Targeting proliferative retinopathy: Arginase 1 limits vitreoretinal neovascularization and promotes angiogenic repair.

Current therapies for treatment of proliferative retinopathy focus on retinal neovascularization (RNV) during advanced disease and can trigger adverse side-effects. Here, we have tested a new strategy for limiting neurovascular injury and promoting repair during early-stage disease. We have recently shown that treatment with a stable, pegylated drug form of the ureohydrolase enzyme arginase 1 (A1) provides neuroprotection in acute models of ischemia/reperfusion injury, optic nerve crush, and ischemic stroke. Now, we have determined the effects of this treatment on RNV, vascular repair, and retinal function in the mouse oxygen-induced retinopathy (OIR) model of retinopathy of prematurity (ROP). Our studies in the OIR model show that treatment with pegylated A1 (PEG-A1), inhibits pathological RNV, promotes angiogenic repair, and improves retinal function by a mechanism involving decreased expression of TNF, iNOS, and VEGF and increased expression of FGF2 and A1. We further show that A1 is expressed in myeloid cells and areas of RNV in retinal sections from mice with OIR and human diabetic retinopathy (DR) patients and in blood samples from ROP patients. Moreover, studies using knockout mice with hemizygous deletion of A1 show worsened RNV and retinal injury, supporting the protective role of A1 in limiting the OIR-induced pathology. Collectively, A1 is critically involved in reparative angiogenesis and neuroprotection in OIR. Pegylated A1 may offer a novel therapy for limiting retinal injury and promoting repair during proliferative retinopathy.

Preclinical investigation of Pegylated arginase 1 as a treatment for retina and brain injury.

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.

Pegylated Recombinant Human Arginase 1 Induces Autophagy and Apoptosis via the ROS-Activated AKT/mTOR Pathway in Bladder Cancer Cells.

Bladder cancer is one of the most commonly diagnosed cancers worldwide, especially in males. Current therapeutic interventions, including surgery, radiation therapy, chemotherapy, and immunotherapy, have not been able to improve the clinical outcome of bladder cancer patients with satisfaction. Recombinant human arginase (rhArg, BCT-100) is a novel agent with great anticancer effects on arginine-auxotrophic tumors. However, the effects of BCT-100 on bladder cancer remain unclear. In this study, the in vitro anticancer effects of BCT-100 were assessed using four bladder cancer cell lines (J82, SCaBER, T24, and 5637), while the in vivo effects were evaluated by establishing T24 nude mice xenograft models. Intracellular arginine level was observed to be sharply decreased followed by the onset of apoptotic events. Furthermore, BCT-100 was found to induce H2O2 production and mitochondrial membrane depolarization, leading to the release of mitochondrial cytochrome c and Smac to the cytosol. Treatment with BCT was observed to upregulate the expression of LC3B and Becllin-1, but downregulate the expression of p62 in a time-dependent manner. Autophagic flux was also observed upon BCT-100 treatment. Besides, the phosphorylation of the AKT/mTOR pathway was suppressed in a time-dependent fashion in BCT-100-treated T24 cells. While N-acetyl-L-cysteine was shown to alleviate BCT-100-induced apoptosis and autophagy, chloroquine, MK-2206, and rapamycin were found to potentiate BCT-100-triggered apoptosis. Finally, BCT-100 was demonstrated to induce autophagy and apoptosis via the ROS-mediated AKT/mTOR signaling pathway in bladder cancer cells.

Contactin 1 modulates pegylated arginase resistance in small cell lung cancer through induction of epithelial-mesenchymal transition.

Drug resistance is a major hurdle in the treatment of small cell lung cancer (SCLC). Previously we demonstrated the potential anticancer effect of pegylated arginase BCT-100 in SCLC cell lines and xenograft models. To facilitate future clinical application of BCT-100 in SCLC treatment, we elucidated the potential mechanisms that underlie acquired drug resistance to BCT-100. H446 and H526 SCLC cells were serially cultured in stepwise increasing concentrations of BCT-100 until stable BCT-100-resistant cell lines emerged (H446-BR and H526-BR). Compared with parent cells, H446-BR and H526-BR displayed stronger migration ability, anoikis resistance and EMT progression. Gene chip assay was employed to select three potential targets (CDH17, CNTN-1 and IGF2BP1). Silencing CNTN-1 rather than CDH17 or IGF2BP1 in H446-BR and H526-BR cells re-sensitized resistant cells to BCT-100 treatment and attenuated the epithelial-mesenchymal transition (EMT) phenotype. The AKT signaling pathway was activated in H446-BR and H526-BR cells accompanied by EMT progression, and AKT inhibitor LY294002 reversed the EMT progression in resistant cells.

