Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Angiotensin II type 1 receptor agonistic autoantibody blockade improves postpartum hypertension and cardiac mitochondrial function in rat model of preeclampsia.

Women with preeclampsia (PE) have a greater risk of developing hypertension, cardiovascular disease (CVD), and renal disease later in life. Angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE during pregnancy and up to 2-year postpartum (PP), and in the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of AT1-AA with a specific 7 amino acid peptide binding sequence ('n7AAc') improves pathophysiology observed in RUPP rats; however, the long-term effects of AT1-AA inhibition in PP is unknown. Pregnant Sprague Dawley rats were divided into three groups: normal pregnant (NP) (n = 16), RUPP (n = 15), and RUPP + 'n7AAc' (n = 16). Gestational day 14, RUPP surgery was performed and 'n7AAc' (144 µg/day) administered via osmotic minipump. At 10-week PP, mean arterial pressure (MAP), renal glomerular filtration rate (GFR) and cardiac functions, and cardiac mitochondria function were assessed. MAP was elevated PP in RUPP vs. NP (126 ± 4 vs. 116 ± 3 mmHg, p < 0.05), but was normalized in in RUPP + 'n7AAc' (109 ± 3 mmHg) vs. RUPP (p < 0.05). PP heart size was reduced by RUPP + 'n7AAc' vs. RUPP rats (p < 0.05). Complex IV protein abundance and enzymatic activity, along with glutamate/malate-driven respiration (complexes I, III, and IV), were reduced in the heart of RUPP vs. NP rats which was prevented with 'n7AAc'. AT1-AA inhibition during pregnancy not only improves blood pressure and pathophysiology of PE in rats during pregnancy, but also long-term changes in blood pressure, cardiac hypertrophy, and cardiac mitochondrial function PP.

Zafirlukast is a broad-spectrum thiol isomerase inhibitor that inhibits thrombosis without altering bleeding times.

BACKGROUND AND PURPOSE: Multiple members of the thiol isomerase (TI) family of enzymes are present in and released by platelets. Inhibition of these enzymes results in diminished platelet responses, aggregation, adhesion and thrombus formation. Recently, the therapeutic potential of TI inhibition has been recognised and drug-development technologies were used to identify selective small molecule inhibitors. To date, few pan-TI inhibitors have been characterised and the most studied, bacitracin, is known to be nephrotoxic, which prohibits its systemic therapeutic usage. EXPERIMENTAL APPROACH: We therefore sought to identify novel broad-spectrum inhibitors of these enzymes and test their effects in vivo. A total of 3,641 compounds were screened for inhibitory effects on the redox activity of ERp5, protein disulphide isomerase (PDI), ERp57, ERp72 and thioredoxin in an insulin turbidity assay. Of the lead compounds identified, zafirlukast was selected for further investigation. KEY RESULTS: When applied to platelets, zafirlukast diminished platelet responses in vitro. Zafirlukast was antithrombotic in murine models of thrombosis but did not impair responses in a model of haemostasis. Since TIs are known to modulate adhesion receptor function, we explored the effects of zafirlukast on cell migration. This was inhibited independently of cysteinyl LT receptor expression and was associated with modulation of cell-surface free thiol levels consistent with alterations in redox activity on the cell surface. CONCLUSION AND IMPLICATIONS: We identify zafirlukast to be a novel, potent, broad-spectrum TI inhibitor, with wide-ranging effects on platelet function, thrombosis and integrin-mediated cell migration. Zafirlukast is antithrombotic but does not cause bleeding.

DNA methylation changes in CD4+ T cells isolated from multiple sclerosis patients on dimethyl fumarate.

BACKGROUND: Dimethyl fumarate is an oral treatment for multiple sclerosis, whose mechanism of action is not fully understood. OBJECTIVE: To investigate the effects of dimethyl fumarate on DNA methylation in the CD4+ T cells of multiple sclerosis patients. METHODS: We performed Illumina EPIC arrays to investigate the DNA methylation profiles of CD4+ T cells derived from multiple sclerosis patients before and after dimethyl fumarate treatment. RESULTS: Treatment with dimethyl fumarate resulted in 97% of differentially methylated positions showing hypermethylation. Four genes, SNORD1A, SHTN1, MZB1 and TNF had a differentially methylated region located within the transcriptional start site. CONCLUSION: This study investigates the effect of dimethyl fumarate on DNA methylation in multiple sclerosis patients.

