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[14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells
肝癌细胞中[14C]色氨酸代谢示踪法   

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Abstract

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for many NAD+-consuming proteins with diverse biological functions. Oscillations in NAD+ levels may influence several cellular signaling pathways. NAD+ synthesis via Preiss-Handler route (salvage reactions) has been extensively reported. However, the contribution of L-tryptophan/kynurenine catabolism in de novo NAD+ synthesis is poorly understood. Using L-[14C]-tryptophan tracing in four liver cancer cell lines and siRNA-mediated silencing of arylformamidase (AFMID), a key enzyme involved in L-tryptophan degradation, we demonstrate the contribution of L-tryptophan catabolism in de novo synthesis of NAD+ pools. NAD+ modulation is therefore important in maintaining cellular homeostasis and appropriate cellular functions according to nutrients availability.

Keywords: Kynurenine pathway(犬尿氨酸途径), De novo NAD+ synthesis(从头合成NAD +), [14C]-Tryptophan metabolic tracing([14C] -色氨酸代谢示踪), Arylformamidase (AFMID)(芳基甲酰胺酶(afmid)), Liver cancer(肝癌)

Materials and Reagents

  1. Liver cancer cell lines: Huh-7 (JCRB, catalog number: JCRB0403 ), HepG2 (ATCC, catalog number: HB-8065 ), SNU-398 (ATCC, catalog number: CRL-2233 ), and SNU-449 (ATCC, catalog number: CRL-2234 )
  2. DMEM (Life technologies)
  3. RPMI (Life technologies)
  4. Amino acid free medium and without fetal bovine serum called Hanks’ Balanced Salt Solution (HBSS) (Sigma-Aldrich)
  5. Fetal bovine serum (Cultek)
  6. Penicilin and streptocmycin (Life Technologies, Gibco®)
  7. 100x Non-essential amino acids’ solution (Life technologies)
  8. RNAiMAX (Life Technologies, InvitrogenTM, catalog number: 13778030 )
  9. OptiMem (Life Technologies, catalog number: 11058021 )
  10. Non silencing small interference RNA control (siCtl) and small interference RNA Arylformamidase (siAFMID) (GE Healthcare Dharmacon, catalog number: L-HUMAN-XX-0005 5 nmol )
  11. NAD+-[carbonyl-14C] (Perkin Elmer, catalog number: NEC831010UC )
  12. L-[benzene-ring-U-14C]-tryptophan (Moravek, catalog number: MC 2335 )
  13. PEI-cellulose thin layer chromatography (TLC) (F plates of 20 x 20 cm) (Merk Millipore catalog number: 105725 )
  14. Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
  15. Ethanol and methanol (Sigma-Aldrich)
  16. NAD+-[carbonyl-14C] (see Recipes)
  17. L-[benzene-ring-U-14C]-tryptophan (see Recipes)
  18. Cell density (around 60-80%) (see Recipes)

Equipment

  1. Phosphor-image reader (Molecular Dynamics, model: Storm820 )
  2. Phosphor-image screen (GE Healthcare Dharmacon, catalog number: 63-0034-79 )
  3. Speedvac centrifuge (Thermo Fisher Scientific)
  4. 4 °C table top centrifuge (Eppendorf, model: 5424R )
  5. Fume hood to handle radioactive materials
  6. Cell scrapers (Corning Incorporated, catalog number: 3008 )
  7. Ruler

