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The role of a novel copper complex in overcoming doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

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The role of a novel copper complex in overcoming doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo
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  Chemico-Biological Interactions 159 (2006) 90–103 The role of a novel copper complex in overcoming doxorubicinresistance in Ehrlich ascites carcinoma cells in vivo S. Majumder, P. Dutta, A. Mookerjee, S.K. Choudhuri ∗  Department of Environmental Carcinogenesis and Toxicology, Chittaranjan National Cancer Institute,37, S.P. Mukherjee Road, Calcutta 700026, India Received 27 August 2005; received in revised form 1 October 2005; accepted 4 October 2005Available online 10 November 2005 Abstract One of the important pathways of resistance to anthracyclines is governed by elevated levels of glutathione (GSH) in cancercells. Resistant cells having elevated levels of GSH show higher expression of multidrug-resistant protein (MRP); the activity of glutathione  S  -transferases (GSTs) group of enzymes have also been found to be higher in some drug-resistant cells. The gen-eral mechanism in this type of resistance seems to be the formation of conjugates enzymatically by GSTs, and subsequent effluxby active transport through MRP (MRP1–MRP9). MRPs act as drug efflux pump and can also co-transport drugs like doxoru-bicin (Dox) with GSH. Depletion of GSH in resistant neoplastic cells may possibly sensitize such cells, and thus overcomemultidrug resistance (MDR). A number of resistance modifying agents (RMA) like  dl -buthionine ( S  ,  R ) sulfoxamine (BSO) andethacrynic acid (EA) moderately modulate resistance by acting as a GSH-depleting agent. As most of the GSH-depleting agentshave dose-related toxicity, development of non-toxic GSH-depleting agent has immense importance in overcoming MDR. Thepresent study describes the resistance reversal potentiality of novel copper complex, viz., copper  N  -(2-hydroxy acetophenone)glycinate (CuNG) developed by us in Dox-resistant Ehrlich ascites carcinoma (EAC/Dox) cells. CuNG depletes GSH in resis-tant (EAC/Dox) cells possibly by forming conjugate with it. Depletion of GSH results in higher Dox accumulation that maylead to enhanced rate of apoptosis in EAC/Dox cells. In vivo studies with male Swiss albino mice bearing ascitic growth of EAC/Dox showed tremendous increase in life span (treated/control, T/C=453%) for the treated group with apparent regressionof tumor. Resistance to Dox in EAC/Dox cells is associated with over expression of GST-P1, GST-M1 (enzymes involved inphase II detoxification) and MRP1 (a transmembrane ATPase efflux pump for monoglutathionyl conjugates of xenobiotics). CuNGcauses down regulation of all these three proteins in EAC/Dox cells. The effect of CuNG as RMA is better than BSO in manyaspects.© 2005 Elsevier Ireland Ltd. All rights reserved. Keywords:  Copper  N  -(2-hydroxy acetophenone) glycinate; Doxorubicin; Drug resistance; Resistance modifying agent; Glutathione  Abbreviations:  ABC, ATP binding cassette; BSO,  dl -buthionine ( S  ,  R ) sulfoxamine; CuNG, copper  N  -(2-hydroxy acetophenone) glycinate;Dox, doxorubicin; EAC, Ehrlich ascites carcinoma; MDR, multiple drug resistance; MRP, multidrug resistance protein ∗ Corresponding author. Tel.: +91 33 2476 5101/2/4x317; fax: +91 33 2475 7606.  