Overcoming Drug-Resistant Cancer by a Newly Developed Copper Chelate through Host-Protective Cytokine-Mediated Apoptosis

Overcoming Drug-Resistant Cancer by a Newly Developed Copper Chelate through Host-Protective Cytokine-Mediated Apoptosis
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  Overcoming Drug-Resistant Cancerbya Newly Developed CopperChelate through Host-Protective Cytokine-Mediated Apoptosis AnandaMookerjee, 1 JayatiMookerjeeBasu, 6 PranabanandaDutta, 1 SurajitMajumder, 1 SankarBhattacharyya, 7 JaydipBiswas, 2 SmarajitPal, 2 PratimaMukherjee, 4 SanghamitraRaha, 8 RathindraN.Baral, 5 TaniaDas, 7 ThomasEfferth, 9 GourisankarSa, 7 ShyamalRoy, 6 andSoumitraK.Choudhuri 1 Abstract  Purpose:  Previously, we have synthesized and characterized a novel Cu(II) complex, copper N  -(2-hydroxyacetophenone)glycinate(CuNG).Herein,wehavedeterminedtheefficacyofCuNGinovercomingmultidrug-resistantcancerusingdrug-resistantmurineandhumancancercelllines. Experimental Design:  Action of CuNG following single i.m. administration (5 mg/kg bodyweight) was tested  in vivo  on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)^ bearingmiceanddoxorubicin-resistant sarcoma180^bearingmice.Tumor size, asciticload, andsurvival rates were monitored at regular intervals. Apoptosis of cancer cells was determinedby cell cycle analysis, confocal microscopy, AnnexinV binding, and terminal deoxynucleotidyltransferase^mediated dUTP nick endlabeling assay  ex vivo . IFN- g  and tumor necrosis factor- a were assayed in the culture supernatants of  in vivo  and  in vitro  CuNG-treated splenic mono-nuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Sourceof IFN- g  and changes in number ofTregulatory marker-bearing cells in the tumor site followingCuNG treatment were investigated by flow cytometry. Supernatants of  in vitro  CuNG-treatedcultures of peripheral blood mononuclear cells from different drug-insensitive cancer patientswere tested for presence of the apoptogenic cytokine IFN- g  andits involvement ininduction ofapoptosisofdoxorubicin-resistantCEM/ADR5000cells. Results: CuNGtreatmentcouldresolvedrug-resistantcancersthroughinductionofapoptogeniccytokines, such as IFN- g  and/or tumor necrosis factor- a , from splenic mononuclear cells orpatientperipheralbloodmononuclearcellsandreducethenumberofTregulatorymarker-bearingcellswhileincreaseinfiltrationofIFN- g -producingTcellsintheascetictumor site. Conclusion:  Our results show the potential usefulness of CuNG in immunotherapy of drug-resistantcancersirrespectiveofmultidrugresistancephenotype. M ultidrug resistance (MDR) is a complex phenomenon wheremalignant cells manifest resistance toward a range of unrelatedchemotherapeutic agents and is a major obstacle to thesuccessful pharmacologic treatment of cancers (1, 2). Many different mechanisms have been suggested to explain thedevelopment of MDR phenotype in cancer cells, such as achange in the specific target of a drug (1), the reduced uptake or increased efflux of a drug (3, 4), a differential compartmental-ization (5, 6), an increased rate of detoxification of a drug (4, 7), an increased ability to repair DNA damage (4, 8), geneamplification (9, 10), and an increase in the activity of survivalproteins and reduced capacity to enter apoptosis (11, 12). Insome cases of MDR, one or more of these mechanisms may act at a time. In cancer cell models, the most studied of thesemechanisms has been the overexpression of several energy-dependent drug efflux pumps belonging to the ATP-binding cassette family of transporters, such as the P-glycoprotein,MDR-associated