This work was supported by grants provided by the National Institutes of Health (NIH), R01 CA200669 to JF and R01 CA121107 to GRJT, and by a postdoctoral fellowship grant from Susan G. Komen for the Cure to IK (PDF12229484).

1.阿司匹林富马酸前药GTCpFE的合成<ol><li>使用塑料柱塞注射器，测量甲醇为0.81毫升（20毫摩尔）和它在水（10毫升）在圆底烧瓶中混合。将烧瓶在冰水浴冷却所得混合物至0℃。添加4-羟基苄基醇（2.48毫克，20毫摩尔），搅拌反应混合物，直到溶液澄清。 </li><li>通过称量的O- -acetylsalicyloyl氯化物所需量，并在一个单独的烧瓶中它溶解在溶剂中制备的无水甲苯（10ml）中的O- -acetylsalicyloyl酰氯（3.77毫克，19毫摩尔）。使用塑料柱塞注射器，这种溶液添加到在步骤1.1中制备的混合物中，离开该反应在0℃下搅拌。 </li><li>监视用薄层色谱（TLC）的反应。的TLC板应具有的二氧化硅作为固定相。 <ol><li>制备20:80乙酸乙酯（AcOEt）纯化/己烷组成的流动相。在TLC室（或小容器）加入5 mL流动相以覆盖所述室的底部。 注:流动相的量取决于所选的腔室中。随时添加足以覆盖底部，但一旦TLC放在里面，溶剂线不应超过在哪里发现的化合物的高度。 </li><li>采取用注射器（0.2ml）中的反应的样品，将其放置在一个小管形瓶中，并用乙酸乙酯（2-3滴）稀释。随着TLC去污剂，小心地稀释反应混合物的样品，并在TLC发现它。 注:点应是1-2毫米，这应该在TLC板的下端1/4英寸来完成。 </li><li>在相同的薄层，放置的O-乙酰水杨酰氯的溶液的光斑（0.2毫克0.5毫升乙酸乙酯）作为比较。一旦点是干的，将其放置在室内，并让它运行，直到溶剂前沿几乎已经达到了TLC的顶部。 </li><li>就拿出来TLC，待其干燥和紫外线灯下进行可视化。该REAC化完成后，将O -acetylsalicyloyl氯化物点从反应混合物中消失。 </li><li>当反应完成时，除去冰 - 水浴，并且使反应在室温下搅拌20小时。 </li></ol></li><li>筛选使用布氏漏斗具有中等孔隙率的多孔盘的沉淀物。放置固体在闪烁瓶中，并留下在真空中化合物在室温下的干燥器过夜。用五氧化二磷（P 2 O 5）作为干燥剂。 </li><li>通过设置该反应前，在80℃将其放置在烘箱中至少两天干燥的圆底烧瓶中。可替代地，密封件用隔膜的烧瓶中，并与连接到真空泵系统针戳。打开真空泵上，并用空气加热枪干燥烧瓶中。使其冷却，然后重复该步骤2次以上。 </li><li>使用氩气充气气球，它连接到一个塑料注射器（其柱塞ħ作为被移除）用针。关掉真空泵和除去附着其在针被插入到现在干燥的圆底烧瓶中。插入通过隔膜密封圆底烧瓶连接到氩气球针头。 <ol><li>添加4-羟甲基苯酚酯的2- acetyloxybenzoic酸（100毫克，0.349毫摩尔），4-二甲氨基吡啶（4毫克，0.033毫摩尔）和三乙胺（53毫克，0.523毫摩尔）到一个单独的烧瓶中，然后溶于它们在无水四氢呋喃（5毫升）中。与附连到针上的塑料注射器中，添加这种溶液到密封圆底烧瓶中。通过将烧瓶置于冰水浴冷却该混合物至0℃。 </li></ol></li><li>到先前的混合物中，加入在无水四氢呋喃（5毫升）的乙基fumaroyl酰氯（68毫克，0.419毫摩尔），逐滴历时10分钟。搅拌在0-5℃2-4小时将所得溶液。 </li><li>用乙酸乙酯萃取（100毫升）和盐水（50毫升）的反应混合物中。&lt;登记/&gt;注:盐水是氯化钠（NaCl）饱和溶液。为了制备它，放置200ml水在一个干净的玻璃容器中，然后开始加入NaCl（同时搅拌），直到它已不溶解。 <ol><li>萃取后，除去水相，并重复后面的步骤两次以上。 </li><li>加入硫酸钠，以将有机相干燥混合物，直到当玻璃器皿搅拌该固体不结块的。使用另一个布氏漏斗有烧结盘，将混合物过滤至一个圆底烧瓶中，除去硫酸钠，然后用设定在40℃的温度下在旋转蒸发器蒸发至干。 </li></ol></li><li>使用薄层色谱法，确定一个合适的流动相在柱色谱法中使用。 注意:在这个实验中，使用20:80乙酸乙酯/己烷的梯度。 </li><li>准备用硅胶和适当的溶剂相的柱。一旦化合物已被添加，添加硫酸钠（或沙）的一个层至Pr努力保护的列作为溶剂加入。 <ol><li>作为列运行时，监视通过TLC洗脱液。因为它们是从该柱收集的斑点每隔试管。当感兴趣的产物洗脱，混合含有纯产品成一个大圆底烧瓶中的所有样品，并使用与所述浴温设定在40℃的旋转蒸发器干燥。 注:产品应显示为白色固体。 </li></ol></li><li>以确认GTCpFE的结构，收集质子和碳（1 H和13 C，分别地）核磁共振（NMR）光谱按照制造商的说明。 注:在本研究中一个400 MHz的FT NMR光谱仪用来收集1 H和13 C谱。运行在室温下至少25次扫描，以获得足够的数据的分辨率。所观察到的NMR峰是以下，1 H核磁共振（CDCl 3）:δ1.29（叔，3H，3 J = 7.0赫兹，CH 3）; 2.31（S，3H，CH 3）; 4.26（Q，2H，3 J = 7.0赫兹，COOCH 2）; 5.25（S，2H，OCH 2）; 6.89（S，2H，HC = CH）; 7.19（M，3H中，Ar）; 7.42（M，3H中，Ar）; 7.65（M，1H，氩）; 8.22（DD，1H，3 J = 7.8，4J = 1.2赫兹，氩）。 13 C核磁共振（CDCl 3）δ:169.70，164.86，164.75，162.89，151.28，150.69，134.76，134.32，133.26，133.16，132.25，129.81，126.26，124.11，122.47，122.04，66.40，61.43，21.05，14.15。 </li><li>另外，使用液相色谱质谱结合精确质量测量根据制造商的说明离子阱和时间的飞行技术作为（LCMS-IT-TOF）收集高分辨率质谱。 注:在本研究（M + NH 4 +）计算:430.1496;观察:430.1477。 </li></ol> 2. GTCPFE抑制NFĸB活动乳腺癌细胞<ol><li> 细胞培养条件 <ol><li>维持人体乳腺癌细胞系MCF-7和MDA-MB-231按照标准细胞尽头TURE技术和传播中，在37℃的潮湿培养箱，用5％ 的 CO 2。 <ol><li>从罗斯韦尔园区纪念研究所（RPMI）用补充有10％胎牛血清（FBS）的酚红，1％非必需氨基酸，2mM的L-谷氨酰胺，1％抗生素青霉素 - 链霉素1640培养基制备的MCF-7细胞培养基和6纳克/ ml胰岛素。准备从改进的最小必需培养基（IMEM），补充了5％FBS，1％非必需氨基酸，2mM的L-谷氨酰胺和1％抗生素青霉素 - 链霉素的MDA-MB-231细胞中。 </li></ol></li></ol></li><li> NFκB-RE荧光素酶报告分析 <ol><li> Trypsinize乳腺癌MCF-7细胞用0.25％胰蛋白酶在37℃5分钟，人工计数，每孔的密度90000细胞使用血细胞计数器和种子在24孔板中。 </li><li>翌日，共转染细胞与NFκB应答元件（NFκB-RE）的萤光素酶构建体，1微克的质粒DNA /孔，人翁与启动子Renilla荧光素酶构建体，0.2微克/孔。为一式三份各治疗组进行转染。 <ol><li>洗涤细胞两次，用PBS，并用质粒DNA的混合物在无血清培养基每转染试剂（ 例如脂质体Lipofectamine）孔的1微升孵育。 6小时后，改变培养基到1ml正规培养基（无抗生素青霉素和链霉素）。 </li></ol></li><li> 16小时后，加入1微升车辆或GTCpFE不同储备溶液或ASA药物的每个孔中。在1000倍的浓度溶解在二甲基亚砜药物储备溶液（100，50，20，10和1毫米）。添加药物后，孵育细胞2小时，在37℃。 </li><li>以激活NFκB途径，添加促炎细胞因子TNFα（10μg/ ml的储备液）到每个孔中的10毫微克/ ml的终浓度，孵育4小时。包括TNFα单独控制。吸出介质并存储单元■在-80℃。 </li><li>使用根据制造商的说明萤光素酶报道测定系统测量荧光素酶。 注意:使用在第一次的荧光素酶测定系统（ 例如双荧光素酶检测系统），通过重新悬浮它在10ml缓冲液中，并在1毫升等份在-80℃下将其存储制备萤光素酶测定试剂的溶液。 <ol><li>通过稀释5倍的股票缓冲液与水准备1X裂解液。裂解细胞，每孔用100μl的1×裂解缓冲液。孵育轨道摇床在板15分钟，以中速。 </li><li>标记每采样1 1.5mL管中。解冻萤光素酶测定试剂到室温（将需要每个样品50微升），并保持在箔。在玻璃小瓶中制备1×淬火试剂，1点49稀释在缓冲液中并涡旋它（将需要每个样品50微升）。 </li><li>添加10微升的细胞裂解物，以标记的离心管中。然后加入50微升荧光素酶检测试剂和轻轻的旋涡。紧接着，将管中的支架和测量经由在光度计的双荧光素酶程序的发光。选择，然后按下列操作:运行Promega公司的协议→双→格洛OK。 </li><li>添加50微升猝灭试剂至试管中并轻轻涡旋。测量发光。重复这些步骤每个样品。 </li><li>通过标准化NFκB-RE的海肾内部控制（NFκB-RE /海肾）分析使用电子表格数据。比较抑制剂数据TNFα仅控制（TNFα仅被设定在100％）。 </li></ol></li></ol></li><li> NFκB-靶基因的转录 <ol><li>种子MCF-7细胞在6孔板中，每孔250,000个细胞在2ml培养基体积如在第2.1节描述的方法制备。 </li><li>次日，加入TNFα为10ng / ml的另外2小时之前添加2微升不同GTCpFE 1000倍股（100，50，20，10和11毫米）2小时的。运行间隔治疗一式三份。包括车辆和TNFα单独控制。 </li><li>使用硫氰酸胍-苯酚-氯仿提取方法根据制造商22的指示的RNA分离。确定RNA浓度（在焦碳酸二乙酯（DEPC）处理的水），并用分光光度计RNA的纯度。 RNA应放在冰上或储存在-80℃。处理RNA时，只能使用无RNase障碍提示。 </li><li>扭转使用莫洛尼鼠白血病病毒（M-MLV）逆转录酶的市售的试剂盒转录0.5微克总RNA。 注:该套件包括5倍与dNTP混合液，和随机六聚体混合物缓冲液和二硫苏糖醇（DTT），在一起。 <ol><li>加入RNA的0.5微克至200个单位M-MLV，0.5毫米的dNTP，100ng的随机六聚体，10毫摩尔DTT，1×缓冲液和DEPC水至最终的10微升反应体积。进行使用50分钟的PCR仪的逆转录酶反应在37℃，并在70然后15分钟＃176;下加热-酶失活。 </li></ol></li><li>稀释得到的cDNA产物至100μl用双蒸水，并使用2微升每个后续定量聚合酶链反应（PCR）反应。 <ol><li>混合的正向和反向引物的兴趣在每个1.25微米浓度的基因。制备主混合物与2微升双蒸水，1.25μM引物混合物1和5μl染料2倍的，以使双链DNA的检测。加载cDNA的2微升和8微升主混合物到96孔PCR板。 </li></ol></li><li>使用实时PCR系统会根据制造商的说明并手动分析数据（40个循环，95-60℃）进行定量PCR。 注意:使用已经过验证的和以前23日报道所有PCR引物。 </li><li>通过ΔΔCT方法，使用电子表格计算基因表达倍数变化，与核糖体蛋白36B4表达作为内部控制23。 </li></ol></li></ol> 3. GTCpFE抑制乳腺癌干细胞的体外 <ol><li> 微球体形成分析 <ol><li>通过补充的Dulbecco改良Eagle培养基制备微球体（MS）培养基:用1％甲基纤维素营养混合物F-12（DMEM / F12）无酚红培养基。允许通过在4℃下轻轻摇动过夜以溶解。过滤消毒介质和补充有B27 1倍，1％青霉素和链霉素，5微克/毫升胰岛素，1微克/ ml氢化可的松和20纳克/ ml重组人表皮生长因子。 </li><li>通过（在37℃的胰蛋白酶0.25％，5分钟）单层培养的胰蛋白酶消化制备的MDA-MB-231细胞系的单细胞和通过目筛进行筛选。在400个细胞的密度手动计数单个游离细胞和板在96孔超低附着板/孔。第二天，加入不同浓度的GTCpFE（ 例如 ，最终GTCpFE浓度:1，10，20，50μM）至最终体积为100微升。进行一式三份的每个治疗。 </li><li>后在培养箱培养7天后，获得使用倒置显微镜通过成像软件图像。手动计数直径数MS&gt; 75微米。比较药物治疗车辆控制。 注意:使用成像软件被确定的MS&gt; 75微米的直径。 </li></ol></li><li> CD44 + CD24 - CSC免疫表型 <ol><li> Trypsinize的MDA-MB-231细胞用0.25％胰蛋白酶进行5分钟，在37℃。伯爵在10毫升中使用10 cm的培养皿血球计数器和种子在每盘300万细胞2.1节所述。添加车辆（10微升二甲基亚砜）或GTCpFE（10微升50毫米股票）细胞72小时。 </li><li>用于车辆或GTCpFE处理组，trypsinize细胞（如在步骤3.2.1），并分发万个单元格在"测试＆＃39;或含2ml 1X Hank氏平衡盐溶液（HBSS）中的"控制"5毫升聚苯乙烯管缓冲补充有2％FBS的。 </li><li>染色为表面标志物CD44和CD24，旋细胞向下并添加每个缀合的抗体的20微升和HBSS + 2％FBS中，以测试管为一个最终100μl的总体积（1:5稀释）。加入100微升HBSS + 2％FBS来控制管。包括CD44-APC结合的抗体和CD24-PE抗体单染控制或IgG的immunoisotype控制。 </li><li>孵育细胞在黑暗中在4℃下30分钟。降速细胞5分钟，在400 XG和在200μlHBSS缓冲液，用2％FBS的重建。继续在黑暗中冰细胞。 </li><li>执行使用根据制造商的说明在FACS分析仪仪器活细胞的荧光激活细胞分选（FACS）。收集每个管至少50,000个事件。运行处理一式三份。 注意:门控是基于从没有染色对照（钴ntrol管），IgG的immunoisotypes和CD44-APC和CD24-PE单污渍。 </li><li>分析使用可用的流式细胞仪软件数据。 注:与CD44 + CD24 GTCpFE处理的细胞的百分比-免疫估计相比，车辆控制。 </li></ol></li></ol>