A Novel Macroscale Acoustic Device for Blood Filtration.

Retransfusion of a patient's own shed blood during cardiac surgery is attractive since it reduces the need for allogeneic transfusion, minimizes cost, and decreases transfusion related morbidity. Evidence suggests that lipid micro-emboli associated with the retransfusion of the shed blood are the predominant causes of the neurocognitive disorders. We have developed a novel acoustophoretic filtration system that can remove lipids from blood at clinically relevant flow rates. Unlike other acoustophoretic separation systems, this ultrasound technology works at the macroscale, and is therefore able to process larger flow rates than typical micro-electromechanical system (MEMS) scale acoustophoretic separation devices. In this work, we have first demonstrated the systematic design of the acoustic device and its optimization, followed by examining the feasibility of the device to filter lipids from the system. Then, we demonstrate the effects of the acoustic waves on the shed blood; examining hemolysis using both haptoglobin formation and lactate dehydrogenase release, as well as the potential of platelet aggregation or inflammatory cascade activation. Finally, in a porcine surgical model, we determined the potential viability of acoustic trapping as a blood filtration technology, as the animal responded to redelivered blood by increasing both systemic and mean arterial blood pressure.

Improved Oviduct Transfer Surgery for Genetically Modified Rat Production.

Rat embryo transfer surgeries are becoming more common with targeted nucleases increasing the demand for rat models. This protocol details pre-surgical preparation, improved surgical techniques for placing embryos into the oviduct, and post-surgical care of rats to parturition. Direct application of mouse oviduct transfer protocols results in limited success in the rat. By combining techniques from several widely used protocols in the field, increased yield of live pups born to healthy dams is possible. This protocol is distinct from previously published protocols by the use of a modified anesthesia protocol (Smith et al., 2004), use of analgesia, the addition of peritoneal sutures (Filipiak and Saunders, 2006), incision location and number of transfers per animal (Krinke et al., 2000).

A novel application of radiomimetic compounds as antibiotic drugs.

OBJECTIVE: This study aims to examine the potential of radiomimetic compounds as antimicrobial therapeutics, as the recent advances in radiomimetic targeting as well as rapid increase of multidrug resistant bacteria make these compounds attractive for future development. METHODS: Representative radiomimetics from each of the three major categories was examined; C-1027 and neocarzinostatin from the protein-chromophore enediyne family; Calicheamicin from the non-protein chromophore enediyne family and Bleomycin and Tallysomycin S10b from the glycopeptide family. The activity of these compounds was examined against 12 distinct bacteria species. Inhibition was determined using disc diffusion assays and a subsequent examination of minimum inhibitory concentration of a representative organism. The onset of action of the compounds was also determined by incubating the organisms with drug in liquid media, before plating, and then determining if growth occurred. RESULTS: We found that the radiomimetic glycopeptides were more active against Gram-negative species, while the enediynes were more effective against Gram-positive species. The radiomimetics also maintained their rapid onset of action, working as quickly as 5 min. CONCLUSIONS: Radiomimetic compounds have activity against a wide variety of microorganisms and would support the development of radiomimetic-antibody conjugates as potential antibiotics as an option against severe bacterial infections.

The radiomimetic enediyne, 20'-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance.

The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027's induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism. In this study, an analog of C-1027, 20'-deschloro-C-1027 was examined for its ability to induce DNA ICLs under hypoxic conditions. Deschloro-induced ICLs were detected under hypoxic cell-free conditions, with a concomitant reduction in the induction of DNA strand breaks. In cells deschloro behaved similarly, inducing cellular ICLs under hypoxic conditions with a reduction in DNA breaks. The cytotoxicity of deschloro treatment was similar in normoxic and hypoxic cells, suggesting that the ICL induction allows deschloro to retain its cytotoxic activity under hypoxia. It appears that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with the hypoxic environment associated with solid tumors.