proteins, and breast cancer resistance protein/ ABCG2 (13–15). Overexpression of these integral membraneproteins causes cancer cells to become resistant to a variety of drugs, such as anthracyclines,  Vinca  alkaloids, taxanes, epipo-dophyllotoxins, etc. (1, 2, 16–18).Doxorubicin is the most commonly used drug in the therapy for solid tumors, many ascitic tumors, and some leukemia.Involvement of ATP-binding cassette transporters and differen-tial compartmentalization of drugs have been reported to causeresistance to doxorubicin and other drugs in different cell lines Cancer Therapy: Preclinical  Authors’Affiliations:  Departments of  1 Environmental Carcinogenesis andToxicology,  2 Surgical Oncology and Medical Oncology, Hospital Unit,  3 ClinicalBiochemistry, Hospital Unit,  4 In vitro Carcinogenesis and Cellular Chemotherapy,and  5 Immunoregulation and Immunodiagnostics, Chittaranjan National CancerInstitute;  6 Department of Immunology, Indian Institute of Chemical Biology; 7 DepartmentofAnimalPhysiology,BoseInstitute; 8 DepartmentofCrystallographyand Molecular Biology, Saha Institute of Nuclear Physics, Calcutta, India and 9 GermanCancerResearchCenter,Heidelberg,GermanyReceived1/18/06;revised3/19/06;accepted4/12/06. Grant support:  Indian Council of Medical Research, New Delhi, grants 5/13/17/2001-NCD-IIIand5/13/18/2004-NCD-III.Thecostsofpublicationof thisarticleweredefrayedinpartby thepaymentofpagecharges.This article must thereforebehereby marked  advertisement   inaccordancewith18U.S.C.Section1734solely toindicatethisfact. Requests for reprints:  Soumitra K. Choudhuri, Department of EnvironmentalCarcinogenesis andToxicology, Chittaranjan National Cancer Institute, 37, S.P.Mukherjee Road, Calcutta 700 026, India. Phone: 91-33-2476-5101/02/04, ext.317;Fax: 91-33-2475-7606;E-mail:soumitra01 @  F  2006AmericanAssociationforCancerResearch.doi:10.1158/1078-0432.CCR-06-0001 Clin Cancer Res 2006;12(14) July15, 20064339  (5, 19, 20). Therefore, to overcome doxorubicin resistanceefficacy of glutathione, depletors (21) and inhibitors (22, 23)of efflux pumps were studied as resistance-modifying agents for induction of apoptosis of resistant cells with doxorubicin.Because no resistance-modifying agent that has been highly successful clinically has emerged thus far, recently, immuno-modulators and cytokines are being tested  in vivo  and  in vitro against various drug-resistant cancers (24–28).Earlier, we have synthesized a novel Schiff’s base chelate of Cu(II), copper   N  -(2-hydroxy acetophenone) glycinate (CuNG)and studied its chemical nature as well as its toxicity (29). Later, we have shown that i.p. administration of CuNG at a dose of 10 mg/kg body weight in doxorubicin-resistant Ehrlich ascitescarcinoma (EAC/Dox)–bearing mice could reverse doxorubicinresistance and allowed doxorubicin to induce apoptosis  in vivo and  in vitro  (30). Interestingly, single i.m. administration of CuNG alone at a lower dose (5 mg/kg body weight) disclosedthat CuNG possessed immunomodulatory activity. Herein, wereport that CuNG alone could resolve doxorubicin-resistant cancers through induction of host protective cytokines, such asIFN- g  and tumor necrosis factor- a  (TNF- a ), which are reportedto have anticancer properties (27, 31). Moreover, CuNG wasfound to induce peripheral blood mononuclear cells (PBMC)from different drug-insensitive and radiation-insensitivepatients to secrete protective cytokines that caused apoptosisof the doxorubicin-resistant human T lymphoblastic leukemiacell line, CEM/ADR5000. Because no highly effective resistance-modifying agent is available clinically, this immunomodulator holds immense promise for treatment of drug-resistant cancer. Materials and Methods  Materials.  