In silico and in vitro analyses to investigate the effects of vitamin C on VEGF protein

Abstract

Objectives: This study was conducted to evaluate the effects of vitamin C on apoptotic and proliferative genes in injured HepG2 cells.

Methods: In silico analysis was performed using molecular docking of chemical compounds with vascular endothelial growth factor (VEGF). The different computational tools used were AutoDock Vina, BIOVIA DISCOVERY studio, and PyMOL. Drug likeness and toxicity were analyzed by SWISS ADMET. Cells that were 60-70% confluent were treated with different concentrations of hydrogen peroxide (H₂O₂) (100-2000 μM) and ascorbic acid (30, 60, 90 μg/mL). The MTT cell proliferation assay was performed to compare the proliferative potential of HepG2 cells treated with H₂O₂ or ascorbic acid with untreated HepG2 cells using 96-well plates.

Results: The lowest binding energy of VEGF with vitamin C -5.2 kcal/mol and L-ascorbic acid-2 glycoside -4.7 kcal/mol was observed by in silico analysis. Vitamin C was selected because it exhibited a high interaction with VEGF and fulfilled Lipinski's rule, and had better oral viability and pharmacokinetics compared to L-ascorbic acid-2 glycoside. Cell viability assays showed that vitamin C had significant apoptotic effects (P < 0.0001). After treating HepG2 cells with ascorbic acid, reduced VEGF (angiogenesis) was observed as determined by apoptotic and proliferative gene expression. Ascorbic acid treatment of HepG2 cells led to downregulation of the proliferation markers, proliferating cell nuclear antigen, Ki67, and DNA topoisomerase II alpha. Increased apoptosis after treatment with vitamin C was observed due to upregulation of p53 and annexin V.

Conclusion: The results of this study showed that vitamin C inhibited the growth of cancer cells, thus protecting HepG2 cells from oxidative stress. Vitamin C exhibited antiproliferative activity as observed in silico and in vitro, as well as by the inhibited expression of genes involved in protein synthesis.

Keywords: Apoptosis; Cell cycle; Gene upregulation; Proliferative genes; Vitamin C.

Figure 1

Flow chart of the Methodology.

Figure 2

Protein structure validation.

Figure 3

In silico modeled structure representation of complex VEGF/vitamin C. (a): 3D Spherical cartoon structure of VEGF represent the interaction with ligand and binding pocket cavity interacting of residue of VEGF with vitamin C. (b): Surrounding interacting residue of VEGF included GLU-64, LEU-66, CYS-63, ASP-63, GLU-67, ASN-62, CYS-63, and GLY-59. (c): 2D structure of VEGF with vitamin C. Dotted line shows the polar interaction.

Figure 4

In silico modeled structure representation of complex VEGF/AA2G. (a): 3D spherical structure of VEGF represents the interaction with ligand and binding pocket cavity interacting residue of VEGF with vitamin C. (b): Interacting residue of VEGF included ASP-34, ASN-62, PHE-36, GLY-69, GLU-64, LEU-66, and GLU-67. (c): 2D structure of VEGF with LAA2G. Dotted line shows polar interaction.

Figure 5

Analysis of cell viability. (a) MTT assay (b) IC₅₀. H₂O₂ to produce oxidative stress that targets malignant cells. The values are presented as the mean ± standard error of the mean (SEM) where P < 0.0003 and ∗ indicate significant differences between the different concentrations of H₂O₂-treated and untreated cells.

Figure 6

Analysis of cell viability: (a). H₂O₂-pretreated HepG2 cells (injured) after ascorbic acid treatment. (b). The IC₅₀ value of vitamin C was calculated as 2.25 mg/mL in hepatocellular carcinoma. The values are presented as the mean ± SEM where P < 0.0001 and ∗ indicate significant differences between different concentrations of vitamin C and untreated cells.

Figure 7

Cell viability assay. (a) Trypan blue (b) Crystal violet. (a). The bar graph shows a lower number of dead cells treated with ascorbic acid. (b)The bar graph shows an increase in the viability of cells treated with ascorbic acid. The values are shown as the mean ± SEM. P < 0.0008 and P < 0.0001 and ∗ indicate significant differences between different concentrations of vitamin C and untreated cells.

Figure 8

Expression of VEGF. The effect of ascorbic acid at 90 μg/mL in HepG2 cells showed significantly reduced VEGF gene expression in the presence of H₂O₂, where P < 0.0001 and ∗ indicate significantly different treatments from untreated cells.

Figure 9

Analysis of apoptotic proteins via ELISA. (a) p53 (b) Annexin V. Compared with the control group, the bar graph shows higher apoptotic gene expression after ascorbic acid treatment at 90 μg/mL. P < 0.0001 and ∗ indicate significantly different treatments from untreated cells.

Figure 10

Gene expression analysis. (a) TOP2A (b) Ki-67 (c) PCNA. The bar graph represents the decrease in proliferative gene expression compared to that in the untreated group where P < 0.0001 and ∗ shows significant differences between treated and untreated cells.

Figure 11

Analysis of gene expression. (a) p53 (b) B-cell lymphoma 2-associated X protein (c) Caspase-3. More apoptotic gene expression was shown in the bar graph than in the untreated group, where P = 0.0001 and ∗ indicate that the treated and untreated cells significantly differed from one another.

Figure 12

Analysis of antioxidants. (a) Glutathione reductase (b) catalase (c) superoxide dismutase (d) Ascarbate peroxidase. Compared with the untreated group, the ascorbic acid-treated group showed both a decline and an increase in antioxidant levels in the bar graph. P < 0.0001 and ∗ indicate a significant difference between treated and untreated cells.