Tài liệu Báo cáo khoa học: Competition between innate multidrug resistance and intracellular binding

Competition between innate multidrug resistance and

intracellular binding of rhodamine dyes

Daniella Yeheskely-Hayon, Ronit Regev, Hagar Katzir and Gera D. Eytan

Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel

A major obstacle impeding the success of chemother￾apy is multidrug resistance (MDR). MDR in patients

is exhibited as a resistance to a wide variety of struc￾turally unrelated drugs. This is caused by several

factors, one of which is ‘classical’ MDR characterized

by diminished cellular uptake of the drugs due to

active export by one or more ABC transporters. The

ABC transporter most often over-expressed in vitro in

cells exposed to increasing drug concentrations is

plasma glycoprotein (Pgp, ABCB1) [1–3]. This protein

is an active efflux pump for chemotherapeutic drugs,

natural products and hydrophobic partially positive

dyes. The ABC transporter superfamily is represented

in humans by 48 members [4,5], of which 24 are

known to function as drug transporters relevant to

various diseases. In addition to Pgp, the multidrug

resistance-associated protein (MRP1, ABCC1) [6] and

breast cancer resistance protein (BCRP⁄MXR ⁄ABCP,

Keywords

innate multidrug resistance; MDR; MRP1;

multidrug resistance; rhodamines

Correspondence

G. D. Eytan, Department of Biology, The

Technion – Israel Institute of Technology,

Haifa, Israel

Fax: +972 4 8225153

Tel: +972 4 8293406

E-mail: [email protected]

Website: http://biology.technion.ac.il

(Received 7 September 2008, revised 12

November 2008, accepted 20 November

2008)

doi:10.1111/j.1742-4658.2008.06812.x

The present study aimed to elucidate the contribution of the intracellular

binding of drugs to multidrug resistance. For this purpose, uptake of rhod￾amines was studied in cells whose mitochondria had been uncoupled with

carbonyl cyanide m-chlorophenylhydrazone. Surprisingly, in a variety of

drug-untreated cells, presumed to be sensitive to multidrug resistance-type

drugs, rhodamines were excluded from entering the cells. Thus, the amount

of rhodamine 123 taken up into parental untreated K562 cells was less than

the amount bound to the cell exterior. Rhodamine uptake was prevented

by an active efflux pump. The efflux was inhibited by 4-chloro-7-nitro￾2,1,3-benzoxadiazole (NBD-Cl) and MK571 and, to a lesser extent, by

ATP depletion, indomethacin, probenecid and vanadate. All the inhibitors,

apart from NBD-Cl, are known to modulate multidrug resistance-associ￾ated protein (MRP) 1. Because MRP1 was expressed in all the cell lines

tested and the efflux of rhodamines in MRP1 over-expressing cells was

abolished by NBD-Cl, it appears that rhodamines are excluded from these

cells by MRP1. On the other hand, the uptake of rhodamines into cells

respiring with their coupled mitochondria demonstrated diminished sensi￾tivity to NBD-Cl and MK571. Thus, active pumping into the mitochondria

allowed enhanced uptake into the cells, overcoming the innate resistance.

The innate resistance provided by MRP1 to cells prevents rhodamine dyes,

and possibly drugs such as doxorubicin, from achieving equilibration of

their concentration in the cytoplasm with their concentration in the exter￾nal medium. The protection provided to multidrug resistance cells by ABC

transporters has to overcome competition by passive uptake of the drugs

and binding ⁄ uptake of the drugs into intracellular targets.

Abbreviations

CCCP, carbonyl cyanide m-chlorophenylhydrazone; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; NBD-Cl,

4-chloro-7-nitro-2,1,3-benzoxadiazole; Pgp, plasma glycoprotein; TMR, tetramethylrhodamine; TMRM, tetramethylrhodamine methyl ester.

FEBS Journal 276 (2009) 637–648 ª 2008 The Authors Journal compilation ª 2008 FEBS 637