Mazur, Lidia

Opydo-Chanek, Małgorzata

Oszczapowicz, Irena

Stojak, Marta

Łukawska, Małgorzata

Anticancer Research

English

Jagiellonian Univeristy Repository

Abstract. Background/Aim: The comparative effects ofdaunorubicin, and its new formamidine derivatives containingeither a morpholine moiety (DAUFmor) or a hexamethyle-neimine moiety (DAUFhex) in the amidine group, on inductionof programmed cell death were determined. Materials andMethods: The experiments were performed on human acutelymphoblastic leukemia MOLT-4 cells and human acutemyeloblastic leukemia ML-1 cells. The research wasconducted using the flow cytometry annexin V–fluorescein(FITC)/propidium iodide (PI) method and tetramethylrho-damine ethyl ester (TMRE) assay. Results: The variouspatterns of temporary changes of early apoptotic cells, lateapoptotic and necrotic cells, and in the frequency of the acuteleukemia cells with high values of mitochondrial membranepotential (MMP) were found. Phosphatidylserineexternalization, plasma membrane disruption, and changes inMMP occurring in the leukemia cells were dependent on theagent tested, its concentration, the time intervals afterdaunorubicin, DAUFmor, and DAUFhex application, and onthe leukemia cell line used. Conclusion: The structuralmodifications of daunorubicin producing two new analogs,DAUFmor and DAUFhex, induced the different responses ofMOLT-4 and ML-1 cells to triggering of programmed death.Daunorubicin, an anthracycline antibiotic, is widely used inanticancer therapy (1, 2). Nevertheless, its clinical efficacy islimited by cardiotoxicity and cellular drug resistance (3). Inthe search for new derivatives of daunorubicin withadvantageous biological properties, a series of analogs havebeen synthesized (4, 5). The transformation of the aminogroup at position 3’of the daunosamine moiety into aformamidino group (-N=CH-N<) (Figure 1) resulted in aseries of new derivatives with different anticancer activitycompared to that of daunorubicin (6, 7). It is accepted thatanticancer potential is dependent on the effective induction ofprogrammed cell death in cancer cells. However, to ourknowledge, there are no published data on induction ofprogrammed cell death by formamidinodaunorubicinscontaining either a morpholine moiety (DAUFmor) orhexamethyleneimine moiety (DAUFhex) in the amidine group.Apoptosis and necrosis are two major types of programmedcell death (8-10). Changes in the cell membrane are one of theevents occurring during apoptotic and necrotic cell death. It isknown that loss of phospholipid asymmetry leading toexposure of phosphatidylserine on the outside of the plasmamembrane is an early event of apoptosis (11). Moreover, theintegrity of plasma membrane is compromised during a latestage of apoptosis and throughout necrosis. Combinationstaining with fluorescein (FITC)–conjugated annexin V andpropidium iodide (PI) allows for the identification of earlyapoptotic cells, late apoptotic and necrotic cells (12).Mitochondria play a pivotal role in the regulation ofprogrammed cell death (13, 14). The mitochondrial membranepotential (MMP) is a key indicator of cell viability and death.Changes in MMP directly reflect on cell transition from lifeto death (15, 16). The MMP plays an important role inmaintaining the physiological function of the respiratory chaingenerating ATP (13). The ATP level is a main determinant ofwhether apoptotic or necrotic cell death occurs (17-19). Thus,evaluation of the MMP is of critical importance for theassessment of apoptosis and necrosis.The aim of the present study was to determine and comparethe in vitro influence of the parent compound daunorubicinand its two analogs DAUFmor, and DAUFhex on human acutelymphoblastic leukemia MOLT-4 cells and human acutemyeloblastic leukemia ML-1 cells. The