E-mail addresses:  majumder surajit@yahoo.co.in (S. Majumder), pranab76in@yahoo.co.uk (P. Dutta), anandamookerjee@yahoo.com(A. Mookerjee), soumitra01@vsnl.net, soumitra01@yahoo.com (S.K. Choudhuri).0009-2797/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2005.10.044  S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103  91 1. Introduction The phenomenon of drug resistance is a major obsta-cle to successful application of cancer chemotherapy.Drug resistance may be acquired or innate. In bothforms of resistance, neoplastic cells become refractoryto multiple drugs, different structurally and function-ally. Multiple drug resistance (MDR) is a basic prob-lem in cancer biology and sometimes a number of mechanisms operate in a single drug-resistant system[1]. In some systems of MDR, ATP-binding cassette(ABC) membrane transporter proteins extrude a widerange of drugs used in modern chemotherapy. Mul-tidrug resistance protein (MRP), a plasma membraneprotein of 190kDa ABC efflux pump is found to be overexpressedinnumerousdrug-resistantcelllines[2].MRP comprises a family of nine proteins (MRP1–MRP9)[3,4] and is expressed in different tissues at low lev-els [5,6]. In normal physiology, MRPs play an impor- tant role in various secretory and transport process [7].MRPs are involved in cellular detoxification processtogether with the glutathione  S  -transferase (GST) groupof enzymes and reduced glutathione (GSH) the twokey members of the phase II detoxification machin-ery [8]. GSTs catalyze the process of conjugation of many amphipathic drugs with GSH that renders theminto high affinity substrates for MRP. These monoglu-tathionyl conjugates are then transported out of the cellin an ATP-dependant manner [9,10]. Most of the drug-resistant cells that over express MRP and/or GST main-tain an elevated intracellular pool of GSH and therebyreduce the effective intracellular concentration of thechemotherapeutic drug, leading to decreased cytotoxi-city [11–13].To overcome MRP/GST/GSH-mediated drug resis-tance, this molecular cascade needs to be disrupted. Oneof the approaches to this end is to utilize compoundsthatdepletecellularGSHlevelsleadingtoimpaireddrugefflux machinery with concomitant rise in the sensitivityof drug-resistant cells to antineoplastic agents [14,15].Compounds that inhibit GST can also overcome MDRasconjugationofxenobioticswithGSHisaffectedintheprocess.Quiteafewagentslike dl -buthionine( S  ,  R )sul-foxamine (BSO), ethacrynic acid (EA), sulphosalazine,  N  -ethylmaleimide utilize either of the two routes [16].However, the major drawback with these resistance-modifying agents (RMA) is that the doses required toachieve a positive response in patients is fraught withtoxic side effects [17]. Under this perspective, we aimedat developing novel RMA capable of overcoming MDRin vivo. The manuscript describes the activity of a novelRMA, viz., copper  N  -(2-hydroxy acetophenone) glyci- Fig. 1. Structure of CuNG. nate(CuNG)[18,19]toovercomeMDRinvivoinascitic tumor model.CuNG is copper coordinated Schiff’s base complex(Fig. 1) and we had earlier reported the synthesis and characterization of it [18,19]. It has low toxicity and depletes GSH in time-dependent manner. This observa-tion prompted us to investigate its potentiality as RMAwhere drug resistance is due to elevated level of GSTand/or GSH. The present report describes that CuNGindeed conforms to our hypothesis, as it reverses Doxresistance in Ehrlich ascites carcinoma in vivo. Theresistance modifying properties of CuNG as RMA arecompared with respect to BSO, well known for its resis-tance reversal capability. 