Leukemia (2002) 16, 820–825  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Dexrazoxane has no impact on sensitivity of childhood leukemic blasts to daunorubicin J Styczynski1, M Wysocki2, W Balwierz2 and JR Kowalczyk3

1Department of Pediatric Hematology and Oncology, Medical University, Bydgoszcz, Poland; 2Department of Pediatric Hematology, Collegium Medicum Jagiellonian University, Krako´w, Poland; and 3Department of Pediatric Hematology and Oncology, Medical University, Lublin, Poland

Dexrazoxane (DEX) prevents the formation of free radical, lipid Materials and methods peroxidation and cardiotoxicity caused by anthracyclines. Due to a concern about its possible interference with anthracyclin cytotoxicity, the in vitro effect of DEX on daunorubicin (DNR) Cell culture and patient samples cytotoxicity, cell cycle and induction of apoptosis by annexin- V was investigated. The sensitivity to DEX, DNR and their com- The cytotoxicity of DEX, DNR and their combination was bination was tested by the MTT assay in human promyelocytic tested in human promyelocytic leukemia HL60, and the leukemia HL-60, the erythroid blast crisis CML K562 cell lines erythroid blast crisis CML K562 cell lines (both were a kind and in 45 children with ALL and AML. Cell cycle analysis and annexin-V expression were performed by flow cytometry. It has gift of Dr Alina Grzanka, Department of Pathology, Medical been observed that DEX itself weakly, but significantly caused University, Bydgoszcz) and in 45 children, including 38 ALL cytotoxicity in both cell lines and in patient samples, especially (28 de novo, 10 relapsed) and seven AML children. The in initial ALL samples. DEX sensitized K562 and HL60, but not median age of patients was 8 years (range 0.4–18 years). Leu- patient samples, to cytotoxicity of DNR. The percentage of kemic cells were isolated by Ficoll–Hypaque (Aqua Medica, necrotic/apoptotic cells, as detected in cell cycle analysis and Ło´dz´, Poland) density gradient separation and resuspended in annexin V staining, was higher after exposure to DEX ± DNR, when compared to respective samples not treated with DEX, in RPMI-1640 (Sigma-Aldrich, Schnelldorf, Germany) with 20% both cell lines but not in patient samples. Expression of fetal calf serum (FCS; Gibco BRL Life Technologies, UK). The annexin V induced by DEX in both cell lines was enlarged, morphology was evaluated from cytospine slide preparations regardless of the presence of DNR. This difference was not with May–Grunwald-Giemsa staining, and the viability was observed in patient samples, however, the number of cells ± assessed by trypan blue dye exclusion. Only fresh leukemic expressing annexin V was higher after exposure to DEX DNR samples were taken for the study and the percentage of blasts in comparison to respective samples not treated with DEX. In conclusion, it seems that DEX possibly has no impact on the in the sample was over 90%. sensitivity of childhood leukemic blasts to DNR, however, has weak cytotoxic properties itself. Leukemia (2002) 16, 820–825. DOI: 10.1038/sj/leu/2402474 Drugs Keywords: daunorubicin; dexrazoxane; sensitivity; resistance DNR (Cerubidine) was purchased from Bellon Rhone-Poul- enc-Rhorer (Neuilly Sur Seine, France) and DEX (Cardioxan) Introduction from Chiron BV (Amsterdam, The Netherlands). DEX was used in concentration always in the ratio of 20:1 to DNR, as indi- The bisdioxopiperazines, including dexrazoxane (DEX), are cated by the manufacturer. Control concentration of DEX catalytic or noncleavable complex-forming inhibitors of DNA without DNR was 20 ␮g/ml. topoisomerase II that do not produce DNA strand breaks.1 It has been suggested that the product of dexrazoxane (ICRF- 187) hydrolysis, ICRF-198, chelates and removes free iron and iron associated with the doxorubicin–iron complex and, there- MTT viability assays fore, prevents the formation of free radical, lipid peroxidation and cardiotoxicity. As an inhibitor of topoisomerase II, it pre- Cytotoxicity was measured by a viability and cell proliferation vents the inactivation of cytochrome C oxidase by Fe3+ and assay by measuring the ability of the cells to cleave the soluble increases the levels of transferrin receptor mRNA and cellular compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazol- iron uptake.2 However, there is a concern about its possible ium bromide (MTT; Serva, Heidelberg, Germany) into an interference with anthracyclin cytotoxicity. In a study of the insoluble salt. The ability of cells to cleave MTT is indicative effect of DEX and a radical scavenger, butylated hydroxyani- of the degree of mitochondrial/cellular respiration within sole (BHA) on in vitro cytotoxicity of topo-II inhibitors, dauno- those cells. Conditions of the assay were similar as