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The role of copper in drug-resistant murine and human tumors

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The role of copper in drug-resistant murine and human tumors
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  The role of copper in drug-resistant murine and humantumors S. Majumder   S. Chatterjee   Smarajit Pal   J. Biswas   T. Efferth   Soumitra Kumar Choudhuri Received: 21 March 2008/Accepted: 14 October 2008/Published online: 28 October 2008   Springer Science+Business Media, LLC. 2008 Abstract  Multidrug resistance (MDR) is still amajor threat to successful clinical application of cancer chemotherapy. Copper plays an important rolein biological systems, and copper is also involved incarcinogenesis. In the present investigation, weaddressed the question whether metal copper mightbe involved in drug resistance of murine and humantumors. By means of atomic absorption spectroscopy,we determined serum copper concentrations. Wefound that the blood serum of tumor-bearing micecontained higher amounts of copper than healthymice with tumors. Secondly, mice bearing doxo-rubicin-resistant Ehrlich ascites carcinoma- orcyclophosphamide-resistant Lewis lung carcinomacontained more copper in their serum than micebearing the corresponding drug-sensitive parentaltumors. Furthermore, the analysis of patients withbreast cancer, colon carcinoma or lung cancershowed that the serum copper contents were higherin patients not responding to chemotherapy whencompared to patients whose tumors responded totreatment. The copper levels in serum of healthyvolunteers were lower than in cancer patientsirrespective of their response to chemotherapy. Ourresults imply that the level of serum copper may beconsidered as a biomarker for treatment response. Keywords  Biomarker    Copper   Multidrug resistance    Serum copper Introduction Multidrug resistance (MDR) is still a major threat tosuccessful clinical application of cancer chemother-apy and also is a topic in basic cancer research. Drugresistance occurs at the cellular level, and a numberof molecular mechanisms account for this phenom-enon. Drug-resistant cells differ from the drug-sensitive cells in many ways, e.g., by (1) reducedaccumulation of cytotoxic drugs, due to decreaseddrug influx and or increased drug efflux; (2) alteredexpression and activity of certain cellular detoxifica-tion proteins and (3) physiological changes that alterthe intracellular milieu (German 1996). Several S. Majumder    S. Chatterjee    S. K. Choudhuri ( & )Department of In-Vitro Carcinogenesis and CellularChemotherapy, Chittaranjan National Cancer Institute(CNCI), 37, S. P. Mukherjee Road,Kolkatta 700 026, Indiae-mail: soumitra01@vsnl.net; soumitra01@yahoo.comS. PalClinical Biochemistry, Hospital Unit CNCI,Kolkatta, IndiaJ. BiswasSurgical Oncology and Medical Oncology, Hospital Unit,CNCI, Kolkatta, IndiaT. EfferthPharmaceutical Biology (C015), German Cancer ResearchCenter, Heidelberg, Germany  1 3 Biometals (2009) 22:377–384DOI 10.1007/s10534-008-9174-3  proteins have been found to be over-expressed inmultidrug-resistant human cancer cells, including themultidrug resistant  mdr1  gene product  P -glycoprotein(Juliano and Ling 1976), the multi-drug resistance- associated protein (MRP) (Cole et al. 1992) and enzymes associated with the glutathione (GSH)metabolism (O’Brien and Tew 1996; Eijdems et al.1995; Broxterman et al. 1993; Edincott and Ling 1989; Roninson 1991). Moreover atypical multi-drug resistance has been ascribed to decreased expressionor altered activity of topoisomerase II (Efferth andVolm 2005). Although each of these proteins hasbeen associated with a unique profile of cellular drugresistance, the drug resistance patterns may bepartially overlapping. In many human tumors, severalmechanisms are involved in drug resistance (Volmet al. 2004).Copper plays an important role in human and otherbiological systems, e.g., copper is essential for theproper functioning of copper-dependent enzymes(cytochrome C oxidase, superoxide dismutase, tyros-inase, dopamine hydroxylase, lysyl oxidase, clottingfactor V, ceruloplasmin) (Shing 1998; Watanbe