TNF- a -neutralizing monoclonal antibodies, IFN- g -neu-tralizing monoclonal antibodies (murine and human), murine recom-binant IFN- g  (rIFN- g ), murine recombinant TNF- a  (rTNF- a ), Opt EIA kits for assay of murine and human IFN- g  and TNF- a , anti-CD4peridinin-chlorophyll-protein complex–conjugated monoclonal anti-body, anti-CD8 phycoerythrin-conjugated monoclonal antibody, anti-IFN- g  FITC-conjugated antibody, and anti-CD25 FITC-conjugatedantibody were obtained from BD Biosciences (San Diego, CA). Anti-Foxp3 phycoerythrin-conjugated monoclonal antibody (murine) wasobtained from eBioscience (San Diego, CA). Penicillin, streptomycin,RPMI 1640, trypan blue, propidium iodide (PI), brefeldin A, concanav-alin A, phorbol 12-myristate 13-acetate (PMA), and ionomycin wereobtained from Sigma (St. Louis, MO). All radioactive chemicals werepurchased from New England Nucleotide (Boston, MA) unless other- wise mentioned. Annexin V-FITC and Apo-Direct kit were procuredfrom Becton Dickinson immunocytometry system (San Jose, CA).  Animals and cell lines.  Swiss albino mice, srcinally obtained fromNational Institute of Nutrition (Hyderabad, India) and reared in theinstitute animal facilities, were used for experimental purposes withprior approval of the institutional animal ethics committee. EAC/Dox, which is also resistant against cisplatin, cyclophosphamide, and vinblastine (32), and doxorubicin-resistant sarcoma 180 (S180/Dox) were developed and maintained according to the methods describedpreviously (32). Doxorubicin-resistant human acute T lymphoblasticleukemia cell line CEM/ADR5000 (33), derived from the parentalCCRF-CEM cell line (34), was provided by T. Efferth. This slow-growing cell line displayed >800-fold resistance to doxorubicin and overex-pressed ABCB1/MDR1 (35).  Peripheral blood samples of patients.  Leftover excesses of blooddrawn for routine examinations of terminal cancer patients insensitiveto various chemotherapeutics as well as toward radiation therapy insome cases (certified by the Department of Surgical Oncology andMedical Oncology, Hospital Unit, Chittaranjan National Cancer Institute) were collected as samples from the Department of ClinicalBiochemistry, Hospital Unit, Chittaranjan National Cancer Institute. The patient profile in brief is presented in Table 1. Treatments.  EAC/Dox-bearing mice were either kept untreated or treated with a single dose of CuNG (5 mg/kg body weight) 7 daysfollowing peritoneal inoculation with 1    10 6 EAC/Dox cells derivedfrom EAC/Dox-bearing mice treated with doxorubicin (48 hours beforeacquisition of cells). S180/Dox cells were maintained in peritonealcavity of doxorubicin-treated mice. These mice were again treated withdoxorubicin 48 hours before acquisition of cells from their peritonealcavity for inoculation. Animals inoculated with 5    10 6 S180/Dox cells in right hind leg were either kept untreated or treated with CuNG(i.m., single administration) 5 mg/kg body weight in left hind leg after 15 days.In some experiments, EAC/Dox cells were cultured in the presence of 2.5  A g/mL CuNG or 100 units/mL rIFN- g  (36) or 20 ng/mL rTNF- a (37) or nonadherent population of splenic mononuclear cells (SPMC)from CuNG-treated EAC/Dox-bearing mice (2    10 6 EAC/Dox cells with 1  10 5 nonadherent SPMC in 1 mL). In some other experiments,EAC/Dox cells were cultured in the presence of SPMC culturesupernatants derived from CuNG-treated EAC/Dox-bearing mice(100  A L/mL) and/or neutralizing anti-IFN- g  (10  A g/mL) and/or anti- TNF- a  (10  A g/mL). In some experiments, CEM/ADR5000 cells werecultured in the presence of culture supernatants (200  A L/mL) of   in vitro CuNG-treated (or untreated) PBMC derived from patients and in thepresence or absence of neutralizing anti-IFN- g  (10  A g/mL). Isolation of EAC/Dox cells from peritoneal cavity of mice.  