Fluorescence methods to assess multidrug resistance in individual cells

Area of Science:

• Cell biology
• Pharmacology

Background:

• Multidrug resistance (MDR) is a significant challenge in treating diseases like cancer.
• Drug extrusion systems, such as P-glycoprotein (Pgp), are key mechanisms contributing to MDR.
• Accurate measurement of efflux pump activity in individual cells is crucial for understanding and overcoming MDR.

Purpose of the Study:

• To develop and validate microscopic methods for measuring the activity of drug extrusion systems in individual cells.
• To assess the utility of fluorescent calcium-sensing dyes for detecting Pgp-mediated drug efflux.
• To evaluate the effectiveness of chemosensitizers in enhancing dye accumulation in MDR cells.

Main Methods:

• Utilized fluorescent calcium-sensing dyes (fura2, indo1, fluo3) in their acetoxymethylester (AM) forms.
• Incubated multidrug-resistant (MDR) and parental cell lines with dye AM forms.
• Employed fluorescence microscopy to quantify intracellular dye accumulation and efflux.
• Tested the impact of chemosensitizers (cyclosporin A, verapamil, quinidine, progesterone) on dye retention.

Main Results:

• MDR cells showed minimal accumulation of dye free acid forms unless chemosensitizers were present.
• Chemosensitizers, particularly cyclosporin A, significantly increased intracellular dye fluorescence in MDR cells.
• Fluorescence microscopy successfully visualized and quantified drug efflux activity.
• Unlike rhodamine 123, fura2 free acid fluorescence remained stable in MDR cells overexpressing Pgp, indicating effective efflux measurement.

Conclusions:

• Developed fluorescence-based microscopic methods to detect and quantify drug extrusion activity in individual cells.
• These methods provide a functional readout of efflux pump activity, complementing protein or mRNA level studies.
• The techniques offer a rapid and quantifiable approach for screening potential multidrug resistance reversing agents.