Effect of S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro

Abstract

Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1 – 3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time-and concentration-dependent modulatory effects on cell proliferation. At 0.1–1 mM SAM increased HCT116 proliferation between 0–12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12–24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1–1 mM, while 3 mM was inhibitory. Longer-term (72h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects.

The Therapeutic Potential of Cystathionine β-Synthetase/Hydrogen Sulfide Inhibition in Cancer

“…Taken together, these data indicate that the stimulatory effects of SAM are mainly related to the stimulation of the CBS pathway, while the inhibitory effects seen with higher concentrations/longer exposures of SAM are due to a significant CBS/H 2 S-independent cytotoxic component. Based on these data, inhibition of cell proliferation by SAM does not appear to represent a tumor cell-selective approach (79). The shRNA approach inhibited the expression of both CBS and CSE genes at the protein level, as shown by Western blotting (inset).…”

“…Aminooxyacetic acid (AOAA) (1 mM) blocked the H 2 S-producing activity of CBS in the tissue extracts (*p < 0.05 vs. corresponding from normal mucosa and # p < 0.05 vs. vehicle), whereas the CSE inhibitor propargylglycine (PAG) (3 mM) had no significant effect. HCT116 cells exhibited the highest rate of H 2 S production, as measured by the methylene blue method in cell lysates (* We have studied the proliferative and bioenergetic effects of the CBS activator, SAM, in HCT116 cells and the nontumorigenic colonic epithelial cell line NCM356 (79). As expected, SAM exerted only minor stimulatory effects on the proliferation and energetics of NCM356 cells, which expressed low levels of CBS and produced low levels of H 2 S, which was increased by SAM to a slight degree.…”

S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase

“…Defects in this regulatory mechanism are currently considered as playing a role in the pathogenesis of classic homocystinuria (27,28), the inherited error of metabolism associated with CBS deficiency. More recently, AdoMet has been shown to markedly modulate the proliferation of colorectal cancer cells as compared with non-tumorigenic cells (29).…”

Down-regulation of cystathionine-γ-lyase/H2S system inhibits cell growth in human breast cancer MDA-MB-231 cells

“…Studies on H 2 S are involved in many scientific fields, including many physiological and pathological processes Current studies on H2S also mainly focus on its functions in physiological and pathophysiological processes [11]. We do not have a good understanding on the functions of H 2 S in cancer field and previous studies possess a little controversy, which is exogenous H 2 S could induce HCT116 cell proliferation, but inhibit WiDr cell growth [12][13]. So the functions of H 2 S in cancer field need to be further investigated.…”