effects of thesecompounds on triggering phosphatidylserine externalization,plasma membrane disruption, and changes in MMP in theleukemia cells were analyzed.4439Correspondence to: Lidia Mazur, Ph.D., D.Sc., Department ofExperimental Hematology, Jagiellonian University, Gronostajowa9, 30-387 Cracow, Poland. Tel: +48 126645237, Fax: +48126645101, e-mail: lidia.mazur@uj.edu.plKey Words: Daunorubicin derivatives, human acute leukemia cells,apoptosis, necrosis, mitochondrial membrane potential.ANTICANCER RESEARCH 33: 4439-4444 (2013)In Vitro Induction of Apoptosis and Necrosis by New Derivatives of DaunorubicinMARTA STOJAK1, LIDIA MAZUR1, MAŁGORZATA OPYDO-CHANEK1, MAŁGORZATA ŁUKAWSKA2 and IRENA OSZCZAPOWICZ21Department of Experimental Hematology, Jagiellonian University, Cracow, Poland;2Department of Modified Antibiotics, Institute of Biotechnology and Antibiotics, Warsaw, Poland0250-7005/2013 $2.00+.40Materials and MethodsCells. Human acute lymphoblastic leukemia MOLT-4 cells andhuman acute myeloblastic leukemia ML-1 cells were obtained fromthe European Collection of Cell Cultures (European Collection ofCell Cultures, Salisbury, Wiltshire, UK). MOLT-4 and ML-1 cellswere cultured in RPMI 1640 medium (Gibco BRL Life Technologies,Warsaw, Poland) supplemented with 10% fetal calf serum (GibcoBRL Life Technologies), 2 mM L-glutamine (Sigma Aldrich, Poznań,Poland), and antibiotic antimycotic solution (Sigma Aldrich)containing 20 units of penicillin, 20 μg streptomycin and 0.05 μgamphotericin B. Every third day, the acute leukemia cells werepassaged. The cells were grown at 37˚C in an atmosphere of 5% CO2in air (HERAcell incubator; KendroLab, Warsaw, Poland). Chemicals. Daunorubicin and its two analogs, DAUFmor andDAUFhex, were synthesized (20, 21) at the Institute ofBiotechnology and Antibiotics (Warsaw, Poland). The anthracyclinecompounds were dissolved in “aqua pro injectione” (Polpharma,Starogard Gdański, Poland). Stock solutions of these agents at aconcentration of 0.5 mM were stored in the dark at −20˚C. Allsolutions were prepared directly before treatment of the MOLT-4and ML-1 cells. Anthracycline doses and cell treatment. After a dilution of the cellsuspension to a density of 15×104 cells/ml medium, MOLT-4 andML-1 cells were treated with anthracycline agents. MOLT-4 cellswere exposed to the action of daunorubicin, DAUFmor, andDAUFhex, at concentrations of 25, 50 and 75 nM, and ML-1 cellswere treated with these agents, at the concentrations of 75 nM,150 nM, and 225 nM (7). The control consisted of untreatedMOLT-4 and ML-1 cells.Analyses of leukemia cells after the anthracycline application.Temporary changes occurring in MOLT-4 and ML-1 cells wererecorded at 24 h, 48 h, and 72 h after exposure to anthracyclines. Atthese three time points, plasma membrane diversity and impairment,and MMP changes were analyzed.Flow cytometric annexin V-FITC/PI assay. The cell surface exposureof phosphatidylserine and the plasma membrane impairment of cellswere assessed using Annexin V–FITC Apoptosis Detection Kit(Calbiochem Merck Millipore, Warsaw, Poland). By staining cellswith a combination annexin V–FITC and PI, it was possible toidentify non-apoptotic live cells (annexin V–FITC–negative/PI–negative), early apoptotic cells (annexin V–FITC–positive/PI–negative), and late apoptotic and necrotic cells (annexinV–FITC–positive/PI–positive) (12). The procedure of dual staining ofcells with annexin V–FITC and PI was performed according to themanufacturer’s instruction (Calbiochem Merck Millipore). Briefly,suspension of treated/control leukemia cells, containing 5x105 cells,were washed with PBS (BioMed, Lublin, Poland) and resuspended in0.5 ml cold binding buffer. Then 1.25 μl of annexin V–FITC wasadded and the cells were incubated in the dark for 15 min at