A phase II clinical study on the efficacy and predictive biomarker of pegylated recombinant arginase on hepatocellular carcinoma.

BACKGROUND: Pegylated recombinant human arginase (PEG-BCT-100) is an arginine depleting drug. Preclinical studies showed that HCC is reliant on exogenous arginine for growth due to the under-expression of the arginine regenerating enzymes argininosuccinate synthetase (ASS) and ornithine transcarbamylase (OTC). METHODS: This is a single arm open-label Phase II trial to assess the potential clinical efficacy of PEG-BCT-100 in chemo naïve sorafenib-failure HCC patients. Pre-treatment tumour biopsy was mandated for ASS and OTC expression by immunohistochemistry (IHC). Weekly intravenous PEG-BCT-100 at 2.7 mg/kg was given. Primary endpoint was time to progression (TTP); secondary endpoints included radiological response as per RECIST1.1, progression free survival (PFS) and overall survival (OS). Treatment outcomes were correlated with tumour immunohistochemical expressions of ASS and OTC. RESULTS: In total 27 patients were recruited. The median TTP and PFS were both 6 weeks (95% CI, 5.9-6.0 weeks). The disease control rate (DCR) was 21.7% (5 stable disease). The drug was well tolerated. Post hoc analysis showed that duration of arginine depletion correlated with OS. For patients with available IHC results, 10 patients with ASS-negative tumour had OS of 35 weeks (95% CI: 8.3-78.0 weeks) vs. 15.14 weeks (95% CI: 13.4-15.1 weeks) in 3 with ASS-positive tumour; expression of OTC did not correlate with treatment outcomes. CONCLUSIONS: PEG-BCT-100 in chemo naïve post-sorafenib HCC is well tolerated with moderate DCR. ASS-negative confers OS advantage over ASS-positive HCC. ASS-negativity is a potential biomarker for OS in HCC and possibly for other ASS-negative arginine auxotrophic cancers. TRIAL REGISTRATION NUMBER: NCT01092091. Date of registration: March 23, 2010.

Microarray analyses of oral punch biopsies from acute myeloid leukemia (AML) patients treated with chemotherapy.

OBJECTIVE: Understanding the pathogenesis of chemotherapy-induced oral mucositis (CIOM) is vital to develop therapies for this common, dose-limiting side effect of cancer treatment. We investigated molecular events in CIOM from buccal mucosa tissue collected before and 2 days after chemotherapy from patients with acute myeloid leukemia (AML) and healthy controls by microarray analysis. METHODS: Microarray analysis was performed using Human Genome U133 Plus 2.0 Array on buccal mucosa punch biopsies from patients with AML before (n = 4) or after chemotherapy (n = 4), and from healthy controls (n = 3). Following Robust Multichip Average (RMA) normalization, we applied Linear Models for Microarray data (LIMMA) and Significance Analysis of Microarrays (SAM) for data analysis using the TM4/TMeV v4.5.1 program. RESULTS: LIMMA and SAM identified genes potentially affected by the presence of AML, including homeodomain-interacting protein kinase 1 (HIPK1), mex-3 homolog D (MEX3D), and genes potentially affected by chemotherapy, including argininosuccinate synthase 1 (ASS1), notch homolog 1 (NOTCH1), zinc transporter ZIP6 (SLC39A6), and TP53-regulated inhibitor of apoptosis 1 (TRIAP1). The expression of 2 genes with potential biological significance in oral mucositis, ASS1 and SLC39A6 (alias LIV-1), was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). CONCLUSIONS: Our results suggest that AML-specific deregulated immune responses and inflammatory tissue damage to the oral mucosa caused by chemotherapy may not be overcome by the natural cellular repair processes and therefore contribute to CIOM.

Effects of acute ammonia toxicity on nitric oxide (NO), citrulline-NO cycle enzymes, arginase and related metabolites in different regions of rat brain.

Nitric oxide (NO) is involved in many pathophysiological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS) enzymes. Citrulline formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) via the citrulline-NO cycle. Hyperammonemia is known to cause poorly understood perturbations of the citrulline-NO cycle. To understand the role of citrulline-NO cycle in hyperammonemia, NOS, ASS, ASL and arginase activities, as well as nitrate/nitrite (NOx), arginine, ornithine, citrulline, glutamine, glutamate and GABA were estimated in cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of rats subjected to acute ammonia toxicity. NOx concentration and NOS activity were found to increase in all the regions of brain in acute ammonia toxicity. The activities of ASS and ASL showed an increasing trend whereas the arginase was not changed. The results of this study clearly demonstrated the increased formation of NO, suggesting the involvement of NO in the pathophysiology of acute ammonia toxicity. The increased activities of ASS and ASL suggest the increased and effective recycling of citrulline to arginine in acute ammonia toxicity, making NO production more effective and contributing to its toxic effects.

Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNgamma, and LPS.

There is evidence that nitric oxide (NO) may mediate some of the functional myocardial changes caused by bacterial LPS and inflammatory cytokines. The expression of the inflammatory or inducible NO synthase (iNOS) in human cardiac myocytes, however, has not been well characterized. Therefore, we treated cultured, dedifferentiated human ventricular cardiac myocytes with the combination of TNF-alpha (500 U/ml), IL-1beta (30U/ml), IFNgamma (100 U/ml), and LPS (E.coli 0111:B4, 10 microg/ml). Northern blot analysis revealed a approximately 4.5 kb transcript for inducible NOS (iNOS) in the stimulated human heart cells but not in untreated cells. RT-PCR confirmed that iNOS mRNA was only present in stimulated cells. However, treatment of the myocytes for up to 96 h with cytokines and LPS did not result in NO synthesis as measured by nitrite + nitrate accumulation in the culture medium, and no iNOS enzymatic activity could be detected in the cell lysates. Western blot analysis failed to detect iNOS protein. Thus, despite high and persistent levels of iNOS mRNA in cytokine-treated cells, iNOS protein was absent in this experimental model. GTP-cyclohydrolase I was induced both at the mRNA and protein levels and resulted in increased biopterin levels, indicating sufficient amounts of the cofactor tetrahydrobiopterin (BH4) were present, and that the failure to express an inducible protein was specific to iNOS. To determine if the absence of iNOS protein was due to a novel cardiac iNOS gene or modified iNOS transcript in human myocytes, we cloned an iNOS cDNA from cytokine-treated myocytes. Sequencing and expression of the clone revealed a functional iNOS cDNA with >99% identity to other human iNOS cDNA clones. When human cardiac cells were transduced with a retroviral vector carrying only the coding region of the human hepatocyte iNOS cDNA, both iNOS mRNA and protein could be detected. In conclusion, these cells derived from cultured human cardiac myocytes lacked the capacity to express an endogenous iNOS protein, the basis of which appears to be a cell-specific suppression or failure of iNOS translation.