EGCG对肺癌细胞WIF-1基因启动子区的去甲基化作用

目的 Wnt信号通路在许多恶性肿瘤包括肺癌的发生发展过程中发挥作用,WIF-1可以抑制Wnt通路的活性,但启动子区高甲基化导致WIF-1基因沉默,使Wnt信号通路异常激活并引起肿瘤.EGCG是一种去甲基化因子,本实验拟验证其去甲基化和对WIF-1基因的再激活作用.方法 利用甲基化特异性PCR,DNA测序及RT-PCR分析技术来测定WIF-1基因启动子区的去甲基化及WIF-1的表达,利用Westernblotting分析技术测定细胞质β-catenin蛋白的表达.结果 EGCG的浓度在0～50 μM时,在72 h培养期内对H460、A549及HCT116细胞系无细胞毒作用.在H460及A549细胞系内发现启动区域的甲基化.EGCG处理H460后WIF-1启动区域甲基化水平由77.6％降至27.6％ （P ＜0.05）,而处理A549后则由76.5％降至28.6％ （P ＜0.05）.使用EGCG后显示肺癌细胞系WIF-1启动子区去甲基化及WIF-1表达恢复.结论 EGCG在逆转WIF-1基因启动子甲基化并再激活WIF-1基因中有潜在的治疗作用.     

Wnt信号通路为靶点的肿瘤治疗新进展

Wnt信号传导通路与肿瘤的关系一直都是近年来肿瘤研究的热点,Wnt信号通路包含许多信号成员蛋白,其中任何一个成员蛋白的突变或异常均可激活Wnt信号通路引起细胞异常增殖而导致肿瘤的发生。为此,针对Wnt信号通路不同基因靶点的高特异性基因药物开发以及肿瘤的分子诊断也相继出现。目前,Wnt信号通路为靶点的肿瘤基因治疗包括细胞膜水平、胞内通路成员蛋白水平、β-catenin水平和核内TCF/LEFs-β-catenin复合体水平。     

经典Wnt信号通路在原发性肝癌发生中的作用

原发性肝癌（HCC）是世界第5大肿瘤,死亡率高,其发病率呈上升趋势。研究表明,多种信号通路参与了HCC的发生发展如p53信号通路、Rb信号通路、经典Wnt信号通路等[2]。Wnt基因家族编码蛋白的异常表达可引起经典Wnt信号通路的激活,进而导致核心因子β-catenin稳态并定位于核内,激活下游靶基因的转录,与肿瘤发生有关。了解该通路在HCC发生发展中的作用有重要意义。     

大肠癌发生与分泌型卷曲相关蛋白2基因甲基化的关系

异常的Wnt信号通路在90%的大肠癌中是个早期事件,它促成肿瘤细胞的成长、增殖和凋亡[1].Wnt拮抗物分泌型卷曲相关蛋白(SFRPs)具有抗Wnt/Frizzled(FZ)信号的功能,激活Wnt信号并持续整个大肠癌进展过程[2].我们应用甲基化荧光定量聚合酶链反应(PCR)分析了大肠癌发生发展过程中SFRF2基因的甲基化状态,并与大肠癌临床病理学特征进行了比较.     

膀胱癌WIF-1启动子甲基化状态及其与膀胱癌临床病理关系的研究

目的:研究Wnt抑制因子1(WIF-1)启动子甲基化状态对WIF-1蛋白表达的影响及其与膀胱癌的临床病理关系。方法:采用甲基化特异性PCR方法和免疫组织化学方法检测57例膀胱癌WIF-1启动子甲基化状态和WIF-1蛋白表达情况。以正常膀胱黏膜及腺性膀胱炎组织为对照。结果:膀胱癌、腺性膀胱炎和正常膀胱组织WIF-1启动子甲基化率分别为57.9%,15.0%和0,差异有统计学意义(P<0.01)。WIF-1启动子甲基化阳性率随肿瘤分级、分期的增加逐渐升高(G1、G2、G3级分别为20.0%、56.3%和86.7%;浅表性和浸润性肿瘤分别为43.8%和76%,P<0.01)。膀胱癌组织WIF-1基因启动子甲基化时WIF-1蛋白表达率明显低于未甲基化组(P<0.001,Kappa值为0.783)。WIF-1甲基化与未甲基化组膀胱癌患者之间的5年总生存率分别为60.6%和91.7%(P<0.05)。结论:膀胱癌WIF-1蛋白表达缺失或减少与WIF-1启动子甲基化密切相关。WIF-1启动子甲基化可能是膀胱肿瘤发生的早期事件,而且与肿瘤的进展有关,可作为膀胱癌患者预后判断分子标志物。     

