Delphinidin Increases the Sensitivity of Ovarian Cancer Cell Lines to 3-Bromopyruvate

Abstract

3-Bromopyruvic acid (3-BP) is a promising anticancer compound. Two ovary cancer (OC) cell lines, PEO1 and SKOV3, showed relatively high sensitivity to 3-BP (half maximal inhibitory concentration (IC₅₀) of 18.7 and 40.5 µM, respectively). However, the further sensitization of OC cells to 3-BP would be desirable. Delphinidin (D) has been reported to be cytotoxic for cancer cell lines. We found that D was the most toxic for PEO1 and SKOV3 cells from among several flavonoids tested. The combined action of 3-BP and D was mostly synergistic in PEO1 cells and mostly weakly antagonistic in SKOV3 cells. The viability of MRC-5 fibroblasts was not affected by both compounds at concentrations of up to 100 µM. The combined action of 3-BP and D decreased the level of ATP and of dihydroethidium (DHE)-detectable reactive oxygen species (ROS), cellular mobility and cell staining with phalloidin and Mitotracker Red in both cell lines but increased the 2',7'-dichlorofluorescein (DCFDA)-detectable ROS level and decreased the mitochondrial membrane potential and mitochondrial mass only in PEO1 cells. The glutathione level was increased by 3-BP+D only in SKOV3 cells. These differences may contribute to the lower sensitivity of SKOV3 cells to 3-BP+D. Our results point to the possibility of sensitization of at least some OC cells to 3-BP by D.

Keywords: 3-bromopyruvate; anthocyanidins; delphinidin; glycolytic inhibitors; ovarian cancer.

Figure 1

The chemical structure of 3-bromopyruvic acid (a) and delphinidin (3,3′,4′,5,5′,7-hexahydroxyflavylium) (b).

Figure 2

Effect of 3-bromopyruvic acid (3-BP) (a) and delphinidin (D) (b) on the survival of PEO1, SKOV3 and MRC-5 cells estimated by the Neutral Red assay. The cells were treated with D in the concentration range of 10–100 μM and 3-BP at concentrations of 5–25 μM for PEO1 and 10–50 μM for MRC-5, as well as SKOV3. Nontreated cell were used as a control. * p < 0.05 with respect to the control (Kruskal-Wallis test; n ≥ 6).

Figure 3

Effect of the combined action of 3-BP and D on the viability of the PEO1, SKOV3 and MRC-5 cells. Survival of the cells treated with 3-BP at a concentration corresponding to ~half maximal inhibitory concentration (IC₅₀) (SKOV3 and MRC-5: 40 μM and PEO1: 20 μM) and various concentrations of D indicated on the axis of the abscissae (a). Plot of the combination index (CI) as a function of the effect level (Fa) for the combined action of 3-BP and D. Concentrations of 3-BP and D: 20, 40, 60 and 80 μM (b). * p < 0.05 with respect to the control (Kruskal-Wallis test; n ≥ 6).

Figure 4

Effect of 3-BP and delphinidin (D) on the ATP (a) and GSH content (b) of PEO1, as well as SKOV3 and MRC-5 cells. *p < 0.05 with respect to the control (Kruskal-Wallis test; n ≥ 6).

Figure 5

Effect of 3-BP and delphinidin (D) on the level of reactive oxygen species (ROS) estimated with dihydroethidium (DHE, (a) and 2’,7’-dihydrodichlorofluorescein diacetate (DCFDA, (b). * p < 0.05 with respect to the control (one-way ANOVA and Least Significant Difference (LSD) post-hoc test; n ≥ 3).

Figure 6

Effect of 3-BP and delphinidin (D) on mitochondrial staining with the JC-1 probe (a) and mitochondrial mass (b) of PEO1 and SCOV3 cells. * p < 0.05 with respect to the control (one-way ANOVA and LSD post-hoc test; n ≥ 3).

Figure 7

Morphological changes in PEO1 and SKOV3 cells (confocal images, 65×). Effect of 3-BP, D and D plus 3-BP on the staining of PEO1 and SKOV3 cells with phalloidin (a) and Mitotracker Red (b).

Figure 7

Morphological changes in PEO1 and SKOV3 cells (confocal images, 65×). Effect of 3-BP, D and D plus 3-BP on the staining of PEO1 and SKOV3 cells with phalloidin (a) and Mitotracker Red (b).

Figure 7

Morphological changes in PEO1 and SKOV3 cells (confocal images, 65×). Effect of 3-BP, D and D plus 3-BP on the staining of PEO1 and SKOV3 cells with phalloidin (a) and Mitotracker Red (b).

Figure 7

Morphological changes in PEO1 and SKOV3 cells (confocal images, 65×). Effect of 3-BP, D and D plus 3-BP on the staining of PEO1 and SKOV3 cells with phalloidin (a) and Mitotracker Red (b).

Figure 8

Effect of 3-BP and delphinidin (D) on the migration of PEO1 and SCOV3 cells. * p < 0.05 with respect to the control (Kruskal-Wallis test; n = 12).

Figure 9

The induction of apoptosis and necrosis by 3-BP, D and 3-BP+D in PEO1 and SKOV3 cells (after 24 h). Fluorescence and luminescence, respectively, of control cells at 0 time assumed as 1. ** p < 0.01 and *** p < 0.001 with respect to the control (one-way ANOVA and LSD post-hoc test; n = 3).