Reversal of drug resistance in P-glycoprotein-expressing T-cell acute lymphoblastic CEM leukemia cells by copper N-(2-hydroxy acetophenone) glycinate and oxalyl bis (N-phenyl) hydroxamic acid

Reversal of drug resistance in P-glycoprotein-expressing T-cell acute lymphoblastic CEM leukemia cells by copper N-(2-hydroxy acetophenone) glycinate and oxalyl bis (N-phenyl) hydroxamic acid
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  Reversal of drug resistance in P-glycoprotein-expressing T-cell acutelymphoblastic CEM leukemia cells by copper  N  -(2-hydroxyacetophenone) glycinate and oxalyl bis (  N  -phenyl) hydroxamic acid S. Majumder a , P. Dutta a , P. Mukherjee b , E.R. Datta b ,T. Efferth c , S. Bhattacharya d , S.K. Choudhuri a, * a  Department of Environmental Carcinogenesis and Toxicology (ECT), Chittaranjan National Cancer Institute (CNCI),37, S.P. Mukherjee Road, Calcutta 700026, India b  Department of In Vitro Carcinogenesis and Cellular Chemotherapy (IVCCC), CNCI, Calcutta, India c German Cancer Research Center, Heidelberg, Germany d  Department of Chemistry, Banaras Hindu University, Varanasi, Uttar Pradesh, India Received 19 July 2005; received in revised form 7 October 2005; accepted 27 November 2005 Abstract Multiple drug resistance (MDR) represents a major obstacle to successful application of chemotherapy and a basic problem incancer biology. MDR occurs at the cellular level and is multi-factorial in nature. The multidrug resistance gene,  MDR1 , and itsgene product P-glycoprotein (P-gp) are now well known as an important determinant of MDR. Much effort has been devoted todevelop P-gp inhibitors to modulate resistance. However, most of these resistance-modifying agents (RMA) are too toxic at therequired doses. Therefore, the development of novel RMAs to overcome MDR represents a major challenge to modern cancerchemotherapy. In the present investigation, we describe the effect of oxalyl bis (  N  -phenyl) hydroxamic acid (OBPHA) and copper  N  -(2-hydroxy acetophenone) glycinate (CuNG) on multidrug-resistant P-gp-expressing CEM/ADR5000 T-cell acutelymphoblastic leukemia cells. CuNG, a known depleting agent for glutathione (GSH) and inhibitor of glutathione S-transferase(GST) and multidrug resistance-related protein 1 (MRP1), also inhibited P-gp-mediated doxorubicin accumulation and retention.The resistance-modifying effects of OBPHA were stronger than that of CuNG. Both novel RMAs overcame drug resistance moreefficiently than verapamil, a well-known P-gp inhibitor. OBPHA and CuNG exposure resulted in an increased doxorubicinaccumulation after 1–3 h incubation by down-regulation of P-gp expression after 24 h incubation. This is a clue that differentmechanisms may contribute to modulation of P-gp-mediated drug resistance by these compounds. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords:  Oxalyl bis (  N  -phenyl) hydroxamic acid (OBPHA); Copper  N  -(2-hydroxy acetophenone) glycinate (CuNG); Doxorubicin (Dox);Multiple drug resistance (MDR); ChemosensitivityCancer Letters 244 (2006) 16–$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.canlet.2005.11.030  Abbreviations  CuNG, copper  N  -(2-hydroxy acetophenone) glycinate; Dox, doxorubicin hydrochloride; EAC, Ehrlich ascites carcinoma; GSH,reduced glutathione; MDR, multiple drug resistance; MRP, multidrug resistance-related protein; NBT-BCIP, nitro blue tetrazolium chloride and5-bromo-4-chloro-3-indolylphosphate-  p -toluidine salt; OBPHA, oxalyl bis (  N  -phenyl) hydroxamic acid; P-gp, P-glycoprotein; RMA, resistancemodifying agent; SRB, sulforhodamine B; Vera, ( G ) verapamil hydrochloride. *  Corresponding author. E-mail address: (S.K. Choudhuri).  