The effect of 3-bromopyruvate on the properties of cathepsin B in the aspect of metastatic potential of colon cancer cells

Background: Cathepsin B (CTSB, EC 3.4.22.1) is a protease that physiologically resides in lysosomes and whose biosynthesis, cell surface location, intracellular distribution, and enzymatic activity undergo changes during the pathogenesis of cancer; it plays an important role in metastasis. Due to its active center structure, it is theoretically susceptible to the action of 3-bromopyruvate - an analogue of pyruvic acid and an alkylator that has been studied in depth in recent years for its anti-cancer activity, mainly through the inhibition of glycolytic enzymes.

Objectives: To investigate the effects of 3-bromopyruvate on the tumor cell properties in selected colorectal carcinoma cell lines that are widely attributed to the dysregulation of CTSB. Moreover, the effect of direct action of 3-bromopyruvate on the CTSB molecule was investigated in vitro.

Material and methods: The research on the effect of 3-bromopyruvate on Caco-2/HCT 116 cells and purified human CTSB included a scratch/wound healing assay, a cell invasion assay, spectrofluorimetric measurements of enzymatic activity of cathepsin B, indirect immunofluorescence and flow cytometry, zymography, and liquid chromatography/mass spectrometry methods.

Results: 3-bromopyruvate reduced the activity and secretion of active CTSB and lowered the motility and invasiveness of Caco-2/HCT 116 human colorectal cancer cells. It decreased the exposure of CTSB on the outer surface of the cell membrane in both cell lines. 3-bromopyruvate inhibited the activity of CTSB reversibly and did not alkylate the molecule of the enzyme.

Conclusions: This is the first report on the effect of 3-bromopyruvate directly on CTSB and indirectly on the mechanisms leading to its distinct pathophysiological properties, resulting in increased metastatic potential of cancer cells, among others. Although detailed mechanisms of the interaction between 3-bromopyruvate and the active site of CTSB require further research, the results provide a new perspective from which to study the antitumor effect of 3-bromopyruvate.

Keywords: 3-bromopyruvate; Caco-2 cells; HCT 116 cells; cathepsin B; protease inhibitor.