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Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

ABSTRACT: 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.

SUBMITTER: Murota Y

PROVIDER: S-EPMC5097359 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Murota Yoshitaka Y Tabu Kouichi K Taga Tetsuya T

BMC cancer 20161104 1

<h4>Background</h4>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 va ...[more]

PMID: 27814696

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Project description:Plants are master regulators of rhizosphere ecology, secreting a complex mixture of compounds into the soil, collectively termed plant root exudate. Root exudate composition is highly dynamic and functional, mediating economically important interactions between plants and a wide range of soil organisms. Currently we know very little about the molecular basis of root exudate composition, which is a key hurdle to functional exploitation of root exudates for crop improvement. Root expressed transporters modulate exudate composition and could be manipulated to develop beneficial plant root exudate traits. Using Virus Induced Gene silencing (VIGS), we demonstrate that knockdown of two root-expressed ABC transporter genes in tomato cv. Moneymaker, ABC-C6 and ABC-G33, alters the composition of semi-volatile compounds in collected root exudates. Root exudate chemotaxis assays demonstrate that knockdown of each transporter gene triggers the repulsion of economically relevant Meloidogyne and Globodera spp. plant parasitic nematodes, which are attracted to control treatment root exudates. Knockdown of ABC-C6 inhibits egg hatching of Meloidogyne and Globodera spp., relative to controls. Knockdown of ABC-G33 has no impact on egg hatching of Meloidogyne spp. but has a substantial inhibitory impact on egg hatching of G. pallida. ABC-C6 knockdown has no impact on the attraction of the plant pathogen Agrobacterium tumefaciens, or the plant growth promoting Bacillus subtilis, relative to controls. Silencing ABC-G33 induces a statistically significant reduction in attraction of B. subtilis, with no impact on attraction of A. tumefaciens. By inoculating selected differentially exuded compounds into control root exudates, we demonstrate that hexadecaonic acid and pentadecane are biologically relevant parasite repellents. ABC-C6 represents a promising target for breeding or biotechnology intervention strategies as gene knockdown leads to the repulsion of economically important plant parasites and retains attraction of the beneficial rhizobacterium B. subtilis. This study exposes the link between ABC transporters, root exudate composition, and ex planta interactions with agriculturally and economically relevant rhizosphere organisms, paving the way for new approaches to rhizosphere engineering and crop protection.

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Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Publications

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

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