Abstract
γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 μm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.
Highlights
␥-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis
In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(N)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 M, when we fixed the concentrations of the donor substrate
Cellular process–specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells
Summary
Enzyme assays were carried out using both human GGT (hGGT) isolated from membranes of human liver cancer cell line HepG2 or chronic B leukemic cells HG3 cells and the commercial equine kidney GGT (eqGGT; with a high percentage of identity with hGGT), maintaining fixed and saturating concentrations of ␥-glutamyl-para-nitroanilide (GpNa) and the acceptor glycyl-glycine (GlyGly) and varying the concentrations of the different testing compounds. Residual GGT activity in the presence of ovothiol A (ovo), isolated from P. lividus eggs in its disulfide form [41], was compared with that in the presence of the trimethyl-2-thiohistidine ergothioneine (erg) stabilized in thione form, the previously characterized GGT inhibitor DON [15], and dithiothreitol (DTT), used as a negative control (Fig. 2). Under these conditions, 50% GGT inhibition was obtained at 16 M for ovo compared with 282 M for DON, which was abandoned in clinical trials for toxicity [21], and with 297 M for erg. Similar results were obtained with eqGGT in the presence of the different compounds. Data of apparent Ki were obtained as reported under “Experimental procedures” and are expressed as mean Ϯ S.D. (n ϭ 3)
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