Role of 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation, Migration, and Bioenergetics in Murine Colon Cancer Cells

Abstract

3-mercaptopyruvate sulfurtransferase (3-MST) has emerged as one of the significant sources of biologically active sulfur species in various mammalian cells. The current study was designed to investigate the functional role of 3-MST's catalytic activity in the murine colon cancer cell line CT26. The novel pharmacological 3-MST inhibitor HMPSNE was used to assess cancer cell proliferation, migration and bioenergetics in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). 3-MST expression was detected by Western blotting; H 2 S production was measured by the fluorescent dye AzMC. The results show that CT26 cells express 3-MST protein and mRNA, as well as several enzymes involved in H 2 S degradation (TST, ETHE1). Pharmacological inhibition of 3-MST concentration-dependently suppressed H 2 S production and, at 100 and 300 µM, attenuated CT26 proliferation and migration. HMPSNE exerted a bell-shaped effect on several cellular bioenergetic parameters related to oxidative phosphorylation, while other bioenergetic parameters were either unaffected or inhibited at the highest concentration of the inhibitor tested (300 µM). In contrast to 3-MST, the expression of CBS (another H 2 S producing enzyme which has been previously implicated in the regulation of various biological parameters in other tumor cells) was not detectable in CT26 cells and pharmacological inhibition of CBS exerted no significant effects on CT26 proliferation or bioenergetics. In summary, 3-MST catalytic activity significantly contributes to the regulation of cellular proliferation, migration and bioenergetics in CT26 murine colon cancer cells. The current studies identify 3-MST as the principal source of biologically active H 2 S in this cell line.

Role of 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation and Cellular Bioenergetics in Human Down Syndrome Fibroblasts

“…Moreover, 3-MST silencing in adipocytes was recently reported to increase the extracellular acidification rate [46], while in endothelial cells, 3-MST silencing or HMPSNE decreased various glycolytic parameters [39]. In murine colon cancer cells, HMPSNE exerted a bell-shaped effect on glycolytic parameters (increases at the lowest inhibitor concentration used and decreases at higher concentrations) [14]. Different cell types in culture may rely to a different extent on oxidative phosphorylation vs. glycolysis, and the biochemical mechanisms regulating these processes may also be cell-type different, which may explain the lack of HMPSNE's significant effect on glycolysis in the current study.…”

“…To address this question, we employed the recently discovered 3-MST inhibitor HMPSNE, which, to date, is the most potent and most selective pharmacological inhibitor of this enzyme [10,36]. HMPSNE has been successfully employed in cell-based studies in various cell types, at concentrations similar (or higher) [14,[37][38][39] than those used in the current project. Pharmacological inhibition of 3-MST in healthy control fibroblasts only had a slight effect on cellular bioenergetic parameters, but it reduced their proliferation rate.…”

“…Extracellular flux (XF) analysis was employed for real-time quantification of oxygen consumption owing to ATP turnover, proton leak, and maximal respiratory capacity in live cells as described [13,14]. The assay was formatted in Seahorse XF24 cell-culture microplates, where diploid and 21-trisomic fibroblasts were seeded at a density of 2 × 10 4 cells per well in a total of 200 µL for 2 h. Cells were subsequently treated as described above for 24 h, and mitochondrial respiration was assessed as previously described [13,14].…”

Piperine Inhibits TGF-β Signaling Pathways and Disrupts EMT-Related Events in Human Lung Adenocarcinoma Cells

“…We also observed that the administration of sublethal concentrations of piperine disrupted classical events related to TGF-β1 induced-EMT processes, such as morphology changes, phenotypic alterations, increased cell motility and expression of metalloproteinases [66,76]. In order to monitor the effect of piperine on cell motility, we initially used the wound assay, a widely used technique [55,[77][78][79]. The results showed that piperine abrogated the increased cell motility induced by TGF-β1.…”

Cysteine Aminotransferase (CAT): A Pivotal Sponsor in Metabolic Remodeling and an Ally of 3-Mercaptopyruvate Sulfurtransferase (MST) in Cancer

“…In fact, several cancer cell lines and tumor specimens have been shown to overexpress one or more of the H 2 S-synthesizing enzymes, including MST, resulting in increased H 2 S levels, which have been proposed to promote carcinogenesis through the regulation of various cancer-related processes [ 97 ]. In the last decade, a novel concept emerged in the field of cancer biology, demonstrating that various cancer cells can increase their endogenous H 2 S levels and use it in an autocrine and paracrine manner to promote cell proliferation, cytoprotective signaling, angiogenesis, and stimulate cellular bioenergetics [ 131 ]. In fact, several biological roles regarding H 2 S in cancer cells have been proposed [ 131 ], which reinforces the importance of the CAT:MST system in cancer context.…”