Wnt信号通路对软骨和软骨下骨双靶向调控及其在骨关节炎进展中的作用

目的综述Wnt信号通路在骨关节炎(osteoarthritis,OA)发生发展中的活性变化,及其对软骨、软骨下骨的双靶向调控和两者间信息交流对OA进程的影响和机制。方法查阅近年在体内外实验研究及临床研究中,OA和非OA状态下Wnt信号通路对关节软骨、软骨下骨调控作用及软骨与软骨下骨间信息交流的相关文献,并对其作用机制进行分析总结。结果 Wnt信号可通过依赖β-catenin的经典或不依赖β-catenin的非经典Wnt信号通路及其与其他信号通路的交联,调控软骨细胞、成骨细胞的分化和功能,进而影响软骨及骨的代谢。过度激活Wnt信号可加重软骨OA样退变,并且Wnt信号通路可激活下游蛋白Wnt1诱导的信号通路蛋白1调控OA进展,还可通过软骨及软骨下骨中不同细胞间建立的缝隙连接,直接进行分子交流调控OA的发生发展。关节腔内注射Wnt信号通路抑制剂SM04690可以抑制OA进程;在成骨细胞中过表达Wnt信号通路抑制剂Dickkopf可以拮抗VEGF对软骨细胞的作用,并抑制其基质的分解代谢。结论 Wnt信号通路及其与其他信号分子的交互作用,可调控软骨和软骨下骨的代谢和功能及两者间信息交流,因此在OA的发生发展中发挥重要作用,有望成为OA治疗的新靶点。     

P120连环蛋白/转录抑制因子Kaiso与Wnt信号通路在肿瘤发病机制中的作用

异常的经典的Wnt信号通路的激活可以促进肿瘤的发生。P120连环蛋白/转录抑制因子Kaiso对Wnt信号通路的靶基因具有调控作用,影响Wnt信号通路的活动,从而影响肿瘤发生的过程。     

Wnt/β-catenin信号通路与骨病——作为药物靶标的潜力

分泌性蛋白家族Wnt蛋白在调节细胞生长、分化、功能及死亡等方面起着重要作用。Wnt蛋白家族激活信号时,它的经典通路Wnt/β-catenin信号通路通过一系列机制调节骨和软骨代谢,如刺激干细胞复制、促进软骨细胞成熟、诱导成骨细胞发生、阻碍成骨细胞和骨细胞凋亡等。该文就骨生物学中Wnt/β-catenin通路的研究现状、与该通路相关骨病、将该通路作为药物靶点的潜力等作一综述。     

Wnt/β-catenin信号通路与骨病——作为药物靶标的潜力

分泌性蛋白家族Wnt蛋白在调节细胞生长、分化、功能及死亡等方面起着重要作用。Wnt蛋白家族激活信号时,它的经典通路Wnt/β-catenin信号通路通过一系列机制调节骨和软骨代谢,如刺激干细胞复制、促进软骨细胞成熟、诱导成骨细胞发生、阻碍成骨细胞和骨细胞凋亡等。该文就骨生物学中Wnt/β-catenin通路的研究现状、与该通路相关骨病、将该通路作为药物靶点的潜力等作一综述。     

