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

Abstract

LysoTracker and MitoTracker Red are fluorescent probes widely used for viable cell staining of lysosomes and mitochondria, respectively. They are utilized to study organelle localization and their resident proteins, assess organelle functionality and quantification of organelle numbers. The ATP‐driven efflux transporter P‐glycoprotein (P‐gp) is expressed in normal and malignant tissues and extrudes structurally distinct endogenous and exogenous cytotoxic compounds. Thus, once aromatic hydrophobic compounds such as the above‐mentioned fluorescent probes are recognized as transport substrates, efflux pumps including P‐gp may abolish their ability to reach their cellular target organelles. Herein, we show that LysoTracker and MitoTracker Red are expelled from P‐gp‐overexpressing cancer cells, thus hindering their ability to fluorescently mark target organelles. We further demonstrate that tariquidar, a potent P‐gp transport inhibitor, restores LysoTracker and MitoTracker Red cell entry. We conclude that LysoTracker and MitoTracker Red are P‐gp transport substrates, and therefore, P‐gp expression must be taken into consideration prior to cellular applications using these probes. Importantly, as MitoTracker was a superior P‐gp substrate than LysoTracker Red, we discuss the implications for the future design of chemotherapeutics evading cancer multidrug resistance. Furthermore, restoration of MitoTracker Red fluorescence in P‐gp‐overexpressing cells may facilitate the identification of potent P‐gp transport inhibitors (i.e. chemosensitizers).

Alteration of the Mitochondrial Effects of Ceria Nanoparticles by Gold: An Approach for the Mitochondrial Modulation of Cells Based on Nanomedicine

“…After 24 h, different NPs (AuCeO 2 or TPP-AuCeO 2 ) at 20 µg/mL were added and incubated for 24 h (37 • C, 5% CO 2 atmosphere (Linde, Barcelona, Spain)). Nuclei (Hoechst 33342 dye, 1:20,000, blue) (Life Technologies, Madrid, Spain), cell membrane (CellMaskTM, 1:10,000, green) (Life Technologies, Madrid, Spain), and mitochondria (MitoTracker™, 1:2,000, red) (Thermo Fisher Scientific, Sant Cugat del Vallés, Spain) were stained following the manufacturer's protocol [39,40]. Fluorescence images were obtained in vivo using a FV1000-spectral confocal microscope (Olympus, Hamburg, Germany).…”

DrosophilaSex Peptide Controls the Assembly of Lipid Microcarriers in Seminal Fluid

“…Microcarriers were also detected using high concentrations of the acidophilic, but partially hydrophobic, LysoTracker dyes (Fig. 1D; (30)). By contrast, in fixed tissue, microcarriers exclude access to antibodies raised against soluble secreted AG proteins, such as angiotensin I-converting enzyme (ANCE; Fig.…”

Centrosome amplification mediates small extracellular vesicles secretion via lysosome disruption

“…Next, we investigated if ROS could prevent fusion between MVBs and lysosomes, thereby promoting MVB fusion with the plasma membrane and release of SEVs ( Figure 3A). Using an antibody against phospholipid lysobisphosphatidic acid (LBPA), a lipid enriched at the membranes of late endosomes and MVBs [42], and lysotracker as a pH-based dye for functional lysosomes [43], we quantified the co-…”