described rubicin (DNR) and etoposide (VP-16) in leukemia cells from previously.4 Briefly, cells were incubated for 4 days with vari- children, it has been observed that DEX increased resistance ous concentrations of DNR (0.001–1 ␮g/ml), DEX (20 ␮g/ml) to DNR and VP-16 in AML and T-ALL, however, it did not or in a combination of both drugs for 4 days in 96-well plates. affect apoptosis induction in T-ALL.3 In an ongoing study, the Ten ␮l of MTT (5 ␮g/ml) was added to each well. The reaction effect of DEX on in vitro DNR cytotoxicity, cell cycle and was stopped after 4 h incubation by adding 100 ␮l of 0.04 N induction of apoptosis by annexin V in leukemia cells from HCl in isopropanol (Sigma-Aldrich), and the optical density children with ALL and AML, was investigated. (OD) was measured at 550 nm (reference wavelength 720 nm) with a Multiscan Bichromatic plate reader (Asys Hitech, Eug- endorf, Austria) and DigiWin software (Asys Hitech). All Correspondence: J Styczynski, ul. Gersona 17–8, 85–305 Bydgoszcz, experiments were performed in triplicate, and the data were Poland; Fax: +48 52 585 4867 confirmed to be reproducible. The cytotoxicity was expressed Received 30 May 2001; accepted 19 December 2001 as LC50, lethal concentration for 50% of cells. When LC50 Effect of DEX on in vitro DNR cytotoxicity J Styczynski et al 821 for DNR was above 1 ␮g/ml, the percentage of cells surviving Results in the highest concentration was calculated. MTT assay: DEX increased DNR cytotoxicity in K562 and HL60 cell lines and had no influence in patient samples Cell cycle analysis At the DNR concentration of 1 ␮g/ml, survival of K562 cells For DNA content analysis, cells were stained with hypotonic incubated with DNR alone or in the combination of DNR + propidium iodine solution (20 ␮g/ml; DNA-Prep Kit, Lot num- DEX, was 63% ± 5% and 54% ± 4%, respectively (P Ͻ 0.01), ber PN 6607055; Coulter, Miami, FL, USA) and 20000 events in comparison to cells incubated without any drug (Figure 1). were analyzed with an Epics XL flow cytometer (Coulter) after K562 cells treated with DEX at the concentration of 20 ␮g/ml 96 h of incubation. This flow cytometer is equipped with an showed survival of 88 ± 4%, when compared to the cells cul- argon laser with an excitation wavelength of 488 nm. Cell tured without drugs (P Ͻ 0.001). For HL60 cell line, incu- cycle was calculated by Multicycle software (Phoenix Flow bation of cells with DEX resulted in 93 ± 4% survival (P Ͻ Systems, San Diego, CA, USA). The percentage of cells in the 0.001), while LC50 for cells incubated with DNR alone or in G1, G2 and S phase was expressed as mean ± s.d. the combination of DNR + DEX, was 0.24 ± 0.03 ␮g/ml and 0.18 ± 0.03 ␮g/ml, respectively (P Ͻ 0.01), in comparison to cells incubated without any drug. For patient samples (Figure 2), LC50 values for DNR with Apoptotic assay using annexin V/propidium iodide and without DEX showed strong correlation (Spearman’s rho = 0.818, P = 0.001) and LC50 for DNR did not differ from LC50 for DNR + DEX (Wilcoxon matched paired samples test Apoptotic cells were determined by double staining with = ± annexin V (FITC) and propidium iodide (PI) after 48 h of incu- P 0.766). When pairs of cytotoxicity values of DNR DEX bation by using a staining kit purchased from PharMingen (lot for each sample were analyzed, the cell survival in the pres- number 556547; Becton Dickinson Biosciences, San Diego, ence of DNR without DEX was higher in 22 cases (ie possible cytotoxic DEX effect) and in the other 23 cases the cell sur- CA, USA). Five thousand to 10000 events were analyzed on + Epics XL flow cytometer (Coulter) and % apoptosis was vival was higher in the presence of DNR DEX (ie possible determined using System II v 3.0 software (Coulter). suppression of cytotoxicity by DEX). Median LC50 values for DNR without DEX was 0.46 ␮g/ml, while with DEX it was 0.48 ␮g/ml, and the differences did not show any statistical difference (P = 0.642 Mann–Whitney U test). When the num- Statistical analysis ber of living control patient cells were taken as 100%, then the survival of cells cultured with DEX only, was 82 ± 20% (P Ͻ 0.001) in comparison to drug-free cultured cells. We did Results are given as mean values ± s.d. and also as median not observe any impact of disease type and status on differ- values + quartiles. For continuous variables, the Mann– ences between cytotoxicity of DNR alone and in combination Whitney U test was used for unpaired comparisons and the Wilcoxon signed rank test was used for paired comparisons. Correlations were analyzed by Spearman’s rho coefficient. The mean values were compared with Student’s t-test. All reported P values are two-sided. Differences were considered to be statistically significant when P values were Ͻ0.05.

