Andrew J. Norris

Ionizing Radiation Activates the Nrf2 Antioxidant Response

The transcription factor NF-E2-related factor 2 (Nrf2) binds the antioxidant DNA response element (ARE) to activate important cellular cytoprotective defense systems. Recently several types of cancers have been shown to overexpress Nrf2, but its role in the cellular response to radiation therapy has yet to be fully determined. In this study, we report that single doses of ionizing radiation from 2 to 8 Gy activate ARE-dependent transcription in breast cancer cells in a dose-dependent manner, but only after a delay of five days. Clinically relevant daily dose fractions of radiation also increased ARE-dependent transcription, but again only after five days. Downstream activation of Nrf2-ARE-dependent gene and protein markers, such as heme oxygenase-1, occurred, whereas Nrf2-deficient fibroblasts were incapable of these responses. Compared with wild-type fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of reactive oxygen species that increased greatly five days after radiation exposure. Further, in vitro clonogenic survival assays and in vivo sublethal whole body irradiation tests showed that Nrf2 deletion increased radiation sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our results indicate that the Nrf2-ARE pathway is important to maintain resistance to irradiation, but that it operates as a second-tier antioxidant adaptive response system activated by radiation only under specific circumstances, including those that may be highly relevant to tumor response during standard clinical dose-fractionated radiation therapy.

Nitroxyl inhibits breast tumor growth and angiogenesis

Nitroxyl (HNO) can inhibit the glycolytic enzyme glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Because of the importance of glycolysis in many malignant cells, we thus propose that HNO can adversely affect tumor growth. This hypothesis was tested using in vitro and in vivo models of breast cancer. We report here for the first time that HNO suppresses the proliferation of both estrogen receptor (ER)‐positive and ER‐negative human breast cancer cell lines, in a dose dependent manner. Mice treated with HNO either injected into the tumor itself or via the intraperitoneal approach had smaller xenograft tumor size. In addition to significantly decreased blood vessel density in the HNO‐treated tumors, we observed lower levels of circulating serum vascular endothelial growth factor (VEGF). Accordingly, there was a decrease in total HIF‐1α (hypoxia‐inducible factor) protein in HNO‐treated tumor cells. Further studies showed inhibition of GAPDH activity in HNO‐treated human breast cancer cell lines and in HNO‐treated tumor tissue derived from xenografts. One explanation for the multiplicity of actions observed after HNO treatment could be the effect from the initial inhibition of GAPDH, providing a potential therapeutic avenue based upon blocking glycolysis resulting in decreased HIF‐1α, thus leading to angiogenesis inhibition. Therefore, HNO appears to act via mechanism(s) different from those of existing breast cancer drugs, making it a potential candidate to overcome known and emerging drug resistance pathways.

High-Throughput Screening Identifies Two Classes of Antibiotics as Radioprotectors: Tetracyclines and Fluoroquinolones

Purpose: Discovery of agents that protect or mitigate normal tissue from radiation injury during radiotherapy, accidents, or terrorist attacks is of importance. Specifically, bone marrow insufficiency, with possible infection due to immunosuppression, can occur after total body irradiation (TBI) or regional irradiation and is a major component of the acute radiation syndrome. The purpose of this study was to identify novel radioprotectors and mitigators of the hematopoietic system. Experimental Design: High-throughput screening of small-molecule libraries was done using viability of a murine lymphocyte line as a readout with further validation in human lymphoblastoid cells. The selected compounds were then tested for their ability to counter TBI lethality in mice. Results: All of two major classes of antibiotics, tetracyclines and fluoroquinolones, which share a common planar ring moiety, were radioprotective. Furthermore, tetracycline protected murine hematopoietic stem/progenitor cell populations from radiation damage and allowed 87.5% of mice to survive when given before and 35% when given 24 h after lethal TBI. Interestingly, tetracycline did not alter the radiosensitivity of Lewis lung cancer cells. Tetracycline and ciprofloxacine also protected human lymphoblastoid cells, reducing radiation-induced DNA double-strand breaks by 33% and 21%, respectively. The effects of these agents on radiation lethality are not due to the classic mechanism of free radical scavenging but potentially through activation of the Tip60 histone acetyltransferase and altered chromatin structure. Conclusions: Tetracyclines and fluoroquinolones can be robust radioprotectors and mitigators of the hematopoietic system with potential utility in anticancer radiotherapy and radiation emergencies. (Clin Cancer Res 2009;15(23):7238–45)

High throughput screening of small molecule libraries for modifiers of radiation responses.

