Human malignant mesothelioma (MM) is an aggressive cancer linked to asbestos and erionite exposure. We previously reported that High-Mobility Group Box-1 protein (HMGB1), a prototypic damage-associated molecular pattern, drives MM development and sustains MM progression. Moreover, we demonstrated that targeting HMGB1 inhibited MM cell growth and motility in vitro, reduced tumor growth in vivo, and prolonged survival of MM-bearing mice. Ethyl pyruvate (EP), the ethyl ester of pyruvic acid, has been shown to be an effective HMGB1 inhibitor in inflammation-related diseases and several cancers. Here, we studied the effect of EP on the malignant phenotype of MM cells in tissue culture and on tumor growth in vivo using an orthotopic MM xenograft model. We found that EP impairs HMGB1 secretion by MM cells leading to reduced RAGE expression and NF-κB activation. As a consequence, EP impaired cell motility, cell proliferation, and anchorage-independent growth of MM cells. Moreover, EP reduced HMGB1 serum levels in mice and inhibited the growth of MM xenografts.Our results indicate that EP effectively hampers the malignant phenotype of MM, offering a novel potential therapeutic approach to patients afflicted with this dismal disease.

The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 59-phosphate but not a-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 59-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent K m and V max values of 1.16 3 10 24 M and 5.05 3 10 25 mol s 21 mg 21 , respectively. The presence of other aromatic amino acids, including L-tyrosine, L-phenylalanine, and L-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.

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