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INTRODUCTION Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However,one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line,MCF-7:5C,which undergoes apoptosis in the presence of estradiol. In contrast,another estrogen deprived cell line,MCF-7:2A,appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study,we evaluated whether buthionine sulfoximine (BSO),a potent inhibitor of glutathione (GSH) synthesis,is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis. METHODS Estrogen deprived MCF-7:2A cells were treated with 1 nM 17beta-estradiol (E2),100 microM BSO,or 1 nM E2 + 100 microM BSO combination in vitro,and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model. RESULTS Exposure of MCF-7:2A cells to 1 nM E2 plus 100 microM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E2 plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun N-terminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E2 plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E2 significantly reduced tumor growth of MCF-7:2A cells. CONCLUSIONS Our data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E2-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer. View Publication

Excessive receptor activator of NF-kappaB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. Thus,down-regulation of RANKL expression or its downstream signals may be a therapeutic approach to the treatment of pathological bone loss. In this study,we investigated the effects of Trolox,a water-soluble vitamin E analogue,on osteoclastogenesis and RANKL signaling. Trolox potently inhibited interleukin-1-induced osteoclast formation in bone marrow cell-osteoblast coculture by abrogating RANKL induction in osteoblasts. This RANKL reduction was attributed to the reduced production of prostaglandin E(2) via a down-regulation of cyclooxygenase-2 activity. We also found that Trolox inhibited osteoclast formation from bone marrow macrophages induced by macrophage colony-stimulating factor plus RANKL in a reversible manner. Trolox was effective only when present during the early stage of culture,which implies that it targets early osteoclast precursors. Pretreatment with Trolox did not affect RANKL-induced early signaling pathways,including MAPKs,NF-kappaB,and Akt. We found that Trolox down-regulated the induction by RANKL of c-Fos protein by suppressing its translation. Ectopic overexpression of c-Fos rescued the inhibition of osteoclastogenesis by Trolox in bone marrow macrophages. Trolox also suppressed interleukin-1-induced osteoclast formation and bone loss in mouse calvarial bone. Taken together,our findings indicate that Trolox prevents osteoclast formation and bone loss by inhibiting both RANKL induction in osteoblasts and c-Fos expression in osteoclast precursors. View Publication

Introducing a reactive carbonyl to a scaffold that does not otherwise have an electrophilic functionality to create a reversible covalent inhibitor is a potentially useful strategy for enhancing compound potency. However,aldehydes are metabolically unstable,which precludes the use of this strategy for compounds to be tested in animal models or in human clinical studies. To overcome this limitation,we designed ketone-based functionalities capable of forming reversible covalent adducts,while displaying high metabolic stability,and imparting improved water solubility to their pendant scaffold. We tested this strategy on the ferroptosis inducer and experimental therapeutic erastin,and observed substantial increases in compound potency. In particular,a new carbonyl erastin analog,termed IKE,displayed improved potency,solubility and metabolic stability,thus representing an ideal candidate for future in vivo cancer therapeutic applications. View Publication

Two groups recently reported the in vitro differentiation of human embryonic stem cells into insulin-secreting cells,achieving an elusive goal for regenerative medicine. Herein we provide a perspective regarding these developments,compare phenotypes of the insulin-containing cells to human $\beta$ cells,and discuss implications for type 1 diabetes research and clinical care. View Publication

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation,injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate,Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence,we hypothesized that CDDO-Me would reduce lung inflammation,fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis,mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation,while fibrosis and lung function were evaluated on day 21. On day 7,CDDO-Me reduced total BALF protein by 50{\%},alveolar macrophage infiltration by 40{\%},neutrophil infiltration by 90{\%} (p≤0.01),inhibited production of the inflammatory cytokines KC and IL-6 by over 90{\%} (p≤0.001),and excess production of the pro-fibrotic cytokine TGF$\beta$ by 50{\%}. CDDO-Me also inhibited $\alpha$-smooth muscle actin and fibronectin mRNA by 50{\%} (p≤0.05). On day 21,CDDO-Me treatment reduced histological fibrosis,collagen deposition and $\alpha$SMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me,as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases. View Publication

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines,with inhibition of CDC7 resulting in cell cycle arrest. In this paper,we disclose the discovery of a potent and selective CDC7 inhibitor,XL413 (14),which was advanced into Phase 1 clinical trials. Starting from advanced lead 3,described in a preceding communication,we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model. View Publication