Software

  1. Fiji software (www.fiji.sc/Fiji)

Procedure

  1. Four liver cancer cell lines: Huh-7, HepG2, SNU-398 and SNU-449.
    1. Huh-7, HepG2, SNU-398 and SNU-449 cells were cultured in 10 cm cell culture dishes in presence of complete DMEM (Huh-7 and HepG2 cells) and RPMI medium (SNU-398 and SNU-449). Both media were supplemented with 10% fetal bovine serum and 100 units/ml of penicillin and 0.1 mg/ml of streptomycin.
    2. Volumes provided are for one well of 12 well plate, and should be scaled according to the size of the well: 200 μl of Optimem is gently mixed with 1.2 μl of either siAFMID or siCtr. This mixture was added to 1.6 μl of RNAiMAX and incubated at room temperature for 20 min in the well.
    3. Gently resuspend Huh-7, HepG2 (8.0 x 104) cells in 1,000 μl of DMEM or SNU398, SNU449 (3.5 x 104) cells in 1,000 μl of RPMI media and add these cells to the RNAiMAX mixture. The different cell numbers used in this step is to circumvent the variable growth rates in our cell culture conditions to obtain similar cell numbers after the transfection experiment.
    4. After 24 h, the cell medium was changed to a DMEM or RPMI medium containing 10% fetal bovine serum and 100 units/ml of penicillin and 0.1 mg/ml of streptomycin after removing the supernatant.
    5. After 4 h, the media was removed, and 1 ml of HBSS medium supplemented with non-essential amino acids (1x) was added to the cell culture.
  2. 2.5 mM of L-[benzene-ring-U-14C]-tryptophan was added to the cells culture in HBSS and in presence of non-essential amino acids. Cells were incubated at 37 °C for 5 h.
  3. Cell culture medium was removed by discarding the supernatant and, cells were then washed 3 times with ice-cold PBS.
  4. Lyophilic metabolites were extracted using 200 μl of 80% methanol and 20% water mixture, by scrapping and pipetting vigorously. The mixture was incubated for 10 min at 4 °C.
  5. Whole cell extracts were then centrifuged at 20,000 x g for 20 min at 4 °C.
  6. The extracts were concentrated in a speedvac centrifuge to a final volume of 40 μl from 200 μl of 80% methanol and 20% water (step 4) (which might take one hour, you can decrease the time by increasing the vacuum pressure) and subsequently spotted on the PEI-cellulose thin layer chromatography (TLC) F plates.
    1. Annotating the PEI-cellulose thin layer chromatography (TLC) F plates
      1. Draw a straight line 1 cm above the bottom of the plate with a pencil.
      2.  Make markings for spotting the metabolic samples equidistant from each other with 2 cm interval as shown in Figure 1.


        Figure 1. Preparing the PEI-cellulose thin layer chromatography (TLC) F plate for the assay

      3. 1 μl of NAD+-[carbonyl-14C] was used as a positive control and spotted in the last lane to calibrate the relative migration of labeled metabolites.
      4. Add 5 μl of metabolic extracts from step 6 and let the spot dry. Repeat this cycle for all the samples until the whole extract is spotted.
    2. Mounting the TLC migration chamber
      1. Use an apparatus that serves to cast gels for big Western blots as a TLC chamber.
      2.  Wipe the TLC chamber with 70% ethanol.
      3.  Fill the lower tray representing a chamber with migration buffer as described in point-7 of the procedure and as shown in Figure 2.


        Figure 2. Mounting the TLC migration chamber

    3. Setup the TLC chamber with the PEI-cellulose TLC plate containing the migration buffer


      Figure 3. Setup the TLC chamber with the PEI-cellulose TLC plate containing the migration buffer (composition in step 7)

      1. Carefully mount the TLC plate in which the bottom part of the plate submerses in the migration buffer (composition in step 7). However the spotted samples should not directly be in contact with the migration buffer.
      2. The plate is mounted vertically to the migration buffer (composition in step 7) and restrained from any movements by sticking it to the top using a tape.
  7. Migration buffer composed of 1 M ammonium acetate (pH 5) (30%) and absolute ethanol (70%) used in the bottom reservoir touching 0.5 cm of the PEI-cellulose TLC F plates was used for the mobile phase.
  8. After 5-6 h of migration reaching about 15 cm from the starting line, the cellulose plate was air-dried and exposed to the radiosensitive phosphor-imager screen for over night. Screen was revealed using Strom-820 phosphor-image reader.


    Figure 4. TLC of radiolabelled metabolites derived from L-[14C]-tryptophan in four different liver cancer cell lines. Metabolites from siCtr and siAFMID were loaded simultaneously next to each other. [14C]-NAD+ was used as the calibration control for relative migration of labeled NAD+ derived from L-[14C]-tryptophan (Tummala et al., 2014).