Wnt拮抗物分泌型卷曲相关蛋白2与大肠癌的研究进展

Wnt蛋白对正常细胞增殖起调整促进作用,改变发生在大多数大肠癌的Wnt信号途径导致β-链蛋白的积聚,分泌型卷曲相关蛋白(SFRP)结合Wnt并通过这条途径抑制信号。当SFRP基因甲基化时,Wnt信号途径激活,蛋白质的机能随之出现异常,使癌细胞不断增殖,导致大肠癌发生。90％的大肠癌患者与SFRP基因甲基化有关,而甲基化SFRP2是最敏感的大肠癌诊断标志物之一。在人类粪便中检测SFRP2基因的甲基化为检测和研究大肠癌提供一种新的策略。     

Inhibition of Wnt signaling downregulates Akt activity and induces chemosensitivity in PTEN-mutated prostate cancer cells

BACKGROUND: The cross-talk between Wnt signaling and the Akt pathway in prostate cancer (Pca) is still unclear. In the present study, we found that WIF-1 downregulates the Akt pathway and also enhances chemosensitivity in PTEN-null Pca cells. METHODS: Wnt inhibitory factor-1 (WIF-1), an inhibitor of Wnt proteins, was transfected into PC-3 and DU145 Pca cells. RESULTS: Akt was phosphorylated in PTEN-null PC-3 cells but underphosphorylated in PTEN-expressed DU145 cells. The levels of phosphorylated Akt in WIF-1 overexpressing PC-3 cells were lower than those in native or control vector-transfected PC-3 cells. However, WIF-1 showed no additional inhibition of already reduced Akt activity in DU145 cells. Overexpression of WIF-1 resulted in sensitizing PC-3 cells for paclitaxel to induce apoptosis. DU145 cells were more sensitive to paclitaxel but were not affected by WIF-1 transfection. The PI3K inhibitor LY294002 seemed to restore the chemosensitivity of native PC-3 cells like WIF-1 did. CONCLUSIONS: Our results show that Wnt signaling is involved in Akt activation in Pca cells. Our data also indicate the possibility that Wnt and its signaling pathway can be therapeutic targets for PTEN-mutated advanced Pca.     

赖氨酸特异性去甲基化酶1介导的经典Wnt信号通路在肿瘤领域的研究进展

作为全球性公共卫生问题,肿瘤是当今世界危害人类健康的主要疾病之一。根据2019年国家癌症中心发布的全国最新癌症数据统计,2015年我国共新发恶性肿瘤392.9万例,死亡约为233.8万例,发病率约为285.83/10万,病死率约为170.05/10万。肿瘤的发生、发展是一个多因素、多基因、多阶段渐进性累积的演变过程,涉及肿瘤的转化、生存、增殖、侵袭、血管生成和转移。在这个过程中伴随着遗传基因和表观遗传的变化:致癌基因、抑癌基因、错配修复基因、细胞黏附分子等在DNA、RNA和蛋白质水平发生改变。虽然近年来肿瘤诊断与治疗技术不断取得进步,但大多数患者就诊时已处于晚期状态,总体预后较差。因此探索肿瘤的发病机制,寻找更为有效的预防治疗手段具有重要意义。现有研究表明,表观遗传学改变在肿瘤发生、发展及侵袭转移中意义重大。目前已知的表观遗传修饰主要包括组蛋白修饰、DNA甲基化、核小体重塑、非编码RNA等。在真核生物中,组蛋白修饰包括了乙酰化、甲基化、磷酸化、核糖化及泛素化等。同其它组蛋白修饰一样,组蛋白甲基化是一个动态可逆的过程。赖氨酸特异性去甲基化酶1(LSD1)能够特异性催化组蛋白H3第4位赖氨酸(H3K4)和第9位赖氨酸(H3K9)的脱一甲基、二甲基反应,并与组蛋白去乙酰化酶相互作用,起到转录阻遏物的作用。该酶对哺乳动物生长发育至关重要并参与多种生物学过程,包括细胞分化、异染色质的形成、细胞内DNA甲基化状态的合理维持以及诱导多能干细胞形成等。目前证实LSD1在多种恶性肿瘤组织中高度表达,在肿瘤的发生、发展及耐药性产生中起到重要作用。Wnt信号通路是一条在进化上高度保守的信号通路,对细胞增殖、分化、迁移及凋亡起着重要作用,Wnt信号通路关键分子的基因突变在肿瘤的发生、发展过程中具有重要作用。虽然LSD1和Wnt信号通路都与肿瘤发生、发展有关,但两者之间是否存在联系尚未阐明。近年来越来越多的研究表明,LSD1可通过调节经典Wnt信号通路的活性影响肿瘤的发生、发展。笔者就LSD1介导的经典Wnt信号通路在肿瘤领域的研究进展作一综述。     