The EAC/Dox cells were isolated from the peritoneal cavity of EAC/Dox-bearing mice (control or treated). Sterile PBS (2-3 mL) was injected into theperitoneal cavity of the mice and the peritoneal fluid containing thetumor cells was withdrawn, collected in sterile Petri dishes, and Table1.  Resistance profile of cancer patients from whom PBMC were isolated Patientno.Typeofcancer Insensitive to 1 Prostatecancer,prostate(prostate-specificantigen:879.45ng/mL;osteoblasticmetastasisinpelvis)Chemotherapy(cyclophosphamide)andantiandrogentherapy2 Breastcancer(metastatic) Chemotherapy(5-fluorouracil+doxorubicin+cyclophosphamide)3 Rectalcancer(metastatic) Chemotherapy(5-fluorouracil)andradiation4 Breastcancer(metastatic) Chemotherapy(5-fluorouracil+cyclophosphamide)andradiation5 Nasopharyngealcancer(metastatic) Chemotherapy(cisplatin+5-fluorouracil)andradiation6 Seminomaof testis(metastasistoothertissues) Chemotherapy(cisplatin+etoposide)7 Breastcancer(metastatic) Chemotherapy(5-fluorouracil+doxorubicin+cyclophosphamide) Cancer Therapy: Preclinical  www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 2006 4340  incubated at 37 j C for 2 hours. The cells of macrophage lineage adheredto the bottom of the Petri dishes. The nonadherent population wasaspirated out gently and washed repeatedly with PBS. EAC/Dox cells were then separated from other nonadherent contaminating cells by fluorescence-activated cell sorting. More than 98% of this separated cellpopulation was CD3 (T cell)/CD14 (macrophage)/CD19 (B cell)/CD56(natural killer cell) negative as was determined by flow cytometer.Moreover, these cells were morphologically characterized as EAC by  Wright staining (38) and viability was assessed to be >95% by trypanblue dye exclusion. The viable EAC/Dox cells were processed for further experiments.  Derivation of mononuclear cells from spleen and lymph node of miceand blood of patients.  Mice [normal or EAC/Dox-bearing (untreated or CuNG treated)] were euthanized, and their spleen and lymph nodes(axillary, inguinal, and cervical) were removed. Spleens were homog-enized separately in ice-cold RPMI 1640. Heparinized peripheral bloodof patients was taken and diluted with equal volume of RPMI 1640.Lymphocyte-enriched mononuclear cells were isolated by Histopaque1077 (Sigma) density gradient centrifugation of murine spleen cellsuspension and diluted blood samples of patients, washed, and finally resuspended in cold RPMI 1640 supplemented with 15% heat-inactivated fetal bovine serum (RPMI-FBS). Lymph nodes from mice were teased over no. 80 steel screen (Sigma) to obtain lymph node cellsuspension in RPMI-FBS (39). Cell viability (>95%) was checked by thetrypan blue dye exclusion method. For certain experiments, the SPMCsuspension, thus obtained, was kept in 35-mm-diameter plastic tissueculture plates for   f 4 hours at 37 j C under a 5% CO 2 -95% air atmosphere to allow attachment of adherent cells. Nonadherent cells(95% lymphocytes) were subsequently removed by aspiration,harvested by centrifugation, and resuspended in RPMI-FBS.  Preparation of SPMC culture supernatant.  