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The authors have nothing to disclose.

In this protocol, we demonstrated the synthesis of an ASA prodrug, GTCpFE, where the fumarate pharmacophore was incorporated to improve the anti-NF&#312;B activity in breast cancer cells. GTCpFE is an effective NF&#312;B inhibitor, whereas ASA itself is not, even at much higher concentrations. The fumarate moiety has anti-inflammatory properties as shown by its ability to inhibit NF&#312;B signaling in a variety of cell lines and tissues14,25-29. The prodrug strategy described herein, is amendable to other malignancies where multiple inflammatory pathways are active and contribute to the pathology. Therefore, GTCpFE may be the prototype for developing new fumarate-based anti-inflammatory and anti-CSC class of drugs. Besides ASA, other non-steroidal anti-inflammatory drugs may also be used. In this case, the synthetic route should be re-designed as well. Ensuring that the new prodrug maintains its anti-NF&#312;B and anti-cyclooxygenase activity via the assays describe herein is critical. 
GTCpFE abrogated MS formation and inhibited the CD44+CD24- immunophenotype. Together these findings suggest that GTCpFE may also be a promising, clinically relevant anti-inflammatory molecule for eradicating breast CSCs by exploiting CSC's reliance on multiple inflammatory pathways, including the NF&#312;B pathway and the cyclooxygenase 2-prostaglandin E2 axis16-21,30. Targeting breast CSCs is important because they are thought to contribute to therapy resistance, recurrence and metastasis31-34. 
The anti-CSCs properties described here, the mammosphere formation and the CD44+CD24- immunophenotype, are based on typical in vitro assays to assess effect on stemness. Mammosphere formation is a functional assay because it exploits the unique property of stem-like/progenitor cells to survive and grow in serum-free suspension, while more differentiated cells undergo anoikis and die in these conditions35,36. However, because a reduction in mammosphere formation may represent (i) increased apoptosis, (ii) decreased proliferation of early progenitor cells or stem cells, (iii) a reduction in stem cell self-renewal, or (iv) interference with anchorage-independent growth, it is best used in conjunction with other anti-CSC assays. The choice of the well-established breast CSC CD44+CD24- immunophenotype is based on the pioneering study by Clarke and colleagues who used breast cancer xenografts to isolate a population of cells capable of initiating tumors in immunodeficient mice24. Similar to the CD44+CD24- immunophenotype, expression of other cell surface markers can be used to isolate stem cells via flow cytometry, but the choice of marker can greatly vary depending on tissues or species (human vs murine model). Alternative assays to measure effects on breast CSCs in vitro are (i) the side population technique, which is based on the ability of stem cells to exclude vital dyes via transmembrane transporters37, and (ii) the ALDEFLUOR assay, which is based on the enzymatic activity of aldehyde dehydrogenase 1 (ALDH1)38. Lastly, a suppression of pluripotency genes, transcription factors, or key stemness pathways may also be indicative of anti-CSCs effects. Promising in vitro anti-CSC properties should be followed up by in vivo characterization. The "gold standard" for assaying anti-CSC properties is in vivo tumorigenicity, wherein the ability of drug-treated cells to initiate or seed a xenograft tumor is examined39,40.

