Prominent tasks of cysteine cathepsins involve endo-lysosomal proteolysis and turnover of extracellular matrix constituents or plasma membrane proteins for maintenance of intestinal homeostasis. Here we report on enhanced levels and altered subcellular localization of distinct cysteine cathepsins in adenocarcinoma tissue in comparison to adjacent normal colon. Immunofluorescence and immunoblotting investigations revealed the presence of cathepsin L in the nuclear compartment in addition to its expected endo-lysosomal localization in colorectal carcinoma cells. Cathepsin L was represented as the full-length protein in the nuclei of HCT116 cells from which stefin B, a potent cathepsin L inhibitor, was absent. Fluorescence activated cell sorting analyses with synchronized cell cultures revealed deceleration of cell cycle progression of HCT116 cells upon inhibition of cathepsin L activity, while expression of cathepsin L-enhanced green fluorescent protein chimeras accelerated S-phase entry. We conclude that the activity of cathepsin L is high in the nucleus of colorectal carcinoma cells because of lacking stefin B inhibitory activity. Furthermore, we hypothesize that nuclear cathepsin L accelerates cell cycle progression of HCT116 cells thereby supporting the notion that cysteine cathepsins may play significant roles in carcinogenesis due to deregulated trafficking.
Kidney-type glutaminase [KGA/isoenzyme
glutaminase C (GAC)] is
becoming an important tumor metabolism target in cancer chemotherapy.
Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics
but limited efficacy in in vivo cancer models. To improve the in vivo
activity, we explored a bioisostere replacement of the sulfur atom
in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839
analogues with selenium using a novel synthesis of the selenadiazole
moiety from carboxylic acids or nitriles. The resulting selenadiazole
compounds showed enhanced KGA inhibition, more potent induction of
reactive oxygen species, improved inhibition of cancer cells, and
higher cellular and tumor accumulation than the corresponding sulfur-containing
molecules. However, both CB839 and its selenium analogues show incomplete
inhibition of the tested cancer cells, and a partial reduction in
tumor size was observed in both the glutamine-dependent HCT116 and
aggressive H22 liver cancer xenograft models. Despite this, tumor
tissue damage and prolonged survival were observed in animals treated
with the selenium analogue of CB839.
Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.
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