Notes

The experiment has been carried out using the 4 different liver tumor cell lines for at least 4 times and this blot is representative of these 4 different experiments. Consistent incorporation of label has been observed. Fiji software (www.fiji.sc/Fiji) was used to quantify the pixilation of different bands in the image and the relative band intensities were calibrated in respect to the siCtr samples. Data interpretation should be done based on the intensity of the bands at the right size, greater is the band intensity, and greater is the NAD+ content.

Recipes

  1. NAD+-[carbonyl-14C] used here as a relative migration standard, however it is not mandatory to use this specific isotope, any other phosphor-imager sensitive NAD+ isotope can be used for this purpose.
  2. L-[benzene-ring-U-14C]-tryptophan has been chosen specifically because of the known contribution of the tryptophan’s benzene ring carbon atom for the NAD+ backbone.
  3. Cell density (around 60-80%) at the time of starvation yielded most consistent results. The cells are not freshly thawed but maintained in the culture for at least 2-3 passages after thawing.
    Note: This method is not validated in other cell lines however, it can be easily adapted to analyze the dependence of different cancer cell lines on de novo NAD+ synthesis.

Acknowledgments

This protocol was adapted as previously reported (Merdanovic et al., 2005). N.D is a recipient of the Spanish Ramón y Cajal fellowship. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2010-18518), the Association for International Cancer Research AICR-UK (11-0242), CNIO (BC1104-08) and the European Foundation for the Study of Diabetes (EFSD).

References

  1. Merdanovic, M., Sauer, E. and Reidl, J. (2005). Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae. J Bacteriol 187(13): 4410-4420.
  2. Tummala, K. S., Gomes, A. L., Yilmaz, M., Grana, O., Bakiri, L., Ruppen, I., Ximenez-Embun, P., Sheshappanavar, V., Rodriguez-Justo, M., Pisano, D. G., Wagner, E. F. and Djouder, N. (2014). Inhibition of de novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage. Cancer Cell 26(6): 826-839.

简介

烟酰胺腺嘌呤二核苷酸(NAD + )是许多具有不同生物功能的NAD +消耗蛋白的辅酶。 NAD + 水平的振荡可能影响几种细胞信号传导途径。 通过Preiss-Handler途径(补救反应)的NAD +合成已被广泛报道。 然而,对L-色氨酸/犬尿氨酸分解代谢在从头NAD +合成中的贡献知之甚少。 在四种肝癌细胞系中使用L- [14 C] - 色氨酸示踪和siRNA介导的芳基甲酰胺酶(AFMID)的沉默,其是参与L-色氨酸降解的关键酶,我们证明L-色氨酸分解代谢在从头合成中的贡献 NAD + 池。 因此,NAD + 调节在维持细胞稳态和根据营养物可用性的适当的细胞功能中是重要的。

关键字:犬尿氨酸途径, 从头合成NAD +, [14C] -色氨酸代谢示踪, 芳基甲酰胺酶(afmid), 肝癌

材料和试剂

  1. 肝癌细胞系:Huh-7(JCRB,目录号:JCRB0403),HepG2(ATCC,目录号:HB-8065),SNU-398(ATCC,目录号:CRL-2233)和SNU-449 目录号:CRL-2234)
  2. DMEM(生命技术)
  3. RPMI(生命技术)
  4. 无氨基酸培养基,不含胎牛血清,称为Hanks平衡盐溶液(HBSS)(Sigma-Aldrich)
  5. 胎牛血清(Cultek)
  6. 青霉素和链霉素(Life Technologies,Gibco )
  7. 100x非必需氨基酸解决方案(生命技术)
  8. RNAiMAX(Life Technologies,Invitrogen TM ,目录号:13778030)
  9. OptiMem(Life Technologies,目录号:11058021)
  10. 非沉默小干扰RNA对照(siCt1)和小干扰RNA芳基甲酰胺酶(siAFMID)(GE Healthcare Dharmacon,目录号:L-HUMAN-XX-00055nmol)
  11. NAD + - [羰基-14C](Perkin Elmer,目录号:NEC831010UC)
  12. L- [苯环-U-14C] - 色氨酸(Moravek,目录号:MC 2335)
  13. PEI-纤维素薄层色谱(TLC)(20×20cm的F板)(Merk Millipore目录号:105725)
  14. 乙酸铵(Sigma-Aldrich,目录号:A1542)
  15. 乙醇和甲醇(Sigma-Aldrich)
  16. NAD + - [羰基-14C](参见配方)
  17. L- [苯环-U-14C] - 色氨酸(参见配方)
  18. 细胞密度(约60-80%)(参见配方)