Purpose: An unbiased approach of drug discovery through high-throughput screening (HTS) of libraries of chemically defined and bioactive small molecule compounds was used to identify modulators of radiation injury with an emphasis on radioprotectors and mitigators rather than radiosensitisers. Assay system endpoints included radiation-induced genotoxicity and DNA damage in yeast and apoptosis in murine lymphocytes. Large-scale data mining of chemically diverse libraries identified agents that were effective with all endpoints. HTS of bioactive compound libraries against murine lymphocytes profiled tetracycline and fluoroquinolone antibiotics and cyclopiazonic acid as having activity, and structure-activity analysis showed a common pharmacophore. Purine nucleosides, the interferon inducer tilorone, and linoleic acid were also identified as potential mitigators of radiation damage that often were also radioprotective. Many of these compounds enhance DNA repair, have anti-inflammatory activity, and stimulate hematopoiesis. Selected compounds within these initial verified hits from both types of libraries identified potent mitigators of lethal whole body irradiation (WBI) in mice. Conclusion: In spite of the fact that in vitro HTS has limitations and is unable to fully recapitulate all aspects of the complex in vivo acute radiation response, it identified several classes of molecules that had activity as radioprotectors and radiomitigators of the hematopoietic system in vivo. In the future, addition of 3-dimensional (3-D) or stem cell cultures or pathway analysis, may improve the power of HTS, but our findings indicate that common, evolutionary conserved, canonical pathways can be identified that could be exploited to mitigate radiation-induced defects.

The Novel Gene EG-1 Stimulates Cellular Proliferation

We recently discovered a novel gene and named it endothelial-derived gene 1 (EG-1). Previously, we have shown that the expression of EG-1 is significantly elevated in the epithelial cells of breast cancer, colorectal cancer, and prostate cancer. Here, we report that EG-1 can stimulate cellular proliferation. Transfection experiments which overexpressed the full-length EG-1 gene in human embryonic kidney HEK-293 cells or human breast cancer cell lines resulted in significantly increased in vitro proliferation, in comparison with transfection with empty vectors. On the other hand, small interfering RNA cotransfection resulted in inhibition of proliferation. S.c. xenograft assays were carried out in a severe combined immunodeficient mouse model. We found that injection of high EG-1 expressing HEK-293 clones resulted in significantly larger tumors, in comparison with clones carrying the empty vectors. To further clarify the function of this gene, we investigated its interaction with Src and members of the mitogen-activated protein kinase (MAPK) family. Immunoprecipitation with anti-Src antibody, followed by immunoblotting with anti-EG-1 antibody, showed an association between these two molecules. Overexpression of EG-1 was correlated with activation of the following kinases: extracellular signal-regulated kinases 1 and 2, c-jun-NH2-kinase, and p38. These observations collectively support the hypothesis that the novel gene EG-1 is a positive stimulator of cellular proliferation, and may possibly be involved in signaling pathways involving Src and MAPK activation.

Targeted inhibition of EG-1 blocks breast tumor growth.

EG-1 is a gene product that is significantly elevated in human breast cancer tissues. Previously, we have shown that EG-1 overexpression stimulates cellular proliferation both in vitro and in vivo. Here, we ask whether this molecule can be targeted for experimental therapeutic purpose. siRNA lentivirus and polyclonal antibody were designed to suppress EG-1 expression. These agents were then used in cell culture proliferation assays and breast tumor xenograft models. Serum and urine from breast cancer patients were also analyzed for the presence of EG-1 peptide. We report here for the first time that endogenous EG-1 can be targeted to inhibit breast tumor growth. This inhibition, whether delivered via siRNA lentivirus or polyclonal antibody, resulted in decreased cellular proliferation in culture and smaller xenografts in mice. The effects were shown in both ER (estrogen receptor)-positive human breast cancer MCF-7 cells, as well as in ER-negative MDA-MB-231 cells. Furthermore, we detected soluble EG-1 in serum and urine of breast cancer patients. These observations demonstrate that EG-1 is relevant to human breast cancer, and is a molecular target worthy of translational efforts into effective breast cancer therapy.

A Concise and Stereoselective Synthesis of C-Glycosyl Analogues of β-L-Fucopyranosyl Phosphate and β-L-Rhamnopyranosyl Phosphate

ABSTRACT The isosteric C-glycosyl analogues of β-L-fucopyranosyl phosphate 1 and β-L-rhamnopyranosyl phosphate 2 have been stereoselectively synthesized from the corresponding glycono-1,5-lactones 3 and 10 via methylphosphonylation, dehydration and catalytic hydrogenation, followed by deprotection.

Chapter 1 An Introduction to the Basic Principles and Concepts of Mass Spectrometry

Publisher Summary This chapter introduces the basic principles and concepts of mass spectrometry (MS). If a molecule can be converted into a gas phase ion, it can be interrogated by this technique. Making possible the study of proteins by MS required the development of methods to convert them into the gas phase ions and of techniques to separate the ions and detect them. The principles of these processes are discussed in the chapter. Amino acids, peptides, and proteins have several unique characteristics that make them particularly suitable to MS analysis. First, their ionizable functionalities render these molecules excellent candidates for electrospray ionization (ESI) and laser desorption (LD) ionization. Second, with the exception of isoleucine and leucine, the unique masses of 18 of the 20 common protein amino acids allow for identification on the basis of their mass alone. Third, the universal amide bond that links the amino acids means that the characterization of polymers of amino acids is not confounded with complications that arise from linkage heterogeneity, as is the case with carbohydrate characterization.

4-(Nitrophenylsulfonyl)piperazines mitigate radiation damage to multiple tissues

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.