et al.1990). Copper (Cu 2 ? ) is also involved in carcinogenic(Hu 1986;Raju etal.1982)aswell asanticarcinogenic (Hu 1986; Brem et al. 2005) processes. Copper stimulates endothelial cell proliferation (Hu 1986).Copper reduction inhibits the antiangiogenic response(Brem et al. 2005). Copper administration suppressesthe development of rat hepatoma induced by chemicalcarcinogens (Raju et al. 1982). Copper (II) complexes induce the re-differentiation of tumor cells to normalcells (Kawamoto et al. 1973). Although the role of  copper in physiological systems is controversial, thereis no doubt that copper is an essential component of several endogenous antioxidant enzymes (Sorenson1987; Yosmi et al. 2001). Although copper transporters have been found totransport anti-neoplastic drugs such as cisplatin (Kuoet al. 2007), the role of copper itself for drugresistance is still poorly understood (Engleka andMaciag 1994; Majumder et al. 2005). We have reported that a copper chelate overcomes drugresistance in vivo (Choudhuri and Chatterjee 1998;Majumder et al. 2006). In the present investigation,we addressed the question for the first time whethermetal copper might be involved in drug resistanceof murine and human tumors. For this reason, wefirst analyzed serum copper levels in mice bearingdrug-sensitive or drug-resistant tumors and comparedthe copper concentrations with healthy mice withouttumors. Then, we determined the levels of copper inthe serum of patients suffering from breast, colon, orlung carcinoma. Again, copper concentrations in theserum of healthy subjects were measured forcomparison. Material and methods Biological materialsAll animals (male Swiss albino and male C57BL6/Jmice) were collected from our animal colony. Allexperiments were done in accordance with the legalregulations for animal experimentation in India andwith official permission of the Chittaranjan NationalCancer Institute (Kolkatta, India).Cell linesThe Ehrlich ascites carcinoma (EAC) cell line wasmaintained in male Swiss albino mice, weighing18–20 g (6–8 weeks old) and water was supplied adlibidum. A doxorubicin-resistant sub-line (EAC/ DOX) was developed by sequential transfer of EACcells to subsequent generation of host mice withcontinuous doxorubicin (Dox) treatment as describedpreviously (Majumder et al. 2005; Choudhuri andChatterjee 1998; Majumder et al. 2006; Friche et al. 1992). The treatment regime consisted of 2.0 mg/kg/ week Dox intraperitoneally (i.p.). The daily treatmentdose was 0.4 mg/kg for 5 days. The drug was applied24 h after inoculation of 1  9  10 6 ascites tumor cellsi.p. to mice (Majumder et al. 2005). The Lewis lung carcinoma (3LL) cell line is a giftfrom Professor Per H. Basse, University of Pittsburg,Cancer Institute, Pittsburg, USA. 3LL cells weremaintained in C57BL6/J mice weighing 18–20 g(6 weeks old). A cyclophosphamide (CTX)-resistantsub-line was developed following the method of Teicher et al .  (Teicher et al. 1990) by sequentialtransfer of 1  9  10 6 3LL cells subcutaneously (s.c.) inthe dorsal hind scapula. The treatment regimeconsisted of 300 mg/kg/week CTX i.p. (single dose).The drug was injected 24 h prior to cell collection.Primary subcutaneous tumors were observed within24  ±  1.9 days (number of animals,  n  =  20) with cell 378 Biometals (2009) 22:377–384  1 3  yields of 26.5  9  10 7 ( ± 2.69). A representative imageof a C57BL/6J mouse bearing a 3LL/CTX primarytumor s.c. is shown in Fig. 1. Secondary tumors(metastatic tumors) of 3LL/CTX-bearing miceappeared in the lung. Fig. 2 shows 3LL/CTXmetastases in the lung. Normal lung is shown forcomparison.Selection of patientsPatients with cancer either in the breast, colon or lungin the advanced stage (histologically confirmed stageII–IV) were treated by Dr. J. Biswas (Hospital of Chittaranjan National Cancer Institute, Kolkatta,India). The median age of the patients was 54 years(range 28–70 years). Patients with significant periph-eral neuritis or congestive heart failure were excludedfrom this study.Collection of serum from normal and canceroushuman beingsBlood (2 ml) was collected directly from the heartand kept for clotting. Serum was collected from bloodof