Carrier-Mediated Uptake of Rhodamine 123: Implications on Its Use for MDR Research Cheong-Weon Cho,* , † Yang Liu,* , ‡ Xiangdong Yan,* , † Thomas Henthorn,† and Ka-Yun Ng* ,1 *Department of Pharmaceutical Sciences, School of Pharmacy, †Department of Anesthesiology, and ‡Department of Neurosurgery, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received October 30, 2000 We have examined the effects of verapamil and PSC 833 on cellular uptake and release of rhodamine 123 (R123) in two human cancer cell lines. Both verapamil and PSC 833 were able to increase R123 accumulation in the multidrug resistant (MDR) MV522/Q6 and KB- 8-5 lines in the release study. However, the effects of these drugs on R123 accumulation during accumula- tion study were quite different. Incubation with PSC 833 increased R123 accumulation in both MDR lines. By contrast, incubation with verapamil only increased R123 accumulation in the KB-8-5 line. The failure of verapamil to increase R123 accumulation in the MV522/Q6 cells can be attributed to the presence of a carrier system in the parent MV522 cells that recog- nizes both R123 and verapamil, but not PSC 833, as substrates. These results imply that performing R123 accumulation study without first ascertaining possi- ble role of a carrier system for cellular uptake of R123 and putative P-gp modulators might inadvertently lead one to draw improper conclusions on P-gp activity. © 2000 Academic Press Key Words: P-glycoprotein; rhodamine 123; multi- drug resistance; MDR; efflux; uptake; cancer; lung carcinoma. Multidrug resistance (MDR) represents a major ob- stacle to successful chemotherapy of metastatic dis- eases (1). The nature of the drug resistance is com- plex, but it is generally believed that cells become multidrug-resistant by either developing mechanisms which interfere with programmed cell death or by ex- pression of energy-dependent pump systems which ex- clude or extrude anti-cancer drugs from cells (1). Of these pumps, the MDR1 gene-encoded multidrug transporter or P-glycoprotein (P-gp) (2, 3) and the MRP gene-encoded MDR-associated proteins (4) have been most extensively studied. Both transporters are ATP- dependent transporters, and belong to a larger family of ATP-binding cassette (ABC) proteins (5). The MDR1-encoded P-gp is known to recognize and transport many structurally and functionally unre- lated anticancer drugs, including vinca alkaloids (6), anthracyclines (7), epipodophyllotoxins (8), and tax- anes (9, 10). In addition to anticancer drugs, a variety of other chemicals have been found to be substrates of P-gp. One such chemical is a lipophilic cationic fluores- cent dye, rhodamine 123 (R123). R123, which selec- tively locates in mitochondria (11), has been found to be relatively non-toxic (11, 12) and effluxed more effi- ciently by MDR cells (13, 14). Moreover, such efflux has also been found to be inhibited by established modula- tors of MDR (14, 15). Thus, by following cellular R123 accumulation or retention in the presence or absence of P-gp modulators, valuable information on P-gp activity as well as efficiency of these modulators in reversing P-gp activity can be obtained. Since R123 is considered a permeant cationic fluo- rescent probe (12), its uptake into cells has generally been assumed to occur via a passive diffusion pro- cess (16). Here, utilizing the parent and P-gp over- expressing MDR human metastatic lung carcinoma (MV522) and epidermoid carcinoma (KB) cell lines, we have information to suggest that cellular uptake of R123 in the MV522 cells might occur via a carrier system. In addition, verapamil, which is an established P-gp modulator, might serve as competitive inhibitor for such a system. These results have important impli- cations on how one should use R123 for P-gp evaluation in that performing R123 accumulation analysis with- out first knowing whether the P-gp modulators use the same carrier system as R123 for uptake into cells might inappropriately rule out the presence of P-gp activity and thereby underestimates the effective- ness of modulators in reversing MDR activity. R123 1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Campus Box C238, 4200 E. 9th Avenue, Denver, CO 80262. Fax: 303-315-0274. E-mail: mailto:Lawrence.Ng@UCHSC.edu. Biochemical and Biophysical Research Communications 279, 124 –130 (2000) doi:10.1006/bbrc.2000.3916, available online at http://www.idealibrary.com on 124 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.