Metabolic influence of walnut phenolic extract on mitochondria in a colon cancer stem cell model
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.
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.
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).
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.
KeywordsColon cancer stem cell Glycolysis Metabolic reprogramming Mitochondria Walnut
Walnut phenolic extracts
Cancer stem cell
Fluorescence-activated cell sorting
Differentially expressed genes
Oxygen consumption rate
Extracellular acidification rate
The Database for Annotation, Visualization and Integrated Discovery
Kyoto Encyclopedia of Genes and Genomes
Fragments per kilobase of transcript per million mapped reads
Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone
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.
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