2. Materials and methods 2.1. Materials dl -Buthionine ( S  ,  R ) sulfoxamine, 1-chloro 2,4-dinitrobenzene(CDNB),dimethylsulphoxide(DMSO),doxorubicin hydrochloride (Dox), 5,5 ′ -dithio bis (2-nitrobenzoic acid) (DTNB), May-Grunwald-Giemsa(MGG), polyvinylidene difluoride (PVDF) membranewere purchased from Sigma Chemical Company (St.Louis,MO,USA).Nitrobluetetrazoliumchloride(NBT)and 5-bromo, 4-chloro 3-indolylphosphate (BCIP) werepurchased from SRL, India. Trichloro acetic acid (TCA)and DPX mountant were from Merck, India. FITC con- jugated active Caspase-3 antibody were from BD Pher-mingen(CA,USA).PolyclonalRabbitAnti-RatGST-P1antibody purchased from Biotrend (Alpha DiagnosticInternational, Germany). MRP1 (Goat polyclonal IgG,N-19, sc-7774) and Rabbit Anti-Goat IgG-AP (sc-2771)were from Santa Cruz Biotechnology (Europe). RabbitAnti-Goat IgG-FITC conjugate, Goat Anti-Rabbit IgG-AP, Goat Anti-Rabbit IgG-FITC and Protein MolecularWeight Marker (3–43 and 29–200kDa) were from Ban-galore Genei (Bangalore, India). Other chemicals usedwere of highest purity available.  92  S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103 2.2. Synthesis of the ligand  The ligand, potassium  N  -(2-hydroxy acetophenone)glycinatewaspreparedaccordingtothereportedmethod[20,21].Inbrief,acoldaqueoussolutionofKOH(1.03gin 12ml) was mixed with cold aqueous solution of glycine (1.38g in 12ml) and held at 15–20 ◦ C in an icebathwithcontinuousstirring.Anethanolicsolutionof2-(hydroxyl)acetophenone(2.5gin25ml)wasaddeddropwise. Deep yellow color was developed and stirring wascontinued for 1h followed by 5h at room temperature.Rotaryevaporatorremovedthesolvent.Theyellowmasswas washed with petroleum ether and precipitated withmethanol-diethyl ether mixture. The crude product wasrecrystallized from methanol to yield potassium  N  -(2-hydroxy acetophenone) glycinate. Yield 75%, meltingpoint 258–260 ◦ C. 2.3. Synthesis of CuNG CuNG was synthesized by the reaction of potassium  N  -(2-hydroxy acetophenone) glycinate and copper sul-phate according to the reported method [18]. In brief, 0.68g CuSO 4 · 5H 2 O was dissolved in 5ml deionizedwater. 0.785g potassium  N  -(2-hydroxy acetophenone)glycinate was dissolved in 25ml ethanol. The solu-tionofpotassium  N  -(2-hydroxyacetophenone)glycinate(yellow color) was slowly added to CuSO 4  solution(blue color) at room temperature with continuous stir-ring by magnetic stirrer for 1h at 45–50 ◦ C. The colorof the mixture changed to deep green. The mixture wascooledatroomtemperatureandthegreenprecipitatewasseparated by filtration. The compound was dried andrecrystallized from DMSO. Yield 40%, melting point242 ◦ C. Anal. Calc. C 10 H 15 O 6 NCu: C, 39.0; H, 4.87; N,4.54; Cu. 20.45. Found: C, 40.57; H, 5.04; N, 4.65; Cu,21.24%. 2.4. Synthesis of CuNG–GSH conjugate CuNG–GSH conjugate was synthesized by the reac-tion of CuNG and GSH [19]. In brief, 2mg CuNG and 2mg GSH were dissolved in 2ml DMSO and 2ml dis-tilled water, respectively. The clear solution of CuNGwas slowly added to the solution of GSH. Gray coloredprecipitate was formed. The mixture was made up tothe volume of 10ml and rotated in a magnetic stirrer for30minandthemixturecooledto4 ◦ C;theprecipitatefil-tered, dried and recrystallized from water–alcohol, yield80%, melting point 160 ◦ C (decomposed). Anal. Calc.C 20 H 31 N 4 O 12 SCu: C, 29.15; H, 4.8; N, 9.3. Found: C,28.82; H, 4.75; N, 8.99%. UV (water)  λ max  226, 357,600; IR (film) 4378, 4281, 4243, 4170, 4134, 4044 (sh),3986(sh),3903(sh),3835(sh),3805,3751,3611,3547,3497, 3443, 3392, 3315, 3241, 3180 (b), 2998 (s), 2932(b), 2803, 2039 (s), 1729 (sh), 1630 (sh, b), 1531 (b),1410 (sh), 1229 (b), 1131 (s), 1096 (b), 1022 (b) cm − 1 ; 1 H NMR (300MHz, D 2 O)  δ  7.87–7.68 (m, 1H), 7.5(s, 1H), 6.98–6.89 (m, 2H), 4.03 (s, 2H), 3.91 (s, 3H),3.49 (s, 2H), 3.28–3.04 (m, 5H), 2.91–2.85 (m, 2H),2.62–2.58 (d, 7H), 2.44 (s, 3H), 2.09 (s, 2H). LC–MScalc. 