Cellular Pharmacology of Mitoxantrone in P

Leukemia (1997) 11, 2066–2074 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines U Consoli, NT Van, N Neamati, R Mahadevia, M Beran, S Zhao and M Andreeff Department of Hematology, Section of Experimental Hematology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Previous reports suggest that resistance to mitoxantrone in dif- protein is particularly important because it confers cross-resist- ferent tumor cell lines is unrelated to the overexpression of p- ance to several structurally unrelated drugs.9 The mechanisms glycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry of action of mitoxantrone resistance are not well known. and confocal microscopy were used to study two human Although mitoxantrone seems to select for a unique drug- myeloid leukemia cell lines selected for resistance to mitoxan- resistance phenotype, some cross-resistance with anthracy- trone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the clines has been shown in vitro.10,11 Human colon carci- detection of intracellular mitoxantrone, we determined the noma,12 leukemia13 and gastric carcinoma14 cell lines selec- maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cyto- ted by continuous exposure to increasing concentrations of metric conditions using 568.2 nm laser emission for excitation mitoxantrone had no p-glycoprotein overexpression. Clinical and a 620 nm long pass filter for fluorescence collection cross-over studies have not demonstrated complete cross- resulted in a 1-log increase in sensitivity, compared with stan- resistance between mitoxantrone and doxorubicin,15,16 pro- dard 488-nm laser excitation. Uptake and retention of mitoxan- viding rationale for the use of mitoxantrone in disease- trone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results relapsed patients following anthracycline treatment. In con- showed no change in uptake and retention of the drug in p- trast to these reports, mitoxantrone like the anthracyclines, has glycoprotein-negative mitoxantrone-resistant HL-60MX2 cells also been shown to select for cells exhibiting the MDR pheno- and in its sensitive parental line, HL-60s. In contrast, 3.1- and type mediated by p-glycoprotein overexpression.17 Further- 2.4-fold increases were found in uptake and retention of mitox- more, a resistance mechanism associated with enhanced antrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug dis- efflux involving a pathway distinct from the MDR-1-encoded 18 tribution demonstrated reduced nuclear uptake, which could be p-glycoprotein has also been reported. reversed by verapamil, in HL-60DOX. A characteristic punctate Flow cytometry and confocal laser microscopy have been pattern was observed for the intracytoplasmic drug distribution successfully used to analyze cellular pharmacology and the in HL-60DOX and HL-60MX2 cells and was partially modified by intracellular distribution of anthracyclines based on their flu- the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX orescence emission after laser excitation. This method pro- cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our vides a powerful tool to evaluate the role of p-glycoprotein in study demonstrates that the cellular pharmacology of mitoxan- the cellular pharmacology of anthracylines and in the devel- trone is affected by p-glycoprotein and can be reversed at least opment of MDR in various systems in vitro19,20 and in in part by verapamil. Other mechanisms of resistance however, vivo.21,22 Although mitoxantrone shares only part of its chemi- seem to play a determinant role in the modulation of mitoxantrone cytotoxicity. cal structure with other anthracyclines, like doxorubicin, it has Keywords: mitoxantrone; p-glycoprotein; multidrug resistance a planar electron-rich chromophore of a polycyclic dye, which is responsible for its fluorescent properties. In this study, we used a fluorimetric method to evaluate the Introduction intracellular content of mitoxantrone in order to clarify the influence of p-glycoprotein on the cellular pharmacology of Mitoxantrone is an anticancer anthraquinone drug1,2 that has mitoxantrone and in the development of MDR. Uptake and shown therapeutic efficacy comparable with that of standard retention of mitoxantrone were evaluated in two human cell induction and salvage treatment regimens in advanced breast lines selected for resistance to mitoxantrone (HL-60MX2, p- cancer,3 acute leukemia,4,5 non-Hodgkin’s lymphoma,6 and glycoprotein negative) and doxorubicin (HL-60DOX, p-glyco- chronic myeloid leukemia in blastic transformation.7 Although protein positive) and their drug-sensitive parent cell line (HL- mitoxantrone is structurally similar to anthracycline doxorub- 60s, p-glycoprotein negative) in the presence of verapamil, a icin, it has an improved tolerability profile,8 and the mech- calcium channel blocker known to inhibit MDR possibly by anism of action appears to be quite