Figure 1 K562 and HL60 cell line survival without any drug, with Figure 2 A histogram of LC50 values for DNR and the combi- DEX 20 ␮g/ml, DNR 1 ␮g/ml, and the combination of DNR 1 ␮g/ml nation of DNR + DEX for ALL and AML patient samples. Both axes plus DEX 20 ␮g/ml. The values are the mean of four experiments express concentration of DNR in ␮g/ml. Concentration of DEX was performed by the MTT assay. P values, Student’s t-test. respectively 20-fold higher.

Leukemia Effect of DEX on in vitro DNR cytotoxicity J Styczynski et al 822 with DEX. In spite of the fact that AML and relapsed ALL In our study, DEX itself exerted a cytotoxic effect and samples were, in general, more resistant to DNR, we did not induced the expression of annexin V in both cell lines. Its observe any specific differences in DEX influence on DNR affect on cell cycle arrest was very weak. When combined cytotoxicity. Only five leukemic samples showed more than with DNR in cell lines, DEX caused higher cytotoxicity of a two-fold difference in sensitivity between DNR and DNR, higher induction of annexin V, higher percentage of DNR+DEX (Figure 2). In four cases (one pro-B-ALL, one com- necrotic cells and cell cycle arrest by increasing the number mon-ALL, one T-ALL and one AML) cells were more resistant of cells in S-phase. Since cytotoxicity of DNR was significantly to combination DNR + DEX, and in one case (T-ALL), were influenced by DEX in K562 and HL60 cell lines, this finding more resistant to DNR alone. indicates weak cytotoxicity and either additive or synergistic effect of DEX on DNR cytotoxicity in both cell lines. DEX is known to cause cell cycle arrest at G2/M,6,7 however, this Cell cycle: DEX weakly potentiated the effect of DNR effect was almost not observed in our study, regardless of the in cell lines but not in patient samples type of cells which were analyzed. When patient samples were taken into account, in all three DEX had almost no effect on cell cycle, both in cell lines and assays the interpatient results have shown great variability; the in patient samples, while DNR itself caused cell cycle arrest differences in range were wider for the cytotoxicity assay and increased the number of cells in S-phase (P Ͻ 0.05 for results and smaller both for annexin V expression and cell cell lines and P Ͻ 0.01 for patient samples). The percentage cycle arrest. In the group of 45 leukemic patient samples, the of cells in S-phase in the presence of DEX + DNR was higher effect of DEX was observed on survival of cells not being when compared to the percentage of cells in S-phase in treated with DNR. The cell survival was lower when the effect respective samples treated with DNR without DEX, in K562 caused by DEX was measured by the MTT assay and annexin and HL60 cell lines (P Ͻ 0.05), but not in patient samples. V expression, but the difference was not obvious when cell The number of necrotic/apoptotic cells, as detected by cell cycle was analyzed. The impact of DEX on DNR cytotoxicity, cycle analysis, was not significantly higher after exposure to cell cycle and induction of annexin V was minimal and did DEX ± DNR both for cell lines and patient samples (Figure 3). not show a clear tendency. The cytotoxicity of DNR was No differences in cell cycle were found between leukemia weakly higher without DEX, but expression of annexin V, the subtypes, both with regard to influence of DNR and the percentage of necrotic cells and inhibition of cell cycle was impact of DEX on DNR-induced cell cycle arrest. weakly higher in the presence of DEX. This might indicate that, in general, DEX neither limits nor potentiates the cytotox- icity of DNR against childhood