613.5, found 613.3. 2.5. LC–MS studies CuNG was dissolved in DMSO. GSH andCuNG–GSH were dissolved in water. Two hundredmicromolar of each was employed for liquid chromato-graphy–mass spectroscopy (LC–MS) analysis. LC–MSanalysis was performed on Shimadzu LC10 ADVPseries HPLC and Applied Biosystems Q trap systemwith a Turbo ion spray as a source. Samples wereinjected (20  l) into LC–MS system through LC10ADVP series auto sampler. Separations were carriedout on 5  m YMC pro C18 column (4.6mm × 50mm)at 35 ◦ C with a flow rate of 1ml/min using HPLCmethod. Aqueous samples were diluted in methanol,water or mixture of both the solvents and analyzed byflow injection analysis method in both positive andnegative mode techniques (Q1 multiple ion scan methodcovering mass range of 50–800Da) using acetonitrileand water as mobile phase. 2.6. Animals Adult male Swiss albino mice weighing 18–20gwere obtained from our animal-breeding colony. Ani-mals were kept for a quarantine period of 1 week at atemperature of 25 ± 2 ◦ C, relative humidity of 55 ± 2%and with photo cycle of 12-h light/12-h dark. Water andfood pellets were provided ad libitum .2.7. Effect of RMA singly and in combination with Dox on hematological parameters CuNG (at 8 and 10mg/kg) and BSO (at 20mg/kg)were injected singly and in combination with Dox (at2mg/kg)tomaleSwissalbinomice.Bloodwascollectedfrom untreated as well as from treated mice 10 days(10D) after treatment. Blood was obtained via closedcardiac puncture with the help of a 22-guage hypoder-mic needle by subxiphoid approach. Blood from eachgroup (CuNG, BSO treated and untreated) was pooledinto separate glass tubes and treated with anticoagulant  S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103  93 (heparin). The average value of haemoglobin (Hb), totalcount (T.C.) and differential count (D.C.) of three inde-pendent experiments were shown in Table 3. 2.8. Preparation of spleen cell suspension Normal and drug (CuNG, BSO singly and in combi-nation with Dox) treated male Swiss albino mice wereanaesthetized and 70% alcohol was sprayed on abdom-inal region. Spleen was removed aseptically and smallamount of PBS was injected to it. Spleen was rubbedagainst the fine wire mesh of tissue grinder. The cellsuspensioncentrifugedat1000rpmfor5min.Thesuper-natant was discarded and the cells centrifuged in PBStwice at room temperature for washing.Cell viability was tested by trypan blue extrusionmethodandcellswerecountedinaphasecontrastmicro-scope. The experiment was repeated thrice. The averagevalue of number of spleen cell of three independentexperiments is shown in Table 4. 2.9. Separation of bone marrow cells Normal and drug (CuNG, BSO singly and in com-bination with Dox) treated mice were anaesthetized andthefemurbonewascutwiththehelpofavertebratescis-sor. Bone marrow was flushed with 0.56% KCl solutionand centrifuged at 3000rpm for 15min at 37 ◦ C.Drug treated and untreated cells were counted undercompoundmicroscope.Theexperimentwasrepeatedforthrice.Theaveragevalueofnumberofbonemarrowcellof three independent experiments is shown in Table 4. 