1. Introduction A major impediment to successful application of cancer chemotherapy is the occurrence of multiple drugresistance (MDR) in cancer patients. MDR is multi-factorial in nature and represents a basic problem of cancer biology. Studies on the molecular basis of MDRhave revealed that MDR-cells differ from drug-sensitive ones by (i) reduced accumulation of cytotoxicdrugs, due to decreased drug influx and/or increaseddrug efflux; (ii) altered expression and/or activity of certain intracellular detoxification proteins and (iii)physiological changes that alter the intracellular milieu[1]. Several proteins have found to be over-expressed inMDR human cancer cells including the multidrugresistance gene,  MDR1 , and its gene product P-glyco-protein (P-gp) [2], the multidrug resistance-relatedprotein 1 (MRP1) [3], or the breast cancer resistanceprotein (BCRP) [4]. Attempts have been made toincrease the activity of drugs, which are usuallyexported by ABC transporters, including doxorubicin(Dox), one of the most useful anticancer drugs [5,6]. Anumber of resistance modifying agents (RMAs)operating through P-gp have been developed so far[7,8]. Previously, we described a novel RMA, oxalylbis (  N  -phenyl) hydroxamic acid (OBPHA), whichsensitizes Dox-resistant Ehrlich ascites carcinoma(EAC/Dox) cells, to Dox in vitro [9]. OBPHA alsoinhibits P-gp in preneoplastic rat hepatocytes and HeLacells [10,11]. We had previously reported the chemicaland spectroscopic properties of OBPHA [12]. Slightvariations in the structure of OBPHA had no effect onP-gp inhibition [10]. However, the effect of OBPHAhas not been studied on drug-resistant human cancercells so far. In the present work, we describe the effectof OBPHA as RMA in multidrug-resistant P-gpexpressing T-cell acute lymphoblastic CEM/ ADR5000 leukemia cells. Another important RMA,copper  N  -(2-hydroxy acetophenone) glycinate (CuNG)that overcomes MDR in drug-resistant Ehrlich ascitescarcinoma (EAC/Dox) cells in vivo has also been testedin the present investigation. CuNG overcomes MDR bydepleting glutathione (GSH) and inhibiting glutathioneS-transferase (GST) and MRP1 [13,14]. Whether,CuNG has any effect on P-gp is not known so far.Therefore, the effect of CuNG on P-gp-expressing cellswas studied. Beside OBPHA, our aim was to decipherwhether CuNG might also inhibit P-gp. The resistance-modifying effects of these two RMAs were compared toverapamil (Vera), a well-known P-gp inhibitor, whichwas used as positive control. 2. Materials and methods 2.1. Cell culture The human T-cell acute lymphoblastic CCRF-CEM andCEM/ADR5000 leukemia cell lines were maintained in RPMImedium (GIBCO Invitrogen Corp., Carlsbad, California,USA) supplemented with 5% fetal bovine serum (FBS),additional glutamine (0.15%), HEPES (25 mM) and 50  m g/mlgentamycin. Cells were grown in plastic tissue culture flasks(Greiner Bio-One, Germany) in a 5% CO 2  atmosphere at37  8 C. Cells were passaged twice weekly. The doxorubicin-resistant CEM/ADR5000 cell line was generated by treatingCCRF-CEM cells with doxorubicin doses up to a finalconcentration of 5000 ng/ml doxorubicin [15]. The CEM/ ADR5000 cells selectively express the MDR1/P-gp, but notother MDR-mediating transporters such as MRP1 and BCRP[16]. Dr Axel Sauerbrey, University of Jena, Germany, kindlyprovided both CCRF-CEM and CEM/ADR5000 cell lines.Cells from exponentially growing cultures were used for allexperiments. All experiments were repeated three times. 2.2. Resistance modifying agents (RMAs) and drugs Oxalyl bis (  N  -phenyl) hydroxamic acid (OBPHA) wassynthesized and characterized by the method describedpreviously [12]. As OBPHA is not soluble in water, DMSO was used at non-toxic concentration.Copper  N  -(2-hydroxy acetophenone) glycinate (CuNG)was synthesized by the reaction of potassium  N  -(2-hydroxyacetophenone) glycinate with copper sulfate according to themethod described previously [13]. As CuNG is not totally soluble in water, DMSO was used at non-toxic concentration.The structures of CuNG and OBPHA were simulated by forcefield calculations. Studies have proven that such computationcan be successfully employed to transition metal complexes[14]. Molecular geometry optimization was carried out usinga modified version of Allinger’s MM2 force field [17] formolecular