炎症是肿瘤underlies的多个方面，如发病率和推广，并转移1最终进展的标志。在乳腺癌中，这进一步通过流行病学观察显示，经常使用经典的非类固醇抗炎药阿司匹林（乙酰水杨酸，ASA）与在两个乳腺癌发病率的降低，并转移和复发的风险相关联的支持2,3。 ASA主要是通过抑制环氧合酶-2的活性，这是常常在乳腺癌4,5-上调作用。然而，ASA的抗癌作用，也可以通过抑制异常核因子κB（NFκB）信令6-8介导的。这是重要的，因为一个失调NFκB途径促进肿瘤细胞存活，增殖，迁移，侵入，血管发生和抗治疗9-11。 NFκB途径激活也用于安装临界的免疫应答。因此，对于其中需要延长的NFκB抑制作用抗癌治疗时，必须考虑的有害副作用涉及长效免疫抑制。因此，ASA可以作为一个很好的起点治疗优化。 用于癌症治疗的ASA应用之一限制是对环加氧酶2和NFκB抑制所需的升高的剂量，这是与胃肠道毒性相关联，如溃疡和胃出血12,13。然而，转换成ASA作为酯前体药物，可降低ASA的胃肠毒性。为了进一步增强效价和/或增加功能性，另外的结构元件或辅助药效也可以并入酯前药设计。一个这样的药效加入以增强ASA效力针对NFκB通路是富马酸盐，这是我们先前已经显示出对于NFκB途径抑制14,15重要。 15，GTCpFE，并推测这种杂交分子是安全而对NFκB途径有力。我们测试在乳腺癌细胞中的抗NFκB活动及其阻断乳腺癌干细胞（CSCS）。15，依赖于NFκB信令生存和发展16-21能力。我们发现，GTCpFE兑NFκB途径的效力超过ASA 15被显著改善。此外，GTCpFE块形成微球体和衰减CSC表面标志物CD44 + CD24 -免疫，表明GTCpFE能够消灭肿瘤干细胞15。这些结果建立了阿司匹林富马酸前药作为有效抗炎剂也可以定位乳房的CSCs。在乳腺癌治疗而言，GTCpFE可能具有治疗侵略性和致命的疾病的潜力。

