Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells

Abstract

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (Δ Ψ m), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell–rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence ( V i) can be used to estimate Δ Ψ m using the Nernst equation: Δ Ψ m=−61.54 log V i−258.46. The Δ Ψ m values determined were −160±4 mV for K562 cell, −146±6 mV for K562/ adr cell, −161±10 mV for GLC4 cell and −168±2 mV for GLC4/ adr cell. An increase or a decrease in Δ Ψ m consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.