mechanical calculations.Doxorubicin hydrochloride (Dox) and ( G ) verapamilhydrochloride (Vera) were purchased from Sigma ChemicalCompany, St Louis, Missouri, USA. Stock solutions of CuNG, OBPHA, Vera and Dox were initially prepared indimethyl sulphoxide (DMSO), and final concentrations (10 K 4 to 10 K 8 M) were prepared by diluting drug stocks in completegrowth media. In all cases, control experiments were carriedout in medium with 0.25% DMSO. 2.3. Sulforhodamine B assay for cellular growthinhibition study To assess the role of RMAs on reversal of Dox-resistancein CEM/ADR5000 cells, the sulforhodamine B assay (SRB)was performed with minor modification as described byMonk et al. [18]. The Dox-sensitive, parental cell line, CCRF-CEM, was used as control. For SRB assays, CCRF-CEM and S. Majumderet al. / Cancer Letters 244 (2006) 16–23  17  CEM/ADR5000 cells (40,000 cells/well) were seeded in 96-well microtiter plates (Nalge Nunc International Corp.,Naperville, USA). After 24 h of cell plating, CEM/ ADR5000 cells were exposed to different RMAs, e.g.CuNG (10 K 5 M), OBPHA (10 K  4 M) and Vera (10 K 4 M) intwo separate plates. After 2 h, Dox (10 K 6 M) was added toone of the two plates treated with different RMAs and theother plate was remained as RMA control. Simultaneously,CCRF-CEM and CEM/ADR5000 cells were treated with Doxalone as control. After 48 h of drug exposure, cells were fixedto the bottom by cold 80% (wt/vol) trichloro acetic acid(TCA) (final concentration, 16% TCA) and incubated for60 min at 4  8 C. The fixative was then discarded; plates werewashed with deionized water and air-dried. Fixed cells werethen stained with SRB (0.4% SRB wt/vol in 1% acetic acid)using 100  m l/well followed by 10 min incubation at roomtemperature. Unbound SRB was removed by washing with1% acetic acid and air-dried. Bound stains were solubilizedwith10 mMTris baseand optical density (OD)ofall the wellswere read on an automated spectrophotometric plate reader ata single wavelength of 515 nm. Using these OD values,cellular viability and cellular growth inhibition in thepresence of test materials were calculated using the formula100 ! [( T  K T   Z  )/( C  K T   Z  )] where  T   Z  , time zero value of SRBprotein content at the beginning of drug incubation;  T  , a set of drug treated test values at the end of 48 h drug incubationperiod; and C, a control value (vehicle control) at the end of 48 h drug incubation period.IC 50  values (i.e. 50% inhibition concentration) of Doxseparately and in combination on both CCRF-CEM andCEM/ADR5000 were determinedfrom dose–response curves.The resistance index (RI) represents a measure for the degreeof resistance. RI values were determined by dividing the IC 50 value for drug-resistant CEM/ADR5000 cells by the IC 50 value for drug-sensitive CCRF-CEM cells. RI values of RMAs were not calculated, as they are not drugs. 2.4. Determination of dox penetration/retention inCCRF-CEM and CEM/ADR5000 cell lines CCRF-CEM and CEM/ADR5000 cells (5 ! 10 6 cells)were seeded in four separate 35 mm petri dishes (Nalge NuncInternational Corp., Naperville, USA) with 1.5 ml completegrowth media. After 24 h, three petri dishes withCEM/ADR5000 cells were incubated with CuNG (10 K 5 M),OBPHA (10 K 4 M) and Vera (10 K 4 M), respectively. Twohours after addition of RMAs, Dox (10 K 6 M) was adminis-tered to RMA-treated CEM/ADR5000 cell-bearing petridishes as well as to CCRF-CEM and CEM/ADR5000 cell-bearing petri dishes. 10 6 cells were then collected at differenttime points (1st, 2nd and 3rd h) in chilled PBS at dark andwashed thrice with cold PBS. Collected cell samples werethen analyzed using a FACSCalibur and processed by Cellquest software (Becton Dickinson) for determination of Doxpenetration [19]. The excitation and emission wavelengths were 495 and 590 nm, respectively. 2.5. Immunoblotting for P-gp CCRF-CEMandCEM/ADR5000cells(5 ! 