Exploiting drug synergism to extend the application of oestrone analogues

Abstract

Most successful treatment strategies for neosplasms comprise combinations of anti-cancer drugs with different mechanisms of action and side effect profiles. This strategy allows for the use of anti-cancer agents at the lowest possible concentrations, translating into fewer adverse events reported in the clinical setting. To identify synergistic combinations in vitro, the Median Effect equation of Chou and Talalay is often employed. In this study two characteristics which distinguish malignant cells from non-malignant cells were targeted using combination therapy: enhanced proliferation rate and altered energy production. The increased proliferation rate of tumourigenic cells were targeted using antimitotic oestrone analogues, ESE-15-ol and ESE-16. These oestrone analogues were in silico designed from the potent anti-cancer compound 2-methoxyoestradiol. Six glycolysis inhibitors were selected for this study in order to selectively target the aerobic glycolysis preferred by malignant cells for energy production. The glycolysis inhibitors included the glucose analogue 2-deoxyglucose, the hexokinase inhibitor 3-bromopyruvate, the anti-spermatogenic agent lonidamine, the small molecule fasentin, the flavonoid quercetin (QUER) and the HIV-protease inhibitor indinavir (IND). The potential synergy of combinations of anti-mitotic oestrone analogues and glycolysis inhibitors were investigated after 72 h using the sulforhodamine B cell enumeration assay and in vitro breast cancer models presenting the three main phenotypic subtypes of clinical breast cancer. Using CalcuSYN software based on the Median Effect equation, a combination index value is calculated for each tested combination indicating the degree of synergy achieved. Based on the combination index values and published toxicity reports, combinations of ESE- 15-ol and ESE-16 with either IND or QUER were selected for further study on the MCF-7 and MDA-MB-231 breast adenocarcinoma cells. The sequence of toxicity induced by the selected synergistic combinations was investigated by evaluating various intracellular parameters. It was demonstrated that selected combinations of oestrone analogues and glycolysis inhibitors resulted in generation of reactive oxygen species after 6 h, which is regarded as an indicator of early toxicity. After 24 h the catabolic adaptive mechanism of autophagy was not induced by any combination. However, as autophagy is a dynamic process its role later in the sequence of toxicity cannot be excluded. Ultrastructure studies indicate the formation of multivesicular bodies in combination treated cells. Data indicate that combinations of oestrone analogues with IND caused hyperpolarisation of the mitochondrial membrane potential which suggests inhibition of ATP synthesis, activation of the extrinsic pathway and the induction of apoptotic and necrotic cell death after 72 h. Treatment with combinations of oestrone analogues with QUER resulted dissipation of mitochondrial membrane potential, activation of caspases 9 and -3/7 which culminated in the induction of apoptotic and necrotic cell death after 72 h. As tumourigenic cells readily induce the formation of new blood vessels and invade nontumourigenic tissue, the ability of the selected combinations to inhibit angiogenesis was investigated. Combinations of oestrone analogues with IND or QUER resulted in decreased secretion of pro-angiogenic vascular endothelial growth factor, inhibited the formation of complex tubule networks of endothelial cells and reduced migration of breast adenocarcinoma and endothelial cells. This suggests that these combinations have anti-angiogenic properties. During this study the potential synergy of oestrone analogues and glycolysis inhibitors were investigated as a means of effectively eradicating breast cancer using low doses of anticancer agents. Synergistic combinations of oestrone analogues and IND or QUER were identified on MCF-7 and MDA-MB-231 breast adenocarcinoma cells. Data indicate that these combinations activate different molecular pathways to induce cell death via apoptosis and necrosis and inhibit various stages of angiogenesis. It is therefore proposed that these combinations have potential as anti-metastatic therapy.