roomtemperature. Following incubation, the cells were centrifuged at 100 ×g for 5 min and the supernatant was removed. The cell pelletwas resuspended in 0.5 ml cold binding buffer, and 10 μl of the 30μg/ml PI solution was added. Cell samples were placed on ice, awayfrom light, and FITC and PI fluorescence was immediately measuredusing FACSCalibur flow cytometer (Becton Dickinson, New Jersey,USA). Data were analyzed using CellQuest Pro software (BectonDickinson). The frequency of live cells, early apoptotic cells, lateapoptotic and necrotic cells was determined. Analysis of mitochondrial membrane potential. MMP was analyzed bymeans of the lipophilic cationic dye tetramethylrhodamine ethyl esterperchlorate (TMRE; Sigma Aldrich). TMRE will accumulate withinmitochondria in inverse proportion to the MMP according to the Nernstequation. More greatly polarized mitochondria accumulate more cationicdye, whereas depolarized mitochondria accumulate less dye (22).A TMRE stock solution was prepared at a concentration of 10 mMin dimethyl sulfoxide (Sigma Aldrich) and stored at −20˚C. The finalconcentration of TMRE staining solution used was 100 nM. Thesuspension of treated/control leukemia cells was washed twice inHank’s balanced salt solution (HBSS; Gibco Life Technologies),resuspended in 100 μl of 100 nM TMRE, and incubated for 20 min at37˚C in the dark. The cells were then washed twice with HBSS, andfinally resuspended in a total volume of 0.5 ml HBSS. TMREfluorescence was detected by FACSCalibur flow cytometer (BectonDickinson). Data were analyzed using the CellQuest Pro software(Becton Dickinson).Statistical evaluation. All experiments were repeated three times withduplicate or triplicate determinations. All the data are presented as themean values±standard deviation. Statistical analyses were performedusing STATISTICA 10 (StatSoft, Cracow, Poland). Data wereanalyzed by one-way analysis of variance (ANOVA) followed byTukey’s honestly significant difference (HSD) multiple range test. Adifference with p<0.05 was considered statistically significant. ANTICANCER RESEARCH 33: 4439-4444 (2013)4440Figure 1. Chemical structures of daunorubicin (DAU) and its newderivatives containing either a morpholine moiety (DAUFmor) or ahexamethyleneimine moiety (DAUFhex) in the amidine group.Stojak et al: Cell Death Induction by Daunorubicin Derivatives4441Figure 2. Effects of daunorubicin (DAU), and its new derivatives containing either a morpholine moiety (DAUFmor) or a hexamethyleneimine moiety(DAUFhex) in the amidine group, on induction of apoptosis and necrosis in MOLT-4 and ML-1 cells. Representative dot plots for MOLT-4 cells (A)and ML-1 cells (B): i - apoptotic cells, ii - late apoptotic and necrotic cells, iii - live cells. The frequency of early apoptotic, late apoptotic andnecrotic, and live MOLT-4 (C) and ML-1 cells (D). Values not significantly different at p<0.05 according to the Tukey’s multiple range test: , , between the groups of leukemia cells treated with the anthracycline agents; # compared to controls; + between the time points.Results The influence of daunorubicin and its two analogs DAUFmorand DAUFhex on alterations in the frequency of MOLT-4(Figure 2A, 2C) and ML-1 (Figure 2B, 2D) cells undergoingearly apoptosis, late apoptosis and necrosis (Figure 2) and thefrequency of the leukemia MOLT-4 (Figure 3A) and ML-1(Figure 3B) cells expressing the high values of MMP (Figure3) were determined. Daunorubicin induced apoptotic and necrotic death ofhuman leukemia cells, and affected their MMP to a higherdegree than did its analogs DAUFmor and DAUFhex.Moreover, among the daunorubicin derivatives, DAUFmorappeared to be more active in cell death induction andreduction of MMP in ML-1 cells than did DAUFhex.Differences between the effects of DAUFmor