Fluorescent cellular indicators are extruded by the multidrug resistance protein

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Multidrug resistance (MDR) is a major challenge in cancer therapy.
• P-glycoprotein (P-gp), encoded by the MDR1 gene, is a key efflux pump responsible for MDR.
• Fluorescent indicators are widely used in cell biology research.

Purpose of the Study:

• To investigate the interaction between fluorescent dye acetoxymethyl ester (AM) derivatives and the human multidrug transporter (MDR1).
• To determine if MDR1 affects the cellular loading of fluorescent indicators.
• To explore the potential of this interaction for detecting MDR1-expressing cells and screening drug interactions.

Main Methods:

• NIH-3T3 mouse fibroblasts stably expressing human MDR1 were used.
• Comparison of dye extrusion in MDR1-expressing cells versus control cells.
• Inhibition studies using known MDR1 substrates and inhibitors (verapamil, vincristine, etc.).
• ATPase activity assays in isolated cell membranes.

Main Results:

• MDR1-expressing cells actively extruded hydrophobic AM derivatives of fluorescent indicators.
• Dye extrusion was blocked by MDR1 inhibitors and competing substrates.
• Hydrophilic free acid forms of the indicators were not exported by MDR1.
• AM derivatives stimulated MDR1-ATPase activity, while free acid forms did not.

Conclusions:

• MDR1 actively extrudes AM derivatives of fluorescent indicators, potentially before cytoplasmic esterase cleavage.
• This interaction can be exploited for efficient detection of MDR1-expressing tumor cells.
• The findings offer a screening method for evaluating drug interactions with the MDR1 transporter.