MOLECULAR PROFILE AND CELL CYCLE IN MCF-7 CELLS RESISTANT TO CISPLATIN AND DOXORUBICIN

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1 Experimentl Onology 31, 87 91, 29 (June) 87 Exp Onol 29 Originl ontriutions 31, 2, MOLECULAR PROFILE AND CELL CYCLE IN MCF-7 CELLS RESISTANT TO CISPLATIN AND DOXORUBICIN N.Yu. Lukynov*, N.V. Rusetsky, N.A. Treguov, V.F. Chekhun R.E. Kvetsky Institute of Experimentl Pthology, Onology nd Rdioiology NAS of Ukrine, Kyiv 322, Ukrine Aim: To ompre ultrstruture, phenotypi profile nd ell yle progression of MCF-7 humn rest ner ells nd MCF7 sulines resistnt to ispltin (MCF-7/DDP) nd doxoruiin (MCF-7/DOX). Methods: MTT-test, immunoytohemistry, flow ytometry, eletron mirosopy. Results: The development of drug resistne to ispltin nd doxoruiin in MCF-7 ells upon the ulturing of the initil ells with the rising onentrtions of ytosttis ws ompnied y the inrese in ells dhesion, the inresing differentition grde nd the loss of steroid hormone reeptors. Besides, it ws shown tht ntipoptoti mehnisms (derese of Bl-2 expression) nd intrellulr glutthione detoxifying system re involved in the proess of ispltin resistne development in MCF-7 ells. At the sme time, P-glyoprotein overexpression in ells resistnt to doxoruiin suggests MDR-dependent mehnism. Both doxoruiin- nd ispltin-resistnt ells re hrterized y the hnges in the expression of severl ell yle regultors Ki-67, ylin D1, pr nd р21). Conlusion: The long-time ulture of MCF-7 ells with ytostti drugs results in the deresed ylin D1, pr, nd Ki-67 expression nd inresed р21 expression with the inresing differentition grde of the resistnt ells. The underlying mehnisms of resistne to ispltin nd doxoruiin in MCF-7 ells my e different. Key Words: drug resistne, ispltin, doxoruiin, humn rest ner, MCF-7 ells, immunoytohemistry, ell yle. Tumor drug resistne is one of the most importnt prolems in ner tretment in generl s well s rest ner tretment [1 3]. Mny hemotherpeuti shedules used in rest ner tretment inlude nthrylines nd pltinum derivtives [2, 3]. Cispltin elongs to the group of lkylting gents. It inds to DNA ses using rosslinks nd reks in DNA strnds interfering with DNA replition [4]. The ntitumor effets of doxoruiin re ssoited with DNA interltion nd degrdtion of the tuulr pprtus used y the tive free rdils [5]. The mehnisms of drug resistne development for these two drugs re different [4 7]. It is elieved tht ispltin resistne is used y n expression of proteins involved in glutthione-mediting detoxifying pthwys suh s glutthione-s-trnsferse, glutthione-redutse nd glutthione itself [8, 9]. DOX resistne, s elieved, results from the overexpression of 17 kd Pgp glyopeptide, whih is n energydependent pump tht effluxes xenoiotis wy from ells [1, 11]. It is lso known tht the development of drug resistne phenotype ould e ompnied y hnges in morphologil struture, prolifertive potentil nd dhesion properties of ells s well s the hnges in expression of proteins involved in poptosis nd ell yle ontrol [12 15]. Studies on mehnisms of ntitumor drug resistne development re of prmount importne for further understnding of fundmentl proesses in formtion of drug resistne phenotype in tumors with the im of serhing the wys for overoming suh resistne. The im of the study ws to ompre the ultrstruture, phenotypi profile nd ell yle of