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Exosomes, abundant in blood, deliver various molecules to recipient cells. Endothelial cells are directly exposed to circulating substances. However, how endothelial cells respond to serum exosomes (SExos) and the implications in diabetes-associated vasculopathy have never been explored. In the present study, we showed that SExos from diabetic db/db mice (db/db SExos) were taken up by aortic endothelial cells, which severely impaired endothelial function in nondiabetic db/m⁺ mice. The exosomal proteins, rather than RNAs, mostly account for db/db SExos-induced endothelial dysfunction. Comparative proteomics analysis showed significant increase of arginase 1 in db/db SExos. Silence or overexpression of arginase 1 confirmed its essential role in db/db SExos-induced endothelial dysfunction. This study is a demonstration that SExos deliver arginase 1 protein to endothelial cells, representing a cellular mechanism during development of diabetic endothelial dysfunction. The results expand the scope of bloodborne substances that monitor vascular homeostasis.

This study used in vitro experiments and an orthotopic glioblastoma (GBM) mouse model to test the efficacy of human pegylated arginase I formulation, BCT-100, against the incurable cancer, GBM. Arginine auxotrophy in GBM was verified in silico and in vitro by absence of OTC and ASS1 expression. BCT-100 inhibited growth and induced cell death in four GBM cell lines in vitro. Transcriptomics of U87 and U373 treated with BCT-100 responded differently. BCT-100-treated U87 showed autophagy induction. Cytotoxicity enhancement was observed in four cell lines treated with BCT-100 + chloroquine (CQ). CQ + BCT-100 induced caspase-dependent and caspase-independent cell death in luciferase-transfected U87 (U87(lf+)). BCT-100 or CQ monotherapy, but not BCT-100+ CQ, prolonged survival of intracranial U87(lf+)-bearing mice similarly without suppressing tumor growth. Mouse microglia cell BV2 protected U87(lf+) from BCT100-induced cytotoxicity in transwell co-culture. Etoposide suppressed BV2’s protection to U87(lf+) upon BCT-100 treatment by suppressing the growth and inducing cell death of BV2, suggesting microglial suppression as a strategy for enhancing the efficacy of BCT-100. Microglial protection may explain the in vitro and in vivo discrepancies. Further investigation into microglia/GBM interactions may help improve the efficacy of arginine deprivation therapy against GBM.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Drug resistance is a major obstacle in the clinical management of small cell lung cancer (SCLC), we have proved the promising anticancer effect of recombinant human arginase (rhArg, BCT-100) in SCLC in vitro and in vivo. In order to promote the clinical application of recombinant human arginase, it is necessary to explore the underlying resistant mechanisms of BCT-100 in SCLC. Here, we cultured and obtained the acquired drug-resistant SCLC cell line (H446-BR), which displayed different cellular phenotypes (enhanced migration ability) compared with the parental cell line (H446). sestrin3 (SESN3) was confirmed with high expression in resistant cell line. Knockdown SESN3 could re-sensitize resistant cells to BCT-100 treatment and reverse the aggressive feature of H446-BR. The Akt-mTOR signal pathway and ASS1, which were highly expressed in resistant cells, were down-regulated after silencing SESN3. MK-2206 and rapamycin suppressed the expression of ASS1 in H446-BR cell. In xenograft model, BCT-100 has little anti-tumor effect on H446-BR compared with H446 as well as H446-BR silenced sestrin3. Collectively, these results elucidate SESN3 plays an essential role in resistant mechanism, which will provide a valuable source of information for translational research.

Summary This study was designed to evaluate the efficacy, safety profile, pharmacokinetics, pharmacodynamics and quality of life of pegylated recombinant human arginase 1 (Peg-rhAgr1) in patients with advanced hepatocellular carcinoma (HCC). Patients were given weekly doses of Peg-rhAgr1 (1600 U/kg). Tumour response was assessed every 8 weeks using RECIST 1.1 and modified RECIST criteria. A total of 20 patients were recruited, of whom 15 were deemed evaluable for treatment efficacy. Eighteen patients (90 %) were hepatitis B carriers. Median age was 61.5 (range 30–75). Overall disease control rate was 13 %, with 2 of the 15 patients achieving stable disease for >8 weeks. The median progression-free survival (PFS) was 1.7 (95 % CI: 1.67–1.73) months, with median overall survival (OS) of all 20 enrolled patients being 5.2 (95 % CI: 3.3–12.0) months. PFS was significantly prolonged in patients with adequate arginine depletion (ADD) >2 months versus those who had ≤2 months of ADD (6.4 versus 1.7 months; p  = 0.01). The majority of adverse events (AEs) were grade 1/2 non-hematological toxicities. Transient liver dysfunctions (25 %) were the most commonly reported serious AEs and likely due to disease progression. Pharmacokinetic and pharmacodynamic data showed that Peg-rhAgr1 induced rapid and sustained arginine depletion. The overall quality of life of the enrolled patients was well preserved. Peg-rhAgr1 is well tolerated with a good toxicity profile in patients with advanced HCC. A weekly dose of 1600 U/kg is sufficient to induce ADD. Significantly longer PFS times were recorded for patients who had ADD for >2 months.

