Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells

Abstract

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 μg/mL melittin + 2 μg/mL cisplatin) and A2780CR (combination treatment 2 μg/mL melittin + 10 μg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R²) and goodness of prediction (Q²), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

Keywords: cisplatin; melittin; metabolomics; sensitive and resistant ovarian cancer cells; synergy.

Figure 1

Examination of cell viability after treatment with either cisplatin or melittin alone with various concentrations on (A) A2780 and (B) A2780CR cell lines. * Significantly different from zero. concentration at <0.05; *** Significant at <0.001.

Figure 2

Effects of melittin in combination with cisplatin on (A) cell viability of A2780 cell lines and (B) combination index. (A) The A2780 cells were treated with various concentrations of the melittin + cisplatin combination for 24 h. Bar graphs represent mean ± SD values. (B) Combination index (CI) analysis was generated using the method of Chou and Talalay [21,22] to determine the extent of synergy, if any, for the melittin + cisplatin combination on A2780 cell lines.

Figure 3

Effect of melittin in combination with cisplatin on (A) cell viability of A2780CR cell lines and (B) combination index. (A) The A2780CR cells were treated with various concentrations of the melittin + cisplatin combination for 24 h. Bar graphs represent mean ± SD values. (B) Combination index (CI) analysis was generated using the method of Chou and Talalay [21,22] to determine the extent of synergy, if any, for the melittin + cisplatin combination on A2780CR cell lines.

Figure 4

(A) Principal components analysis (PCA) vs. (B) Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). PCA and OPLS-DA scores plot generated from PCA and OPLS-DA using LC-MS normalised data of cells after exposure to the combination (melittin + cisplatin) and controls of A2780 and A2780CR cell lines. A2780-treated cells at 5 μg/mL melittin + 2 μg/mL cisplatin (SOS); untreated A2780 cells (C); A2780CR-treated cells at 2 μg/mL melittin + 10 μg/mL cisplatin (SOR); untreated A2780CR (CR); pooled quality control (QC) samples (P).

Figure 5

OPLS-DA score plot of (A) A2780 cells and (B) A2780CR before and after treatment with melittin + cisplatin respectively. Separation of the treated and untreated cells in both cell lines in the model suggests that there are significant metabolite differences induced by treatment in both cell lines. The metabolites responsible for the observed separation are shown in Table 1.