MCF-7 humn Reeived: My 3, 29. *Correspondene: Fx: E-mil: onom@ononet.kiev.u Arevitions used: DOX doxoruiin. rest ner ells nd sulines resistnt to ytotoxi effets of ispltin (MCF-7/DDP) nd doxoruiin (MCF-7/DOX). MATERIALS AND METHODS Cell lines nd drug tretment. For our studies we used humn rest ner ell line MCF-7 nd its sulines resistnt to ytotoxi effets of ispltin (MCF-7/DDP) nd doxoruiin (MCF-7/DOX). The ells of the initil MCF-7 line were ultivted in modified Duleo s medium ISCOV ( Sigm, Germny) with ddition of 1% of fetl lf serum ( Sngv, Ukrine) t the temperture of 37 C nd CO 2 onentrtion of 5%. Cells were reseeded twie week t the density 2 4 x 1 4 ells/m 2, when ell lyer overed out hlf of the flsk surfe. The resistnt vrints MCF-7/DOX nd MCF-7/DDP were originted y growing initil MCF-7 ells with rising onentrtions of ispltin (from.1 to 6 µg/ml) or doxoruiin (from.1 to 32 µg/ml), respetively. Cispltin nd doxoruiin were dded twie week fter reseeding. Every two months, ell survivl ws nlyzed y MTT ssy. IC5 vlues for MCF-7 nd MCF-7/DDP ells were.25 nd 1 µg/ml of ispltin, respetively, nd for MCF-7 nd MCF-7/DOX ells.5 nd 8 µg/ml of doxoruiin, respetively. Therefore, MCF-7/DDP were 4 times s muh resistnt to the ytotoxi effet of ispltin nd MCF-7/DOX ells were 16 times s muh resistnt to the ytotoxi effet doxoruiin s ompred with the initil MCF-7 ells. MTT ssy. Sensitivity to ntitumor drugs (ispltin nd doxoruiin) ws mesured every two months using stndrd MTT-olorimetri test with 3-[4,5,dimethylthisol-2-1]-2,5-diphenyltetrsolium romide ( Sigm, Germny) [16]. Immunoytohemistry. Expression of surfe nd intrellulr ntigens ws studied immunoytohemi-

2 88 Experimentl Onology 31, 87 91, 29 (June) lly using mouse monolonl ntiodies to P-glyoprotein (Pgp), glutthione-s-trnsferse (GST), RE, PR, p53, Bl-2, E-dherin, Ki-67, ylin D1, pr, -my, p21 ( Dko Cytomtion, Denmrk). Flow ytometry. For ell yle nlysis of MCF-7 ells nd its sulines, resistnt to ispltin (MCF-7/DDP) nd doxoruiin (MCF-7/DOX), the ell suspension (1 6 ells per 2 µl of sline) ws wshed, 2 ml of old 7% ethnol ws dded, nd suspension ws fixed on ie for t lest 3 min. Speimens were entrifuged for 5 min t 3 g with further superntnt denttion. After resuspending the smple in 4 µl of sline, 5 µl of RNAse (25 mg/ml) nd 1 µl of propidium iodide (.5 mg/ml) were dded. Smples were nlyzed on PAS Prte flow ytometer (Germny) using red filter [17]. Eletron mirosopy. The ells were fixed in 1.6% gluthrldehyde solution in.1 M odylte uffer (ph 7.3) for 1 h followed y wshing in.1 M odylte uffer solution for h. To hieve the isotoni stte, surose (5 mg/ml) ws dded. Cell postfixtion ws done in 2% osmium tetroxide with further dehydrttion in lohols nd emedding in rldite s desried elsewhere. The ultrthin setions prepred on LKB-88 ultrtome nd ontrsted y urnil ette nd led itrte were exmined in JEM-1B eletron mirosope with 6 kv elertion voltge with further speimen photogrphy [18]. Sttistil nlysis. Sttistil nlysis ws done using STATISTICA 6. softwre (SttSoft In., USA) with quisition of men vlues nd stndrd devition (SD). Student s t-test ws used to evlute the signifine of the differenes etween groups. p >.5 ws onsidered s the signifint differene. RESULTS AND DISCUSSION Morphologil fetures of sensitive nd resistnt to ispltin nd doxoruiin MCF-7 ells. It is known, tht