设备

  1. 荧光图像读取器(Molecular Dynamics,型号:Storm820)
  2. 荧光图像筛(GE Healthcare Dharmacon,目录号:63-0034-79)
  3. Speedvac离心机(Thermo Fisher Scientific)
  4. 4℃台式离心机(Eppendorf,型号:5424R)
  5. 处理放射性物质的通风橱
  6. 细胞刮刀(Corning Incorporated,目录号:3008)
  7. 标尺

软件

  1. 斐济软件(www.fiji.sc/Fiji)

程序

  1. 四种肝癌细胞系:Huh-7,HepG2,SNU-398和SNU-449。
    1. Huh-7,HepG2,SNU-398和SNU-449细胞在10cm细胞中培养 培养皿在完全DMEM(Huh-7和HepG2细胞)和 RPMI培养基(SNU-398和SNU-449)。 两种培养基补充有10%   胎牛血清和100单位/ml青霉素和0.1mg/ml 链霉素
    2. 所提供的体积是12孔板的一个孔,   并且应该根据孔的大小缩放:200μl 将Optimem与1.2μl的siAFMID或siCtr轻轻混合。 这个 混合物加入1.6μlRNAiMAX中并在室温下温育   在井中20分钟。
    3. 轻轻重悬Huh-7,HepG2(8.0 x 10×10 4个)细胞在1,000μl的DMEM或SNU398,SNU449(3.5×10 4个)细胞中的 1,000μlRPMI培养基,并将这些细胞添加到RNAiMAX混合物中。 的 在该步骤中使用的不同单元格数目是绕过变量 生长速率在我们的细胞培养条件下获得类似的细胞 转染实验后的数字
    4. 24小时后,细胞 培养基更换为含有10%胎牛的DMEM或RPMI培养基 血清和100单位/ml的青霉素和0.1mg/ml的链霉素   除去上清液。
    5. 4小时后,除去培养基   1ml补充有非必需氨基酸(1x)的HBSS培养基 加入到细胞培养物中。
  2. 在HBSS中和在非必需氨基酸存在下将2.5mM的L- [苯环-U-14C] - 色氨酸加入细胞培养物中。将细胞在37℃下孵育5小时
  3. 通过弃去上清液除去细胞培养基,然后将细胞用冰冷的PBS洗涤3次
  4. 使用200μl的80%甲醇和20%的水混合物,通过废弃和用力吸取提取亲液代谢物。将混合物在4℃下孵育10分钟
  5. 然后将全细胞提取物在4℃以20,000×g离心20分钟。
  6. 将提取物在speedvac离心机中浓缩,从200μl的80%甲醇和20%水(步骤4)(其可能需要一小时,通过增加真空压力可以减少时间)的最终体积为40μl,随后点在PEI-纤维素薄层色谱(TLC)F板上
    1. 注释PEI-纤维素薄层色谱(TLC)F板
      1. 用铅笔在平板底部上方1厘米处画一条直线。
      2.  如图1所示,以2厘米的间隔,将代谢样品彼此等距离放置标记。


        图1.准备PEI-纤维素薄层色谱(TLC)F板进行测定

      3. 使用1μlNAD + - [羰基-14C]作为阳性对照 斑点在最后一个泳道中以校准标记的相对迁移 代谢物
      4. 添加5微升的代谢提取物从步骤6,让 现场干燥。 对所有样品重复此循环,直到整体 提取物被点样。
    2. 安装TLC迁移室
      1. 使用一种装置,用于将凝胶用于大型Western印迹作为TLC室
      2.  用70%乙醇擦拭TLC室。
      3.  填充代表迁移缓冲区的下层托盘 如过程的第7点所述,如图2所示。