different. Like other interfering with p-glycoprotein function.23 Cytotoxicity assays anthracyclines, mitoxantrone intercalates into DNA and in the presence and absence of verapamil were also perfor- causes DNA-protein cross-links and protein-associated dou- med to analyze the role of p-glycoprotein in protecting cells ble-stranded and single-stranded breaks in the DNA. In from mitoxantrone cytotoxicity. Lastly, to evaluate the cellular addition, this drug is able to stabilize the DNA topoisomerase pharmacology of mitoxantrone better, we studied the different II cleavable complex and to prevent DNA synthesis.8 patterns of intracellular distribution using confocal laser scan- The development of cellular resistance to anticancer drugs ning microscopy with optical sectioning capability. In this greatly limits the activity of cancer chemotherapy against study, we provide evidence that mitoxantrone is a substrate malignant tumors. In this regard, overexpression of p-glyco- for p-glycoprotein and that higher intracellular concentrations of mitoxantrone in p-glycoprotein-positive cells incubated in the presence of verapamil are associated with increased cyto- Correspondence: U Consoli, Institute of Hematology, Ospedale Ferra- toxicity, circumventing, at least in part, p-glycoprotein- rotto, Via Citelli 6, Catania 95124, Italy mediated MDR. Received 23 November 1996; accepted 8 September 1997 P-glycoprotein and mitoxantrone U Consoli et al 2067 Materials and methods Cell uptake and retention studies Drugs Cells were incubated at the concentration of 2 × 106/ml in medium plus FCS 5% with or without verapamil (10 ␮M) for Mitoxantrone and verapamil were obtained as pharmaceutical 2 h at 37°C with mitoxantrone at the concentration of 5 ␮g/ml. preparations from Lederle Parenterals (Carolina, Puerto Rico) For uptake studies, aliquots of 5 × 105 cells were taken after and American Regent Laboratory (Shirley, NY, USA). 15, 30, 60, 90 and 120 min of incubation. Cells were washed in ice-cold PBS without Ca2+ or Mg2+, resuspended in 0.5 ml ice-cold PBS, and mitoxantrone fluorescence was measured within 5 min. For retention studies, at the end of a 2 h Cell lines exposure, every sample was washed twice in ice-cold PBS and resuspended for 2 h in fresh medium, with or without 10 ␮M The human acute myeloid leukemia HL-60s and its mitoxan- verapamil. Cells were incubated at 37°C, and aliquots of cells trone-selected HL-60MX2 cell lines were generously provided were analyzed at 15, 30, 60, 90 and 120 min in the same by Dr Graydon Harker (VA Medical Center, Salt Lake City, conditions used for the uptake studies. Experiments were per- UT, USA). The multiple myeloma parent cell line 8226/S and formed in triplicate, and a representative experiment is shown. its doxorubicin-resistant mutants 8226/DOX6 and The uptake and retention were calculated by dividing the 8226/DOX40 were kindly provided by Dr WS Dalton median level fluorescence channel, after 2 h of incubation (University of Arizona, Tucson, AZ, USA). Those cell lines with verapamil, by the fluorescence signal of the cells incu- were grown in RPMI 1640 supplemented with 10% FCS, 1% bated with the drug alone, measured at the same time point. penicillin, 1% streptomycin and 1% L-glutamine (all from The level of drug fluorescence increase corrected for autoflu- GIBCO-BRL, Gaithersburg, MD, USA). Doxorubicin-resistant orescence was expressed as the mean of three experiments. selected HL-60DOX cells were grown in IMDM supplemented with 10% FCS, 1% penicillin, 1% streptomycin and 1% L- glutamine. Cell cultures were maintained at 37°C in a humidi- fied 5% CO2 atmosphere. Cytotoxicity (MTT assay) Drug cytotoxicity in vitro was assessed for each cell line by RNA extraction and reverse transcriptase PCR using the MTT dye reduction assay (Sigma Chemical Com- (RT-PCR) pany, St Louis, MO, USA). This technique relies on converting MTT (3,(4,-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium RNA was isolated according to the acid-guanidium-phenol- bromide) to a red formazan product by reductive enzymes chloroform method.24 RT-PCR was performed as previously present only in metabolically viable cells. Hence, the quantit- reported.25 ation of insoluble formazan was possible by dissolving it in a lysing buffer and measuring the optical density of each sample with a microplate spectrophotometer (Molecular Devices USA, Menlo Park, CA, UA) at a 570 nm wavelength. Cells Antibody staining (135 ␮l) were plated in 96-well microassay culture plates × 4 ° (2 10 cells/well) and grown for 24 h at 37 CinaCO2 incu- For the study of p-glycoprotein MoAb 4E3 (kindly provided bator. Drugs were then added to the wells (15 ␮l) to achieve by Dr R Arceci, Dana-Farber Cancer Center, Boston, MA, a final drug concentration of 0.025 to 5 ␮g/ml (eight wells USA) was used as previously described.26 were used for each different concentration).

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