Walnut Phenolic Extracts Reduced both Telomere Length and Telomerase Activity in Colon Cancer Stem Cells
Authors: Shin Phil‐Kyung , Zoh Yoonchae , Kim Yuri , Choi Sang‐Woon 
Published: November 2023

Telomeres, TTAGGG tandem repeats at chromosomal ends, protect the linear chromosomal ends from degradation during cell division. The steady shortening of telomeres with each replication in somatic cells may play a critical role in cell senescence. Telomerase is a RNA‐dependent DNA polymerase that synthesizes telomeric DNA sequences. In most healthy human somatic cells, telomerase activity is undetectable or low, but it appears in most immortalized cells, stem cells and cancer cells for unlimited proliferative potential. In our previous study, walnut phenolic extract (WPE) demonstrated a decrease in cell viability with an increase in cell differentiation in colon cancer stem cells (CSC). We, therefore, investigated the effect of WPE on telomere length and telomerase activity in colon CSCs. From the human colon cancer cell line HCT116, CD133+CD44+ cells were sorted by FACS and treated with WPE at 0ug/mL, 10ug/mL, 20ug/mL and 40ug/mL for 6 days. Telomere lengths were assessed by quantitative real‐time PCR using telomere specific primers and DNA extracted from cells. The telomere length was represented by the ratio of telomere repeat copy number (T) to single copy gene copy number (S), which was further adjusted with T/S ratio of a reference DNA sample. The 36B4 gene was used for single copy number gene and the human embryonic kidney 293 (HEK 293) cell line was used as a reference DNA. The T/S ratio (dCt) for each sample was calculated by subtracting the average 36B4 Ct value from the average telomere Ct value. The relative T/S ratio (ddCt) was adjusted by Ct of HEK 293 cells. Telomerase activity was measured by the SYBR Green method of detection. The PCR reaction mixture consisted of SYBR Green master mix, EGTA, each of primers TS and ACX, protein extract of cells. The PCR reaction mixture was incubated at room temperature for 30 min to allow the telomerase in the protein extracts to elongate the TS primer by adding TTAGGG repeat sequences. Thereafter, quantitative real time PCR was conducted. TS was a forward primer and ACX was a reverse primer. The HEK 293 cell line was used as a reference DNA and telomerase activity (mean ± SE) was represented as dCT. Differences between variables were tested using ANOVA and correlations between telomere length and telomerase activity were assessed using Spearman's correlation. Telomere length of WPE treated cells was significantly decreased in a dose‐dependent manner (5.16±0.13 at 0ug/ml, 4.79±0.12 at 10ug/ml, 3.24±0.08 at 20ug/ml and 3.99±0.09 at 40ug/ml, p<0.0276). Interestingly, telomerase activities were concurrently decreased with telomere length (1.47±0.04 at 0ug/ml, 1.09±0.01 at 10ug/ml, 0.76±0.08 at 20ug/ml and 0.88±0.06 at 40ug/ml, p<0.0067). There was a significant positive correlation between telomere length and telomerase activity (r=0.9090, p<0.0001). In the present cell culture model, WPE down‐regulates telomere maintenance by reducing the activity of telomerase, which may result in the shortening of telomere length. This observation may provide a mechanistic link to the effect of walnut on cell viability and differentiation of colon CSCs.

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