Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-b-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro Katalin Módis a,1,2 , Ciro Coletta a,1 , Antonia Asimakopoulou a,b,1 , Bartosz Szczesny a , Celia Chao c , Andreas Papapetropoulos a,b , Mark R. Hellmich c , Csaba Szabo a,⇑ a Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA b Department of Pharmacology, University of Patras, Patras, Greece c Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA article info Article history: Available online xxxx Keywords: Proliferation Bioenergetics Mitochondria Colorectal cancer Allosteric modulation abstract Recent data show that colon cancer cells selectively overexpress cystathionine-b-synthase (CBS), which produces hydrogen sulfide (H 2 S), 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 H 2 S 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 H 2 S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1–1 mM SAM increased HCT116 proliferation between 0 and 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 (1 h) exposure of HCT116 cells to SAM induced a concentration-depen- dent increase in oxygen consumption and bioenergetic function at 0.1–1 mM, while 3 mM was inhibitory. Longer-term (72 h) 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 bioen- ergetics 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 H 2 S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H 2 S 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 H 2 S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Hydrogen sulfide (H 2 S) is an important signaling molecule in- volved in the regulation of vascular tone, angiogenesis and cellular bioenergetics [1–5]. With respect to its vascular effects, multiple http://dx.doi.org/10.1016/j.niox.2014.03.001 1089-8603/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Address: Department of Anesthesiology, The University of Texas Medical Branch at Galveston, 601 Harborside Drive, Galveston, TX 77555, USA. Fax: +1 409 772 6409. E-mail address: szabocsaba@aol.com (C. Szabo). 1 Equally contributed. 2 Present address: Cardiovascular and Metabolic Research Unit, Department of Biology, Lakehead University, Thunder Bay, Ontario P7B 5E1, Canada. Nitric Oxide xxx (2014) xxx–xxx Contents lists available at ScienceDirect Nitric Oxide journal homepage: www.elsevier.com/locate/yniox Please cite this article in press as: K. Módis et al., Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-b-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro, Nitric Oxide (2014), http://dx.doi.org/10.1016/j.niox.2014.03.001