Endogenous arginase 2 as a potential biomarker for PEGylated arginase 1 treatment in xenograft models of squamous cell lung carcinoma.

Depletion of arginine induced by PEGylated arginase 1 (ARG1) (BCT-100) has shown anticancer effects in arginine auxotrophic cancers that lack argininosuccinate synthetase (ASS1) and ornithine transcarbamylase (OTC). High levels of endogenous arginase 2 (ARG2) have been previously reported in human lung cancers. Although a high-ARG2 level neither causes immunosuppression nor affects disease progression, it may theoretically affect the efficacy of PEGylated ARG1 treatment. ARG2 was shown to be highly expressed in H520 squamous cell lung carcinoma (lung SCC) xenografts but undetectable in SK-MES-1 and SW900 lung SCC xenografts. We propose that high-endogenous expression of ARG2 could impede the anti-tumor effect of PEGylated ARG1 in lung SCC. The in vivo effect of PEGylated ARG1 was investigated using three xenograft models of lung SCC. PEGylated ARG1 (60 mg/kg) suppressed tumor growth in SK-MES-1 and SW900 but not H520 xenografts. ASS1 was expressed in SK-MES-1 and SW900 xenografts while OTC expression remained low in all xenografts. A high-endogenous ARG2 level was detected only in H520 xenografts. Serum arginine level was decreased significantly by PEGylated ARG1 in all xenografts. Nonetheless intratumoral arginine level was decreased by PEGylated ARG1 in SK-MES-1 and SW900, not H520 xenografts. In SK-MES-1 xenografts, PEGylated ARG1 treatment induced G1 arrest, downregulation of Ki67 and Mcl-1 and activation of apoptosis. In SW900 xenografts, upregulation of Bim and activation of apoptosis were observed upon PEGylated ARG1 treatment. Silencing of ARG2 re-sensitized the H520 xenografts to PEGylated ARG1 treatment, partially mediated through arginine depletion via G1 arrest and apoptosis. PEGylated ARG1 treatment (BCT-100) was effective in lung SCC xenografts with low-endogenous levels of ASS1/OTC and ARG2. High-endogenous ARG2 expression may cause resistance to PEGylated ARG1 treatment in lung SCC xenografts. ARG2 may serve as a third predictive biomarker in PEGylated ARG1 treatment in lung SCC.

Inhibition of ornithine decarboxylase 1 facilitates pegylated arginase treatment in lung adenocarcinoma xenograft models.

Arginine depletion has shown anticancer effects among arginine auxotrophic cancers. An anti­proliferative effect of pegylated arginase (BCT­100) has been shown in acute myeloid leukaemia, hepatocellular carcinoma and mesothelioma. The aim of the present study was to evaluate the effect of BCT­100 in lung adenocarcinoma. A panel of lung adenocarcinoma cell lines and xenograft models were used to investigate the effect of BCT­100. 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. BCT­100 converts arginine to ornithine. BCT­100 reduced in vitro cell viability across different lung adenocarcinoma cell lines and suppressed tumour growth in an HCC4006 xenograft, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 xenografts. Upon BCT­100 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 (α­difluoromethylornithine, DFMO) was studied in the restoration of the anticancer effects of BCT­100 in lung adenocarcinoma. In both H1650 and H1975 xenografts, a combination of DFMO and BCT­100 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 xenografts. Nonetheless spermidine was reduced only following DFMO/BCT­100 treatment in the H1650 and H1975 xenografts. Apoptosis was enhanced in the combined treatment arm in both H1650 and H1975 xenografts. In the HCC4006 xenograft, addition of DFMO did not alter the tumour suppressive effect of BCT­100. In conclusion, inhibition of ODC1 by DFMO was crucial in facilitating BCT­100 treatment in lung adenocarcinoma that was partially mediated by depleting arginine and polyamines with consequent apoptosis.

Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer.

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.

Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts.

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 < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms. CONCLUSIONS: BCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

Deprivation of arginine by recombinant human arginase in prostate cancer cells.

BACKGROUND: Recombinant human arginase (rhArg) has been developed for arginine deprivation therapy in cancer, and is currently under clinical investigation. During pre-clinical evaluation, rhArg has exhibited significant anti-proliferative activity in cancer cells deficient in the expression of ornithine carbamoyl transferase (OCT). Interestingly, a variety of cancer cells such as melanoma and prostate cancer deficient in argininosuccinate synthetase (ASS) are sensitive to arginine deprivation by arginine deiminase. In this study, we investigated levels of gene expression of OCT and ASS, and the effects of rhArg in human prostate cancer cells: LNCaP (androgen-dependent), PC-3 and DU-145 (both androgen-independent). RESULTS: Quantitative real-time PCR showed minimal to absent gene expression of OCT, but ample expression of ASS expression in all 3 cell lines. Cell viability assay after 72-h exposure of rhArg showed all 3 lines had half maximal inhibitory concentration less than or equal to 0.02 U/ml. Addition of ornithine to cell culture media failed to rescue these cells from rhArg-mediated cytotoxicity.Decreased phosphorylation of 4E-BP1, a downstream effector of mammalian target of rapamycin (mTOR), was noted in DU-145 and PC-3 after exposure to rhArg. Moreover, there was no significant apoptosis induction after arginine deprivation by rhArg in all 3 prostate cancer cell lines. CONCLUSION: rhArg causes significant cytotoxicity in LNCaP, DU-145 and PC-3 prostate cancer cells which all demonstrate decreased OCT expression. Inhibition of mTOR manifested by hypophosphorylation of 4E-BP1 suggests autophagy is involved as alternative cell death mechanism. rhArg demonstrates a promising novel agent for prostate cancer treatment.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC).

BACKGROUND: Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable. METHODS: The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo. RESULTS: Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 microM from approximately 170 microM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared. CONCLUSION: Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.

Pegylated recombinant human arginase (rhArg-peg5,000mw) inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion.

Hepatocellular carcinoma (HCC) is believed to be auxotrophic for arginine through the lack of expression of argininosuccinate synthetase (ASS). The successful use of the arginine-depleting enzyme arginine deiminase (ADI) to treat ASS-deficient tumors has opened up new possibilities for effective cancer therapy. Nevertheless, many ASS-positive HCC cell lines are found to be resistant to ADI treatment, although most require arginine for proliferation. Thus far, an arginine-depleting enzyme for killing ASS-positive tumors has not been reported. Here, we provide direct evidence that recombinant human arginase (rhArg) inhibits ASS-positive HCCs. All the five human HCC cell lines we used were sensitive to rhArg but ADI had virtually no effect on these cells. They all expressed ASS, but not ornithine transcarbamylase (OTC), the enzyme that converts ornithine, the product of degradation of arginine with rhArg, to citrulline, which is converted back to arginine via ASS. Transfection of HCC cells with OTC resulted in resistance to rhArg. Thus, OTC expression alone may be sufficient to induce rhArg resistance in ASS-positive HCC cells. This surprising correlation between the lack of OTC expression and sensitivity of ASS-positive HCC cells shows that OTC-deficient HCCs are sensitive to rhArg-mediated arginine depletion. Therefore, pretreatment tumor gene expression profiling of ASS and OTC could aid in predicting tumor response to arginine depletion with arginine-depleting enzymes. We have also shown that the rhArg native enzyme and the pegylated rhArg (rhArg-peg(5,000mw)) gave similar anticancer efficacy in vitro. Furthermore, the growth of the OTC-deficient Hep3B tumor cells (ASS-positive and ADI-resistant) in mice was inhibited by treatment with rhArg-peg(5,000mw), which is active alone and is synergistic in combination with 5-fluorouracil. Thus, our data suggest that rhArg-peg(5,000mw) is a novel agent for effective cancer therapy.