DiOC2(3) is not a substrate for multidrug resistance protein (MRP)-mediated drug efflux

Abstract

Multidrug resistance (MDR) is often related to expression of P‐glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp‐mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine‐123 (Rh‐123), and 3,3′‐diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP‐expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh‐123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug‐free medium), no retention difference of the three agents was detected between parental and MRP‐expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh‐123 and Dox in the resistant lines became apparent and even more pronounced after 24 h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK‐104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh‐123 can be used to evaluate MRP‐function, but DiOC2(3) can not. © 1996 Wiley‐Liss, Inc.

Idarubicin DNA intercalation is reduced by MRP1 and not Pgp

“…Rhodamine is a better substrate for Pgp than MRP1, but the rhodamine efflux assay with a long (2 h) efflux is a sensitive method to detect both Pgp-and MRP1-mediated efflux. 19,24,25 Comparison of H33342 and rhodamine efflux may provide another functional assay for discriminating Pgp from MRP1.…”

“…The adherent Pgp expressing ovarian carcinoma cell line A2780/dox5 16,17 and the leukemia cell line HL60/ADR [18][19][20] displaying the MRP1 phenotype and their parent cell lines A2780/WT and HL60/WT were cultured in Iscove's medium (Life Technologies, Paisley, UK) supplemented with 10% heatinactivated fetal calf serum (FCS; Hyclone, Logan, UT, USA), 2 mM glutamine and 100 IU/ml penicillin, at 37°C in a humidified atmosphere with 5% CO 2 . The cells were used in an exponential growth phase and all cultures were routinely checked for mycoplasma contamination.…”

“…This MRP1-mediated rhodamine efflux is less pronounced and can only be detected after a prolonged efflux of at least 2 h as used in our experiments. 19 H33342 is also a substrate of Pgp efflux, 21,22 but currently no data are available about the influence of MRP1 expression on H33342 accumulation. Therefore, H33342 efflux was studied in A270 and HL60 cells by means of the H33342 efflux assay (without exposure to anthracyclines).…”

Role of MRP1 in multidrug resistance in acute myeloid leukemia

“…59,60 Another substrate, DiOC2(3), which is a substrate of Pgp, is not a substrate of MRP1. 57 Among some of these compounds are substrates of both MRP1 and Pgp, such as calcein-AM, Rh123, DNR. Although MRP1 has been identified as an organic anion transporter and Pgp as a transporter of certain positively charged compounds, there was considerable overlap in resistance spectrum, suggesting that both proteins transport important anticancer agents in AML treatment such as the anthracyclines and etoposide.…”

Is modulation of multidrug resistance a viable strategy for acute myeloid leukemia?

“…Measuring drug efflux from these AML blast cells using the efflux of fluorescent dyes 3, 3 0 diethyloxacarbocyanine iodide (DiOC 2 (3)) and rhodamine indicated that about two-thirds of these older patients did show efflux that could be inhibited by PSC833, and one-third of patients did not have inhibitable efflux of daunorubicin or of DiOC 2 (3). 17,39,40 The CR rate was 91% in the small number of patients studied whose cells did not efflux anthracycline-like drugs and who had received the ADE regimen. This was significantly higher than the 41% CR rate seen with the same treatment in the efflux positive patients (P ¼ 0.03).…”