leukemic cells. All three assays Apoptosis: DEX induces apoptosis in patient samples have shown that DEX itself exerts certain cytotoxic effects on and in HL-60 and K562 cell lines leukemic cells. The results of these assays gave comparable results. Cells became positive for annexin V staining after exposure to The effect of DEX on cells can be related to its molecular DEX in K562 cell line (24.5% vs 16.5%, P Ͻ 0.05), in HL60 mechanism of activity, with the target in topoisomerase II, cell line (21.1% vs 14.9%, P Ͻ 0.05) but not in patient which is the same target as for DNR. However, when DEX samples (40.2% vs 35.7%, NS). After exposure to DEX + DNR, was added to DNR, the effect of DEX was very weak. This in comparison to cell exposure to DNR only, induction of was probably because of sufficient topoisomerase II blockade annexin V was higher in K562 cell line (46.0% vs 30.2%, P already caused by DNR. Ͻ 0.05), in HL60 cell line (93.5% vs 86.4%, P Ͻ 0.05) but For patient samples we did not see the influence of DEX on again, not in patient samples (63.4% vs 61.2%, NS). The per- cytotoxicity of DNR, which we could observe in cell lines. centage of necrotic cells was higher in both cell lines (P Ͻ As there is no pharmacokinetic interaction of dexrazoxane on 0.05 for each cell line) in the presence of DEX vs no drug anthracycline metabolism,8 the explanation for these discrep- and DEX+DNR vs DNR (Figure 4). This difference for patient ancies between cell lines and patient samples is not clear. This samples was significant for the effect of DEX without DNR vs may be caused by differences in the blockage of topoisomer- drug-free cells (P Ͻ 0.01), and did not reach significance ase II by DNR; by differences in the level of expression of when DNR + DEX was compared to DNR without DEX. The topoisomerase II between K562 and HL60 cell lines and percentage of cells expressing annexin V after exposure to patient samples, or by variability in topoisomerase II DNR ± DEX did not show statistical differences between leu- expression in human cells, especially between leukemia sub- kemia subtypes, however, the number of cells with annexin types. It can be related to differences in apoptosis pathways, V expression after treatment with DNR without DEX, showed especially caspase activity. In a previous study, it was also a tendency to be higher in initial ALL samples than in relapsed suggested that the acquired resistance to DEX in the DZR cell ALL and in AML patients. line was caused mainly through an alteration in the topoiso- merase II target.9 In experimental conditions, DEX was also shown to cause a decrease in DNA synthesis by interaction Discussion with topoisomerase I–DNA covalent complexes.10 DEX combined with DOX (plus 5-fluorouracil plus cyclo- Dexrazoxane (DEX) which is currently used as a cardioprotec- phosphamide in the FAC regimen) led to a significant decrease tive agent in combination with DNR and doxorubicin (DOX) in doxorubicin cardiotoxicity and a significant increase in was originally developed as an antitumor agent in the 1970s.5 median survival time for patients with advanced breast cancer Previous experiments have explored the ability of DEX to responsive to FAC.11 As DOX resistance was not induced in inhibit the catalytic activity of topoisomerase II and it has been the K562 cell line in the presence of DEX for several months, found that prolonged exposures to DEX are cytotoxic to this suggested that DEX could delay the development of human leukemic cells at concentrations that are clinically MDR1, but did not act as a conventional modulator of P-gly- achievable.5,6 coprotein, thus allowing responders to the FAC regimen to

