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. 2014 Sep 15:41:146-56.
doi: 10.1016/j.niox.2014.03.001. Epub 2014 Mar 22.

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

Katalin Módis  1 , Ciro Coletta  1 , Antonia Asimakopoulou  2 , Bartosz Szczesny  1 , Celia Chao  3 , Andreas Papapetropoulos  2 , Mark R Hellmich  3 , Csaba Szabo  4
Affiliations

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

Katalin Módis et al. Nitric Oxide. .

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 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-dependent 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 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.

Keywords: Allosteric modulation; Bioenergetics; Colorectal cancer; Mitochondria; Proliferation.

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Figures

Figure 1
Figure 1. CBS is highly expressed in colon cancer cells and is downregulated by SAM
Representative Western blot analysis of CBS expression in cultured NCM356 (left lanes) and HCT116 (right lanes) cells. Please note the high level of expression of a native (65 kDa) band and lower level of expression of an additional, lower intensity band at 45 kDa. The Western blot analysis was done at 72 hours after culturing in the presence of either vehicle or the indicated concentrations of SAM. A blot representative of three independent determinations is shown.
Figure 2
Figure 2. SAM significantly enhances the activity of CBS in vitro.
(A) The effect of SAM (1 mM) is shown on H2S production by the recombinant human CBS enzyme, as detected by the methylene blue method, in the presence of either L-cysteine (10 mM) or the combination of L-cysteine (10 mM) and L-homocysteine (0.5 mM). Data represent mean±SEM of n=3 experiments; **p<0.01 shows a significant enhancement of H2S production by SAM. (B) The effect of SAM (0.1–3 mM) is shown on H2S production in HCT116 cell homogenates, as detected by a fluorescent H2S method, in the presence of the combination of L-cysteine (10 mM) and L-homocysteine (0.5 mM). Data represent mean±SEM of n=3 experiments; **p<0.01 shows a significant enhancement of H2S production by SAM.
Figure 3
Figure 3. Visualization of the SAM-mediated increase in H2S production in HCT116 and NCM356 cells
Cells were treated with SAM (3 mM) in the absence or presence of AOAA (1 mM) for 1 hour and intracellular H2S was detected using the AzMC fluorescent probe. Note the increase in H2S signal in response to SAM treatment, and the inhibition of both basal and SAM-stimulated H2S production by AOAA. The figures shown are representative of three independent experiments that were run in duplicates for each end-point.
Figure 4
Figure 4. Dual effect of SAM on the proliferation of colon cancer cells (HCT116) and normal mucosa cells (NCM356)
Colon cancer cell proliferation was monitored over a period of 24 hours (A and B). Results are also expressed as the area under the curve of the Cell Index (CI) over time (C and D). Short-time exposure (12 hours) to SAM (0.1–1 mM) significantly increased HCT116 cell proliferation rate in a concentration-dependent manner, but tended to decline as the concentration of SAM was further increased to 3 mM (A and C) (*p<0.05 vs corresponding vehicle). The stimulatory effect of SAM on HCT116 cell proliferation was abolished by AOAA (1 mM) (B and C). Long-term exposure (12 to 24 hours) of HCT 116 cells to relatively high concentrations of SAM caused a drop of the CI values, indicating inhibition of cell proliferation/cell death (#p<0.05 vs corresponding vehicle). Please also note that AOAA treatment reduced the basal HCT116 cell proliferation (C and D). NCM356 cells (which express relatively low CBS protein levels) grow at considerably slower rate compared to the HCT116 cells, showing almost no growth when seeded at the density of 30,000 cells/ml. A cell titration experiment (E) confirmed that the CI values depend on the density of the cell monolayer. The density of 100,000 cells/ml was then selected for further testing. Addition of SAM had no significant effect on NCM356 cell proliferation in the early phase (0–12 hours) indicating that the stimulatory effect of SAM is preferential for colon cancer cells which express high CBS levels (F and G). On the other hand, longer time exposure to SAM induced a concentration-dependent decrease in NCM356 cell proliferation (E) (#p<0.05 vs vehicle). Data represent mean±SEM of at least n=4.
Figure 5
Figure 5. Stable silencing of CBS in HCT116 cells abolishes the stimulatory effect of SAM on cell proliferation
The effect of SAM on cell proliferation (0–24h) is shown in sham-silenced (shCTL) and CBS-silenced (shCBS) HCT116 cells in the presence of vehicle or low (0.3 mM), intermediate (1 mM) and high (3 mM) concentrations of SAM. (A and B). Results were also expressed as the area under the curve CI/time (C and D). *p<0.05 indicates significant stimulatory effects of low concentration of SAM (0.3 mM) on proliferation in non-targeted cells while #p<0.05 indicates significant inhibitory effect of long-term exposure to a high concentration of SAM (3 mM) on proliferation. Please note that shCBS cells proliferate slower than sham-silenced cells, and their proliferation rate is largely unaffected by SAM. Data represent mean±SEM of n=6.
Figure 6
Figure 6. Short-term exposure to SAM stimulates mitochondrial bioenergetics in HCT116 cells
Extracellular Flux Analysis experiments assessing the effect of acute (1 h) exposure of (A) NCM356 or (B) HCT116 cells to SAM (0.1–3 mM). Panel (C) shows the mean±SEM of the increment in the FCCP-induced OCR in both cell types; #p<0.05 indicates significant differences between the responses of the NCM356 and the HCT116 cells. Data represent mean±SEM of n=9.
Figure 7
Figure 7. Long-term exposure to SAM suppresses oxygen consumption and cell viability, both in HCT116 and NCM356 cells
Data represent the effect of 72 hours of incubation with SAM (0.1, 0.3 or 1 mM) on various parameters of cell respiration (A–C), LDH release, an index of cell death (D) and cellular ATP content (E). *,** indicates significant effects of SAM in NCM356 cells (p<0.05 and p<0.01, respectively), while #,## indicates significant effects of SAM in HCT116 cells (p<0.05 and p<0.01, respectively). Data represent mean±SEM of n=9.
Figure 8
Figure 8. Stable silencing of CBS in HCT116 cells abolishes the short-term stimulatory effect of SAM on cellular bioenergetics
Oxygen consumption rate responses are shown in sham-silenced and CBS-silenced HCT116 cells in the presence of vehicle, and low (0.3 mM) and high (3 mM) concentrations of SAM at 1 hour. *p<0.05 indicates significant stimulatory effects of low concentration of SAM (0.3 mM) on cellular bioenergetics in wild-type cells, while #,## indicates significant inhibitory effect of SAM (0.3, 3 mM) on bioenergetics in cells with stable CBS silencing (p<0.05 and p<0.01, respectively). Please note that SAM no longer induces an increase in bioenergetics in shCBS cells. Data represent mean±SEM of n=9.
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