Effects of galloflavin and ellagic acid on sirtuin 6 and its anti-tumorigenic activities

Abstract

Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates distinct cellular functions; genome stability, DNA repair, and inflammation related diseases. Recently, we demonstrated that anthocyanidins in berries induce the catalytic activity of SIRT6. In this study, we explored the effects of Galloflavin and Ellagic acid, the most common polyphenols in berries, on SIRT6. SIRT6 deacetylation was investigated using HPLC and immunoblotting assays. The expression levels of SIRT6, glycolytic proteins and cellular metabolism were studied on human colon adenocarcinoma cells (Caco2). Molecular docking studies were carried out to study possible interactions of the compounds with sirtuins. Ellagic acid increased the deacetylase activity of SIRT6 by up to 50-fold; it showed moderate inhibition of SIRT1-3. Galloflavin and Ellagic acid showed anti-proliferative effects on Caco2. The compounds also upregulated SIRT6 expression whereas key proteins in glycolysis were downregulated. Galloflavin decreased glucose transporter 1 (GLUT1) expression, and Ellagic acid affected the expression of protein dehydrogenase kinase 1 (PDK1). Interestingly, both compounds caused reduction in glucose uptake and lactate production. Both Galloflavin and Ellagic acid were able to form hydrogen bonds with Asp188 and Gly6 in SIRT6. In this study, we showed that Galloflavin and Ellagic acid increased SIRT6 activity and decreased the expression of SIRT6 associated proteins involved in cancer development. Taken together, Galloflavin and Ellagic acid targeting SIRT6 activity may provide a new insight in the development of anti-cancer therapy.

Keywords: Berries; Cancer; Polyphenol; Sirtuin; Tannin.

Fig. 1.

The chemical structures of Cyanidin, Gallic acid, Galloflavin and Ellagic acid.

Fig. 2.. Galloflavin and Ellagic acid stimulate SIRT6 and inhibit SIRT2 deacetylation activities in vitro.

(A) SIRT6 deacetylation activity in the presence of Gallic acid, Galloflavin and Ellagic acid at 10 μM and 50 μM concentrations. (B) SIRT1–3 deacetylation activity in the presence of Galloflavin and Ellagic acid at 100 μM. The results are presented as a fold change compared to the DMSO control value (mean ± SD; n = 3).

Fig. 3.. Galloflavin and Ellagic acid dose-dependently induce SIRT6 activity.

(A) Dose response effect on SIRT6 deacetylation activity by Galloflavin and Ellagic acid by HPLC assay. (B) Ellagic acid induced a decrease in K_m value of acetylated H3K9Ac substrate. Michaelis-Menten analysis of H3K9Ac (0–225 μM) with (filled square) and without (filled circles) Ellagic acid at 25 μM concentrations. The data are presented as mean ± SEM (n = 3).

Fig. 4.. The docking pose of Ellagic acid at SIRT6′s putative activator site.

The purple dashes indicate hydrogen bonding between amino acids (Gly6, Asp188) and Ellagic acid. The dark grey molecule is ADP-ribose (Adenosine diphosphate ribose) part of cofactor NAD⁺. The surface is colored by the electrostatic potential with blue showing the highest potentials and red the lowest.

Fig. 5.. Galloflavin and Ellagic acid did not show effect on cell viabilitybut decreased relative cell numberafter 24-h treatments.

(A, B) Cell viability is expressed as percent of viable cells from total number of cells, and(C, D) relative cell number as percent of treated cells (gray bars) from DMSO control (light gray bars). The values are means ±SEM from three individual experiments (n = 3) each mean of four replicates on the same 48-well plate. Statistical significance of treated groups to DMSO control groups were analyzed with one-way ANOVA followed Bonferroni and Dunnett post hoc test (*p values < 0.05 vs. control, **p values < 0.01 vs. control).

Fig. 6.. Galloflavin or Ellagic acid up-regulated SIRT6 expression in Caco2 cells.

(A, B) Cells were exposed to 0.5 % DMSO control (light gray bars) or various concentrations of Galloflavin or Ellagic acid (gray bars) for 24-h. The effect of treatment on the SIRT6 expression was determined with one way-ANOVA with Bonferroni and Dunnett post hoc test by comparing treated groups to DMSO control groups. Values are expressed as mean ± SEM of three independent experiments (*p values < 0.05 vs. control, **p values < 0.01 vs. control).

Fig. 7.. Galloflavin and Ellagic acid affected acetylation levels of H3K9Ac and the expression of SIRT6 target genes GLUT1 and PDK1.

(A, B) H3K9Ac levels after Galloflavin and Ellagic acid treatments. (C) GLUT1 expression after Galloflavin treatment. (D) PDK1 expression after Ellagic acid treatment. Caco2 cells were treated with DMSO control (light gray bars) or different concentration of compounds (gray bars) for 24-h. Results are shown as a fold change compared to the control value where ratio between the protein and corresponding loading control is calculated. The effect of treatment on H3K9Ac levels or GLUT1/PDK1 expression was determined with one way-ANOVA with Bonferroni and Dunnett post hoc test by comparing treated groups to DMSO control groups. Data represents the mean ± SEM of three independent experiments (*p < 0.05 vs. control, **p < 0.01 vs. control and ***p < 0.001 vs. control).

Fig. 8.. Galloflavin and Ellagic acid decreased (A, B) glucose uptake and (C, D) lactate production.

Caco2 cells were exposed to DMSO control (light gray bars) or various concentrations of compounds (gray bars) for 24-h. Data represent the mean ± SEM of three independent experiments, and the statistical analysis was carried out with one way-ANOVA with Bonferroni and Dunnett post hoc test by comparing treated groups to DMSO control groups (*p < 0.05 vs. control, **p < 0.01 vs. control and ***p < 0.001 vs. control).

Fig. 9.. Schematic presentation metabolic reprogramming results in abnormal glycolysis in cancer cell.

Process involves the uptake of high levels of glucose, enhanced glycolysis, and the metabolism of pyruvate to lactic acid rather than enter to tricarboxylic acid (TCA). Sirtuins regulated the expression of key genes, pyruvate dehydrogenase kinase 1 (PDK1) and glucose transporter 1 (GLUT1) involved in the process through hypoxia-inducible factor 1α (HIF1α). Galloflavin (GF) decreased the expression of GLUT1 and reduced glucose uptake while Ellagic acid (EA) downregulated PDK1 which subsequently decreased lactate production through pyruvate dehydrogenase (PDH).