5-杂氮脱氧胞苷逆转膀胱癌细胞WIF-1基因转录表达的研究

目的 探讨去甲基化药物5-杂氮脱氧胞苷对膀胱癌WIF-1基因表达的影响及其生物学效应.方法 用5-杂氮脱氧胞苷处理膀胱癌细胞株T24和BIU87,RT-PCR检测WIF-1 mRNA表达情况,免疫细胞化学和Western blot检测WIF-1蛋白表达变化,甲基化特异性PCR检测WIF-1启动子甲基化状态变化,MTT法检测5-杂氮脱氧胞苷对膀胱癌细胞的增殖抑制作用,TUNEL法和流式细胞仪检测细胞凋亡率.结果 5-杂氮脱氧胞苷处理72 h后,WIF-1 mRNA及蛋白表达明显增加,而且恢复为启动子未甲基化状态,膀胱癌细胞增殖明显抑制,FCM法检测T24和BIU87的凋亡率分别为(18.2±2.6)%和(17.3±2.7)%.结论 DNA甲基化是导致WIF-1不表达的重要原因,5-杂氮脱氧胞苷可以通过逆转WIF-1甲基化状态而恢复WIF-1表达,从而抑制膀胱癌细胞的生长并诱导细胞凋亡.5-杂氮脱氧胞苷有望成为膀胱癌的有效辅助化疗药.     

TC-1 (c8orf4) enhances aggressive biologic behavior in lung cancer through the Wnt/β-catenin pathway

Background

The thyroid cancer-1 (TC-1) or c8orf4 gene encodes a 106-residue naturally disordered protein that has been found to be associated with thyroid, gastric, and breast cancer. A recent study has indicated that the protein functions as a positive regulator in the Wnt/β-catenin signaling pathway in human breast cancer. However, no research has been done in the area of lung cancer. Therefore, the goal of the present study was to confirm the relationship among TC-1, lung cancer, and the Wnt/β-catenin signaling pathway.

Materials and methods

The expression of TC-1 was immunohistochemically examined in 147 patients with non–small-cell lung cancer. TC-1–overexpressed and silenced A549 cells were infected using lentivirus and MTT cell proliferation analysis, and Matrigel invasion assays and scratch-wound assays were performed to confirm the biologic behavioral changes in different A549 cell subsets. The Wnt/β-catenin signaling pathway, key gene β-catenin, target genes of vascular endothelial growth factor, cyclin D1, matrix metalloproteinase-7, c-myc, and survivin were tested at the mRNA and protein level.

Results

TC-1 was detected in 97 of the 147 non–small-cell lung cancer primary tumor specimens, and its expression correlated with the TNM stage and regional lymph node metastasis (P < 0.01). In vitro experiments demonstrated that TC-1 expression affected both proliferation and invasion in the A549 cell line. Furthermore, expression of TC-1 protein affected the Wnt/β-catenin signaling pathway’s downstream genes, such as vascular endothelial growth factor and matrix metalloproteinase-7, at the mRNA and protein level.

Conclusions

TC-1 expression is associated with aggressive biologic behavior in lung cancer and might coordinate with the Wnt/β-catenin pathway as a positive upstream regulator that induces these behaviors.     

