Search

Abstract: β-Hexosaminidase, involved in degradation of glycoproteins and glycosphingolipids, is altered in several tumours leading to enhanced migration capacity. To date, the expression of the β-hexosaminidase isoenzymes in prostate cancer cells has not been elucidated. By using PC3, LNCaP, DUCaP, MDAPCa 2b, and hyperplasic prostate (BPH-1) cell lines, we analysed the β-hexosaminidase activity in each cell line and determined β-hexosaminidase α subunit gene expression in PC3, LNCaP, and BPH-1. We then investigated the methylation status of the gene promoter and determined the cellular responses of PC3 and LNCaP after transfection with β-hexosaminidase α subunit. We found that each prostate cancer cell line had a decrease in total hexosaminidase activity and that the lack of hexosaminidase A activity, observed in PC3 and LNCaP cells, was associated with mRNA disappearance. The HEXA promoter region in LNCaP and PC3 cell lines had methylated CpG islands, as confirmed by 5′-Aza-2′-deoxycitidine treatment, in PC3 cells, used as cell cancer model. We also tested, the involvement of hexosaminidase A in the migration capacity by migration assay using Hex α subunit-transfected PC3. Finally, we found that, after Hex α subunit transfection, both PC3 and LNCaP were less susceptible to exogenous ceramide treatment. Results indicate a likely contribution of the lysosomal enzyme to the acquisition of cancerous features. [Copyright &y& Elsevier]

Multiple epidemiological studies have shown that individuals affected by type-2 diabetes mellitus (T2DM) carry a 2-to-5-fold higher risk of developing Alzheimer's disease (AD) when compared to non-diabetic subjects. Thus, biochemical parameters that can be easily and routinely assessed for high-confidence evaluation of diabetic conditions leading to AD (AD-T2DM) are regarded as efficient tools aimed at early diagnosis and, in turn, timely AD treatment. In this regard, the activity of lysosomal glycohydrolases may of use, in light of the implication of these enzymes in early events that underlie AD pathology and an overt correlation, in diabetes, between altered metabolic homeostasis, abnormal glycohydrolase secretion in body fluids, and occurrence of diabetic complications. Based on marked up-regulation previously shown in a peripheral, cell-based model of AD, we selected β-Galactosidase, β-Hexosaminidase, and α-Mannosidase to discriminate T2DM from AD-T2DM subjects. A screen of 109, 114, and 116 patients with T2DM, AD and AD-T2DM, respectively, was performed by testing enzyme activities in both blood plasma and peripheral blood mononuclear cells. Compared to age-matched, healthy controls (n = 122), β-Galactosidase and β-Hexosaminidase activities markedly diverged across the three groups, whereas virtually unchanged values were observed for α-Mannosidase. In particular, plasma β-Galactosidase and β-Hexosaminidase levels were higher in patients with AD-T2DM compared to those with T2DM, suggesting different mechanisms leading to enzyme secretion. Statistical analyses based on ROC curves showed that both β-Galactosidase and β-Hexosaminidase activities, either intracellular or plasma-secreted, may be used to discriminate AD patients from controls and AD-T2DM from T2DM patients. [ABSTRACT FROM AUTHOR]

Disturbed metabolism of glycosaminoglycans (GAGs) has been proposed to play an important role in the pathogenesis of late diabetic complications. The effect of diabetic complications and metabolic control on both total serum GAGs content and the serum activity of lysosomal glycosidases (N-acetyl-β-D-glucosaminidase, α-L-fucosidase, β-D-galactosidase, and α-D-mannosidase) contributing to GAGs degradation, was investigated in 48 patients with type 2 diabetes mellitus. The activity of β-D-glucosidase and acid phosphatase, the lysosomal enzymes unrelated to GAGs metabolism, was determined for comparison. The elevated serum total GAG concentration in diabetic patients was strongly and positively influenced by poor metabolic compensation of diabetes and the presence of vascular complications. A similar tendency has been shown in regard to the activity of enzymes involved in GAG degradation, especially N-acetyl-β-D-glucosaminidase, α-L-fucosidase and β-D-galactosidase. Furthermore, the total serum GAG concentrations, as well as the activity of lysosomal enzymes involved in the extracellular matrix degradation, closely followed metabolic compensation, regardless of diabetic vascular complications. Thus, we suggest that increased values of the investigated parameters may indicate the degree of endothelial cell dysfunction and may be useful to predict the development of diabetic vascular pathology. [ABSTRACT FROM AUTHOR]

The metabolic changes in the connective tissue glycosaminoglycans were studied in tissues of adjuvant induced arthritic rats. Arthritic process was induced in rats with the inoculation of Freund's adjuvant containing heat killed Mycobacterium tuberculosis in paraffin oil. The connective tissue glycosaminoglycans were fractionated into sulfated and non-sulfated glycosaminoglycans by chemical and enzymatic methods. The biosynthesis of sulfated glycosaminoglycans was examined using radioactive labeled (S)-sulfate incorporation measurements into the sulfated glycosaminoglycans in tissues such as liver, kidney, spleen and skin of arthritic rats. The catabolism of glycosaminoglycans was studied by measuring the activity of various connective tissue degrading lysosomal glycohydrolases in tissues of experimental animals. In addition, the changes in the contents of total glycosaminoglycans, mono-sulfated, highly-sulfated and non-sulfated glycosaminoglycans were quantitatively assessed in diseased tissues. Alterations in the metabolism of connective tissue glycosaminoglycans were demonstrated in tissues of arthritic rats. The uptake of (S)-sulfate into the tissue was found to be increased in liver, kidney and spleen, while that of skin decreased during the process of arthritis. The total glycosaminoglycan content was significantly elevated in diseased tissues compared to normal. Similarly, mono-sulfated, highly-sulfated and non-sulfated glycosaminoglycans were found to be increased in arthritic tissues. In addition, the activity of various connective tissue degrading lysosomal glycohydrolases such as β-glucuronidase, β-N-acetylglucosaminidase, cathepsin B, cathepsin L and collagenolytic cathepsin was increased in tissues of arthritic rat. The results presented in this communication indicate that the characteristic alterations were induced in the metabolism of glycosaminoglycans by the dynamic process of adjuvant arthritis. [ABSTRACT FROM AUTHOR]

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of β-hexosaminidase (Hex, EC 3.2.1.52) and β-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane. [ABSTRACT FROM AUTHOR]