Development and application of a UPLC-MS/MS method for P-glycoprotein quantification in human tumor cells

Area of Science:

• Biochemistry and Molecular Biology
• Pharmacology and Toxicology
• Cancer Research

Background:

• Multidrug resistance (MDR) in tumors reduces sensitivity to chemotherapy.
• Overexpression of P-glycoprotein (P-gp) is a primary mechanism of MDR.
• Understanding P-gp regulation is crucial for overcoming drug resistance.

Purpose of the Study:

• To establish a quantitative Ultra Performance Liquid Chromatography and Tandem Mass Spectrometry (UPLC-MS/MS) method for P-gp.
• To determine P-gp expression levels in human tumor cells and their drug-resistant subclones.
• To investigate the relationship between P-gp expression and the level of drug resistance.

Main Methods:

• Developed a UPLC-MS/MS assay using a synthesized P-gp peptide and a stable isotope-labeled internal standard.
• Quantified absolute P-gp expression in parental and multidrug-resistant human tumor cell lines (MCF-7, HepG-2, SMMC-7721).
• Analyzed P-gp mRNA levels and correlated resistance index (RI) with P-gp expression.

Main Results:

• The UPLC-MS/MS method demonstrated linearity and acceptable precision/accuracy for P-gp quantification.
• Most drug-resistant subclones showed significantly increased P-gp expression compared to parental cells.
• P-gp overexpression was not solely regulated by MDR1, and a moderate to high correlation was found between RI and P-gp expression.

Conclusions:

• A robust UPLC-MS/MS method for P-gp quantification was successfully established.
• The study provides quantitative data on P-gp expression in various drug-resistant cancer models.
• This work advances the understanding of MDR mechanisms and expands LC-MS/MS applications for efflux transporter studies.