Wnt通路激活的结直肠癌细胞抑制肿瘤浸润性树突细胞成熟的机制研究

［摘要］ 目的 大部分肠癌存在Wnt通路激活,基础研究发现Wnt通路可影响肿瘤免疫,但其在结肠癌中的作用不明。本研究主要探讨Wnt通路激活对结肠癌免疫微环境的影响以期发现结直肠癌免疫治疗新靶点。方法 免疫荧光技术验证SW480和NCM460 Wnt通路状态;qPCR和Western blot技术对比Wnt通路激活的结肠癌细胞系SW480和正常肠上皮细胞NCM460 IL?1β表达水平的不同;分离人外周血单核细胞,用集落刺激因子（GM?CSF）和IL?4联合诱导形成未成熟树突细胞（iDC）,再加入IL?1β刺激诱导成熟树突细胞（mDC）,用流式细胞技术检测mDC细胞表面成熟分子表达量。结果 SW480细胞核表达beta?catenin,提示Wnt通路激活。使用Wnt通路抑制剂（ICG?001）处理SW480后IL?1β mRNA和蛋白表达水平均升高,且随浓度升高而升高,使用wnt通路激活剂（Licl）处理NCM460后IL?1βmRNA和蛋白表达水平均低于对照组,且随浓度升高而降低,且IL?1β蛋白表达量均与非磷酸化β?连环蛋白（active β?catenin）表达量呈负相关关系。IL?1β可促进iDC成熟,IL?1β 50 ng/mL组HLA?DR、CD86表达率分别为（69±3.62）%、（84.3±4.7）%,与RPMI组及TNF?a 10 ng/mL组相比,表达显著上调（P<0.001）。结论 Wnt通路激活的结直肠癌可能通过减少IL?1β的表达来阻碍肿瘤微环境中DC的成熟。     

Wnt5a as an Effector of TGFβ in Mammary Development and Cancer

Wnt5a is a member of the Wingless-related/MMTV-integration family of secreted growth factors, which are involved in a wide range of cellular processes. Wnt signaling can be broadly divided into two categories the canonical, ß-catenin-dependent pathway and the non-canonical ß-catenin-independent pathway. Wnt5a is a non-canonical signaling member of the Wnt family. Loss of Wnt5a is associated with early relapse of invasive breast cancer, increased metastasis, and poor survival in humans. It has been shown that TGF-ß directly regulates expression of Wnt5a in mammary gland and that Wnt5a mediates the effects of TGF-ß on branching during mammary gland development. Here we review the evidence suggesting Wnt5a acts as an effector of TGF-ß actions in breast cancer. It is suggested that the tumor suppressive functions of TGF-ß involve Wnt5a-mediated antagonism of Wnt/ß-catenin signaling and limiting the stem cell population. Interactions between TGF-ß and Wnt5a in metastasis appear to be more complex, and may depend on specific cues from the microenvironment as well as activation of specific intracellular signaling pathways.     

Wnt Proteins in Mammary Development and Cancer

Secreted proteins of the Wnt family play widespread roles in the regulation of embryonic development, and aberrant activation of the canonical Wnt/beta-catenin pathway is one of the most frequent signaling abnormalities known in human cancer. While the consequences of Wnt signaling in development are diverse at the cellular level, they are often concerned with cell fate determination. Recent data also indicate that Wnt proteins influence the self-renewal of stem cells in certain tissues. In the mammary gland, Wnt signals are strongly implicated in initial development of the mammary rudiments, and in the ductal branching and alveolar morphogenesis that occurs during pregnancy. Transgenic expression of Wnt1 or Wnt10b in the mouse mammary gland leads to lobuloalveolar hyperplasia with a major risk of progression to carcinoma. Recent evidence suggests that this phenotype is associated with expansion of a multipotent progenitor cell population. In human breast cancer, evidence of beta-catenin accumulation implies that the canonical Wnt signaling pathway is active in over 50% of carcinomas. However, specific mutations that might account for this activation of signaling have not yet been identified.