Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines

Abstract

Previous reports suggest that resistance to mitoxantrone in different tumor cell lines is unrelated to the overexpression of pglycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry and confocal microscopy were used to study two human myeloid leukemia cell lines selected for resistance to mitoxantrone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the detection of intracellular mitoxantrone, we determined the maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cytometric conditions using 568.2 nm laser emission for excitation and a 620 nm long pass filter for fluorescence collection resulted in a 1-log increase in sensitivity, compared with standard 488-nm laser excitation. Uptake and retention of mitoxantrone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results showed no change in uptake and retention of the drug in pglycoprotein-negative mitoxantrone-resistant HL-60MX2 cells and in its sensitive parental line, HL-60s. In contrast, 3.1-and 2.4-fold increases were found in uptake and retention of mitoxantrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug distribution demonstrated reduced nuclear uptake, which could be reversed by verapamil, in HL-60DOX. A characteristic punctate pattern was observed for the intracytoplasmic drug distribution in HL-60DOX and HL-60MX2 cells and was partially modified by the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our study demonstrates that the cellular pharmacology of mitoxantrone is affected by p-glycoprotein and can be reversed at least in part by verapamil. Other mechanisms of resistance however, seem to play a determinant role in the modulation of mitoxantrone cytotoxicity.

Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor κB Activation in HL60 Cells

“…Morphological analysis of cells showed a similar result, with mitoxantrone inducing changes indicative of apoptosis in the parental but not in the HL60/MX2 cell line (data not shown). Our data therefore support the reported phenotype of these cells (45,46,52) and show that sensitivity to apoptotic induction correlates with the ability of the same doses of mitoxantrone to activate NFB, the effect being dependent on the presence of functional topoisomerase II isoforms.…”

Topoisomerase II is required for mitoxantrone to signal NFkB activation in HL60 cells

“…Morphological analysis of cells showed a similar result, with mitoxantrone inducing changes indicative of apoptosis in the parental but not in the HL60/MX2 cell line (data not shown). Our data therefore supports the reported phenotype of these cells [45,46,53] and shows that sensitivity to apoptotic induction correlates with the ability of the same doses of mitoxantrone to activate NFκB, the effect being dependent on the presence of functional topoisomerase II isoforms.…”

“…These cells lack topoisomerase II β isofom expression and have a disruption in the carboxy terminus of the α isoform. Furthermore, altered cellular distribution of the α isoform, as detected by immunoflourescence microscopy has been reported for these cells [53]. These changes are the likely explanation for why the DNA cleavage activity of topoisomerase II present in the MX2 cell line is less sensitive to inhibition by mitoxantrone when compared to the parental cell line.…”

“…HL60/MX2 cells were recently confirmed not to exhibit changes in drug uptake or retention compared with the parental cell line, as determined by FACS analysis [53]. In addition, these cells are normally sensitive to other anti-tumour agents such as the vinca alkaloids, resistance to which in combination with some anthracyclines characterizes the multi-drug resistance (MDR) phenotype [45].…”

Multidrug Resistance Protein 1 (MRP1, ABCC1) Mediates Resistance to Mitoxantrone via Glutathione-Dependent Drug Efflux

“…Multiple mechanisms of cancer cell resistance to MX have been described, including altered topoisomerase II and increased efflux (Schurr et al, 1989;Harker et al, 1991Harker et al, , 1995aConsoli et al, 1997;Errington et al, 1999;Zhou et al, 1999). Regarding increased efflux, an association between P-glycoprotein (MDR1, ABCB1) and MX resistance has long been appreciated (Dalton et al, 1986;Schurr et al, 1989;Consoli et al, 1997;Litman et al, 2000), and more recently, an association between high-level MX resistance and overexpression of ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein) has been reported (Doyle et al, 1998;Allen et al, 1999;Brangi et al, 1999;Maliepaard et al, 1999;Ross et al, 1999;Litman et al, 2000). Although ABCG2 clearly mediates MX transport and thereby confers robust cellular resistance, the role of MRP1 (ABCC1) in MX sensitivity has remained controversial; some groups have found no relationship between MRP1 overexpression and MX sensitivity, whereas others have reported low-level resistance associated with increased MRP1 (Mirski et al, 1987;Cole et al, 1994;Schneider et al, 1994;Breuninger et al, 1995;Borst et al, 2000).…”