Assessment and comparison of efflux pumps of cancer cells and MDR bacteria under physiological conditions by a real-time semi-automated system

Assessment of overexpressed efflux pumps (EPs) is usuallyconducted with the common EP substrate fluorochromeethidium bromide (EB) in the absence and presence of agentsthat are believed to inhibit EPs and hence promote theaccumulation of EB. This method is conducted at roomtemperature with a buffer of pH 7 and usually without anysource of metabolic energy. These conditions are far fromthose under which EPs are expected to function optimally. Thesemi-automated method to be presented utilises a buffer whosepH ranges from 5 to 8, contains a source of metabolic energyand is maintained at 37 ̊C. These are conditions that favourefflux and hence should be suitable for the study of EPs ofmultidrug-resistant (MDR) bacteria. Accumulation of EB, itsefflux, and the effects of agents that increase accumulation andinhibit efflux, has been followed on a real-time basis with theaid of the Rotor-Gene 3000™. The method has been appliedfor the study of EPs of MDR strains of Escherichia coli,Salmonella, Enterobacter, Enterococcus, Staphylococcusandmycobacteria. Overall, whereas at pH 5 CCCP, PAβN andphenothiazines do not increase the accumulation of EB orprevent its efflux under conditions that favor efflux, withincreasing pH, these agents cause increased accumulation ofEB, although the medium is highly supportive of efflux. Theresults suggest that at low pH, the energy (protons) that is usedfor driving efflux is supplied by the proton gradient, whereasat high pH the needed protons are provided by metabolicenergy. Evaluation of the efflux pump of cancer cells was alsoconducted by the same methodology. The results presentedshow that the assessment and inhibition of efflux pumps ofcancer cells that render the cells immune to cytotoxic agentscan be conducted on a real-time basis with a degree ofprecision not possible with flow cytometry. Moreover, becauseof the capacity of the system, a large number of cell systemscan be evaluated and compared with any one run of themethod.