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

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.

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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

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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.

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Correspondence to Sang-Woon Choi.

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Electronic supplementary material

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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)

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)

Supplementary material 3 (DOCX 19 KB)

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Choi, J., Shin, P., Kim, Y. et al. Metabolic influence of walnut phenolic extract on mitochondria in a colon cancer stem cell model. Eur J Nutr 58, 1635–1645 (2019). https://doi.org/10.1007/s00394-018-1708-z

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Keywords

  • Colon cancer stem cell
  • Glycolysis
  • Metabolic reprogramming
  • Mitochondria
  • Walnut