cancerous patients undergoing routine check-upsin the Department of Clinical Biochemistry (Hospitalof Chittaranjan National Cancer Institute, Kolkatta,India). At least 2 weeks have elapsed from theapplication of radiotherapy or chemotherapy, beforesamples have been collected for this study, in order toavoid interferences of cancer therapy with copperlevel determination. Number of patients and treat-ment schedule are shown in Table 1. Afterchemotherapy, patients were grouped as responders(complete or partial response) or non-responders (nochange, progression). In addition, blood samples(peripheral blood) were collected from five healthyvolunteers of age group 20–40 years.Measurement of serum copperAliquots of 100  l l serum were added to 3.9 ml of nitric acid (2.5%) and vortexed for 5 min. Thesolutions were kept at 37  C for 6 h with occasionalshaking. The mixture was centrifuged at 2,800 rpmfor 5 min. Copper was measured in the clear super-natant by means of flame atomic absorptionspectrophotometry (AAS) (Varian Spectra 200 FS,Varian Inc, Californa, USA) (hollow cathode lamp,Flame type: Air acetylene; replicate 3; wavelength324.8 nm) as described (US EPA 1994). Results As a first step, we measured serum copper concen-trations in Swiss albino mice bearing sensitive EAC/Sor doxorubicin-resistant EAC/DOX tumors. Highconcentrations of copper were found in EAC/DOX-bearing mice compared with EAC/S-bearing mice orhealthy mice without tumors (Fig. 3).Next, we determined serum copper levels inC57BL/6J mice bearing sensitive 3LL/S or cyclo-phosphamide-resistant 3LL/CTX tumors. The serumcopper levels of 3LL/CTX-bearing C57BL/6J micewere 146% higher than in normal C57BL/6J micewithout tumors (Fig. 4). The copper concentrations inthe serum were 29% higher in 3LL/S-bearing micethan in non-tumor-bearing ones (Fig. 4).Then, we determined the serum copper levels incancer patients and healthy volunteers. Sixty percentof human breast cancer patients contained higherlevels of copper in their serum (average: 3.25  l g/ml)than the normal healthy individuals having 2  l g/mlcopper in the serum. These patients responded tochemotherapy and were, therefore, categorized asbeing sensitive. They contained 63% more serumcopper than normal subjects without cancer (Fig. 5). Fig. 1  Cyclophosphamide-resistant 3LL/CTX primary tumor(48 days growth) in C57BL6/J mouse Fig. 2  Cyclophosphamide-resistant 3LL/CTX metastases(48 days growth) in the lung of C57BL6/J mouseBiometals (2009) 22:377–384 379  1 3  The other 40% patients did not respond to chemo-therapy. The serum of these drug-resistant breastcancer patients revealed 161% more serum copperthan normal human beings (Fig. 5).Fifty-three percent of patients with colon carci-noma were non-responders (no change orprogression). These patients showed 171% higherserum copper levels than healthy control subjects,whereas colon cancer patients, who responded tochemotherapy (complete or partial responders) hadonly 110% more serum copper than normal humanbeings (Fig. 5).Eighty-five percent of patients having non-smallcell lung cancer did not respond to chemotherapeuticdrugs. These resistant patients had 169% more copperin their serum than healthy volunteers (Fig. 5). The15% patients with drug-sensitive lung cancer had109% more serum-copper than normal individuals. Table 1  Clinical data and serum copper determination of cancer patientsSerial number Age/ sexHistology Treatment Chemotherapy Clinical response Serum copperconcentration( l g/ml)Breast Ca1 25/F IDC, stage II S  ?  RT  ?  CT CTX, MTX, 5FU Responder 3.662 35/F IC in mammary tissue,stage IIIS  ?  RT  ?  CT CTX Non-responder 5.223 58/F IDC, stage II CT 5FU, DOX, CTX Responder 3.764 30/F IDC, stage II S  ?  RT  ?  CT CTX Non-responder 4.215 70/F ET, stage II S  ?  RT  ?  CT CTX, MTX, 5FU Responder 1.96 54/F IDC, stage II S  ?  RT  ?  CT CTX, MTX, 5FU Responder 2.007 37/F IDC, stage III S  ?  RT  ?  CT 5FU, DOX, CTX Non-responder 3.768 56/F IDC, stage III S  ?  RT  ?  CT CTX, DOX Non-responder 4.129 28/F IDC, stage III CT CTX Non-responder 3.910 44/F IDC, stage III S  ?  RT  ?  CT CTX, MTX, 5FU Non-responder 3.78Lung Ca1 65/F Adeno Ca, stage II RT  ?  