Rituximab plus chlorambucil as first-line treatment for chronic lymphocytic leukemia: Final analysis of an open-label phase II study.

PURPOSE: Most patients with chronic lymphocytic leukemia (CLL) are elderly and/or have comorbidities that may make them ineligible for fludarabine-based treatment. For this population, chlorambucil monotherapy is an appropriate therapeutic option; however, response rates with chlorambucil are low, and more effective treatments are needed. This trial was designed to assess how the addition of rituximab to chlorambucil (R-chlorambucil) would affect safety and efficacy in patients with CLL. PATIENTS AND METHODS: Patients with first-line CLL were treated with rituximab (375 mg/m(2) on day 1, cycle one, and 500 mg/m(2) thereafter) plus chlorambucil (10 mg/m(2)/d all cycles; day 1 through 7) for six 28-day cycles. For patients not achieving complete response (CR), six additional cycles of chlorambucil alone could be administered. The primary end point of the study was safety. RESULTS: A total of 100 patients were treated with R-chlorambucil, with a median follow-up of 30 months. Median age of patients was 70 years (range, 43 to 86 years), with patients having a median of seven comorbidities. Hematologic toxicities accounted for most grade 3/4 adverse events reported, with neutropenia and lymphopenia both occurring in 41% of patients and leukopenia in 23%. Overall response rates were 84%, with CR achieved in 10% of patients. Median progression-free survival was 23.5 months; median overall survival was not reached. CONCLUSION: These results compare favorably with previously published results for chlorambucil monotherapy, suggesting that the addition of rituximab to chlorambucil may improve efficacy with no unexpected adverse events. R-chlorambucil may improve outcome for patients who are ineligible for fludarabine-based treatments.

Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of ß3 integrins.

Extracellular protein disulfide isomerase (PDI) is required for platelet thrombus formation and fibrin generation after arteriolar wall injury in live mice. PDI is secreted from platelets and endothelial cells on cellular activation, but the mechanism of capture of secreted PDI within the injured vasculature is unknown. We establish that, like the endothelial ß3 integrin α(V)ß(3), the platelet integrin α(IIb)ß(3) binds PDI. PDI also binds to recombinant ß3. Using intravital microscopy, we demonstrate that PDI accumulation at the site of laser-induced arteriolar wall injury is markedly reduced in ß3-null (ß3(-/-)) mice, and neither a platelet thrombus nor fibrin is generated at the vessel injury site. The absence of fibrin after vascular injury in ß3(-/-) mice is because of the absence of extracellular PDI. To evaluate the relative importance of endothelial α(V)ß(3) versus platelet α(IIb)ß(3) or α(V)ß(3), we performed reciprocal bone marrow transplants on wild-type and ß3(-/-) mice. PDI accumulation and platelet thrombus formation were markedly decreased after vessel injury in wild-type mice transplanted with ß3(-/-) bone marrow or in ß3(-/-) mice transplanted with wild-type bone marrow. These results indicate that both endothelial and platelet ß3 integrins contribute to extracellular PDI binding at the vascular injury site.

Facial growth in children with complete cleft of the primary palate and intact secondary palate.