SPMC (4    10 6 ) fromEAC/Dox-bearing mice either untreated or treated with CuNG  in vivo  were cultured in RPMI-FBS for 24 or 60 hours. In some cases, 4    10 6 SPMC from CuNG untreated EAC/Dox-bearing mice were cultured inthe presence of 2.5  A g/mL CuNG in RPMI-FBS for 24 or 60 hours.Supernatants were collected by centrifugation at 500    g  .  Preparation of PBMC culture supernatant.  PBMC (4    10 6 ) fromeach patient were either kept untreated or treated  in vitro  with 1  A g/mL CuNG and maintained in RPMI-FBS for 48 hours. Supernatants werecollected by centrifugation at 500    g  .  Lymphocyte proliferation assay.  Lymphocyte proliferation experi-ments were carried out   in vitro  in 96-well tissue culture plates, each wellof which contained 2    10 5 cells in 200  A L culture. Cells werestimulated with concanavalin A (2.5  A g/mL) or a combination of PMA (20 ng/mL) and ionomycin (500 ng/mL) for 48 hours at 37 j C under 5% CO 2 -95% air. Unstimulated (control) cultures did not receive any concanavalin A or PMA plus ionomycin. Next, cell suspensions werepulsed with [ 3 H]thymidine (0.5  A Ci/well) for another 20 hours. Cells were harvested on glass fiber filter papers (Whatman, Maidstone,United Kingdom) by using a cell harvester (Nunc, Roskilde, Denmark),and incorporation of [ 3 H]thymidine was measured by a liquidscintillation counter (Wallac 1409, Gaithersburg, MD; ref. 39).  Detectionofapoptosisbyflow cytometry.  Forthedeterminationofcellcycle phase distribution, EAC/Dox cells harvested from tumor-bearing mice or CEM/ADR5000 cells were permeabilized and nuclear DNA waslabeled with PI. Cell cycle phase distribution of nuclear DNA wasdetermined on fluorescence-activated cell sorting, fluorescence detector equipped with 488 nm argon laser light source and 623 nm band passfilter (linear scale) using CellQuest software (Becton Dickinson). A totalof 10,000 events were acquired and analysis of flow cytometric data wasdone using ModFit software. A histogram of DNA content (  X   axis, PIfluorescence) versus counts ( Y   axis) has been displayed. To distinguish between apoptosis and necrosis, in a double-labeling system, EAC/Dox cells (1  10 6 in each case) from untreated or CuNG-treated EAC/Dox-bearing mice were harvested and PI and Annexin V-Fluos were added directly to the medium. The mixture was incubatedfor 15 minutes at 37 j C. Excess PI and Annexin V-Fluos were then washed off, and cells were fixed and then analyzed on flow cytometer (equipped with 488 nm argon laser light source; 515 nm band passfilter for FITC fluorescence and 623 nm band pass filter for PIfluorescence) using CellQuest software. Electronic compensation of theinstrument was done to exclude overlapping of the emission spectra. A total of 10,000 events were acquired and the cells were properly gatedfor analysis. By this technique, we could distinguish between apoptoticand necrotic cells. Unfixed apoptotic cells are impermeable to PI, but  Annexin V binds specifically to phosphatidylserine that is translocatedto the outer leaflet of the membrane of apoptotic cells, whereas necroticcells are permeable to both the fluorochromes. To confirm the nature killing of EAC/Dox by CuNG treatment, EAC/Dox cells were fixed, permeabilized, and incubated with terminaldeoxynucleotidyl transferase enzyme and FITC-Br-dUTP. Cells were washed, incubated with PI/RNase solution, and analyzed on fluores-cence-activated cell sorting. Electronic compensation of the instrument  was done to exclude overlapping of the emission spectra. A dot plot of PI fluorescence (  X   axis) versus FITC fluorescence ( Y   axis) has beendisplayed (40). Oligonucleosomal fragmentation.  