        图2.安装TLC迁移室

    3. 用含有迁移缓冲液的PEI-纤维素TLC板设置TLC室


      图3.使用含有迁移缓冲液的PEI-纤维素TLC板(在步骤7中的组成)设置TLC室

      1. 小心地安装TLC板,其中板的底部   浸没在迁移缓冲液中(步骤7中的组成)。 但是 斑点样品不应直接接触迁移 缓冲区
      2. 板垂直安装在迁移缓冲液上  (步骤7中的组合物),并通过粘着抑制任何移动 它到顶部使用磁带。
  7. 在底部储存器中使用由接触0.5cm PEI-纤维素TLC F板的1M乙酸铵(pH 5)(30%)和无水乙醇(70%)组成的迁移缓冲液用于流动相。
  8. 在从起始线达到约15cm的5-6小时的迁移后,将纤维素板空气干燥并暴露于辐射敏感磷光体成像器屏幕过夜。使用Strom-820荧光图像读取器显示屏幕

    图4.在四种不同的肝癌细胞系中衍生自L- [14 C] - 色氨酸的放射性标记的代谢物的TLC。来自siCtr和siAFMID的代谢物同时彼此相邻地加载。 [14 C] -NAD + 用作来自L- [14 C] - 色氨酸的标记的NAD + 的相对迁移的校准对照(Tummala等,2014)。

笔记

使用4种不同的肝肿瘤细胞系进行实验至少4次,并且该印迹是这4个不同实验的代表。已观察到标记物的一致掺入。斐济软件( www.fiji.sc/Fiji )用于量化图像中不同波段的像素化并且相对于siCtr样品校准相对带强度。数据解释应当基于正确大小的条带的强度进行,更大的是条带强度,并且更大的是NAD + 含量。

食谱

  1. 作为相对迁移标准使用的NAD + Supp + + [羰基-14C],然而,并不强制使用这种特定的同位素,任何其它荧光成像剂敏感的NAD + sup/+同位素可用于此目的。
  2. 已经特别选择了L- [苯环-U-14C] - 色氨酸,因为色氨酸的苯环碳原子对NAD + 主链已知的贡献。
  3. 在饥饿时的细胞密度(约60-80%)产生最一致的结果。细胞不是新解冻,而是在解冻后保持在培养物中至少2-3代 注意:这种方法在其他细胞系中没有验证,但是可以容易地适用于分析不同癌细胞系对新细胞系的依赖性 em> +

致谢

该方案根据之前报道的方法改变(Merdanovic等人,2005)。 N.D是西班牙语Ramóny Cajal奖学金的接受者。这项工作由西班牙经济和竞争力部(SAF2010-18518),国际癌症研究协会AICR英国(11-0242),CNIO(BC1104-08)和欧洲糖尿病研究基金会(EFSD )。

参考文献

  1. Merdanovic,M.,Sauer,E。和Reidl,J。(2005)。 NAD + 生物合成与烟酰胺核糖基转运的偶联:NadR的表征 流感嗜血杆菌的核糖核苷酸激酶突变体。细菌 187(13):4410-4420。
  2. Tummala,KS,Gomes,AL,Yilmaz,M.,Grana,O.,Bakiri,L.,Ruppen,I.,Ximenez-Embun,P.,Sheshappanavar,V.,Rodriguez- Justo,M.,Pisano,DG ,Wagner,EF和Djouder,N。(2014)。 通过致癌性URI抑制新的NAD(+)合成导致肝脏 通过DNA损伤的肿瘤发生。癌细胞 26(6):826-839。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tummala, K. S. and Djouder, N. (2015). [14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells. Bio-protocol 5(17): e1582. DOI: 10.21769/BioProtoc.1582.
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Chloe Mica
Creative Peptides
Well, since I know little about liver cancer cells, I think I better read your post again and then try to figure out the connections involved.
11/16/2015 8:54:48 PM Reply