Protein citrullination has been shown to regulate numerous physiological pathways (e.g.,the innate immune response and gene transcription) and is,when dysregulated,known to be associated with numerous human diseases,including cancer,rheumatoid arthritis,and multiple sclerosis. This modification,also termed deimination,is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals,there are five PAD family members (i.e.,PADs 1,2,3,4,and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported,and these efforts guided the development of the two most potent PAD4 inhibitors (i.e.,F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors,we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3,thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated,the development of PAD-selective inhibitors is of paramount importance. To aid that goal,we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein,we report the results of these studies,which suggest that,like PAD4,PADs 1 and 3 employ a reverse protonation mechanism. Additionally,the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds,denoted F4- and Cl4-amidine,are the most potent PAD3 inhibitors ever described. View Publication

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here,we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP),a molecule externalized in the NETs. NZM mice were treated with Cl-amidine,an inhibitor of peptidylarginine deiminases (PAD),to block NET formation and were evaluated for lupus-like disease activity,endothelial function,and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further,Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells,improved endothelium-dependent vasorelaxation,and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall,these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients. View Publication

CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However,circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression,and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically,TE cells treated with TGR undergo metabolic remodelling that,after glucose re-exposure,supports enhanced glucose uptake,increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state-all indicators of a more anabolic programme to support their enhanced functionality. Thus,metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy. View Publication

Human respiratory syncytial virus (RSV) is a major cause of serious respiratory tract infections in infants and the elderly. Recently it was shown that the RSV G glycoprotein mediates attachment to cells using CX3CR1 as a receptor,and that G-specific neutralizing antibodies can be detected using human airway epithelial (HAE) cell cultures. To investigate the contributions of G-specific antibodies to RSV neutralization,we performed HAE neutralization assays on sera from RSV G-immunized mice or RSV-infected infants. We confirmed that G-specific neutralization using serum from mice or humans could only be detected on HAE cultures. We also found that RSV G-specific antibodies in infants were either subgroup specific or cross-neutralizing. Altogether,our results suggest that G is an important target for generating neutralizing antibodies and would be beneficial to include in an RSV vaccine. Further,inclusion of G antigens from both RSV subgroups may enhance the vaccine cross protection potency. View Publication

Perhexiline is a potent prophylactic anti-anginal agent that has been shown to inhibit myocardial utilization of long-chain fatty acids and to inhibit the mitochondrial enzyme carnitine palmitoyltransferase (CPT)-1. We compared the hemodynamic and biochemical effects of perhexiline (0.5 and 2.0 microM) and of another CPT-1 inhibitor,oxfenicine (0.5 mM),in Langendorff-perfused rat hearts subjected to 60 min of low-flow ischemia (95{\%} flow reduction) followed by 30 min of reperfusion. Both perhexiline (2 microM only) and oxfenicine attenuated (p {\textless} 0.003,p {\textless} 0.0002,respectively) increases in diastolic tension during ischemia,without significant effects on developed tension,or on cardiac function during reperfusion. Myocardial concentrations of long-chain acylcarnitines (LCAC),products of CPT-1 action,were decreased (p {\textless} 0.05) by oxfenicine,unaffected by 2 microM perhexiline,and increased slightly by 0.5 microM perhexiline. Perhexiline,but not the active metabolite of oxfenicine,also inhibited cardiac CPT-2 with similar IC50 and Emax,although lower Hill slope,compared with CPT-1. Oxfenicine,but not perhexiline,reduced concentrations of the endogenous CPT-1 inhibitor,malonyl-CoA. Perhexiline,but not oxfenicine,inhibited myocardial release of lactate during normal flow. We conclude that (a) perhexiline protects against diastolic dysfunction during ischemia in this model,independent of major changes in LCAC accumulation and (b) this may result from simultaneous effects of perhexiline on myocardial CPT-1 and CPT-2. View Publication

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix (ECM) changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however,whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production,we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) co-culture model. RSV infection of BEC/HLF co-cultures led to decreased hyaluronidase expression by HLFs,increased accumulation of HA,and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however,BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared to control mice which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared to SPC-Cre(-) RSV-infected littermates. Taken together,these data demonstrate that altered ECM accumulation of HA occurs following RSV infection and may contribute to airway inflammation. Additionally,loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment. View Publication