the development of drug resistne phenotype is ompnied y the hnges in different iologil fetures of mlignnt ells, inluding morphologil ones. In ontrst to spindle-shped MCF-7 nd MCF-7/DOX ells, MCF-7/DDP ells were more spheril with high nuleus/ytoplsm rtio. In ll three lines, the nulei were rounded with 2 4 nuleoli. Cells resistnt to doxoruiin nd ispltin were lrger thn initil MCF-7 ells nd were hrterized y stronger dhesion to the underlying surfe. Eletron mirosopy demonstrted more omplited ultrstruturl orgniztion nd inresed diffe rentition grde upon formtion of drug resistne oth to ispltin nd to doxoruiin (Fig. 1, ). The numer of the mirotuules inresed nd the fiers omprising the mirofilments of vrying width were evident. An tive Golgi pprtus onsisting of 2 3 loi with mny multivesiulr odies ws lso reveled in the resistnt ells (Fig. 1,, ). The formtion of lot of multivesiulr odies nd their positioning ner the plsmti memrne my e inditive of the inresed efflux of the foreign sustnes through ell memrne, whih dereses the mounts of ispltin nd doxoruiin in ytoplsm of resistnt ells nd their ytotoxi tivity. In MCF-7/DDP ells, the system of tin filments loted in the ortil lyer of ytoplsm ws tivted (see Fig. 1, ), while in MCF-7/DOX ells, the intermedite filments were tivted in the entrl re of ytoplsm (see Fig. 1, ). Therefore, the development of resistne to ispltin nd doxoruiin in MCF-7 ells ws ompnied y the signifint hnges in ultrstruturl orgniztion suggestive of the inresing differentition grde. Fig. 1. Eletron mirosopy of MCF-7 ells () nd sulones with drug resistne to ispltin MCF-7/DDP () nd doxoruiin MCF-7/DOX () Immunoy tohemil hrteristis of MCF-7 ells, sensitive nd resistnt to ispltin nd doxoruiin. Mdr1 gene mplifition followed y P-glyoprotein overexpression is one of the mehnisms involved in the development of drug resistne. An lterntive mehnism is onneted with GST isoenzymes expression nd inresed metllothionein expression [19 21]. Glutthione-S-trnsferses re responsile for onjugting glutthione with different xenoiotis. The detoxifition of ntiner drugs y metllothioneins is relted to their linkge with

3 Experimentl Onology 31, 87 91, 29 (June) 89 eletrophili ntitumor drugs from ispltin group, euse free metllothioneins re nuleophili ompounds [21]. In our experiments, P-gp expression ws sent oth in ispltin-sensitive nd resistnt ells (Tle 1, Fig. 2, ) suggesting mdr1-independent wy of drug resistne. In ontrst, in ytoplsm of most doxoruiin-resistnt ells (MCF-7/DOX), P-gp ws overexpressed (see Tle 1; Fig. 2, ). Also we found out the signifint differenes in GST expression mong the sulines studied. In ispltin-resistnt ells, the inrese in GST ontent (Fig. 2, ) long with the derese in the perentge of MT-positive ells (see Tle 1) ws evident. Aording to urrent knowledge, reeptor sttus of rest tumors is prognosti ftor tht is onneted with their sensitivity to neodjuvnt therpy nd rdiotherpy. We hve reported erlier tht the drug resistne in MCF-7 humn rest ner ells developed upon ell ulture in rising ispltin nd doxoruiin onentrtions ws ompnied y the hnges in hormone reeptor expression [22]. Prtiulrly, estrogen nd progesterone reeptors were expressed in 2% nd 4% respetively of the initil ells, while in MCF-7/DDP nd MCF-7/DOX ells these reeptors were sent (see Tle 1). Aording to the linil