2.10. Cell line, tumor implantation and experimental protocol EAC cell was maintained as an ascitic tumor inmale Swiss albino mice. A Dox-resistant sublinewas developed by sequential transfer of Dox treatedEAC cells to the subsequent generation of host micewith continuous Dox treatment [22,23]. Briefly, the treatment regime consisted of 2mg/kg/week Doxintraperitoneally (i.p.). The daily treatment dose was0.4mg/kg for 5 days. Dox was started 24h afterinoculation of 10 6 EAC cells i.p. into mice. The meansurvival time (MST in days) ± S.D. of untreated micebearing EAC cell was 19.4 ± 1.5 days ( n =20). TheMST ± S.D. of the host mice bearing EAC cell andDox was 33.4 ± 2.06 days ( n =22), whereas MST of the 17th generation of host mice was came down to24.8 ± 1.2 days ( n =20). After this degree of resistancehad been developed, the dose of Dox was increasedto 4mg/kg/week (daily treatment dose was 0.8mg/kgfor 5 days), which resulted in 36 ± 2.3 days MST( n =18). When this tumor subline was re-treatedwith Dox after 17th passage the MSTs were sharplydecreased at every generation and came up to 19.2 ± 0.7days ( n =25) at 25th generation. The animals withdrug-resistant cells (EAC/Dox) (25th generation mice)had the survival (19.2 ± 0.7 days) close to that of micewith drug-sensitive cells (EAC/S) (19.4 ± 1.5 days)[24].Eightsetsofanimalsweretakenforstudiesonanimalsurvival (Table 1). Each mouse was inoculated with 10 6 EAC/Dox cells i.p. Both CuNG (8, 10mg/kg bodyweight) and BSO (20mg/kg body weight) were injectedi.p. 24h later of EAC/Dox cell passage to appropriategroups. Dox was injected i.p. after an hour of CuNG orBSO treatment. Animals were checked daily for assess-mentofasciticgrowthandbodyweightsweremeasured.Two weeks after the implantation of EAC cells, totalascitic fluid (TAF) and packed cell volume (PCV) weremeasured; the average value of three independent exper-iments of each set was presented in Table 6. Time of deathwasrecordedforcalculationofmeansurvivaltime(MST). Table 1Treatment schedule for studies on animal survivalGroups a TreatmentGroup I (EAC/Dox) No treatmentGroup II (EAC/Dox+Dox) Only Dox injectedGroup III (EAC/Dox+CuNG at 8) Only CuNG injected at 8mg/kg body weightGroup IV (EAC/Dox+CuNG at 10) Only CuNG injected at 10mg/kg body weightGroup V (EAC/Dox+BSO at 20) Only BSO injected at 20mg/kg body weightGroup VI (EAC/Dox+CuNG at 8+Dox) Dox injected 1h after CuNG injection at 8mg/kg body weightGroup VII (EAC/Dox+CuNG at 10+Dox) Dox injected 1h after CuNG injection at 10mg/kg body weightGroup VIII (EAC/Dox+BSO at 20+Dox) Dox injected 1h after BSO injection at 20mg/kg body weight a Each mouse was inoculated with 10 6 EAC/Dox cells intraperitoneally. Treatment was started 24h after cell inoculation. For each of the groups,Dox was administered at the dose of 2mg/kg body weight.  94  S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103 The MSTs were recorded following various drugtreatment protocols [23,25]. The statistical significance of the survival data, i.e., survival of the drug treatedgroups versus untreated groups was evaluated by  P -values (Student’s  t  -test). Mouse survival times in differ-ent groups were also compared as treated/control (T/C)ratio(percent),i.e.,theratioofthesurvivaltime(indays)fortreatedmicetountreatedcontrolmice.AsinstandardNational Cancer Institute protocols for screening newanticancer drugs, it was considered that the increase insurvival time corresponding to T/C ratios around 120%to “marginal”, T/C ratios between 120 and 150% to be“clear” and T/C ratios equal or superior to 150% to be“marked”. 