For the assessment of chromatincondensation and nuclear blebbing, EAC/Dox and CEM/ADR5000 cells were fixed and nuclear DNA was stained with PI (10  A g/mL) for 15 minutes at room temperature. A Leica model DM 900 (Wetzlar,Germany) fluorescent microscope was used to visualize apoptotic cells.Digital images were captured with cool (  25 j C) CCD cameracontrolled with MetaMorph software (Universal Imaging, Downing-town, PA; ref. 40).  Detection of infiltration of CD4 + , CD8 + , and T regulatory cells andintracellular IFN- g   by flow cytometry.  Ascitic fluids from five untreatedand five  in vivo  CuNG-treated (15 days after treatment) EAC/Dox-bearing mice were drawn. Ascitic fluids from mice of each group werepooled and centrifuged at 100    g   for 5 minutes and supernatants were collected. Supernatants were then centrifuged at 400    g   for 10 minutes. Each pellet was resuspended in 5 mL RPMI-FCS, plated inFCS precoated tissue culture Petri dish, and incubated at 37 j C in 5%CO 2 -95% air for 3 hours for adherence. Nonadherent cells werecollected, washed twice with HBSS, and finally resuspended in 5 mL  Fig.1.  Effectof invivo  CuNG treatmentonsurvivalandtumorloadof mice.TreatmentofEAC/Dox-bearingmicewithi.m. administrationof CuNG (5 mg/kgbody weight) 7 daysfollowinginoculation (day0) increases survivalbeyond180 days( > 70% miceof this group survivedover15months), whereasuntreatedmice didnot survivebeyond45days.Experiment was startedwith120 miceineachgroup. Anotherexperimentshowedthat aboosterdoseof 0.5mgCuNG/kgbody weight (i.m.) 15 days afterinitialtreatment with 5 mgCuNG/kgbody weight(i.m.) furtherincreasedthe survivalrates(datanot shown). ResolutionofDrug-ResistantCancerbyCopperChelate Clin Cancer Res 2006;12(14) July15, 20064341  HBSS. This was then divided into two equal parts. One part wasincubated with brefeldin A. Cells of these parts were incubated withanti-CD4 peridinin-chlorophyll-protein complex–conjugated mono-clonal antibody and anti-CD8 phycoerythrin-conjugated monoclonalantibody for 45 minutes following blocking with 2.5% (v/v) normalmouse serum. Cells were next washed, fixed with 4% paraformaldehydefor 30 minutes, and then washed with 0.1% saponin in FACScan buffer (0.2% bovine serum albumin, 0.02% NaN 3  in PBS). Cells were thenincubated with anti-IFN- g  FITC-conjugated antibody or isotype controlmonoclonal antibodies. Cells were resuspended in FACScan buffer andused for flow cytometry. Cells from another part were incubated withanti-CD4 peridinin-chlorophyll-protein complex–conjugated mono-clonal antibody and anti-CD25 FITC-conjugated antibody following blocking with 2.5% (v/v) normal mouse serum. Next, cells were washed, fixed with 4% paraformaldehyde, and then washed with 0.1%saponin in FACScan buffer as before. Cells were then incubated withanti-Foxp3 phycoerythrin-conjugated monoclonal antibody or isotypecontrol monoclonal antibodies. Cells were resuspended in FACScanbuffer and used for flow cytometry as before. Cytotoxicity assay.  Cytotoxicity was measured in terms of   51 Cr released (41). Target cells (1  10 6 ) were labeled with 100  A Ci Na 2 CrO 4 for 1 hour at 37 j C in 5% CO 2  incubator and washed several times untilno  g -irradiation count was detected in the supernatant. Nonadherent splenocytes from different experimental groups were incubated with Table 2.  