Nutr Res Pract.  2019 Feb;13(1):58-63. 10.4162/nrp.2019.13.1.58.

Walnut phenolic extracts reduce telomere length and telomerase activity in a colon cancer stem cell model

• Shin PK
• Zoh Y
• Choi J
• Kim MS
• Kim Y
• Choi SW

• KMID: 2434077
• DOI: http://doi.org/10.4162/nrp.2019.13.1.58

Abstract

BACKGROUND/OBJECTIVES
Telomeres are located at the chromosomal ends and progressively shortened during each cell cycle. Telomerase, which is regulated by hTERT and c-MYC, maintains telomeric DNA sequences. Especially, telomerase is active in cancer and stem cells to maintain telomere length for replicative immortality. Recently we reported that walnut phenolic extract (WPE) can reduce cell viability in a colon cancer stem cell (CSC) model. We, therefore, investigated the effect of WPE on telomere maintenance in the same model.
MATERIALS/METHODS
CD133+CD44+ cells from HCT116, a human colon cancer cell line, were sorted by Fluorescence-activated cell sorting (FACS) and treated with WPE at the concentrations of 0, 10, 20, and 40 µg/mL for 6 days. Telomere lengths were assessed by quantitative real-time PCR (qRT-PCR) using telomere specific primers and DNA extracted from the cells, which was further adjusted with single-copy gene and reference DNA (ddCt ). Telomerase activity was also measured by qRT-PCR after incubating the PCR mixture with cell protein extracts, which was adjusted with reference DNA (dCt ). Transcriptions of hTERT and c-MYC were determined using conventional RT-PCR.
RESULTS
Telomere length of WPE-treated cells was significantly decreased in a dose-dependent manner (5.16 ± 0.13 at 0 µg/mL, 4.79 ± 0.12 at 10 µg/mL, 3.24 ± 0.08 at 20 µg/mL and 3.99 ± 0.09 at 40 µg/mL; P = 0.0276). Telomerase activities concurrently decreased with telomere length (1.47 ± 0.04, 1.09 ± 0.01, 0.76 ± 0.08, and 0.88 ± 0.06; P = 0.0067). There was a positive correlation between telomere length and telomerase activity (r = 0.9090; P < 0.0001). Transcriptions of both hTERT and c-MYC were also significantly decreased in the same manner.
CONCLUSIONS
In the present cell culture model, WPE reduced telomere maintenance, which may provide a mechanistic link to the effect of walnuts on the viability of colon CSCs.

Keyword

Walnut; colonic neoplasms; stem cells; telomere; telomerase

MeSH Terms

Base Sequence
Cell Culture Techniques
Cell Cycle
Cell Line
Cell Survival
Colon*
Colonic Neoplasms*
DNA
Flow Cytometry
Humans
Juglans*
Phenol*
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Stem Cells*
Telomerase*
Telomere*
DNA
Phenol
Telomerase

Figure

• 

  Fig. 1 WPE reduced both telomere length and telomerase activity. (a) Relative telomere length (ddCt) of HCT116 CD133+CD44+ cells with respect to the WPE concentration. One-way ANOVA was used to determine the association between telomere length and WPE. Values are represented as Mean±SE, P < 0.05. Significant differences are observed for values having bars with different letters. (b) Relative telomerase activity (dCt) of HCT116 CD133+CD44+ cells with respect to the WPE concentration. One-way ANOVA was used to determine the association between telomerase activity and WPE. Values are represented as Mean±SE, P < 0.05. Significant differences are observed for values having bars with different letters. (c) Correlation between telomere length and telomerase activity. Correlation was assessed using Spearman's correlation. The correlation coefficient is observed to be r = 0.9090 and P < 0.0001.

• 

  Fig. 2 WPE reduced the transcriptions of hTERT and c-MYC. To determine the transcription of hTERT and c-MYC genes RT-PCR was conducted with RPLP0 gene as an endogenous control. WPE downregulated the transcriptions of hTERT (a) and c-MYC (b) in a dose dependent manner. Values are represented as Mean±SE, P < 0.05 by one-way ANOVA. Significant differences are observed for values having bars with different letters.

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