2.11. Treatment schedule for enzyme assay,immunoblot and flow cytometry For enzyme assay, immunoblot and flow cytometry,ascitic growth in mice was allowed for a period of 10days. Mice with similar ascitic growth were random-ized into different groups and treated with RMAs and/orDox.RMAsusedintheexperimentsareBSOandCuNG.Whenever treatment schedule included both RMA andDox, Dox was administered 1h after RMA injection.At different time intervals, cells were collected fromthe intraperitoneal cavity and were washed in phosphatebuffer saline (PBS). Cells were then processed accord-ingly as described in the following sections. CuNG ata dose of 10mg/kg body weight showed the highestactivity as RMA, and hence applied in the experiments(Table 2). 2.12. In vitro assay for cell cytotoxicity One-dimensional titrations were performed todetermine the IC 50  value of individual drugs in drugsensitive (EAC/S) and drug-resistant (EAC/Dox) cellsfollowing the method of Choudhuri and Chatterjee Table 2Treatment schedule for evaluation of in vivo GST-P1, GST-M1 andMRP1 modulatory activity of CuNG and BSOGroups a TreatmentGroup I (EAC/S) No treatmentGroup II (EAC/Dox) Dox injected i.p. at 4mg/kgGroup III (EAC/Dox+CuNG) CuNG injected i.p. at 10mg/kgGroup IV (EAC/Dox+BSO) BSO injected i.p. at 20mg/kg a Allanimalsreceived10 6 EACcells(EAC/SandEAC/Dox,respec-tively) and ascitic growth was allowed for a week before being sub- jected to experiments. Cells were aseptically collected after 24h of treatment from the intraperitoneal cavity of mice. [23]. Briefly, ascitic fluid was extracted from the micewithin 10–15 days of inoculation of tumor cells. Notreatment was given during the last passage beforean in vitro experiment. Cells (2 × 10 4 ) were collectedand washed thrice in PBS, and then plated in eachwell of flat bottomed 96-well plate with RPMI 1640medium (Gibco BRL), containing HEPES (Sigma),penicillin–streptomycin and 10% FCS (Gibco BRL).Cells were incubated without drug for 24h. EAC/Doxcells were treated with 3.07ng/ml of CuNG. Afteran hour, different concentrations of Dox [effectiveconcentration (EC) 0.15–5.8  g/ml] were added. Thecells were then incubated for an additional 4 days.Cells were then fixed with TCA (EC 16%), followed bywashing with double distilled water. Fixed cells werestained with crystal violet. Cell-associated dye was thensolubilized with 10% acetic acid and the viable cellnumber was assayed by measuring the concentration of stain in the well with an ELISA reader at 570nm. 2.13. Assessment of apoptotic cells by MGGstaining Morphological assessment of the apoptotic cells wasperformed using the May-Grunwald-Giemsa (MGG)staining method [26]. Briefly, drug treated EAC/Dox cells were collected in cold PBS. After washing, a cellsuspension (10 6 cells/ml PBS) was prepared and 30  lof it was spread over grease free glass slides. The slideswere dried overnight and stained with MGG stain for5minfollowedbyintensePBSwashing.Then,theslideswere again stained with Giemsa for 20min followed byintense wash in deionized water. After air-drying theslidesweremountedwithDPX.Themorphologyofcellswas examined under light microscope. Apoptotic cellswere identified on the basis of nuclear fragmentation[27]. Apoptotic index (AI) was determined as the per-centageofapoptoticcellsfromatleast400countedcells. 2.14. Measurement of cellular glutathione GSH was measured following the method of Sed-lack and Lindsay [28] briefly, 10 6 cells homogenate in0.1ml PBS was mixed with 2.4ml EDTA (0.02M) andkept on ice bath for 10min. Then, 2ml deionized waterand 0.5ml 50% TCA were added. The mixture wasagain kept on ice bath for 10–15min and centrifugedat 3000rpm for 15min at 4 ◦ C. Two milliliters of super-natant was mixed with 2ml 0.4M Tris buffer (pH 8.9).Fifty microliters of DTNB (0.01M) was added to themixtureandvortexed.CelllysatefromeachRMAand/orDox treated animal was used to prepare a corresponding
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