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Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP.

ABSTRACT: Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.

SUBMITTER: Swain BM

PROVIDER: S-EPMC7674423 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP.

Swain Brendan M BM Guo Dawei D Singh Himansha H Rawlins Philip B PB McAlister Mark M van Veen Hendrik W HW

Scientific reports 20201118 1

Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interac ...[more]

PMID: 33208856

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Project description:Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism.The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively.SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable.Inhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

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Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP.

Publications

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP.

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