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European Journal of Nutrition

, Volume 58, Issue 4, pp 1635–1645 | Cite as

Metabolic influence of walnut phenolic extract on mitochondria in a colon cancer stem cell model

  • Jina Choi
  • Phil-Kyung Shin
  • Yuri Kim
  • Chang Pyo Hong
  • Sang-Woon ChoiEmail author
Original Contribution

Abstract

Purpose

Walnut phenolic extract (WPE) reduces proliferation and enhances differentiation of colon cancer stem cells (CSCs). The present study investigated the metabolic influence of WPE on the mitochondrial function of colon CSCs to determine its underlying mechanism.

Methods

CD133+CD44+ HCT116 colon cancer cells were selected by fluorescence-activated cell sorting and were treated with or without 40 µg/mL WPE. RNA-sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs), which were further validated with RT-PCR. WPE-induced alterations in mitochondrial function were investigated through a mitochondrial stress test by determining cellular oxygen consumption rate (OCR), an indicator of mitochondrial respiration, and extracellular acidification rate (ECAR), an indicator of glycolysis, which were further confirmed by glucose uptake and lactate production tests.

Results

RNA-Seq analysis identified two major functional clusters: metabolic and mitochondrial clusters. WPE treatment shifted the metabolic profile of cells towards the glycolysis pathway (ΔECAR = 36.98 mpH/min/ptn, p = 0.02) and oxidative pathway (ΔOCR = 29.18 pmol/min/ptn, p = 0.00001). Serial mitochondrial stimulations using respiration modulators, oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, and rotenone/antimycin A, found an increased potential of mitochondrial respiration (ΔOCR = 111.5 pmol/min/ptn, p = 0.0006). WPE treatment also increased glucose uptake (Δ = 0.39 pmol/µL, p = 0.002) and lactate production (Δ = 0.08 nmol/µL, p = 0.005).

Conclusions

WPE treatment shifts the mitochondrial metabolism of colon CSC towards more aerobic glycolysis, which might be associated with the alterations in the characteristics of colon CSC.

Keywords

Colon cancer stem cell Glycolysis Metabolic reprogramming Mitochondria Walnut 

Abbreviations

WPE

Walnut phenolic extracts

CSC

Cancer stem cell

RNA-Seq

RNA-sequencing

FACS

Fluorescence-activated cell sorting

DEGs

Differentially expressed genes

OCR

Oxygen consumption rate

ECAR

Extracellular acidification rate

2-DG

2-Deoxyglucose

GO

Gene ontology

DAVID

The Database for Annotation, Visualization and Integrated Discovery

KEGG

Kyoto Encyclopedia of Genes and Genomes

FPKM

Fragments per kilobase of transcript per million mapped reads

FCCP

Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone

Notes

Acknowledgements

This work was supported by the California Walnut Commission. All authors have participated in the conception, design, and conduction of the study, as well as interpretation of data and drafting the manuscript. J. C. and P. K. S. also performed cell culture studies and measured endpoints. C. H. conducted RNA-Seq and data analysis. Y. K. and S. W. C. have supervised the study.

Compliance with ethical standards

Conflict of interest

The authors have declared no conflicts of interest.

Supplementary material

394_2018_1708_MOESM1_ESM.pptx (103 kb)
Supplementary material 1. The volcano plot that represents the overall changes in the transcription by WPE treatment. Log2 fold changes of WPE/Ctrl were plotted against –log10 p value. Red dots represent significantly differentially expressed transcripts (p < 0.05, fold change ≥ 1.5). WPE: WPE-treated group. Ctrl: Control group (PPTX 103 KB)
394_2018_1708_MOESM2_ESM.pptx (605 kb)
Supplementary material 2. Functional enrichment analysis and cluster distribution network of up- and down-regulated genes in the WPE-treated CD133+CD44+ HCT116 cells. The circular nodes indicate the ontology terms and pathways of DEGs, which are functionally grouped and interconnected based on the kappa score. The terms and pathways with adjusted p values less than 0.05 were selected for the network construction. The size of the nodes represents the term significance after Bonferroni correction. The significant terms of each group are highlighted. (A) A cluster distribution network of enriched functions and pathways of the 1168 upregulated genes, which are categorized into four major clusters: cluster1 (metabolism), cluster2 (mitochondrion), cluster3 (apoptosis), and cluster4 (cancer pathway), (B) A cluster distribution network of the 815 downregulated genes are representing four major clusters: cluster1 (DNA metabolic process), cluster2 (mitotic cell cycle), cluster3 (RNA metabolic process), and cluster4 (centriole). Both figures are generated by the ClueGO and CluePedia plugin (PPTX 604 KB)
394_2018_1708_MOESM3_ESM.docx (19 kb)
Supplementary material 3 (DOCX 19 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.CHA University School of MedicineSeongnamSouth Korea
  2. 2.Nutritional Science and Food ManagementEwha Womans UniversitySeoulSouth Korea
  3. 3.Teragen EtexSuwonSouth Korea
  4. 4.Chaum Life CenterCHA UniversitySeoulSouth Korea
  5. 5.Jean Mayer USDA Human Nutrition Research Center at Tufts UniversityBostonUSA

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