PURPOSE: Children with unoperated cleft lip/palate have nearly normal facial growth, whereas patients who have had labiopalatal repair often exhibit midfacial retrusion. The aim of this study was to compare cephalometric data in patients with repaired unilateral or bilateral complete cleft lip/alveolus (UCCLA or BCCLA) with patients with repaired unilateral or bilateral complete cleft lip/palate (UCCLP or BCCLP). This study might provide insight into the etiology of impaired facial growth in patients with repaired cleft lip/palate. MATERIALS AND METHODS: This was a retrospective, cross-sectional analysis of nonsyndromic patients with UCCLA, BCCLA, UCCLP, and BCCLP. Angular and linear measurements of the midfacial region were made on traced lateral cephalograms. Paired t tests were used to compare each group with normative controls from the Michigan Growth Study. Multivariate analysis of variance was used to determine possible differences among the groups. RESULTS: There were 77 patients (38 male and 39 female) with a mean age of 11.2 years (range, 6 to 16 years; UCCLA, n = 25; BCCLA, n = 7; UCCLP, n = 18; and BCCLP, n = 27). There was no significant difference in midfacial position between the UCCLA and BCCLA groups and noncleft age-matched controls. In contrast, the maxilla in patients with UCCLP and BCCLP was significantly smaller and more retruded (P < .05) compared with patients with UCCLA and BCCLA and controls. CONCLUSIONS: Children with UCCLA and BCCLA appear to have normal midfacial growth, whereas the maxilla in children with UCCLP and BCCLP is small and retrusive. This study suggests that the presence and/or repair of the secondary palate is responsible for midfacial hypoplasia in these patients.

Validating a nondestructive optical method for apportioning colored particulate matter into black carbon and additional components.

Exposure of black carbon (BC) is associated with a variety of adverse health outcomes. A number of optical methods for estimating BC on Teflon filters have been adopted but most assume all light absorption is due to BC while other sources of colored particulate matter exist. Recently, a four-wavelength-optical reflectance measurement for distinguishing second hand cigarette smoke (SHS) from soot-BC was developed (Brook et al., 2010; Lawless et al., 2004). However, the method has not been validated for soot-BC nor SHS and little work has been done to look at the methodological issues of the optical reflectance measurements for samples that could have SHS, BC, and other colored particles. We refined this method using a lab-modified integrating sphere with absorption measured continuously from 350 nm to 1000 nm. Furthermore, we characterized the absorption spectrum of additional components of particulate matter (PM) on PM(2.5) filters including ammonium sulfate, hematite, goethite, and magnetite. Finally, we validate this method for BC by comparison to other standard methods. Use of synthesized data indicates that it is important to optimize the choice of wavelengths to minimize computational errors as additional components (more than 2) are added to the apportionment model of colored components. We found that substantial errors are introduced when using 4 wavelengths suggested by Lawless et al. to quantify four substances, while an optimized choice of wavelengths can reduce model-derived error from over 10% to less than 2%. For environmental samples, the method was sensitive for estimating airborne levels of BC and SHS, but not mass loadings of iron oxides and sulfate. Duplicate samples collected in NYC show high reproducibility (points consistent with a 1:1 line, R(2) = 0.95). BC data measured by this method were consistent with those measured by other optical methods, including Aethalometer and Smoke-stain Reflectometer (SSR); although the SSR looses sensitivity at filter loadings above 90 ng/mm(2). Furthermore, positive correlations (R(2) = 0.7) were observed between EC measured by NIOSH Method 5040 on quartz filters and BC measured in co-located Teflon filter samples collected from both heating and non-heating seasons. Overall, the validation data demonstrates the usefulness of this method to evaluate BC from archived Teflon filters while potentially providing additional component information.

A randomized phase II trial of fludarabine, cyclophosphamide and mitoxantrone (FCM) with or without rituximab in previously treated chronic lymphocytic leukaemia.

Combination fludarabine (F), cyclophosphamide (C) and rituximab (R) is the standard front-line therapy in chronic lymphocytic leukaemia (CLL), but appropriate treatment of relapsed/refractory CLL is less clear. Combined FC and mitoxantrone (M) has been reported to be effective in a single arm study, and rituximab when added to chemotherapy in CLL is synergistic. A randomized, two-stage, Phase II trial of FCM and FCM-R was conducted in relapsed CLL. The primary endpoint was response rate 2 months after therapy, assessed according to the 2008 International Workshop CLL criteria. In addition, minimal residual disease (MRD) in the marrow was studied 2 months after therapy, with MRD negativity defined as <0·01% CLL cells. Fifty-two patients were entered, 26 in each arm. The overall response rates to FCM and FCM-R were 58% and 65% respectively. Combined complete response (CR) and CR with incomplete marrow recovery [CR(i)] was 15% (95% confidence interval [CI]:4-35%) for FCM and 42% (95%CI:23-63%) for FCM-R, with eight patients achieving MRD negativity (3 FCM; 5 FCM-R). The toxicity of both regimens was acceptable. In conclusion, the addition of rituximab to FCM improves the response rates in relapsed CLL, resulting in more complete remissions and without additional safety concerns. Efficacy and safety should be fully tested in a randomized Phase III trial.

