Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells.
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 (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-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 DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(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 DeltaPsi(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.
Reungpatthanaphong
Paiboon
P
Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131, Thailand.
Dechsupa
Samarn
S
Meesungnoen
Jintana
J
Loetchutinat
Chatchanok
C
Mankhetkorn
Samlee
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005562:Formazans; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013778:Tetrazolium Salts
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate; D004317:Doxorubicin; D005456:Fluorescent Dyes; D005562:Formazans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012235:Rhodamines; D013778:Tetrazolium Salts; C079782:MTT formazan; C027260:pirarubicin; C029773:rhodamine B; C421109:multidrug resistance-associated protein 1