<table><tbody><tr><td>RPMI 1640 Red Medium</td><td>Life Technologies&amp;nbsp;</td><td>11875-093</td><td>Warm up to 37 &amp;deg;C before use</td></tr><tr><td>IMEM</td><td>Corning</td><td>10-024-CV</td><td>Warm up to 37 &amp;deg;C before use</td></tr><tr><td>DMEM / F12 Medium</td><td>ThermoFisher</td><td>21041-025</td><td>Warm up to 37 &amp;deg;C before use</td></tr><tr><td>MEM NEAA 10 mM 100x</td><td>Life Technologies&amp;nbsp;</td><td>11140</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies&amp;nbsp;</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine 100x</td><td>Life Technologies&amp;nbsp;</td><td>25030-081</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-1882</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Trypsin 2.5% 10x</td><td>Invitrogen</td><td>15090046</td><td></td></tr><tr><td>Methyl Cellulose</td><td>Sigma&amp;nbsp;</td><td>M0262</td><td></td></tr><tr><td>B27 Supplement 50x</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>EGF</td><td>Gibco</td><td>PHG0311L</td><td></td></tr><tr><td>NF&amp;kappa;B-RE Luciferase Construct&amp;nbsp;</td><td>Clontech&amp;nbsp;</td><td>pGL4.32</td><td></td></tr><tr><td>Renilla Luciferase Construct&amp;nbsp;</td><td>Promega</td><td>pGL4.70</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>ThermoFisher</td><td>11668-019</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay&amp;nbsp;</td><td>Promega</td><td>120000032</td><td></td></tr><tr><td>NanoDrop Spectrophotometer</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Eppendorf Mastercycler&amp;nbsp;</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>StepOne Real Time PCR System</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Eclipse Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>CyAn ADP Analyzer&amp;nbsp;</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>QCapture Software</td><td>QImaging</td><td></td><td></td></tr><tr><td>Summit Software</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>GraphPad Software</td><td>Prism</td><td></td><td></td></tr><tr><td>TRIzol</td><td>ThermoFisher</td><td>15596-018</td><td></td></tr><tr><td>M-MLV Reverse Transcriptase</td><td>Invitrogen</td><td>28025-013</td><td></td></tr><tr><td>100 mM dNTP Set</td><td>Invitrogen</td><td>10297-018</td><td></td></tr><tr><td>Random Hexamers&amp;nbsp;</td><td>Invitrogen</td><td>48190-011</td><td></td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ThermoFisher</td><td>4385612</td><td></td></tr><tr><td>Costar 96W, ultra low attachment&amp;nbsp;</td><td>Corning</td><td>3474</td><td></td></tr><tr><td>HBSS, 1x</td><td>ThermoFisher</td><td>14025134</td><td></td></tr><tr><td>CD44-APC conjugated antibody&amp;nbsp;</td><td>BD Pharmingen</td><td>560990</td><td>Store at 4 &amp;deg;C, protect from light</td></tr><tr><td>CD24-PE antibody</td><td>BD Pharmingen</td><td>560991</td><td>Store at 4 &amp;deg;C, protect from light</td></tr><tr><td>Recombinant Human TNF&amp;alpha;</td><td>Fisher</td><td>210TA100&amp;nbsp;</td><td></td></tr><tr><td>CCL2 Primer Forward Sequence</td><td></td><td></td><td>AGAATCACCAGCAGCAAGTGTCC</td></tr><tr><td>CCL2 Primer Reverse Sequence</td><td></td><td></td><td>TCCTGAACCCACTTCTGCTTGG</td></tr><tr><td>ICAM1 Primer Reverse Sequence</td><td></td><td></td><td>TGACGAAGCCAGAGGTCTCAG</td></tr><tr><td>ICAM1 Primer Forward Sequence</td><td></td><td></td><td>AGCGTCACCTTGGCTCTAGG</td></tr><tr><td>TNF Primer Forward Sequence</td><td></td><td></td><td>AAGGGTGACCGACTCAGCG</td></tr><tr><td>TNF Primer Reverse Sequence</td><td></td><td></td><td>ATCCCAAAGTAGACCTGCCCA</td></tr><tr><td>36B4 Primer Forward Sequence</td><td></td><td></td><td>GTGTTCGACAATGGCAGCAT</td></tr><tr><td>36B4 Primer Reverse Sequence</td><td></td><td></td><td>GACACCCTCCAGGAAGCGA</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td></td></tr><tr><td>4-Hydroxybenzyl Alcohol</td><td>Sigma-Aldrich</td><td>20806-10G</td><td></td></tr><tr><td>&lt;em&gt;O&lt;/em&gt;-Acetylsalicyloyl Chloride</td><td>Sigma-Aldrich</td><td>165190-5G</td><td></td></tr><tr><td>Phosphorous Pentoxide</td><td>Sigma-Aldrich</td><td>79610-100G</td><td></td></tr><tr><td>Ethyl Fumaroyl Chloride</td><td>Sigma-Aldrich</td><td>669695-1G</td><td></td></tr><tr><td>Sodium Sulfate</td><td>Sigma-Aldrich</td><td>246980-500G</td><td></td></tr><tr><td>4-Dimethylaminopyridine</td><td>Sigma-Aldrich</td><td>714844-100ML</td><td>0.5&amp;nbsp;M in tetrahydrofuran</td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>T0886-100ML</td><td></td></tr><tr><td>Toluene</td><td>Sigma-Aldrich</td><td>244511-100ML</td><td></td></tr><tr><td>Ethyl Acetate</td><td>Sigma-Aldrich</td><td>270989-100ML</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Sigma-Aldrich</td><td>401757-2L</td><td></td></tr><tr><td>400 MHz FT NMR spectrometer&amp;nbsp;</td><td></td><td></td><td>See Bruker&amp;rsquo;s Avance User&amp;rsquo;s Manual, version 000822 for details</td></tr></tbody></table>