Leukemia Effect of DEX on in vitro DNR cytotoxicity J Styczynski et al 823 continue to respond.12 DEX-induced apoptosis in the K562 tion of reactive oxygen species,16 apart from a cardioprotec- cell line is probably associated with a caspase-3 tive effect, also has weak cytotoxic properties. Because the activation/cleavage pathway. The ability of DEX to induce dif- mechanism of its action partially overlaps with the mechanism ferentiation and apoptosis suggests that bisdioxopiperazines of DNR cytotoxicity, it seems that in the presence of DNR, the may be useful in treating some types of leukemia.13 In con- effect of DEX cytotoxicity is very weak, or absent, especially in trast, DEX prevented DNR-induced myocyte apoptosis, but patient leukemic samples. On the other hand, with the three not necrosis caused by higher anthracycline concentrations. methods used in the study we did not observe suppressive The antiapoptotic action of DEX was mimicked by the super- influence of DEX on DNR cytotoxicity. These results suggest oxide-dismutase mimetic porphyrin.14 The recognition that that DEX has no impact on daunorubicin cytotoxicity to child- anthracycline-induced cardiac myocyte apoptosis, perhaps hood leukemic blasts. mediated by superoxide anion generation, occurs at concen- trations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the tox- Acknowledgements icity of these drugs.14 Although the cardioprotective effect of DEX in cancer patients undergoing chemotherapy with anthra- This work was supported by State Committee for Scientific cyclines is well documented, the potential of this drug to Research grant KBN 6 PO5E 082 21 and Cancer Children’s modulate topoisomerase II activity and cellular iron meta- Foundation of Bydgoszcz, Poland. The study was performed bolism may hold the key for future applications of DEX in in Laboratory of Clinical and Experimental Medicine, Depart- cancer therapy, immunology, or infectious diseases.15 ment of Pediatric Hematology and Oncology, Medical Uni- In conclusion, the results of this study show that DEX, versity, Bydgoszcz, Poland. The authors thank The Board of which is an inhibitor of topoisomerase II and prevents forma- Polish Childhood Leukemia Study Group for sending patient

Figure 3 Cell cycle profiles in K562, HL60 and patient samples. Cells were incubated with medium, dexrazoxane, daunorubicin and a combination of both chemicals. Cells were prepared and stained with PI for DNA content. DNA profiles were acquired with flow cytometry. The percentage of the apoptotic/necrotic cells and the cells in S phase of the cell cycle is shown in each histogram. Presented values are the mean of four experiments for cell lines and of all experiments on patient samples. Representative experiments are shown.

Leukemia Effect of DEX on in vitro DNR cytotoxicity J Styczynski et al 824

Figure 4 Detection of apoptotic cells as detected by annexin V–FITC/propidium iodide binding. Cells were treated with medium, dexrazox- ane, daunorubicin or a combination of both chemicals. The lower left quadrant represents the live cells (annexin negative, PI negative). The upper left quadrant represents the early apoptotic cells (annexin positive, PI negative). The upper right quadrant represents the late apoptotic/necrotic cells (annexin positive, PI positive). The lower right quadrant represents the dead cells (annexin negative, PI positive). The mean percentages of cells in each quadrant are indicated next to each quadrant. Presented values are the mean of four experiments for cell lines and of all experiments on patient samples. Representative experiments are shown.