10 6 cells)wereseededinafiveseparate35 mmpetridishes,respectively,with1.5 ml complete growth media. After 24 h, three petri disheswith CEM/ADR5000 cells were incubated with CuNG(10 K 5 M), OBPHA (10 K 4 M) and Vera (10 K 4 M), respec-tively, for 24 h. Cells were then harvested and collected bycentrifugation at 1000 rpm for 5 min. Cells were resuspendedin 125 mM Tris–HCl buffer and lysed by adding an equalvolumeof4%SDStomakeafinalconcentrationof2%SDSinsolution. Cell extracts were boiled for 10 min, chilled on iceand centrifuged at 13,000 rpm for 10 min at 4  8 C beforecollecting the supernatant [20]. The protein content of thesupernatants was quantified following the method of Lowryetal.[21].Sixtymicrograms ofproteinswerethenloadedonto8% SDS-PAGE pre-cast gel (Biotech, Calcutta, India).Proteins were wet-transferred electrophoretically to PVDFmembrane(0.1  m mporesize)at20 mAfor14 h.Blockingwasdoneat 37  8 Cfor1 h with3%BSA inTris buffer saline (TBS).Then the membrane was washed with PBS followed bypolyclonal primary antibody (mdr (Ab-1), Polyclonal RabbitIgG, Oncogene Science, San Diego, CA, USA) reaction in a1:500 dilution or  b -actin (1:2000 dilution, Sigma ChemicalCo., St Louis, USA) for 3 h with continuous shaking.Membrane was washed thrice for 10 min each in buffer(0.1% Tween-20 in TBS) followed by secondary antibody(Goat Anti-Rabbit IgG-AP) reaction in a 1:1000 dilution for1 h at the same temperature. Again the membrane was washedtwice with Tween-TBS followed by washing thrice in TBS.Immunoreactions were detected by NBT/BCIP (nitro bluetetrazolium chloride and 5-bromo-4-chloro-3-indolylpho-sphate-  p -toluidine salt) substrate. Densitometric analysiswas done with the help of Bio Rad Gel Doc. 3. Results 3.1. Chemical structures Both CuNG and OBPHA were synthesized andcharacterized by us on the basis of UV, IR,  1 NMRspectraldata[12,13].2Dand3Dstructures(Hatoms arenot shown) of CuNG and OBPHA are shown in Figs. 1and 2, respectively. The space-filling model of CuNGreveals the twisted shape of the monomer molecule; inOBPHA the aromatic rings are not in planar positions. Fig. 1. Chemical structure of CuNG. (a) 2D structure and (b) 3Dstructure. S. Majumderet al. / Cancer Letters 244 (2006) 16–23 18  3.2. Reversal of doxorubicin resistance CCRF-CEM and multidrug-resistantCEM/ADR5000 cells were treated either with Doxalone, resistance-modifying agents alone (OBPHA,CuNG, or Vera) and combinations of Dox C OBPHA,Dox C CuNG, or Dox C Vera. As shown in Fig. 3, thecombination of Dox C OBPHA and Dox C CuNGinhibitedthegrowthofCEM/ADR5000moreefficientlythan Dox C Vera with similar efficacy as Dox alone didin sensitive CCRF-CEM. For comparison, the dose–response curves of CCRF-CEM for Dox alone andDox C OBPHA, Dox C CuNG, and Dox C Vera weresimilar, indicating that the three RMAs had no or only aminimal effect on the effect of Dox in sensitive CCRF-CEM cells.Resistance index (RI) values decreased from 400 to21.79 in Vera C Dox treated cells, to 11.33 in CuNG C Dox treated cells, and to 5.27 in OBPHA C Dox treatedcells as compared to treatment with Dox alone(Table 1). Low RI values indicate stronger modulatoryeffects of Dox resistance in CEM/ADR5000 cells thanhigher RI values. Hence, OBPHA overcame Doxresistance more efficiently than CuNG, whereas theeffects of the control drug, Vera, were lowest in thispanel of RMAs. The high IC 50  values of OBPHAindicate that this compound is non-toxic. The growthinhibitory effects of combination treatment withVera C Dox, OBPHA C Dox, or CuNG C Dox in CEM/ ADR5000 cells are shown in Fig. 4. As determined by SRB assay after 48 h of Dox exposure, CEM/ADR5000cells showed only 20.19% growth inhibition. When theabove cells were exposed to the same drug incombination with OBPHA, CuNG, and Vera thepercentage of growth inhibition increased to 84.28,36.17 and 65.71%, respectively. These results showthat treatment of CEM/ADR5000 cells with Vera C Dox increased the level of growth inhibition by 3.26-fold compared to treatment with Dox alone. 