Fang Mingzhu, Guo Wei-Ren, Park Youngil, Kang Hwan-Goo, Zarbl Helmut
Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.
School of Public Health, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.
Oncotarget. 2015 Dec 15;6(40):42879-91. doi: 10.18632/oncotarget.6002.
We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis.
我们之前报道过，膳食中的甲基硒代半胱氨酸（MSC）通过在肿瘤发生早期重置被致癌物破坏的昼夜节律基因表达，抑制N-甲基-N-亚硝基脲（NMU）诱导的乳腺肿瘤发生。为了研究其潜在机制，我们构建了一个昼夜节律报告系统，该系统由人乳腺上皮细胞组成，带有由核心昼夜节律基因人周期蛋白2（PER2）启动子驱动的荧光素酶报告基因。在这个体外模型中，NMU以与SIRT1特异性抑制剂类似的模式破坏细胞昼夜节律；相反，MSC恢复了被NMU破坏的昼夜节律，并对SIRT1抑制剂具有保护作用。此外，NMU以剂量依赖的方式抑制细胞内NAD⁺/NADH比值，并降低NAD⁺依赖性SIRT1活性，而MSC恢复了NMU处理细胞中的NAD⁺/NADH和SIRT1活性，表明NAD⁺-SIRT1途径是NMU和MSC的作用靶点。在大鼠乳腺组织中，致癌剂量的NMU也破坏了NAD⁺/NADH振荡并降低了SIRT1活性；膳食中的MSC恢复了NMU处理大鼠乳腺中的NAD⁺/NADH振荡并增加了SIRT1活性。MSC诱导的SIRT1活性与乳腺组织中Per2启动子E-盒处BMAL1乙酰化减少和组蛋白3赖氨酸9乙酰化增加相关。SIRT1活性的变化分别与NMU处理或MSC挽救的大鼠乳腺中节律性Per2 mRNA表达的丧失或恢复在时间上相关。与我们之前的研究结果一起，这些结果表明，增强NAD⁺依赖性SIRT1活性通过在乳腺肿瘤发生早期恢复昼夜节律基因表达的表观遗传调控，有助于MSC的化学预防功效。
