i.e., Asp36-Cys39 and amino acids between 12-20 and 33-40. MsDef1 interacts with drug resistant breast cancer MCF-7R cells, permeates GlcCer-rich plasma membrane and releases apoptotic ceramide. This results in the activation of ceramide pathway involving interaction of the peptide with intracellular thioredoxin (Trx), another tumor specific biomarker. MsDef1 oxidizes Trx through four S-S bonds and in the process, gets reduced to thiols. Oxidation of Trx is correlated with the activation of Apoptosis Stimulating Kinase 1 (ASK1) which is known to sensitize cancer cells to chemotherapeutics including front-line drug Doxorubicin. A combination of MsDef1 and Doxorubicin exhibits 5-10-fold greater apoptosis in vitro in MDR triple negative breast cancer (TNBC) cells compared to either MsDef1 or Doxorubicin alone. Conclusion: An antifungal plant defensin MsDef1 is shown to be a cell permeating peptide (CPP) for MDR cancer cells targeted to two tumor specific targets activating two cell death pathways. That makes MsDef1, potentially a tumor targeted sensitizer neoadjuvant to cancer therapy.

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Cell death plays an integral role in physiology, including turnover of cells in the gastrointestinal tract, the menstrual cycle and the immune system. Imbalance of this process is also associated with disease. Excessive cell death is characteristic of vascular disorders, neurodegenerative diseases, myelodysplastic syndromes, ischemia/reperfusion injury and organ transplant rejection, among others. Cell death also plays a role in the treatment of disease. In cancer for example, most chemotherapeutics, radiation treatments and anti-hormonal agents act by inducing death of cancer cells. Given the prevalence of cell death in normal physiology and disease, non-invasive imaging of this process is likely to have wide application in biological research and ultimately in patient diagnosis and management. Common to apoptotic and necrotic death cell is eventual loss of plasma membrane integrity. We have developed a small, synthetic organoarsenical compound (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) that rapidly accumulates in the cytosol of dying cells, coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90-kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was non-invasively imaged by SPECT/CT using an 111In-tagged compound. This versatile compound has the advantage of chemical stability, exquisite selectivity for dying cells and versatility with respect to the reporting group. The nature and abundance of its cytosolic target, Hsp90, also bodes well for its suitability for the imaging of cell death in most experimental settings. 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 5287. doi:10.1158/1538-7445.AM2011-5287