Metabolomic profiling of the effects of melittin and cisplatin on ovarian cancer cells using high resolution mass spectrometry

Over the last few years, metabolomics has come to play an increasingly important part in many fields of research, notably medical studies. However, there is a dearth of research on metabolomics in the area of ovarian cancer and the increase in anti-cancer (platinum) drug resistance. Thus further studies on the modes of anticancer action and the mechanisms of resistance of ovarian cancer cells at the metabolome level are needed. The aim of this study was to characterise the metabolic profiles of two human ovarian cancer cell lines, A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant), in response to their exposure to melittin, cisplatin and melittin-cisplatin combination therapy. It has been suggested that melittin may have potential as an anti-cancer therapy; combining cisplatin and melittin may increase response and tolerability in cancer treatment, as well as reducing drug resistance.The A2780 and A2780CR cell lines were treated with sub-lethal doses of melittin, cisplatin and melittin-cisplatin combination therapy for 24 hours before extraction and global metabolite analysis of cell lysates by LC-MS using a HPLC system. Phenotype MicroArray™ experiments were also applied in order to test carbon substrate utilisation or sensitivity in both cell lines after exposure to melittin and cisplatin. Data extraction was carried out with MZmine 2.10 with metabolite searching against an in-house database. The data were analysed using univariate and multivariate methods.The changes induced by melittin in the cisplatin-sensitive cells mainly resulted in reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, ATP and NAD+. It was necessary to evaluate the effect of a melittin on lipid activities of ovarian cancer cell lines. In order to do so, an LC coupled to an Orbitrap Exactive mass spectrometer using an ACE silica gel column was employed. The two cell lines had distinct lipid compositions, with the A2780CR cells having lower levels of several ether lipids than the A2780 cells. The changes induced by melittin in both cell lines mainly led to a decrease the level of PC and PE. Lipids were significantly altered in both A2780 and A2780CR cells. The observed effect was much more marked in the cisplatin-sensitive cells, suggesting that the sensitive cells undergo much more extensive membrane re-modelling in responsexviito melittin in comparison with the resistant cells. Regarding the metabolic effects of cisplatin on A2780 cells, these mainly resulted decreased levels of acetylcarnitine, phosphocreatine, arginine, proline and glutathione disulfide, as well as to increased levels of tryptophan and methionine. A number of metabolites were differently affected between the A2780 and A2780CR cells following cisplatin treatment, with A2780CR cells presenting increased levels of lysine, and decreased levels of N-acetyl-glutamate, oxoglutarate and 2-oxobutanoate compared to sensitive cells. However, when the combination treatment was applied, there were significant changes in both cell lines, mainly resulting in a reduction of levels of citrate cycle, oxidative phosphorylation, purine, pyrimidine and arginine/proline pathways. The combination of melittin with cisplatin has a synergistic effect when targeting these pathways. The melittin-cisplatin combination had stronger effect on A2780 cell lines than it had on those of A2780CR.Overall, this study suggests that melittin may have some potential as an adjuvant therapy in cancer treatment. A global metabolomics approach can be a useful tool for evaluating the pharmacological effects of anti-cancer compounds or synergetic sensitisers using mass spectrometry.Over the last few years, metabolomics has come to play an increasingly important part in many fields of research, notably medical studies. However, there is a dearth of research on metabolomics in the area of ovarian cancer and the increase in anti-cancer (platinum) drug resistance. Thus further studies on the modes of anticancer action and the mechanisms of resistance of ovarian cancer cells at the metabolome level are needed. The aim of this study was to characterise the metabolic profiles of two human ovarian cancer cell lines, A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant), in response to their exposure to melittin, cisplatin and melittin-cisplatin combination therapy. It has been suggested that melittin may have potential as an anti-cancer therapy; combining cisplatin and melittin may increase response and tolerability in cancer treatment, as well as reducing drug resistance.The A2780 and A2780CR cell lines were treated with sub-lethal doses of melittin, cisplatin and melittin-cisplatin combination therapy for 24 hours before extraction and global metabolite analysis of cell lysates by LC-MS using a HPLC system. Phenotype MicroArray™ experiments were also applied in order to test carbon substrate utilisation or sensitivity in both cell lines after exposure to melittin and cisplatin. Data extraction was carried out with MZmine 2.10 with metabolite searching against an in-house database. The data were analysed using univariate and multivariate methods.The changes induced by melittin in the cisplatin-sensitive cells mainly resulted in reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, ATP and NAD+. It was necessary to evaluate the effect of a melittin on lipid activities of ovarian cancer cell lines. In order to do so, an LC coupled to an Orbitrap Exactive mass spectrometer using an ACE silica gel column was employed. The two cell lines had distinct lipid compositions, with the A2780CR cells having lower levels of several ether lipids than the A2780 cells. The changes induced by melittin in both cell lines mainly led to a decrease the level of PC and PE. Lipids were significantly altered in both A2780 and A2780CR cells. The observed effect was much more marked in the cisplatin-sensitive cells, suggesting that the sensitive cells undergo much more extensive membrane re-modelling in responsexviito melittin in comparison with the resistant cells. Regarding the metabolic effects of cisplatin on A2780 cells, these mainly resulted decreased levels of acetylcarnitine, phosphocreatine, arginine, proline and glutathione disulfide, as well as to increased levels of tryptophan and methionine. A number of metabolites were differently affected between the A2780 and A2780CR cells following cisplatin treatment, with A2780CR cells presenting increased levels of lysine, and decreased levels of N-acetyl-glutamate, oxoglutarate and 2-oxobutanoate compared to sensitive cells. However, when the combination treatment was applied, there were significant changes in both cell lines, mainly resulting in a reduction of levels of citrate cycle, oxidative phosphorylation, purine, pyrimidine and arginine/proline pathways. The combination of melittin with cisplatin has a synergistic effect when targeting these pathways. The melittin-cisplatin combination had stronger effect on A2780 cell lines than it had on those of A2780CR.Overall, this study suggests that melittin may have some potential as an adjuvant therapy in cancer treatment. A global metabolomics approach can be a useful tool for evaluating the pharmacological effects of anti-cancer compounds or synergetic sensitisers using mass spectrometry