synthesis and characterization of an aspirin-fumarate prodrug that inhibits nf&#954;b activity and breast cancer stem cells

炎症是癌症标志的underlies癌症发病率和推广，并最终发展为转移。因此，加入一种抗炎药物标准癌症团可改善患者的预后。一种这样的药物，阿司匹林（乙酰水杨酸，ASA），已探索用于癌症的化学预防和抗肿瘤活性。除了抑制环氧合酶2 - 前列腺素轴，ASA的抗癌活性也被归因于核因子ĸB（NFĸB）抑制。由于长时间使用ASA可能引起胃肠道毒性，前体药的策略已成功实施。在这个前药设计的ASA的羧酸被掩蔽和附加药效并入。 这个协议描述了我们如何合成的阿司匹林富马酸盐的前药，GTCpFE，并且其特征在于它的NFĸB途径的抑制乳腺癌细胞和癌症的衰减干细胞样正确联系，一个重要的NFĸB依赖性表型。 GTCpFE有效抑制乳腺癌细胞系NFĸB通路而ASA缺乏任何抑制活性，这表明加入富马酸盐向ASA结构显著有助于其活性。此外，GTCpFE示出了通过阻断微球体形成并衰减所述癌症干细胞相关的CD44 + CD24显著抗癌干细胞活性-免疫。这些结果建立一个可行的策略开发改进的抗炎药为化学预防和治疗癌症。

在图1中 ，阿司匹林富马酸盐的前药，GTCpFE，其抑制活性的对细胞因子的化学结构诱导乳腺癌细胞NFĸB通路表示。 GTCpFE既抑制NFĸB端点，NFĸB-RE萤光素酶活性（ 图1B）和表达NFĸB靶基因，如细胞间粘附分子1（ICAM1），趋化因子CC基序配体2（CCL2），以及肿瘤坏死因子（TNF）（ 图1C）在MCF-7乳腺癌细胞。在50％计算出的抑制浓度上的两个端点（IC 50）值是〜20微米。 IC 50值是用绘图软件计算。通过比较ASA本身即使在200微米（10倍GTCpFE的IC 50值）表示在乳腺癌细胞没有抑制活性（ 图1B，蓝线）。这表明，添加富马酸药效到ASA标志意义的前药策略ntly提高其抗NFĸB活动。 为了测量抗CSC活性，我们使用两种测定:所述微球体（MS）的形成分析和表达CD44 + CD24细胞群-免疫，在乳腺癌24 善意的CSC表面标记。 GTCpFE抑制在图2A中所示的剂量依赖性方式的MS形成的MDA-MB-231乳腺癌细胞。类似于在贴壁培养NFĸB通路的抑制作用，对形成微球体的IC 50值是〜20微米。这种一致性的预期，考虑到肿瘤干细胞依赖NFĸB信令的生存和繁殖16-21。此外，GTCpFE预处理导致CD44 + CD24的一个显著耗尽-在MDA-MB-231细胞群体（ 图2B）。总之，这些结果建立GTCpFE的有效地针对乳腺肿瘤干细胞的能力。 <img alt="图1" src="/files/ftp_upload/54798/54798fig1.jpg" /> 图1:GTCpFE抑制乳腺癌细胞的NFĸB通路。 （ 一 ）阿司匹林富马酸药，GTCpFE的化学结构。 （B - C）MCF-7细胞进行预处理2小时，用各种浓度GTCpFE，ASA，或者车辆随后用TNFα（10纳克/ ml）处理2-4小时。 （B）NFκB-RE活动由双荧光素酶报告基因检测测量。用RT-QPCR测量NFκB靶基因，ICAM1，CCL2和TNF的（C）中的表达。药物抑制活性绘制成TNFα的单独％。数据表示为平均值±SEM表示。这个数字已经从15参考修改。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54798/54798fig1large.jpg" target="_blank">请点击此处查看该figu的放大版本</a>回覆。 <img alt="图2" src="/files/ftp_upload/54798/54798fig2.jpg" /> 图2:GTCpFE 抑制乳腺球形成和CD44 + CD24 - 在乳腺癌细胞免疫。 （A）的微球体（MS）形成的MDA-MB-231细胞在处理后测定与不同浓度GTCpFE的。 MS生长（左）和在20X的MS的代表画面的定量（右）中示出。 GTCpFE的效果被绘制为％车辆控制。 （B）中的CD44 + CD24 -群体通过用50μM的GTCpFE 72小时处理的MDA-MB-231细胞的FACS分析确定。每个种群百分比（左）和具有代表性的散点图从FACS（右）的定量被示出。数据表示为平均值±SEM和2因素ANOVA FOLL的统计分析由杜克后测拖欠。 *** P &lt;0.001。这个数字已经从15参考修改。

