engleski

The effects and molecular mechanisms of crown ethers on drug efflux pumps

Cancer stem cells (CSCs) are regarded as a leading cause of cancer relapse due to their ability to reconstitute its heterogeneity even after tumor eradication. It has been suggested that CSC resistance to chemo- and radiotherapy could be related to enhanced survival pathways, decreased levels of reactive oxygen species and increased expression of drug efflux pumps. Nevertheless, related molecular mechanisms remain elusive. Recent research has pinpointed salinomycin, a naturally occurring potassium ionophore, as a promising compound for targeting CSCs. Moreover, several studies indicate that one of the mechanisms of action of salinomycin towards CSCs is the inhibition of P-gp drug efflux pump activity. Pertaining to the mentioned studies and results previously published by our group regarding anticancer activity of proprietary crown ethers that act as K+ ionophores, we hypothesized that these compounds could show selectivity towards CSC. Therefore, we set about to elucidate their mechanism of action, with the focus on the effect towards the function and activity of drug efflux pumps. Materials and Methods: The impact of compounds on the expression of P-gp was evaluated by Western Blot and qRT-PCR. Moreover, MTT, cell cycle and annexin V assays were used to determine the effect of compounds on cancer cells with respect to P-gp expression, while the functional activity of P- gp was evaluated by Rhodamine efflux assay. Mechanisms of action of tested compounds were evaluated by UIC2 shift assay, Western Blot and immunofluorescence. Results and Discussion: Cytotoxicity evaluated by MTT showed increased resistance of P-gp overexpressing cells to P-gp substrates (e.g. doxorubicine, paclitaxel), as expected. Results obtained by Rhodamine efflux assay showed strong P-gp inhibition by proprietary compounds, while combined treatment with paclitaxel indicated cell sensitization towards this chemotherapeutic. Additionally, implications of crown ether molecular mechanisms of action were obtained by UIC2 shift assay and by analysis of the impact of these compounds on P-gp expression and upstream regulators of this drug efflux pump. Conclusions: Results indicate stronger P-gp inhibition obtained by proprietary crown ether treatment than by the commercially available P- gp inhibitor, verapamil. Additionally, plausible molecular mechanisms of action of crown ether compounds will be presented.

tumorske matične stanice ; ABC transporteri ; P-gp ; krunasti eteri

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