samples. The Board members are: Anna Balcerska, Walentyna novel topoisomerase II inhibitor. Semin Oncol 1998; 25 (4 Suppl. Balwierz, Alicja Chybicka, Jacek Wachowiak, Jerzy R Kowal- 10): 93–99. czyk, Michal Matysiak, Roma Rokicka-Milewska, Danuta 7 Scambia G, Della Bitta R, Benedetti Panici P, De Vincenzo R, ContuG, Ercoli A, Bonanno G, Pierelli L, MancusoS. Bisdioxypip- Sonta-Jakimczyk and Mariusz Wysocki. erazine, (+)-1,2-Bis(3,5-dioxypiperazinyl-1-yl)propane (ICRF 187), enhances the antiproliferative effect of cisplatin on human ovarian cancer cells. Gynecol Oncol 1995; 57: 16–22. 8 Hochster HS. Clinical pharmacology of dexrazoxane. Semin References Oncol 1998; 25 (4 Suppl. 10): 37–42. 9 Hasinoff BB, Kuschak TI, Creighton AM, Fattman CL, Allan WP, 1 Hasinoff BB, Abram ME, Chee GL, Huebner E, Byard EH, Barnabe Thampatty P, Yalowich JC. Characterization of a Chinese hamster N, Ferrans VJ, YuZX, Yalowich JC. The catalytic DNA topoisomer- ovary cell line with acquired resistance to the bisdioxopiperazine ase II inhibitor dexrazoxane (ICRF-187) induces endopolyploidy dexrazoxane (ICRF-187) catalytic inhibitor, of topoisomerase II. in Chinese hamster ovary cells. J Pharmacol Exp Ther 2000; 295: Biochem Pharmacol 1997; 53: 1843–1853. 474–483. 10 Hasinoff BB, Chee GL, Thampatty P, Allan WP, Yalowich JC. The 2 Vaidyanathan S, Boroujerdi M. Interaction of dexrazoxane with cardioprotective and DNA topoisomerase II inhibitory agent red blood cells and hemoglobin alters pharmacokinetics of dexrazoxane (ICRF-187) antagonizes camptothecin-mediated doxorubicin. Cancer Chemother Pharmacol 2000; 46: 93–100. growth inhibition of Chinese hamster ovary cells by inhibition of 3 Savasan S, Ozdemir O, Buck S, Ravindranath Y. Differential effect DNA synthesis. Anti-Cancer Drugs 1999; 10: 47–54. of dexrazoxane on the in vitro cytotoxicity of topoisomerase-II 11 Swain SM, Whaley FS, Gerber MC, Ewer MS, Bianchine JR, Gams inhibitors by the cell type in childhood leukemias. Leukemia RA. Delayed administration of dexrazoxane provides cardiopro- 2001; 15: 489 (Abstr. O32). tection for patients with advanced breast cancer treated with 4 Styczynski J, Pieters R, Huismans DR, Schuurhuis GJ, Wysocki M, doxorubicin-containing therapy. J Clin Oncol 1997; 15: 1333– Veerman AJP. In vitro drug resistance profiles in adult versus child- 1340. hood acute lymphoblastic leukaemia. Br J Haematol 2000; 110: 12 Sargent JM, Williamson CJ, Yardley C, Taylor CG, Hellmann K. 813–818. Dexrazoxane significantly impairs the induction of doxorubicin 5 Sharpe HBA, Field EO, Hellmann K. Mode of action of the cytos- resistance in the human leukaemia line, K562. Br J Cancer 2001; tatic agent ‘ICRF 159’. Nature 1970; 226: 524–526. 84: 959–964. 6 Synold TW, Tetef ML, Doroshow JH. Antineoplastic activity of 13 Hasinoff BB, Abram ME, Barnabe N, Khelifa T, Allan WP, Yalow- continuous exposure to dexrazoxane: potential new role as a ich JC. The catalytic DNA topoisomerase II inhibitor dexrazoxane

Leukemia Effect of DEX on in vitro DNR cytotoxicity J Styczynski et al 825 (ICRF-187) induces differentiation and apoptosis in human leuke- 16 Herman EH, Zhang J, Chadwick DP, Ferrans VJ. Comparison of mia K562 cells. Mol Pharmacol 2001; 59: 453–461. the protective effects of amifostine and dexrazoxane against the 14 Sawyer DB, Fukazawa R, Arstall MA, Kelly RA. Daunorubicin- toxicity of doxorubicin in spontaneously hypertensive rats. Cancer induced apoptosis in rat cardiac myocytes is inhibited by dexra- Chemother Pharmacol 2000; 45: 329–334. zoxane. Circ Res 1999; 84: 257–265. 15 Weiss G, Loyevsky M, Gordeuk VR. Dexrazoxane (ICRF-187). Gen Pharmacol 1999; 32: 155–158.

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