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Arginase 1 (A1) is the enzyme that hydrolyzes the amino acid, L-arginine, to ornithine and urea. We have previously shown that A1 deletion worsens retinal ischemic injury, suggesting a protective role of A1. In this translational study, we aimed to study the utility of systemic pegylated A1 (PEG-A1, recombinant human arginase linked to polyethylene glycol) treatment in mouse models of acute retinal and brain injury. Cohorts of WT mice were subjected to retinal ischemia-reperfusion (IR) injury, traumatic optic neuropathy (TON) or brain cerebral ischemia via middle cerebral artery occlusion (MCAO) and treated with intraperitoneal injections of PEG-A1 or vehicle (PEG only). Drug penetration into retina and brain tissues was measured by western blotting and immunolabeling for PEG. Neuroprotection was measured in a blinded fashion by quantitation of NeuN (neuronal marker) immunolabeling of retina flat-mounts and brain infarct area using triphenyl tetrazolium chloride (TTC) staining. Furthermore, ex vivo retina explants and in vitro retina neuron cultures were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (R) and treated with PEG-A1. PEG-A1 given systemically did not cross the intact blood-retina/brain barriers in sham controls but reached the retina and brain after injury. PEG-A1 provided neuroprotection after retinal IR injury, TON and cerebral ischemia. PEG-A1 treatment was also neuroprotective in retina explants subjected to OGD/R but did not improve survival in retinal neuronal cultures exposed to OGD/R. In summary, systemic PEG-A1 administration is neuroprotective and provides an excellent route to deliver the drug to the retina and the brain after acute injury.

Background Bladder cancer is one of the dominant cancers worldwide, especially for male. Currently, the therapeutical regimen of bladder cancer is based on surgery, radiation therapy, chemotherapy and immunotherapy, but the clinical outcome is still needed to improve. Recombinant human arginase (rhArg, BCT-100) is a novel agent to show great anticancer effect on arginine auxotrophic tumor. However, the effect of rhArg on bladder cancer still remains unclear. Methods A panel of six bladder cancer cell lines (BIU-87, EJ-1, J82, SCaBER, T24 and 5637) was employed to assess the anticancer effect of BCT-100 in vitro by MTT assay. T24 nude mice xenograft models were established to evaluate the anticancer effect of BCT-100 in vivo. Protein level (argininosuccinate synthetase 1 (ASS1), ornithine transcarbamylase, cleaved-PARP, PEG, Survivin, p62, Beclin-1, LC3B, p-AKT, p-mTOR) was detected by Western blot. Intracellular, serum and intratumoral arginine concentrations were examined by ELISA. Apoptotic rate, H2O2 and mitochondrial membrane depolarization were tested by flow cytometer. Immunoflorescence on ki67 and TUNEL assay were applied to identify cellular and tumoral apoptotic events. Results BCT-100 displayed anticancer effects on bladder cancer cells in vitro and in vivo. The expression of ASS1 varies in different bladder cancer cell lines, and ornithine transcarbamylase is almost deficient except weakly expressed in SCaBER cell line. Knockdown ASS1 in BIU-87 cells could enhance the cytotoxicity induced by BCT-100. Intracellular arginine level was sharply decreased followed by apoptotic events. Futhermore, BCT-100 induced H2O2 production and mitochondrial membrane depolarization, leading to cytochrome c and smac released from mitochondria to cytosol. The expression of LC3B and Becllin-1 was up-regulated, while p62 was down-regulated in a time dependent manner. Autophagic flux was also observed upon BCT-100 treatment. Besides, the phosphorylation of AKT/mTOR pathway was suppressed in a time dependent fashion in BCT-100-treated T24 cells. N-Acetyl-L-cystein reduced the apoptosis and autophagy induced by BCT-100, while CQ, MK-2206 and rapamycin potentiated the apoptosis triggered by BCT-100. Conclusions The present study demonstrated that BCT-100 induced autophagy and apoptosis via ROS mediated AKT/mTOR signaling pathway in bladder cancer cells.

Small cell lung cancer (SCLC) accounts for approximately 13% of all lung cancer cases. Small cell lung cancer is characterized by frequent relapse, and current treatments lack tumor specificity. Arginine is a non‐essential amino acid for human normal cells but critical to some tumor cells that cannot synthesize arginine. Therefore, arginine deprivation has become a potential therapeutic option for selected tumors. BCT‐100 is a pegylated arginase that has documented anticancer activity in arginine auxotrophic tumors, such as melanoma, hepatocellular carcinoma, and acute myeloid leukemia. One of the resistance mechanisms to arginase treatment is overexpression of argininosuccinate synthetase (ASS1) and ornithine transcarbamylase (OTC), two important enzymes in the urea cycle. We selected 9 SCLC and 1 non‐small cell lung carcinoma cell lines to determine the growth inhibition effects of BCT‐100 and established that cell lines with low expression of ASS1 and OTC are relatively sensitive to BCT‐100 treatment. Knocking down OTC in a H841 cell line could potentiate its sensitivity to BCT‐100 treatment. Arginine concentration was sharply decreased, accompanied by apoptosis through oxidative stress as well as G1 cell cycle arrest. In addition, BCT‐100 showed an anticancer effect on H446 and H510A xenograft models by lowering arginine levels and inducing apoptosis.