Detection of active gastrointestinal hemorrhage with CT angiography: a 4(1/2)-year retrospective review.

PURPOSE: To retrospectively evaluate the performance of computed tomography (CT) angiography in the detection and localization of clinically active gastrointestinal (GI) hemorrhage of an unknown source. MATERIALS AND METHODS: Eighty-six CT angiograms were obtained in 74 patients with the clinical diagnosis of acute GI hemorrhage of an unknown source. Results of CT angiography were recorded, and the patients' electronic medical records were reviewed for documentation of subsequent interventional procedures performed within 24 hours of the reference CT angiogram to diagnose or control ongoing GI hemorrhage. Surgical, endoscopic, and final pathologic reports, if available, were reviewed. RESULTS: Twenty-two of the 86 CT angiograms (26%) were positive for active hemorrhage, with findings confirmed in 19 of the 22 cases (86%). Thirteen cases were confirmed with angiography, five cases were confirmed with surgery, and one case was confirmed with autopsy. Sixty-four of the 86 CT angiograms were negative, and 59 (92%) of the CT angiograms required no further intervention. These patients were discharged without incident. There were no cases in which CT angiography was negative and subsequent angiography within 24 hours was positive. The overall sensitivity, specificity, accuracy, and positive and negative predictive value of CT angiography in the detection of active GI hemorrhage within this study population were 79%, 95%, 91%, 86%, and 92%, respectively. CONCLUSIONS: CT angiography provides valuable information that can be used to determine the appropriateness of catheter angiography and guide mesenteric catheterization if a bleeding source is localized. The authors' experience with this study cohort supports its use before angiography in those patients with acute GI bleeding of an unknown source who are being considered for catheter-directed intervention.

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR.

We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.

Designer enediynes generate DNA breaks, interstrand cross-links, or both, with concomitant changes in the regulation of DNA damage responses.

The ability of the radiomimetic anticancer enediyne C-1027 to induce ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR)-independent damage responses was discovered to reside in its unique ability to concurrently generate robust amounts of double-strand breaks (DSBs) and interstrand cross-links (ICLs) in cellular DNA. Furthermore, a single substitution to the chromophore's benzoxazolinate moiety shifted DNA damage to primarily ICLs and an ATR- but not ATM-dependent damage response. In contrast, single substitutions of the chromophore's beta-amino acid component shifted DNA damage to primarily DSBs, consistent with its induction of conventional ATM-dependent damage responses of the type generated by ionizing radiation and other radiomimetics. Thus, phosphatidylinositol 3-kinase-like protein kinase regulation of DNA damage responses is dictated by the relative proportions of DSBs and ICLs.

Single chemical modifications of the C-1027 enediyne core, a radiomimetic antitumor drug, affect both drug potency and the role of ataxia-telangiectasia mutated in cellular responses to DNA double-strand breaks.

The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7''-desmethyl-C-1027 (desmethyl), 20'-deschloro-C-1027 (deschloro), and 22'-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 2-fold (deschloro) to 55-fold (desmethyl) less than C-1027. The induction of cellular DNA breaks based on pulsed field gel electrophoresis, comet analysis, and gammaH2AX activation was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared with the other analogues. Despite the disparity in inducing DNA DSBs, all of the analogues produced G2-M cell cycle arrest and activated DNA DSB damage response proteins, such as p53-Ser15 and Chk2-Thr68, at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogues, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings show that simple modifications of the chromophore of C-1027 can result in varied induction of, and cellular response to, DNA DSBs.