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
Summary
This procedure will demonstrate how we synthesized and characterized the anti-NFκB and anti-cancer stem cell activity of an aspirin-fumarate prodrug.
Abstract
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。
Introduction
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。
<p class="“jove_content”">我们合成的阿司匹林富马酸药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。Protocol
1.阿司匹林富马酸前药GTCpFE的合成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。 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?…
Representative Results
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…
Discussion
In this protocol, we demonstrated the synthesis of an ASA prodrug, GTCpFE, where the fumarate pharmacophore was incorporated to improve the anti-NFĸB activity in breast cancer cells. GTCpFE is an effective NFĸB 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 NFĸB signaling in a variety of cell lines and tissues14,25-29. The prodrug strategy described herein, is amendable to other mal…
Declarações
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| RPMI 1640 Red Medium | Life Technologies | 11875-093 | Warm up to 37°C before use |
| IMEM | Corning | 10-024-CV | Warm up to 37°C before use |
| DMEM / F12 Medium | ThermoFisher | 21041-025 | Warm up to 37°C before use |
| MEM NEAA 10mM 100X | Life Technologies | 11140 | |
| Penicillin Streptomycin | Life Technologies | 15140-122 | |
| L-Glutamine 100X | Life Technologies | 25030-081 | |
| Insulin | Sigma-Aldrich | I-1882 | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150 | |
| Trypsin 2.5% 10X | Invitrogen | 15090046 | |
| Methyl Cellulose | Sigma | M0262 | |
| B27 Supplement 50X | Gibco | 17504-044 | |
| EGF | Gibco | PHG0311L | |
| NFκB-RE Luciferase Construct | Clontech | pGL4.32 | |
| Renilla Luciferase Construct | Promega | pGL4.70 | |
| Lipofectamine 2000 | ThermoFisher | 11668-019 | |
| Dual-Luciferase Reporter Assay | Promega | 120000032 | |
| NanoDrop Spectrophotometer | ThermoFisher | ||
| Eppendorf Mastercycler | Eppendorf | ||
| StepOne Real Time PCR System | Thermo Scientific | ||
| Eclipse Microscope | Nikon | ||
| CyAn ADP Analyzer | Beckman Coulter | ||
| QCapture Software | QImaging | ||
| Summit Software | Beckman Coulter | ||
| GraphPad Software | Prism | ||
| TRIzol | ThermoFisher | 15596-018 | |
| M-MLV Reverse Transcriptase | Invitrogen | 28025-013 | |
| 100 mM dNTP Set | Invitrogen | 10297-018 | |
| Random Hexamers | Invitrogen | 48190-011 | |
| Fast SYBR Green Master Mix | ThermoFisher | 4385612 | |
| Costar 96W, ultra low attachment | Corning | 3474 | |
| HBSS, 1X | ThermoFisher | 14025134 | |
| CD44-APC conjugated antibody | BD Pharmingen | 560990 | Store at 4°C, protect from light |
| CD24-PE antibody | BD Pharmingen | 560991 | Store at 4°C, protect from light |
| Recombinant Human TNFα | Fisher | 210TA100 | |
| CCL2 Primer Forward Sequence | AGAATCACCAGCAGCAAGTGTCC | ||
| CCL2 Primer Reverse Sequence | TCCTGAACCCACTTCTGCTTGG | ||
| ICAM1 Primer Reverse Sequence | TGACGAAGCCAGAGGTCTCAG | ||
| ICAM1 Primer Forward Sequence | AGCGTCACCTTGGCTCTAGG | ||
| TNF Primer Forward Sequence | AAGGGTGACCGACTCAGCG | ||
| TNF Primer Reverse Sequence | ATCCCAAAGTAGACCTGCCCA | ||
| 36B4 Primer Forward Sequence | GTGTTCGACAATGGCAGCAT | ||
| 36B4 Primer Reverse Sequence | GACACCCTCCAGGAAGCGA | ||
| Sodium Hydroxide | Sigma-Aldrich | S5881-500G | |
| 4-Hydroxybenzyl Alcohol | Sigma-Aldrich | 20806-10G | |
| O-Acetylsalicyloyl Chloride | Sigma-Aldrich | 165190-5G | |
| Phosphorous Pentoxide | Sigma-Aldrich | 79610-100G | |
| Ethyl Fumaroyl Chloride | Sigma-Aldrich | 669695-1G | |
| Sodium Sulfate | Sigma-Aldrich | 246980-500G | |
| 4-Dimethylaminopyridine | Sigma-Aldrich | 714844-100ML | 0.5 M in tetrahydrofuran |
| Triethylamine | Sigma-Aldrich | T0886-100ML | |
| Toluene | Sigma-Aldrich | 244511-100ML | |
| Ethyl Acetate | Sigma-Aldrich | 270989-100ML | |
| Tetrahydrofuran | Sigma-Aldrich | 401757-2L | |
| 400 MHz FT NMR spectrometer | See Bruker’s Avance User’s Manual, version 000822 for details |
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Citar este artigo
Kastrati, I., Delgado-Rivera, L., Georgieva, G., Thatcher, G. R. J., Frasor, J. Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells. J. Vis. Exp. (119), e54798, doi:10.3791/54798 (2017).