Cinnamaldehyde and eugenol change the expression folds of AKT1 and DKC1 genes and decrease the telomere length of human adipose-derived stem cells (hASCs): An experimental and in silico study

Abstract

Objectives: To investigate the effect of cinnamaldehyde and eugenol on the telomere-dependent senescence of stem cells. In addition, to search the probable targets of mentioned phytochemicals between human telomere interacting proteins (TIPs) using in silico studies.

Materials and methods: Human adipose derived stem cells (hASCs) were studied under treatments with 2.5 µM/ml cinnamaldehyde, 0.1 µg/ml eugenol, 0.01% DMSO or any additive. The expression of TERT, AKT1 and DKC1 genes and the telomere length were assessed over 48-hr treatment. In addition, docking study was conducted to show probable ways through which phytochemicals interact with TIPs.

Results: Treated and untreated hASCs had undetectable TERT expression, but they had different AKT1 and DKC1 expression levels (CI=0.95; P<0.05). The telomere lengths were reduced in phytochemicals treated with hASCs when compared with the untreated cells (P<0.05). Docking results showed that the TIPs might be the proper targets for cinnamaldehyde and eugenol. Data mining showed there are many targets for cinnamaldehyde and eugenol in the intracellular environment.

Conclusion: The general effect of cinnamaldehyde and eugenol is their induction of stem cell senescence. Therefore, they could be applicable as chemo-preventive or antineoplastic agents.

Keywords: Aging; Cinnamaldehyde; Eugenol; Stem cells; Telomerase; Telomere.

Figure 1

Tukey-HSD and ANOVA analysis for AKT1 folds comparison, using qPCR. The Figure shows the 0.01% DMSO or 2.5 µM/ml cinnamaldehyde increase the AKT1 expression folds in hASCs. Nevertheless, the 0.1 µg/ml eugenol did not affect AKT1 expression significantly

Figure 2

Tukey-HSD and ANOVA analysis for DKC1 folds comparison. The Figure shows the 0.01% DMSO or 2.5 µM/ml cinnamaldehyde increase the DKC1 expression folds in hASCs. But the 0.1 µg/ml eugenol decreased DKC1 expression significantly

Figure 3

Telomere length comparison between examined groups. The control hASCs, 2.5 µM/ml cinnamaldehyde and 0.1 µg/ml eugenol significantly decreased the telomere length (P-value< 0.05) but not in comparison with 0.01% DMSO group (P-value> 0.05)

Figure 4

Dendrogram of cluster analysis for 22 TIPs plus telomerase (TERT); Left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores. Clustering for docking scores performed by the nearest neighbor method and the measure of Squared Euclidian distance. Some important docking results are shown in Figure 6-14

Figure 5

Comparison of docking scores for different ligand-proteins interactions. The cinnamaldehyde and eugenol docked with TIPs in Molegro Virtual Docker (MVD) software. Analysis of variance (ANOVA) shows there is a significant difference between all scores (CI= 0.95; P= 0.000)

Figure 6

Docking result of RPL22 (PDB ID: 3J3A) protein with eugenol; Above: Binding site of the eugenol (green) on the protein; Below: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -101.80. It is the best score of dockings and belong to the category 5 of clustering

Figure 7

Docking result of TERT (modeled structure) protein with eugenol; Above: Binding site of the eugenol (green) on the protein; Below: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -95.6. This score belongs to category 6 of clustering

Figure 8

Docking result of HSP90AA1 (PDB ID: 3Q6N) protein with eugenol; Above: Binding site of eugenol (green) on the protein; Below: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -89.73. This score belongs to category 3 of clustering

Figure 9

Docking result of KU70-KU80 hetero-complex (PDB ID: 1JEQ) protein with eugenol; Above: Binding site of eugenol (green) on the protein; Below: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -84.95. This score belongs to category 4 of clustering

Figure 10

Docking result of 14-3-3 (PDB ID: 2BR9) protein with eugenol; Above: Binding site of eugenol (green) on the protein; Below: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -80.42. This score belongs to category 1 of clustering

Figure 11

Docking result of KIP (PDB ID: 1Y1A) protein with eugenol; Above: Binding site of eugenol (green) on the protein; Below: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -75.21. This score belongs to category 2 of clustering

Figure 12

Docking result of RNPC2 (PDB ID: 2CQ4) protein with cinnamaldehyde; Above: Binding site of cinnamaldehyde (green) on the protein; Below: binding pocket has not hydrogen bounds. MolDock Score for this interaction= -62.42. This score belongs to category 8 of clustering

Figure 13

Docking result of SMN1 (PDB ID: 1G5V) protein with eugenol; Above: Binding site of eugenol (green) on the protein; Below: Hydrogen bound of ligand-protein interaction (striated line) in binding site pocket. MolDock Score for this interaction= -58.96. This score belongs to category 7 of clustering

Figure 14

Docking result of HNRNPA1 (PDB ID: 1PGZ) protein with eugenol; Left: Binding site of eugenol (green) on the protein; Right: Hydrogen bounds of ligand-protein interaction (striated lines) in binding site pocket. MolDock Score for this interaction= -82.83. The molecule eugenol locates in a deep binding pocket of protein

Figure 15

Association network of 23 TIPs; the thicker lines represent stronger confidence of association. Some abbreviations in the string-db plotted net were not mentioned in the text; they include: RBM38 equals RNPC1; RBM39 equals RNPC2; XRCC6 equals Ku70; XRCC5 equals Ku80; NCL equals nucleolin; La equals SSB; CIB1 equals KIP; YWHAQ equals 14-3-3; TMED10 equals p23

Figure 16

The probable effects of the cinnamaldehyde and eugenol on the senescence and expected events after the cell treatment. The cinnamaldehyde and eugenol may bind to TIPs or act on their gene expression and impel the signal transduction pathways. This effect strength or postponed the cellular aging. The left gray box shows the gene names of proteins, which have positive effect on the telomere or telomerase. The left gray box shows two proteins with negative effects on the telomere or telomerase. In our work, we have seen that phytochemicals induce the senescence in hASCs. Then, the red lines show the probable occurred events in the current work