Decrease of ascites load and solid tumor size following CuNG treatment Group EAC/Dox S180/DoxCellcount (  10 6 ) Asciticfluidvolume (mL) Tumor size (mm 3 ) Secondary tumor Untreated 2,250 F 300 15 F 2 105 F 5 +++CuNGtreated 4 F 0.1 0.5 F 0.05 4.2 F 1.5   NOTE:Volumes (in mL) indicate the amounts ofascitic fluidin the peritoneal cavity. CuNG treatment started 7 days following inoculation with EAC/Dox and15 daysfollowinginoculationwithS180/Dox.Asciticloadwasstudied21daysfollowingtreatment,whereasi.m.tumorwithS180/Doxwasstudied45daysfollowingtreatment.MediansurvivalofuntreatedS180/Dox-bearingmicewas75 F 5,whereasnomortalitywasrecordedevenat120daysaftertreatmentinCuNG-treatedgroupofS180/Dox-bearingmice(datanotshown). Fig. 2.  A,invivo  CuNG treatmentcausedapoptosis ofEAC/Doxcells.The extentof sub-G 0 -G 1 populations(Ml) inuntreated ( R ) and invivo  CuNG-treated ( RC )EAC/Dox-bearingmiceis shownbyhistogramsfrom cellcycle analysis ( a ). EAC/Doxcells fromanimals ofuntreatedand invivo  CuNG-treatedgroupswerelabeledwith P1and AnnexinV-Fluos andthenfixedandanalyzedby flowcytometry. Dual-variable dotplotof FITC fluorescence(  Xaxis ) versus P1fluorescence( Yaxis ) hasbeenshowninlogarithmic fluorescenceintensity. Bottomleftquadrant, live cells; bottom rightquadrant, apoptoticcells; top rightquadrant, necrotic cells ( b ). Nuclear fragmentationinducedby invivo  CuNGtreatmentisshownbyconfocalmicroscopy.MinimalnuclearfragmentationwasobservedinEAC/Doxcellsderivedfromuntreatedanimals,whereas invivo  CuNG treatment resultedinextensivenuclear fragmentation ( c ).Terminaldeoxynucleotidyltransferase^mediateddUTPnickendlabelingassay forconfirmationofapoptosis: apoptotic cells (FITC-dUTPpositive) were analyzedflowcytometrically. Dotplotdisplayof P1fluorescence(  Xaxis,  linear scale) versus FITC fluorescence( Yaxis,  logarithmic scale) hasbeendisplayedforEAC/Doxcells derivedfromanimals ofuntreatedandCuNG-treatedgroups( d  ).  B,  effectof invitro  treatment with CuNG(2.5 A g/mL) onproliferationof EAC/Doxcells derivedfromuntreatedanimals. Invitro  CuNG treatmentofEAC/Doxcells didnotinhibitproliferationofEAC/Doxcells. C,  extentof sub-G 0 -G 1 population (Ml) of EAC/Doxcells treatedwith CuNG invitro . Ml(sub-G 0 -G 1 population) wasonly f 6.58%. Cancer Therapy: Preclinical  www.aacrjournals.orgClin Cancer Res 2006;12(14) July15, 2006 4342  51 Cr-labeled targets (EAC/Dox cells from untreated or doxorubicin-treated mice) in round-bottomed 96-well plates at a different E:T (12:1,25:1, and 50:1) for 4 hours. After 4 hours of incubation with effectors,100  A L cell-free culture supernatant was collected and counted intriplicates in liquid scintillation counter (Tri-Carb 2100TR; PackardInstrument, Meridien, CT). Specific lysis was calculated according to theformula: % specific lysis = [(sample    spontaneous release) /(maximum release  spontaneous release)]  100, where spontaneousrelease represents basal count of cell-free culture supernatant of target cells in the absence of effector cells and maximum release (i.e.,complete lysis) represents count of culture supernatant of target cellsfollowing their lysis with 10% (v/v) Triton X-100. Statistical analysis.  Each experiment was done three to five timesand results are expressed as mean  F  SE or Student’s  t   test for significance was done and  P   < 0.01 was considered significant. Flow cytometric and fluorescence microscopic data show representative dataof at least three independent experiments. Results CuNG treatment resolves both EAC/Dox and S180/Dox.  