CT DDP, DOX, CTX Responder 3.882 66/M PD SCC, stage II RT  ?  CT CTX, VBL, 5FU Non-responder 3.763 55/M Adeno Ca, stage III CT CTX, DDP, DOX Responder 2.144 35/M SCC, stage III RT  ?  CT CTX, VCR, MTX Non-responder 5.385 56/F SCC, stage II RT  ?  CT CTX, DOX, DDP Responder 3.76 55/M SCC, stage III CT DDP, ETO Non-responder 5.36Colon Ca1 54/M Adeno Ca, stage III CT 5FU, Bleo Non-responder 4.882 36/M Adeno Ca, stage III S  ?  RT  ?  CT 5FU, Bleo Non-responder 6.13 32/M PD IC (adeno) Ca, stage II CT 5FU, Bleo Responder 3.664 28/M MD IC(Adeno), stage III CT 5FU, Bleo Non-responder 5.425 43/M AdenoCa, stage III CT 5FU, Bleo Non-responder 5.326 42/M PD IC (Adeno Ca, stage II) CT 5FU, Bleo Responder 3.65  Bleo  bleomycin;  Ca  Carcinoma;  CT   chemotherapy;  CTX   cyclophosphamide;  DDP  cisplatin;  DOX   doxorubicin;  ET   epithelial tumor; ETO  etoposide;  5FU   5-fluorouracil;  IC   infiltrating carcinoma;  IDC   infiltrating adenocarcinoma;  MD  moderately differentiatingtumor;  MTX   methotrexate;  PD  poorly differentiating tumor;  R  radioptherapy;  S   surgery;  SCC   small cell lung carcinoma;  VBL  vinblastine;  VCR  vincristineStudent  t- test for responders versus non responders disclose that: Group I Breast Ca  P  =  0.026; Group II Lung Ca  P  =  0.107; GroupIII Colon Ca  P  =  0.005380 Biometals (2009) 22:377–384  1 3  The clinical response to chemotherapy as well asthe copper levels is given in Table 1. Student  t- testfor responders versus non responders disclose that inGroup I for breast Ca  P  =  0.026; in Group II for lungCa  P  =  0.107 and in Group III for colon Ca P  =  0.005. To prove the statistical significance of the results, we subjected the clinical response tochemotherapy with the serum copper levels of thepatients to Fisher’s exact test. As shown in Table 2,patients responding to chemotherapy had significantlower serum copper levels than non-responders ( P  =0.002) indicating that copper is associated with drugresistance. Discussion In the present investigation, we analyzed the associ-ation between copper and drug resistance with a viewto identify drug resistant patients for better treatment.Firstly, we found that the blood serum of tumor-bearing mice contained higher amounts of copperthan healthy mice with tumors. Secondly, micebearing doxorubicin-resistant EAC- or cyclophospha-mide-resistant 3LL tumors contained more copper intheir serum than mice bearing the correspondingdrug-sensitive parental tumors. Thirdly, we studiedthe level of copper in healthy volunteers and cancerpatients. This indicates that copper is linked both totumor growth and to drug resistance.Animal models of drug resistance have theadvantage that the level of copper can be measuredin each generation (Majumder et al. 2005). In humancancer patients, this kind of measurements is notfeasible. Therefore, we measured the level of serumcopper only in advanced stages of human cancers.Our analysis showed that the concentration of copperin the serum of patients correlated with the responseof the tumors to chemotherapy. Therefore, weconclude that higher levels of serum copper areassociated with drug resistance and refractoriness.The reasons are not yet known, but it can behypothesized that proteins involved in the transportof copper may also be causatively linked to drugresistance. Indeed, it has been shown previously thatmetallothioneins confers drug resistance to multipleanti-neoplastic drugs (Bahnson et al. 1991; Lazo et al.1998; Volm et al. 2002; Efferth and Volm 2004). The stress-induced expression of metallothioneinsdepends on the transcription factor MTF-1, and Fig. 3  Serum-copper level of healthy normal and Ehrlichcarcinoma-bearing Swiss albino mice Fig. 4  Serum-copper level of healthy normal and Lewis lungcarcinoma-bearing C57BL/6J mice Fig. 5  Serum-copper concentration in healthy volunteers(control) and cancer patients.  Resp  Responder;  NResp  Nonresponder Table 2  Relationship of serum copper of cancer patients andresponse to chemotherapyResponder Non-responder \ 4  l g/ml Cu 8 4 [ 4  l g/ml Cu 0 9 P  =  0.002 (Fisher exact test)Biometals (2009) 22:377–384 381  1 3

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