experiene, in third of rest ner ptients the worse prognosis is ssoited with the sene of steroid hormone reeptors. It is known tht ytotoxi tivity of mny ntitumor drugs is linked to their ility to indue poptosis in trget ells [23 25]. We hve ttempted to ompre the ontent of severl poptosis-relted proteins in MCF-7 sulines with quired drug resistne nd in the initil MCF-7 ells. The quisition of resistne to ispltin or doxoruiin ws not ssoited with hnges of p53 expression, whih ws deteted t low level in ll ell lines studied (see Tle 1). Antipoptoti Bl-2 protein ws present in ytoplsm of lmost ll sensitive nd doxoruiin-resistnt ells (Fig. 2, d, f) while the longtime ulture of MCF-7 ells in the presene of ispltin filitted redution of Bl-2-positive ell perentge from 8 to 1% (see Tle 1; Fig. 2, e). Therefore, the Tle 1. Phenotypi fetures of rest ner MCF-7 ells nd sulines with indued resistne to ispltin nd doxoruiin Cell line RE PR Pgp GST Cells, positive for studied proteins, % MT p53 Bl2 Ki-67 E-d С-my p21 рr Cylin D MCF-7 2 2, 3 4 2, , 3 6 2, 3 6 2, 3 4 2, 3 9 2, , 3 4 2, 3 MCF-7/DOX/ , MCF7/DDP/ , In Tle 1 nd 2: 1 signifintly (p <.5) different from MCF-7; 2 signifintly (p <.5) different from MCF-7/DOX/16; 3 signifintly (p <.5) different from MCF-7/DDP/4. d e f g h i Fig. 2. Imunoytohemil fetures of MCF-7 ells with ispltin nd doxoruiin drug resistne phenotype: а loss of Pgp expression in sensitive MCF-7 ells; expression of GST in MCF-7/DDP ells; с Pgp expression in MCF-7/DOX ells; d, е, f Bl-2 expression in MCF-7, MCF-7/DDP nd MCF-7/DOX, respetively; g, h Ki-67 expression in sensitive MCF-7 ells nd MCF-7/DDP ells; i р21 expression in MCF-7/DOX ells

4 9 Experimentl Onology 31, 87 91, 29 (June) development of ispltin resistne in rest ner ells is ssoited with the lowered Bl-2 expression. These dt gree with oth experimentl nd linil findings demonstrting tht high Bl-2 expression predits hemosensitivity in rest nd lung ner. We hve lso found deresed prolifertive tivity of MCF-7 ells with resistne to ispltin nd doxoruiin (Fig. 2, g, h) with three-fold redution of prolifertive potentil in MCF-7/DDP ells nd twofold redution in MCF-7/DOX ells (see Tle 1). The derese in the prolifertive potentil my e orrelted with differentition of the resistnt ells. In ft, oth resistnt vrints (MCF-7/DOX nd MCF-7/DDP) were hrterized y strong expression of E-dherin s ompred to low expression of E-dherin limited to the tight interellulr ontt only in the initil MCF-7 ells (see Tle 1). The deresed prolifertive tivity in ells quiring drug-resistnt phenotype is elieved to e ssoited with hnges in the expression of some ell yle-ontrolling proteins [26]. In our study, ll three MCF-7 sulines expressed low level of -my. The development of ispltin nd doxoruiin resistne in humn rest ner ells ws ompnied y the redution of ylin D1, pr expression nd the overexpression of p21 (Fig. 2, i). These findings orrespond with the ville dt of other uthors [22, 26]. Therefore, the formtion of drug resistne to ispltin nd doxoruiin in humn rest ner MCF-7 ells is hrterized y hnges in expression of proteins involved in ontrol of poptosis, ell yle, prolifertion, nd dhesion. Anlysis of ell yle in sensitive MCF-7 ells nd ells with resistne to ispltin nd doxoruiin. The norml regultion of ell yle is known s one of the hrteristi fetures of the