and DAUFhexon apoptosis and necrosis, and the loss of MMP triggered inMOLT-4 cells were not found.MOLT-4 cells were more sensitive than ML-1 cells totriggering programmed cell death. A greater time- andconcentration-dependent decrease of the frequency of cellswith high MMP was observed in MOLT-4 cells than ML-1cells. Throughout the 72 h period after agent application, adistinctly higher frequency of late apoptotic and necroticMOLT-4 cells was found than ML-1 cells. Taking into consideration the results obtained, we cangenerally state that the patterns of changes in early apoptosis,late apoptosis and necrosis, and in the change of MMPdepended on the agent tested, its concentration, the timeintervals of anthracycline application, and the leukemia cellline used. DiscussionIn the present study, the effects of daunorubicin, and twonew analogs were determined. It was demonstrated that theparent anthracycline antibiotic DAU, and the twoformamidinodaunorubicins, DAUFmor and DAUFhex, invarying degree caused phosphatidylserine externalization,plasma membrane impairment, and changes of MMP inANTICANCER RESEARCH 33: 4439-4444 (2013)4442Figure 3. Effects of daunorubicin (DAU), and its new derivatives containing either a morpholine moiety (DAUFmor) or a hexamethyleneimine moiety(DAUFhex) in the amidine group, on the mitochondrial membrane potential (MMP). Representative dot plots showing MOLT-4 cells (A) and ML-1cells (B) with the high (R1) and low (R2) MMP values. The frequency of MOLT-4 (A) and ML-1 cells (B) with the high MMP values. Values notsignificantly different at p<0.05 according to the Tukey’s multiple range test: , ,  between the groups of leukemia cells treated with theanthracycline agents; # compared to controls; + between the time points.MOLT-4 and ML-1 cells. Moreover, it was found thatmitochondria were involved in the leukemia cell response tothe action of these agents. These are the first data showingthat the formamidinodaunorubicin agents triggerprogrammed cell death of leukemia cells, and comparingtheir cell death-inducing potential. The structural modifications of daunorubicin would seemto be responsible for the different antileukemic activities of thetwo new formamidinodaunorubicins. DAUFmor and DAUFhexdiffer in the size of the cyclic amine ring in the amidine group.DAUFmor contains a six-membered morpholine ring with anoxygen heteroatom in the γ-position in the formamidine group,while DAUFhex contains a seven-membered hexamethyle-neimine ring with a CH2 group in the γ position. TheDAUFmor molecule has an oxygen atom, whereas DAUFhexhas two CH2 groups. DAUFmor is capable of forming anadditional hydrogen bond with proton-donating sites, due to thepresence of the free electron pairs on the oxygen atom. Thesedifferences in the molecular structure of the twoformamidinodaunorubicin agents, DAUFmor and DAUFhex,surely reflect their different biological properties (4-7).It is accepted that programmed cell death induction is onemechanism of anticancer therapy (23). However, therelationship between the efficacy of chemotherapy andprogrammed cell death induction is still an open issue. Anunderstanding of the precise mechanisms of triggering ofprogrammed cell death by new anthracycline derivatives isessential for the development of novel anticancer therapeuticstrategies (3).Conflicts of Interest The Authors declare that there are no conflicts of interest.AcknowledgementsThe study was supported by the Research Projects BW/4/IZ/2010 andK/ZDS/001959.References1 Hortobagyi GN: Anthracyclines in the treatment of cancer. Anoverview. Drugs 54: 1-7, 1997.2 Nadas J and Sun D: Anthracyclines as effective anticancer agents.Expert Opin Drug Discov 1: 539-548, 2006.3 Minotti G, Menna P, Salvatorelli E, Cairo G and Gianni L:Anthracyclines: molecular