Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.
Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.
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DOI：
10.1016/j.niox.2014.03.001
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发表时间：
2014-09-15
期刊：
影响因子：
--
通讯作者：
Szabo C
中科院分区：
文献类型：
--
作者：
Módis K;Coletta C;Asimakopoulou A;Szczesny B;Chao C;Papapetropoulos A;Hellmich MR;Szabo C
最近的数据表明，结肠癌细胞选择性过度表达胱硫醚-β-合酶（CBS），其产生硫化氢（H2S），以维持细胞生物能、支持肿瘤生长并刺激肿瘤微环境中的血管生成和血管舒张。本研究的目的是研究变构 CBS 激活剂 S-腺苷-L-甲硫氨酸 (SAM) 对表达 CBS 的结肠癌细胞系 HCT116 的增殖和生物能的影响。使用未转化的、非致瘤性结肠上皮细胞系NCM356作为对照。为了评估细胞增殖，使用了 xCELLigence 系统。通过细胞外通量分析测量生物能功能。使用人类重组 CBS 或 HCT116 匀浆的实验补充了基于细胞的研究。 SAM 显着增强了 CBS 介导的体外 H2S 产生，特别是当半胱氨酸和同型半胱氨酸组合用作底物时。向 HCT116 细胞中添加 SAM (0.1 – 3 mM) 会导致 H2S 产量呈浓度依赖性增加。 SAM 对细胞增殖发挥时间和浓度依赖性调节作用。 0.1-1 mM SAM 在 0-12 小时内增加 HCT116 增殖，而最高 SAM 浓度 (3 mM) 抑制增殖。在较长的时间段（12-24小时）内，仅使用最低浓度的SAM（0.1 mM）刺激细胞增殖；较高的 SAM 浓度产生浓度依赖性抑制。 SAM 的短期刺激作用可通过 CBS 抑制剂氨氧乙酸 (AOAA) 或 CBS 的稳定沉默来减弱。相反，SAM 对细胞增殖的抑制作用不受 CBS 抑制或 CBS 沉默的影响。与HCT116细胞相比，低CBS表达NCM356细胞的较低增殖率不受SAM影响。 HCT116 细胞短期（1 小时）暴露于 0.1-1 mM 的 SAM 中，可诱导耗氧量和生物能功能出现浓度依赖性增加，而 3 mM 则具有抑制作用。 HCT116 细胞长期（72 小时）暴露于所有测试浓度的 SAM 中，可抑制线粒体耗氧率、细胞 ATP 含量和细胞活力。在 CBS 稳定沉默的细胞中，SAM 对生物能的刺激作用减弱，而抑制作用不受影响。在 NCM356 细胞中，SAM 对细胞生物能量学的影响比在 HCT116 细胞中更小。我们还在 HCT116 和 NCM356 细胞中观察到 CBS 因长期暴露于 SAM 而下调。综上所述，结果表明 HCT116 细胞中 H2S 的产生受到变构 CBS 激活剂 SAM 的刺激。在低到中等水平和早期阶段，产生的 H2S 可作为内源性癌细胞生长和生物能因子。相反，SAM 对细胞增殖和生物能功能的抑制似乎与 CBS 过度刺激引起的 H2S 不利自分泌作用无关，而是与 CBS 无关的药理作用有关。
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.
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DOI：
10.1161/atvbaha.110.209783
发表时间：
2010-10-01
影响因子：
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作者：
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