mlignnt ells. We hve shown tht the ptterns of ell yle distriution in the initil MCF-7 ells re the sme s in ispltin nd doxoruiin-resistnt sulines (Tle 2, Fig. 3). The mehnisms of ytotoxiity upon ell exposure to ispltin nd doxoruiin re diffe rent. It is elieved tht ispltin is not phse-speifi drug euse it uses disorders in DNA trnsription nd replition, whih led to ell yle rrest nd poptosis. In ontrst to ispltin, doxoruiin is phse-speifi drug, ffeting predominntly S nd G 2 phses of the ell yle. It ws of interest to ompre ell yle trverse in the initil nd resistnt MCF-7 ells upon ell exposure to ispltin nd doxoruiin. We hve shown tht in the initil MCF-7 ells, inution with ispltin t dose of IC 1 for 24 h resulted in the signifint derese of S phse perentge (from 28.52% to 19.55%) with G 2 /M rrest (from 16.37% to 33.4%). Menwhile, doxoruiin tretment resulted in the umultion of MCF-7 ells in G /G 1 phse with the G /G 1 ell perentge inresing from 55.11% to 76.8%. In ontrst, exposure to doxoruiin in MCF-7/DOX ells nd exposure to ispltine in MCF-7/DDP ells hd no effet on ell yle trverse (Fig. 4). Tle 2. Cell yle distriution of MCF-7 ells nd sulines with indued resistne to ispltin nd doxoruiin Cell line Perentge of ells in phse of the ell yle G /G 1 S G 2 /M MCF MCF-7/DOX/ MCF7/DDP/ Fig. 3. Distriution of MCF-7, MCF-7/DDP, MCF-7/DOX ells etween ell yle phses 9 MCF-7/Dox % % 61.5% 59.42% 49.3% 27.25% 26.2% 19.55% 11.5% 33.4% 14.38% MCF-7/S + Dox MCF-7/CP MCF-7/S + CP 12.15% 11.73% G /G 1 S G 2 /M Fig. 4. Chnges in ell yle s result of impt of studied ntitumor drugs To sum up, the long-time ulture of humn rest ner MCF-7 ells in the presene of ispltin or doxoruiin in vitro is ompnied y the pronouned

5 Experimentl Onology 31, 87 91, 29 (June) 91 hnges in moleulr-iologil properties of the ells with oth shred nd drug-speifi moleulr mehnisms of the formtion of the resistnt phenotype. In oth doxoruiin-resistnt nd ispltin-resistnt ells, the steroid hormone reeptors hve een lost. The development of ispltin resistne involves the ntipoptoti mehnisms (deresed Bl-2 expression) nd intrellulr glutthione detoxifying system while the development of doxoruiin resistne seems to follow MDR-dependent mehnism suggested y P-glyoprotein overexpression. In ells resistnt to either ispltin or doxoruiin, the dhesive properties re enhned nd the ultrstruturl orgniztion is hrterized of more omplited ptterns implying the inresed differentition grde. At the sme time, in resistnt ells ylin D1, pr, nd Ki-67 expression deresed while р21 expression inresed. The hnged expression pttern suggests the deresed prolifertive potentil of the ells with drug resistnt phenotype. Therefore, the resistnt MCF-7 ells differ from the initil ell line y the expression of proteins ssoited with drug resistne s well s proteins involved in ontrol of poptosis, prolifertion nd ell dhesion. Our dt my e dvntgeous for developing the novel shedules of ntiner tretment ounting for the moleulr-iologil hrteristis of drug-resistnt ells. ACKNOWLEDGMENTS This work ws supported y Fundmentl reserhes stte foundtion ( F28.4/47). We grtefully knowledge Phd.M.P. Zvelevih (R.E. Kvetsky Institute of Experimentl Pthology, Onology nd Rdioiology) for his helpful dvie. REFERENCES 1. O Drisoll L, Clynes M. Moleulr mrkers of multiple drug resistne in rest ner. Chemotherpy 26; 3: Chekhun VF, Shishov YuV. Current view on development of tumor drug resistne. Onology 2; 2: 11 5 (In Russin). 