Watch this Scientific Journal Video about Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÎºB Activity and Breast Cancer Stem Cells at JoVE.com

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

合成与阿司匹林富马酸酯前药的表征抑制NFκB活动和乳腺癌干细胞

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

synthesis-characterization-an-aspirin-fumarate-prodrug-that-inhibits

Nanyang Technological University

Research

JoVE Journal

Cancer Research

12.2K 次观看.  University of Illinois at Chicago. This procedure will demonstrate how we synthesized and characterized the anti-NFκB and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

视频: Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物:用于个性化医疗的机会？

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

科研

化学

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.

We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.

增强细胞凋亡和自噬的诱导合成一种新型的C-1 7-deoxypancratistatin的类似物在人类乳癌细胞和神经母细胞瘤与他莫昔芬

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce ...

医学

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

癌症研究

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

将原发性癌症相关纤维细胞与乳腺癌的合成素 Murine 模型分离，用于靶向纳米粒子的研究

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

生物学

FAM83A 作为宫颈癌靶基因的验证

Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps involved in the identification of a tumor target gene FAM83A in cervical cancer. First, the Cancer Genome Atlas dataset was employed to validate the expression and prognostic significance of FAM83A in women. A small interfering RNA (siRNA) was used for knockdown of the FAM83A gene in HeLa and C33a cells. Next, 5-ethynyl-2&#39;-deoxyuridine (EdU) staining was conducted to determine the effects on the proliferation capabilities of the tumor cells. Wound healing and porous membrane insert assays were performed to evaluate tumor cell migration and invasion abilities.
Western blotting was used to quantify apoptosis-related protein levels. JC-1 staining was employed to evaluate mitochondrial function alterations. Furthermore, cisplatin (diaminedichloroplatinum, DDP) intervention was used to assess the therapeutic potential of the target gene. Flow cytometry and colony formation assays were conducted to further validate the anticancer characteristics of the gene. As a result, FAM83A knockdown was shown to inhibit the proliferation, migration, and invasion of cervical cancer cells and sensitize these cells to cisplatin. These comprehensive methodologies collectively validate FAM83A as a tumor-associated target gene, holding promise as a potential therapeutic target in the prevention and treatment of cervical cancer.

作者聚焦：探索 FAM83A 在宫颈癌中的作用 

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug research.

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps ...

<meta charset="utf8"></head><body>靶向致癌 miRNA-10b 治疗转移性乳腺癌的纳米药物

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

<meta charset="utf8"></head><body>监测小鼠乳腺癌转移灶中纳米药物的积累

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

监测小鼠乳腺癌转移灶中纳米药物的积累

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

合成与阿司匹林富马酸酯前药的表征抑制NFκB活动和乳腺癌干细胞

摘要

探索更多视频

此视频中的章节

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物:用于个性化医疗的机会？

增强细胞凋亡和自噬的诱导合成一种新型的C-1 7-deoxypancratistatin的类似物在人类乳癌细胞和神经母细胞瘤与他莫昔芬

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

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