4.17 and1.79-fold increased growth inhibition rates wereobserved after treatment with OBPHA C Dox andCuNG C Dox, respectively, as compared to applicationof Dox alone. The percentage of growth inhibition Fig. 2. Chemical structure of OBPHA. (a) 2D structure and (b) 3Dstructure. 0.1110100100010000100000100%90%70%50%30%10%Cellular growth (% of Control)    R   M   A  a  n   d   D  o  x  c  o  n  c  e  n   t  r  a   t   i  o  n   (  µ   M   ) CCRF-CEM + DoxCEM/ADR5000 +DoxCCRF-CEM + CuNGCCRF-CEM + CuNG + DoxCEM/ADR5000 +CuNGCEM/ADR5000 +CuNG +DoxCCRF-CEM + VeraCCRF-CEM + Vera+ DoxCEM/ADR5000 +VeraCEM/ADR5000+ Vera +DoxCCRF-CEM + OBPHACCRF-CEM + OBPHA +DoxCEM/ADR5000 +OBPHACEM/ADR5000 +OBPHA +Dox Fig. 3. Dose–response curve of different RMAs and Dox on CCRF-CEM and CEM/ADR5000 cell line. S. Majumderet al. / Cancer Letters 244 (2006) 16–23  19  (84.28%) in drug-resistant CEM/ADR5000 cellsinduced by treatment with OBPHA C Dox was onlyslightly lower than the growth inhibition (91.88%)induced only by Dox alone in drug-sensitive CCRF-CEM cells. 3.3. Doxorubicin accumulation and retention After incubation for 1 h, OBPHA enhanced Doxaccumulation in CEM/ADR5000 cells more thanCuNG or Vera (Fig. 5). The extent of Dox accumu-lation in OBPHA-treated CEM/ADR5000 cells wascomparable to that in CCRF-CEM cells. Furtherincubation for 2 and 3 h resulted in a decrease of intracellular Dox accumulation, which was slower inOBPHA-treated CEM/ADR cells than in those treatedwith CuNG or Vera. This resulted in a higher retentionof Dox after 3 h in OBPHA-treated CEM/ADR5000cells, which was similar to Dox retention observed inparental CCRF-CEM cells. Vera increased Doxaccumulation in CEM/ADR5000 cells by 1.42-fold,whereas the effect of OBPHA was much stronger (2.08-fold increase of Dox accumulation). CuNG treatmentled to a 1.08-fold increase of Dox accumulationin CEM/ADR5000 cells. Dox accumulation andretention (after 1st, 2nd and 3rd h) was highest inDox-treated CCRF-CEM cells. 3.4. Down-regulation of P-gp expression Detection of protein expression by immunoblottingrevealed a band at 170 kDa specific for P-gp. P-gp isoverexpressed in CEM/ADR5000 cells (4.95-fold)compared to CCRF-CEM cells (Fig. 6). CuNG,OBPHA, and Vera down-regulated the expression of P-gp after 24 h of administration. OBPHA down-regulated P-gp with highest efficacy among the threeRMAs tested (1.69-foldexpression compared to CCRF-CEM). The expression level in CEM/ADR5000 cellsafter treatment with CuNG and Vera were 4.15 and2.89-fold compared to CCRF-CEM cells, respectively. 4. Discussion In the present investigation, we found an increase of Dox accumulation and retention by OBPHA, CuNG, orVera in multidrug-resistant P-gp-expressingCEM/ADR5000 cells. We have synthesized the twonovel compounds viz, OBPHA [12] and CuNG [13]; Table 1Doxorubicin sensitivity of CCRF-CEM and CEM/ADR5000 cellsDrugs IC 50  ( ! 10 K 7 M) RI a CCRF-CEM CEM/ADR5000Dox 2.5 G 0.15 1000 G 89 400CuNG 350 G 25.6 390 G 23.81 –CuNG b C dox 2.49 G 0.11 28.2 G 0.13 11.33Vera 380 G 31.4 375 G 29.41 –Vera b C dox 2.35 G 0.13 51.2 G 4.97 21.79OBPHA 520 G 49.5 480 G 30.56 –OBPHA b C dox 2.56 G 0.21 13.5 G 1.14 5.27All the data are representative of three similar experiments. Standarddeviation (SD) was usually within 10% of the mean. Values representmean G SD ( P ! 0.05). a Resistance index (RI) (for details see Section 2). b The IC 50  values after combination treatment with RMA C Doxwere only due to Dox, not for RMAs; as the RMAs used at non-toxicdoses (determined from dose–response curves; Fig. 3). 0102030405060708090100CCRF-CEM + DoxCEM/ADR5000 + DoxCEM/ADR5000 + VeraCEM/ADR5000 + Vera + DoxCEM/ADR5000 + OBPHACEM/ADR5000 + OBPHA + DoxCEM/ADR5000 + CuNGCEM/ADR5000 +CuNG + Dox % Growth inhibition Fig. 4. Percentage of growth inhibition (48 h) after different combination treatments with RMA and Dox in CCRF-CEM and CEM/ADR5000 cells.Values represent mean G SD ( P ! 0.05). The figure is representative of three similar experiments. S. Majumderet al. / Cancer Letters 244 (2006) 16–23 20
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