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

BackgroundElucidating 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.MethodsThe 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.ResultsSP 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.ConclusionInhibiting 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.

The PI3K/AKT signaling pathway regulates ABCG2 expression and confers resistance to chemotherapy in human multiple myeloma

“…SP cells have been detected in numerous solid tumors (36–38) and hematological malignancies (39,40). In an early study on MM, SP cells were detected in 18 out of 21 samples; the inability to detect SP cells in the remaining three samples was likely due to biological polymorphisms (41).…”

LysoTracker and MitoTracker Red are transport substrates of P‐glycoprotein: implications for anticancer drug design evading multidrug resistance

“…P-gp forms a natural barrier against toxins and xenobiotics in various human organs, but slight alterations such as polymorphisms in exons of the MDR1 gene may endow this pump with the ability to recognize different anticancer drugs [31], thus, rendering tumour cells MDR to chemotherapy [32,33]. In addition to various cytotoxins and chemotherapeutic agents, ABC transporters including P-gp were also shown to expel various molecular markers, such as the viable DNA dye Hoechst 33342, the oxidative stress indicator CellROX, the mitochondrial marker MitoTracker Green and the mitochondrial membrane potential probe JC-1 [2,34]. This extrusion of fluorescent molecular markers hinders their application in cells expressing these MDR pumps resulting in misinterpretation of the findings if this extrusion is not taken into account.…”

Apoptotic effects of ε-viniferin in combination with cis-platin in C6 cells

“…Loss of mitochondrial membrane potential (MMP, Wm) is a crucial step in the apoptotic process that kills cells because it leads to the release of various proapoptotic factors from the mitochondria to the cytoplasm (Cao et al 2011). Numerous studies evaluated MMP using flow cytometry in C6 cells (Bobermin et al 2017;Murota et al 2016;Geng et al 2016). The integrity of the mitochondrial membrane was analyzed using JC-1, a cationic dye, in the present study.…”