肝癌细胞中[14C]色氨酸代谢示踪法

Krishna S. Tummala; Nabil Djouder

doi:10.21769/BioProtoc.1582

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Peer-reviewed

[14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells

肝癌细胞中[14C]色氨酸代谢示踪法

Krishna S. Tummala

Nabil Djouder email

发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1582 浏览次数: 9270

评审: Jia LiShannon RuppertYong Teng

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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

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 )
DMEM (Life technologies)
RPMI (Life technologies)
Amino acid free medium and without fetal bovine serum called Hanks’ Balanced Salt Solution (HBSS) (Sigma-Aldrich)
Fetal bovine serum (Cultek)
Penicilin and streptocmycin (Life Technologies, Gibco^®)
100x Non-essential amino acids’ solution (Life technologies)
RNAiMAX (Life Technologies, Invitrogen^TM, catalog number: 13778030 )
OptiMem (Life Technologies, catalog number: 11058021 )
Non silencing small interference RNA control (siCtl) and small interference RNA Arylformamidase (siAFMID) (GE Healthcare Dharmacon, catalog number: L-HUMAN-XX-0005 5 nmol )
NAD+-[carbonyl-14C] (Perkin Elmer, catalog number: NEC831010UC )
L-[benzene-ring-U-14C]-tryptophan (Moravek, catalog number: MC 2335 )
PEI-cellulose thin layer chromatography (TLC) (F plates of 20 x 20 cm) (Merk Millipore catalog number: 105725 )
Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
Ethanol and methanol (Sigma-Aldrich)
NAD⁺-[carbonyl-14C] (see Recipes)
L-[benzene-ring-U-14C]-tryptophan (see Recipes)
Cell density (around 60-80%) (see Recipes)

Equipment

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

Software

Fiji software (www.fiji.sc/Fiji)

Procedure

English

中文翻译

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如何引用

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.