3. Fute MA, Lurent L, Ploton D, et l. Distintive ltertion of invsiveness, drug resistne nd ell-ell orgniztion in 3Dultires of MCF-7, humn rest ner ell line, nd its multidrug resistne vrint. Clin Exp Metstsis 22; 19: MWhinney SR, Golderg RM, MLeod HL. Pltinum neurotoxiity phrmogenetis. Mol Cner Ther 29; 1: Minotti G, Menn P, Slvtorelli E. Anthrylines: moleulr dvnes nd phrmologi developments in ntitumor tivity nd rdiotoxiity. Phrmol Rev 24; 56: Kurpeshev OK, Tsy AF, Mrdynsky YuS, Berdov BA. Mehnisms of development nd wys to overome tumor hemoresistne. Rus J Onol 22; 6: (In Russin). 7. Chekhun VF, Kulik GІ, Yurhenko OV, et l. Role of DNA hypomethyltion in the development of the resistne to doxoruiin in humn MCF-7 rest denorinom ells. Cner Let 26; 231: Alderden RA, Hll MD, Hmley TW. The disovery nd development of ispltin. J Chem Ed 26; 83: Kurokw H, Nishio K, Ishid T, et l. Effet of glutthione depletion on ispltin resistne in ner ells trnsfeted with the γ-glutmylysteine synthetse gene. Jpn J Cner Res 1997; 88: Stordl B, Dvey M. Understnding ispltin resistne using ellulr models. IUBMB Life 27; 59: Sun ZE, Smith MM, Müller M, et l. The mehnism of tion of multidrug-resistne-linked P-glyoprotein. J Bioenerg Biomemr 21; 33: Eytn GD. Mehnism of multidrug resistne in reltion to pssive memrne permetion. Biomed Phrmother 25; 59: Teodori E, Dei S, Mrtelli C, et l. The funtions nd struture of ABC trnsporters: implitions for the design of new inhiitors of Pgp nd MRP1 to ontrol multidrug resistne (MDR). Curr Drug Trgets 26; 7: Chekhun VF, Lukynov NYu, Yurhenko OV, Kulik GI. The role of expression of the omponents of proteome in the formtion of moleulr profile of humn ovrin rinom A278 ells sensitive nd resistnt to ispltin. Exp Onol 25; 27: Chekhun VF, Gnin KP, Kulik GI, et l. Impt of tumor drug resistne phenotype on dynmis of ispltin-indued hnges in hromtin struture of interphse nulei of lymphoytes from peripheril lood from rts with Gueren rinom. Cytology nd Genetis 2; 34: 11 7 (In Russin). 16. Ni J, Hollnder D. Applition of the MTT-ssy to funtionl studies of mouse intestinl intrepithelil lymphoytes. J Clin L Anl 1996; 1: Ormerod MG. Flow ytometry: prtil pproh. Oxford University Press, p. 18. Luft JH. Improvements in epoxy resin emedding methods. J Biophys Biohem Cytol 1961; 9: Cheng G, Zhu H, Sun L. The expression of multiple drug resistne ssoited genes ovrin ner. Zhonghu Fu Chn Ke Z Zhi 2; 35: 87 9 (In Chinese). 2. Dziegiel P. Expression of metllothioneins in tumor ells. Pol J Pthol 24; 55: Fn LZ, Cherin MG. Potentil role of p53 on metllothionein indution in humn epithelil rest ner ells. Br J Cner 22; 87: Keen JC, Dvidson NE. The iology of rest rinom. Cner 23; 97: Lukynov NYu, Kulik GI, Chekhun VF. Role of p53 nd l-2 genes in poptosis nd tumor drug resistne. Onology 2; 46: (In Russin). 24. Yrilin АА. Apoptosis: nture of phenomenon nd its role in the whole orgnism. Pth Physiol Experim Therpy 1998; 1: (In Russin). 25. Stewrt BW. Mehnisms of poptosis: integrtion of geneti, iohemil nd ellulr inditors. J Nt Cner Inst 1994; 86: Shh MA, Shwrtz GK. Cell yle-medited drug resistne аn emerging onept in ner therpy. Clin Cner Res 21; 7: Copyright Experimentl Onology, 29