advances and pharmacologicdevelopments in antitumor activity and cardiotoxicity. PharmacolRev 56: 185-229, 2004.4 Wąsowska M, Oszczapowicz I, Wietrzyk J, Opolski A, Madej J,Dzimira S and Oszczapowicz J: Influence of the structure of newanthracycline antibiotics on their biological properties. AnticancerRes 25: 2043-2048, 2005.5 Wąsowska M, Wietrzyk J, Opolski A, Oszczapowicz J andOszczapowicz I: Effect of structural modification ofanthracyclines on the ability to overcome drug resistance of cancercells. Anticancer Res 26: 2009-2012, 2006.6 Wąsowska-Łukawska M, Wietrzyk J, Opolski A, Oszczapowicz Jand Oszczapowicz I: Biological properties of new derivatives ofdaunorubicin. In Vivo 21: 413-416, 2007.7 Stojak M, Mazur L, Opydo-Chanek M, Łukawska M andOszczapowicz I: Effects of structural modifications ofdaunorubicin on in vitro antileukemic activity. Anticancer Res 32:5271-5278, 2012.8 Guimaraes CA and Linden R: Programmed cell deaths. Eur JBiochem 271: 1638-1650, 2004. 9 De Bruin E.C., Medema J.P. 2008. Apoptosis and non-apoptoticdeaths in cancer development and treatment response. CancerTreatment Reviews 34: 737-749.10 Bialik S, Zalckvar E, Ber Y, Rubinstein AD and Kimchi A:Systems biology analysis of programmed cell death. TrendsBiochem Sci 35: 556-564, 2010.11 Aubry JP, Blaecke A, Lecoanet-Henchoz S, Jeannin P, HerbaultN, Caron G, Moine V and Bonnefoy JY: Annexin V used formeasuring apoptosis in the early events of cellular cytotoxicity.Cytometry 37: 197-204, 1999.12 Darżynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T andTraganos F: Cytometry in cell necrobiology: Analysis of apoptosisand accidental cell death (necrosis). Cytometry 27: 1-20, 1997.13 Kroemer G and JC Reed JC: Mitochondrial control of cell death.Nature Med 6: 513-519, 2000. 14 Bras M, Queenan B and Susun SA: Programmed cell death viamitochondria: Different modes of dying. Biochemistry 70: 231-239, 2005. 15 Desagher S and Martiniou JC: Mitochondria as the central point ofapoptosis. Trends Cell Biol 10: 369-377, 2000.16 Ly JD, Grubb DR and Lawen A: The mitochondrial membranepotential (ΔΨm) in apoptosis; an update. Apoptosis 8: 115-128,2003.17 Tsujimoto Y: Apoptosis and necrosis: intracellular ATP level as adeterminant for cell death modes. Cell Death Differ 4: 429-434,1997.18 Nicotera P, Leist M and Ferrando-May E: Intracellular ATP, aswitch in the decision between apoptosis and necrosis. ToxicolLett 102-103: 139-142, 1998.19 Nicotera P and Melino G: Regulation of the apoptosis-necrosisswitch. Oncogene 23: 2757-2765, 2004. 20 Oszczapowicz I, Wąsowska M, Oszczapowicz J, Owoc A,Dominiczak E, Wietrzyk J and Opolski A: New derivatives ofanthracycline antibiotics, method of their synthesis,pharmaceutical agents containing them, and their use. PolishPatent 210494, 2005 (in Polish).21 Wąsowska M, Oszczapowicz J, Oszczapowicz I and Owoc A:New method of synthesis of anthracycline antibiotic derivatives.Polish Patent Application P. 381654, 2006 (in Polish).22 Perry SW, Norman JP, Barbieri J, Brown EB and Gelbard HA:Mitochondrial membrane potential probes and the proton gradient:A practical usage guide. BioTechniques 50: 98-115, 2011.23 Portugal J, Bataller M and Mansilla S: Cell death pathways inresponse to antitumor therapy. Tumori 95: 409-421, 2009.Received July 31, 2013Revised September 18, 2013Accepted September 20, 2013Stojak et al: Cell Death Induction by Daunorubicin Derivatives4443

In vitro induction of apoptosis and necrosis by new derivatives of daunorubicin

https://ruj.uj.edu.pl/xmlui/bitstream/handle/item/5424/stojak_et-al_in_vitro_induction_of_apoptosis_2013.pdf?sequence=1&isAllowed=y

In vitro induction of apoptosis and necrosis by new derivatives of daunorubicin

Abstract

Similar works

Full text

Available Versions

Jagiellonian Univeristy Repository