白藜芦醇通过SIRT1 抑制假鳃鳉自发肝脏肿瘤的作用及其表观遗传调控机制

Sirtuins represent a promising new class in model organisms for tumorgenesis and aging modulation. They are NAD+-dependent protein deacetylases that are involved in a wide variety of cellular processes from cancer to ageing. SIRT1 is a critical regulator at the crossroads between cancer and aging. Exploring the underlying mechanism is of great importance and may contribute to better understanding of inhibition tumorgenesis and anti-aging for resveratrol to activate SIRT1 and regulate FoxO/p53 epigenetically. According to previous study, the applicant found for the first time that long term of the pharmaceutical treatment of resveratrol, a natural plant-derived polyphenol, prolonged lifespan of the annual fish Nothobranchius Guentheri accompanied with resistance to oxidative stress. There is a high incidence of spontaneous neoplasias in the liver of the old fish, and resveratrol has decreased the incidence of tumorgenesis with increase of SIRT1 expression. Base on these findings, now we intend to elucidate dual mechanisms of resveratrol on tumorgenesis inhibition and anti-aging. Specifically, the study is aimed at (1) defining the special tumor markers such as TP53, K-Ras, FoxO, DLC1, and related signalling pathways during initial, developmental and progression of tumorigenesis in liver of the annual fish with resveratrol supplement; (2) exploring the regulatory pathways of resveratrol on SIRT1-p53/FoxO and SIRT-DLC1 signaling；(3) defining the regulatory pathways of resveratrol on SIRT1-p53/FoxO/DLC1 signaling associated with cell apoptosis using cultured hepatoma cell lines. (4) defining the molecular mechanism of resveratrol on SIRT1/FoxO signal to decrease ROS level and postpone aging process in the short-lived fish. Finally, defining more targeted proteins both related to inhibit the progress of tumorgenesis and to postpone the program of aging by transcriptomic anylasis. The proposal will provide a novel vertebrate model and data for resveratrol as a potential cancer chemopreventive and anti-aging agents in humans.

sirtuins调控模式生物肿瘤和寿命的机理是新兴研究主题，SIRT1是肿瘤和衰老交叉点的调节因子，解析白藜芦醇激活SIRT1表观遗传调控FoxO和p53抑制动物体肿瘤发生和延缓衰老的机理是重要科学问题。申请人首次发现白藜芦醇（RES）在延长一年生贡氏假鳃鳉寿命的同时，抑制了肝脏自发肿瘤，上调了SIRT1和DLC1的表达。在此基础上，本项目拟研究RES抑制假鳃鳉肝脏肿瘤发生的作用及机理：（1）在整体动物水平，研究RES抑制肝脏肿瘤发生的组织形态改变及SIRT1、FoxO、p53、K-Ras等特异性分子变化；（2）探究RES激活SIRT1，去乙酰化FoxO/p53及上调DLC1抑制肿瘤发生的信号通路；（3）在细胞水平检验RES激活SIRT1表观遗传调控肝癌细胞凋亡的分子机制；（4）探究RES通过SIRT1/FoxO信号延缓假鳃鳉衰老的机理，建立假鳃鳉作为自发肝脏肿瘤及老年病研究的动物模型。

白藜芦醇是一种植物多酚，因其副作用小和具有抗癌作用受到广泛关注，但其抑制脊椎动物肝脏自发肿瘤产生的机制尚不清楚。本课题以一年生的贡氏假鳃鳉为模式动物，发现从中年到老年，其肝脏中自发产生结节状肿瘤，肿瘤特异性标记物TP53和K-Ras染色呈阳性，肿瘤的发生率随衰老而增加，长期喂食白藜芦醇明显抑制了肝脏自发肿瘤的发生。白藜芦醇通过激活SIRT1调节K-Ras/PI3K/AKT信号途径和FoxO1/3a的翻译后修饰、上调DLC1的表达及SIRT1与DLC1的相互结合而降低DLC1的磷酸化，促进细胞凋亡而抑制肝脏肿瘤的发生。在培养的肝癌等细胞水平上，白藜芦醇通过上调SIRT1的蛋白水平及其酶活性来减少p-PI3K、p-AKT、p-FoxO3a和Ac-FoxO1的表达水平、上调DLC1的蛋白水平及其磷酸化来诱导肝癌细胞凋亡并抑制其增殖和迁移。诱导癌细胞衰老是一种新兴的抗癌机制，白藜芦醇升高ROS水平下调DNMT1并上调DLC1的表达、以DLC1依赖性的方式诱导癌细胞衰老，也通过DLC1调控SIRT1介导FoxO3a/NF-κB通路和升高ROS水平导致DNA损伤来促进癌细胞衰老。白藜芦醇联合双氢青蒿素显著上调DLC1、下调TCTP的蛋白水平，通过DLC1/TCTP轴降低Cdc42相关的JNK/NF-κB 和 N-WASP信号通路以抑制癌细胞的迁移。在贡氏假鳃鳉的肠道中，白藜芦醇激活SIRT1，减少p-IκB和Ac-RelA/p65的表达而抑制了NF-κB的活性，通过调节细胞因子IL-8、TNF 和IL-10的表达而降低了SASP的分泌，逆转了SA-β-gal活性随衰老进程呈现的上升趋势，延缓了贡氏假鳃鳉的衰老。本项目建立了贡氏假鳃鳉可作为一种肝脏自发肿瘤的模型，阐明了白藜芦醇抑制癌细胞增殖和迁移、诱导癌细胞衰老以及脊椎动物肝脏自发肿瘤细胞凋亡的机制，为白藜芦醇的临床应用提供了依据。