Metabolic anticancer therapy based on enzymatic arginine (Arg) deprivation (ADT) is currently being evaluated in clinical trials. The combination of ADT with low doses of the plant cytotoxic analogs of Arg, canavanine (Cav) or indospicine (Isp), have been proposed as being more efficient and selective against malignant cells. The leguminous plant Indigofera spicata contains one of the highest known amounts of Isp. Here we demonstrate for the first time that the Isp-containing ethanolic extract from I. spicata is growth-inhibiting and toxic for cultured human colorectal and ovarian carcinoma cells. The extract reduces the viability of colorectal carcinoma cells two-fold under Arg-deficient conditions and entirely abrogates their residual proliferative potential (growth recovery) after the treatment. Pre-exposure of the extract to recombinant human arginase I (rhARGI) as a therapeutic Arg-depleting agent did not impact the extract’s efficacy. Further development of Isp as a component of combinatorial anticancer metabolic targeting strategies is discussed.

Alzheimer's disease (AD) is an insidious neurodegenerative disorder representing a serious continuously escalating medico-social problem. The AD-associated progressive dementia is followed by gradual formation of amyloid plaques and neurofibrillary tangles in the brain. Though, converging evidence indicates apparent metabolic dysfunctions as key AD characteristic. In particular, late-onset AD possesses a clear metabolic signature. Considerable brain insulin signaling impairment and a decline in glucose metabolism are common AD attributes. Thus, positron emission tomography (PET) with glucose tracers is a reliable non-invasive tool for early AD diagnosis and treatment efficacy monitoring. Various approaches and agents have been trialed to modulate insulin signaling. Accumulating data point to arginase inhibition as a promising direction to treat AD via diverse molecular mechanisms involving, inter alia, the insulin pathway. Here, we use a transgenic AD mouse model, demonstrating age-dependent brain insulin signaling abnormalities, reduced brain insulin receptor levels, and substantial energy metabolism alterations, to evaluate the effects of arginase inhibition with Norvaline on glucose metabolism. We utilize fluorodeoxyglucose whole-body micro-PET to reveal a significant treatment-associated increase in glucose uptake by the brain tissue in-vivo. Additionally, we apply advanced molecular biology and bioinformatics methods to explore the mechanisms underlying the effects of Norvaline on glucose metabolism. We demonstrate that treatment-associated improvement in glucose utilization is followed by significantly elevated levels of insulin receptor and glucose transporter-3 expression in the mice hippocampi. Additionally, Norvaline diminishes the rate of Tau protein phosphorylation. Our results suggest that Norvaline interferes with AD pathogenesis. These findings open new avenues for clinical evaluation and innovative drug development.

Arginase has therapeutic potential as a cytotoxic agent in some cancers, but this is unclear for precursor B acute lymphoblastic leukaemia (pre-B ALL), the commonest form of childhood leukaemia. We compared arginase cytotoxicity with asparaginase, currently used in pre-B ALL treatment, and characterised the forms of cell death induced in a pre-B ALL cell line 697. Arginase and asparaginase both efficiently killed 697 cells and mature B lymphoma cell line Ramos, but neither enzyme killed normal lymphocytes. Arginase depleted cellular arginine, and arginase-treated media induced cell death, blocked by addition of arginine or arginine-precursor citrulline. Asparaginase depleted both asparagine and glutamine, and asparaginase-treated media induced cell death, blocked by asparagine, but not glutamine. Both enzymes induced caspase cleavage and activation, chromatin condensation and phosphatidylserine exposure, indicating apoptosis. Both arginase- and asparaginase-induced death were blocked by caspase inhibitors, but with different sensitivities. BCL-2 overexpression inhibited arginase- and asparaginase-induced cell death, but did not prevent arginase-induced cytostasis, indicating a different mechanism of growth arrest. An autophagy inhibitor, chloroquine, had no effect on the cell death induced by arginase, but doubled the cell death induced by asparaginase. In conclusion, arginase causes death of lymphoblasts by arginine-depletion induced apoptosis, via mechanism distinct from asparaginase. Therapeutic implications for childhood ALL include: arginase might be used as treatment (but antagonised by dietary arginine and citrulline), chloroquine may enhance efficacy of asparaginase treatment, and partial resistance to arginase and asparaginase may develop by BCL-2 expression. Arginase or asparaginase might potentially be used to treat Burkitt lymphoma.

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. BCT‐100 is one type of pegylated recombinant human arginase. BCT‐100 showed an anticancer effect against small cell lung cancer through oxidative stress, apoptosis, and cell cycle arrest.

Purpose: Colorectal cancer (CRC) is the third most common type of cancer worldwide, and it represents over half of all gastrointestinal cancer deaths. Knowing that cancer cells have a high proliferation rate, they require high amounts of amino acids, including arginine. In addition, several tumor types have been shown to downregulate ASS-1 expression, becoming auxotrophic for arginine. Therefore, Arginine deprivation is one of the promising therapeutic approaches to target cancer cells. This can be achieved through the use of a recombinant human arginase, HuArgI(Co)-PEG5000, an arginine degrading enzyme. Methods: In this present study, the cytotoxic effect of HuArgI(Co)-PEG5000 on CRC cell lines (HT-29, Caco-2, Sw837) is examined though cytotoxicity assays. Wound healing assays, invasion assays, and adhesion assays were also performed to detect the effect on metastasis. Results: Wound healing and invasion assays revealed a decrease in cell migration and invasion after treatment with arginase. Cells that were treated with arginase also showed a decrease in adhesion, which coincided with a decrease in RhoA activation, demonstrated though the use of a FRET biosensor to detect RhoA activation in a single cell assay, and a decrease in MMP-9 expression. Treating cells with both arginase and L-citrulline, which significantly restores intracellular arginine levels, reversed the effect of HuArgI(Co)-PEG5000 on cell viability, migration, and invasion. Conclusion: We can, therefore, conclude that colorectal cancer is partially auxotrophic to arginine and that arginine depletion is a potential selective inhibitory approach for motility and invasion in colon cancer cells.