The inhibitory effect of 6-gingerol and cisplatin on ovarian cancer and antitumor activity: In silico, in vitro, and in vivo

Abstract

Background: Epithelial ovarian cancer is very common in women and causes hundreds of deaths per year worldwide. Chemotherapy drugs including cisplatin have adverse effects on patients' health. Complementary treatments and the use of herbal medicines can help improve the performance of medicine. 6-Gingerol is the major pharmacologically active component of ginger. In this study, we compared the effects of 6-gingerol, cisplatin, and their combination in apoptotic and angiogenetic activities in silico, in test tubes, and in in vivo assays against two ovarian cancer cell lines: OVCAR-3 and human umbilical vein endothelial cells (HUVECs).

Methods: The drug-treated cell lines were evaluated for their cytotoxicity, cell cycle, and apoptotic and angiogenetic gene expression changes.

Results: The proportion of apoptosis treated by 6-gingerol coupled with cisplatin was significantly high. In the evaluation of the cell cycle, the combination therapy also showed a significant promotion of a higher extent of the S sequence. The expression of p53 level, Caspase-8, Bax, and Apaf1 genes was amplified again with combination therapy. Conversely, in both cell lines, the cumulative drug concentrations reduced the expression of VEGF, FLT1, KDR, and Bcl-2 genes. Similarly, in the control group, combination treatment significantly decreased the expression of VEGF, FLT1, KDR, and Bcl-2 genes in comparison to cisplatin alone.

Conclusions: The findings of the present study demonstrated that the cisplatin and 6-gingerol combination is more effective in inducing apoptosis and suppressing the angiogenesis of ovarian cancer cells than using each drug alone.

Keywords: angiogenesis; apoptosis; chick embryo; cisplatin; gingerol; molecular dynamics simulation; ovarian neoplasm.

Figure 1

(I) 3D structure of (A) 6-gingerol and (B) cisplatin as stick and bond types in a position using MVD studies. (II) Docking configuration and dynamic sites of (A) Bax, (B) Bcl-2, (C) Caspase-8, (D) KDR1, and (E) VEGF-A target proteins (PDB ID: 5W5X, 5JSN, 1I4E, 2QU5, and 5T89, respectively) using MVD studies.

Figure 2

Illustration of the finest score docking solution of the 6-gingerol ligands and (A) Bax, (B) Bcl-2, (C) Caspase-8, (D) KDR1, and (E) VEGF-A receptor with the designated crystal construction of 5W5X, 5JSN, 1I4E, 2QU5, and 5T89, respectively, and a ligand map with various chemical bonds courtesy of Discovery Studio.

Figure 3

Illustration of the finest score docking solution of the cisplatin ligands and (A) Bax, (B) Bcl-2, (C) Caspase-8, (D) KDR1, and (E) VEGF-A receptor with the designated crystal construction of 5W5X, 5JSN, 1I4E, 2QU5, and 5T89, respectively, and a ligand map with various chemical bonds courtesy of Discovery Studio.

Figure 4

Representation of the finest score docking solution of the mixture of the two drugs and best ligands and (A) Bax, (B) Bcl-2, (C) Caspase-8, (D) KDR1, and (E) VEGF-A receptor with the designated crystal structure of 5W5X, 5JSN, 1I4E, 2QU5, and 5T89, respectively, and a ligand map with various chemical bonds courtesy of Discovery Studio.

Figure 5

The isobologram analysis of the effects of the drug combination of cisplatin and 6-gingerol. (A) In HUVECs, foci a and b displayed the IC₅₀ value of cisplatin (136.52 ± 21.36 µM) and 6-gingerol (154.2 ± 38.43 µM), respectively. Theoretical IC₅₀ was 145.36 µM and our experimental IC₅₀ was 118.6 ± 18.52 µM. (B) In OVCAR-3 cells, foci a and b displayed the IC₅₀ value of cisplatin (61.23 ± 4.22 µM) and 6-gingerol (79.66 ± 8.63 µM), respectively. Theoretical IC₅₀ was 70.46 µM and our experimental IC₅₀ was 46.33 ± 3.68 µM. Statistical analysis revealed that there was a significant difference between experimental IC₅₀ and theoretical IC₅₀ (p < 0.001).

Figure 6

Characteristics of apoptosis and necrosis of (I) HUVECs and (II) the OVCAR-3 cell line treated with different concentrations of cisplatin, 6-gingerol, and the combination therapy.

Figure 7

Cell cycle characteristics of (I) HUVECs and (II) the cell line treated with different concentrations of cisplatin, 6-gingerol, and the combination therapy [*significant difference with the control group (p < 0.001), **significant difference between drug combination therapy and cisplatin (p < 0.001)].

Figure 8

Evaluation of (I) apoptotic (Bax, Bcl-2, Caspase, p53, and Apaf1) and (II) angiogenetic (KDR, FLT1, and VEGF) gene expression in HUVECs and OVCAR-3 cell lines treated with different concentrations of cisplatin, 6-gingerol, and the combination therapy [*significant difference with the control group (p < 0.001)].

Figure 9

(I) Effect of cisplatin, 6-gingerol, and the combination therapy on the chick embryo’s blood vessels. (A) Control group, (B) cisplatin, (C) 6-gingerol, (D) mixture, and (E) vascular density (error bars show mean ± standard error; *p < 0.05 compared to the control group). (II) Effect of cisplatin, 6-gingerol, and the combination therapy on the apoptotic (Bax, Bcl-2, Apaf1, and TP53) and angiogenetic (VEGF and KDR) mediator gene expression changes treated embryos compared to controls. The expression levels were normalized to GAPDH and HPRT and calibrated to controls (error bars show mean ± standard error; *p < 0.05).

Figure 10

Histopathological changes in H&E and IHC study [apoptotic (Bax and Bcl-2) and angiogenetic (CD34) markers] of the chick embryo treated with cisplatin, 6-gingerol, and the combination therapy.