Arginine depletion has shown anticancer effects among arginine auxotrophic cancers. An anti-proliferative effect of pegylated arginase (BCT-100) has been shown in acute myeloid leukaemia, hepatocellular carcinoma and mesothelioma. The aim of the present study was to evaluate the effect of BCT-100 in lung adenocarcinoma. A panel of lung adenocarcinoma cell lines and xenograft models were used to investigate the effect of BCT-100. Protein expression, arginine level, putrescine level, spermidine level and apoptosis were analyzed by western blotting, ELISA, high performance liquid chromatography, dot blot and TUNEL assay, respectively. BCT-100 converts arginine to ornithine. BCT-100 reduced in vitro cell viability across different lung adenocarcinoma cell lines and suppressed tumour growth in an HCC4006 ×enograft, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 ×enografts. Upon BCT-100 treatment, ornithine decarboxylase 1 (ODC1) was induced in two solid tumour xenografts (H1650 and H1975). It was postulated that the accumulated ornithine could be channeled via ODC1 to produce polyamines that promoted tumour growth. The action of an ODC1 inhibitor (α-difluoromethylornithine, DFMO) was studied in the restoration of the anticancer effects of BCT-100 in lung adenocarcinoma. In both H1650 and H1975 ×enografts, a combination of DFMO and BCT-100 significantly suppressed tumour growth, resulting in doubled median survival compared with the control. Putrescine was decreased in almost all treatment arms in the H1650, H1975 and HCC4006 ×enografts. Nonetheless spermidine was reduced only following DFMO/BCT-100 treatment in the H1650 and H1975 ×enografts. Apoptosis was enhanced in the combined treatment arm in both H1650 and H1975 ×enografts. In the HCC4006 ×enograft, addition of DFMO did not alter the tumour suppressive effect of BCT-100. In conclusion, inhibition of ODC1 by DFMO was crucial in facilitating BCT-100 treatment in lung adenocarcinoma that was partially mediated by depleting arginine and polyamines with consequent apoptosis.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM. METHODS: A panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events. RESULTS: Argininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC50 values at 13-24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p, published_or_final_version

e20538Background: Arginine depletion induced by PEGylated arginase 1 (BCT-100, PEG-BCT-100 or rhArg1peg5000) has shown promising anticancer effects among arginine auxotrophic cancers that are defic...

POSTER SESSION 3 – P3.03: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies

e23184Background: Arginine depletion has shown promising anticancer effects among arginine auxotrophic cancers that are deficient in argininosuccinate synthetase (ASS) and/or ornithine transcarbamylase (OTC). Pegylated arginase (PEG-BCT-100 (rhArg1peg5000)) works as an arginine depletor by converting arginine to ornithine. However, accumulated ornithine can be channeled via ornithine decarboxylase (ODC) to produce polyamines that are known to promote tumor growth. We postulate that ODC inhibition enhances anticancer effects of BCT-100 in lung adenocarcinoma. Methods: The activity of BCT-100 was tested in a panel of lung adenocarcinoma cell lines (H23, H358, HCC827, H1650, H1975, HCC2935 and HCC4006). Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Protein expression was evaluated by Western Blot. Nude mice subcutaneous xenograft models(XMs) derived from these cell lines were used for in vivostudy. An ODC inhibitor (α-difluoromethylornithine or ...

e20083Background: Cancers with deficient argininosuccinate synthetase (ASS) and/or ornithine transcarbamylase (OTC) are biologically vulnerable to arginine depletion. Preliminary clinical data had ...

The current status of digital image processing in fluid flow research is reviewed. In particular, attention is given to a comprehensive approach to the extraction of quantitative data from multivariate databases and examples of recent developments. The discussion covers numerical simulations and experiments, data processing, generation and dissemination of knowledge, traditional image processing, hybrid processing, fluid flow vector field topology, and isosurface analysis using Marching Cubes.

e15137 Background: This study was designed to evaluate the efficacy, safety profile, pharmacokinetics/pharmacodynamics (PK/PD) and quality of life (QoL) of pegylated recombinant human arginase 1(Pe...

Cell-based polygonal isosurface generation is a common and useful technique for visualizing sampled scalar fields. Typically, however, the number of polygons in the surface model can become quite large as the size of the data set increases. In this paper, we present octree pruning techniques designed to compress the isosurface model while maintaining an accurate representation. We show results for several data sets, and demonstrate speed improvements of over two orders of magnitude over our previous results. A key feature of this work is the use of a volumetric error criterion to quantify model accuracy.

Systematic fractionations of crude extracts from various strains of Aspergillus versicolor were carried out by a combination of techniques, including liquid-liquid partition, column chromatography and thin-layer chromatography. As a result of these fractionations a total of eleven metabolites were obtained in pure form. Ten of these were identified as the known compounds, sterigmatocystin, de-0-methylsterigmatocystin, 5-methoxysterigmatocystin, versicolorin A, versicolorin C, aversin, averufin, griseofulvin, dechlorogriseofulvin and 3,8- dihydroxy-6 -methoxy-1-methylxanthone. A new metabolite was isolated and identified as 6,8-di-O-methylnidurufin. Two high-performance liquid chromatography systems were developed for the analysis of these metabolites, using microparticle silica gel and microparticle polar bonded phase columns. Studies were also carried out on the reduction of the hemi-acetal derivatives of sterigmatocystin and versicolorin A. Reduction with a limited amount of sodium borohydride yielded in each case a mixture of two major products. The fully reduced products were identified as diols, while the partially reduced products were found to be new hemiacetals. The structure elucidation of these compounds was carried out with the aid of carbon magnetic resonance spectroscopy and of chemical conversions. A modified pathway for the biosynthesis of versicolorin A from averufin is proposed that incorporates nidurufin as an intermediate. Ph. D.