It has been reported earlier that serum copper concentrationincreases, whereas tissue copper concentration, especially that of liver, decreases in mice bearing drug-resistant cancers (42).Hence, we decided to administer a chelate of Cu(II) [i.e., Cu(II)chelated to an organic backbone] through i.m. route, which canrelease copper in tissues of mice bearing drug-resistant cancers. The i.m. route was found to be more effective than i.v. or i.p.routes (data not shown).Intramuscular administration of CuNG alone elevated tissuecopper concentration (data not shown) and interestingly enough was observed to increase the survivability of peritonealEAC/Dox-carrying mice (Fig. 1). It also increased the longevity of S180/Dox-bearing mice beyond 6 months (data not shown).Moreover, CuNG could reduce the loads of EAC/Dox (Table 2)by >99% ( P   < 0.001) by 21 days after treatment as well as thesize of muscular S180/Dox tumor (Table 2). Actually, CuNGcould resolve both doxorubicin-resistant carcinoma and sarco-ma. Interestingly, CuNG increased urea, serum alanine amino-transferase, and aspartate aminotransferase (which arealarmingly lowered in EAC/Dox-bearing mice) to near normallevel (data not shown) at this dose, and within 24 hours, theserum level of copper was  V 0.5  A g/mL (data not shown). In vivo  but not   in vitro  treatment of CuNG induced apoptosisof EAC/Dox cells.  Because apoptosis is usually the preferredmode of elimination of cancer cells to avoid toxicity (43), weinvestigated whether the mechanism of killing of EAC/Dox cellsby   in vivo  administration of CuNG was due to apoptosis. CuNGtreatment   in vivo  increased the sub-G 0 -G 1  population asobserved by flow cytometric analysis of cell cycle (Fig. 2A, a). To better understand the nature of cell death, we used a double-labeling technique involving Annexin V-Fluos and PI. Our flow cytometric data revealed that in comparison with EAC/Dox cells derived from untreated mice, unfixed EAC/Dox cells fromCuNG-treated mice showed Annexin V-FITC binding but negli-gible (<0.001%) PI staining (Fig. 2A, b). Nuclear fragmentation was observed with confocal microscopy (Fig. 2A, c). For further confirmation of apoptosis, we did terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling assay. Apprecia-ble increase in number of terminal deoxynucleotidyl trans-ferase–mediated dUTP nick end labeling–positive cells wereobserved in EAC/Dox cell population from CuNG-treated micecompared with that from untreated mice (Fig. 2A, d). Theseresults together indicated that CuNG-induced death of cancer-ous cells was through apoptosis. Moreover, the sub-G 0 -G 1 Fig. 3.  A,  effectof invivo  CuNG treatmentonproliferationof splenicmononuclearcellsandlymphnode cells derivedfrom EAC/Dox-bearingmice. Cells from CuNG-treatedEAC/Dox-bearingmicewere eitherkeptuntreatedor treated invitro  withPMA +ionomycin ( Io ) orconcanavalin A ( ConA ).Resultswere comparedwith SPMC orlymphnode cells obtainedfromnormal ( N  )and EAC/Dox-bearingmiceuntreated invivo .  B,  effectofcoculture ofEAC/Doxcells withnonadherent SPMC fromCuNG-treated EAC/Dox-bearingmice.Extensivenuclear fragmentationofEAC/Doxcells representedby sub-G 0 -G 1 population(M1) wasobservedfollowingtheircoculturewithnonadherentsplenocytes from CuNG-treatedEAC/Dox-bearinganimals. M1, sub-G 0 -G 1 ;M2,G 0 -G 1 ; M3, S; M4, G 2 -M.  C , extentofcell-mediatedcytotoxicityofnonadherentsplenocytes from CuNG-treatedEAC/Dox-bearingmice. NonadherentSPMC derived14 daysfollowing invivo CuNG treatmentofEAC/Dox-bearinganimals showednosignificant rapidcytotoxicity invitro  comparedwithnonadherent( NA )splenocytesofuntreatedEAC/Dox-bearinganimals, with EAC/Doxcells fromuntreatedanimals as target. ResolutionofDrug-ResistantCancerbyCopperChelate Clin Cancer Res 2006;12(14) July15, 20064343
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