Research ArticleMorphine Promotes Tumor Angiogenesis and Increases BreastCancer Progression

Sabrina Bimonte Antonio Barbieri Domenica Rea Giuseppe Palma Antonio LucianoArturo Cuomo Claudio Arra and Francesco Izzo

Istituto Nazionale per lo Studio e la Cura dei Tumori ldquoFondazione G Pascalerdquo IRCCS Via Mariano Semmola 80131 Naples Italy

Correspondence should be addressed to Sabrina Bimonte sbimonteistitutotumorinait

Received 31 July 2014 Accepted 14 October 2014

Academic Editor Andrea Vecchione

Copyright copy 2015 Sabrina Bimonte et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Morphine is considered a highly potent analgesic agent used to relieve suffering of patients with cancer Several in vitro and in vivostudies showed that morphine also modulates angiogenesis and regulates tumour cell growth Unfortunately the results obtainedby these studies are still contradictory In order to better dissect the role of morphine in cancer cell growth and angiogenesis weperformed in vitro studies on ER-negative human breast carcinoma cells MDAMB231 and in vivo studies on heterotopic mousemodel of human triple negative breast cancer TNBC We demonstrated that morphine in vitro enhanced the proliferation andinhibited the apoptosis of MDAMB231 cells In vivo studies performed on xenograft mouse model of TNBC revealed that tumoursof mice treated with morphine were larger than those observed in other groups Moreover morphine was able to enhance theneoangiogenesis Our data showed that morphine at clinical relevant doses promotes angiogenesis and increases breast cancerprogression

1 Introduction

Morphine is an opiate-based drug largely used to relievepains of patients with cancer in terminal phases in orderto improve their quality of life [1] It was isolated for thefirst time in 1803 by Friedrich W Serturner [2] It is notedthat morphine explains its function by acting through opioidreceptors localized in the brain named 120583 120575 and 120581 [3 4]Morphine relieves pain by acting directly on central nervoussystem (CNS) although its activity on peripheral tissueleads to many secondary complications including immuno-costipation respiratory depression addiction and toleranceMorphine is still considered the most effective analgesicclinically available used to relieve suffering of patients withcancer [5] Several experimental studies performed on cancercell lines and mouse models showed that morphine can alsoplay a role in regulation of cancer cell growth Unfortunatelyat present the role of morphine in the regulation of tumor cellgrowth is not yet correctly established The results obtainedby these studies are still contradictory Many reports showedthat morphine was able to inhibit the growth of various

human cancer cell lines [6ndash12] or animal models [13ndash16] Onthe contrary other studies proved that morphine increasedtumor cell growth in in vivo [17 18] or in vitro [19] modelsIt has been demonstrated that morphine at clinically relevantdoses stimulates angiogenesis in vitro [20] promotes tumourgrowth in breast cancer mouse model and increases vascularpermeability [21] One explanation for these different resultscould be due to different concentration andor time ofadministration of morphine applied In fact in vitro andin vivo studies showed that tumor suppression occurs afterchronic high doses of morphine [11 15 16] while tumor-enhancing effects with morphine occur after administrationof low daily doses or single dose of morphine [22] Thusthere is a dilemma about the effects of morphine on cancercell growth and angiogenesis [23] Recently it has beendemonstrated that morphine stimulates cancer progressionand mast cell activation and impairs survival in transgenicmice with breast cancer [24]

For these reasons in order to elucidate the role ofmorphine in regulation of tumor growth and angiogenesis intriple negative breast cancer (TNBC) we performed in vitro

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 161508 8 pageshttpdxdoiorg1011552015161508

2 BioMed Research International

and in vivo studies on the ER-negativehuman breast carci-noma cells MDAMB231 Our data showed that morphineat clinical relevant doses promotes tumor angiogenesis andincreases breast cancer proliferation and migration

2 Materials and Methods

21Materials Morphine sulphate used for in vitro and in vivoexperiments was kindly gifted by Dr Arturo Cuomo (IRCCSFondazione Pascale) and was dissolved in distilled water to aconcentration of 100mM as a stock solution Then the drugwas added toMDAMB231 cells in three different doses (1 10and 100 120583M) The antibody against PECAM-1 was obtainedfrom Santa Cruz Biotechnology (Santa Cruz CA) Anti-p53antibody was kindly provided by Imgenex (San Diego CA)The liquid DAB+ Substrate Chromogen System-HRP usedfor immunocytochemistry was obtained from DakoCytoma-tion (Carpinteria CA) Penicillin streptomycin Dulbeccorsquosmodified Eagle medium (DMEM) and fetal bovine serum(FBS) were obtained from Invitrogen (Grand Island NY)Tris glycine NaCl SDS and bovine serum albumin (BSA)were obtained from Sigma Chemical (St Louis MO)

22 Cell Lines ER-negativebreast cancer cell line MDAMB231 was obtained fromAmerican Type Culture Collection(Manassas VA) Cells were cultured in RPMI-1640 sup-plemented with fetal bovine serum (FBS) 10 antibiotics(penicillin 100 unitsmL streptomycin 100 120583gmL) and l-glutamine (2mM) at 37∘C in an atmosphere of 5 of CO

2

23 Proliferation Assay The effect of drug on cell prolifera-tion was determined by using TACS 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) cell prolif-eration assay (Trevigen Gaithersburg) The cells (2000 perwell) were incubated with or without morphine in triplicatein a 96-well plate and then incubated for 2 4 and 6 days at37∘C A MTT solution was added to each well and incubatedfor 2 h at 37∘C An extraction buffer (20 SDS and 50dimethylformamide) was added and the cells were incubatedovernight at 37∘C The absorbance of the cell suspensionwas measured at 570 nm using a microplate reader (DASTechnologies Chantilly VA) This experiment was repeatedtwice and the statistical analysis was performed to obtain thefinal values

24 Wound-Healing Assay MDAMB231 cells were seeded atthe density of 40 times 103 cells per well into a 6-multiwell plateand cultured in DMEMmedium supplemented with 1 FBSAt the time of confluence cells were incubated in the absenceor presence of morphine (1 10 and 100 120583M) for 48 h after aslit made horizontally with a white tip at the center of eachconfluent well Cell invasion on the slit of the confluent wellwas assessed at 0 24 48 hours in each condition by lightmicroscopy

25 Mice Six eight-week-old female Foxn1nunu mice werepurchased by Harlan San Pietro al Natisone Italy Mice werehoused five for cage in the standard mice plexiglass cages and

maintained on a 12 h light 12 h dark cycle (lights on at 700am) in a temperature-controlled room (22 plusmn 2∘C) and withfood and water ad libitum at all times All the experimentsperformed on animal models were in compliance with theguidelines for the Care and Use of Laboratory Animals of theNational Cancer Institute ldquoFondazione G Pascalerdquo IRCCSMoreover all experiments were performed by also followingthe European Directive 632010UE and the Italian Law (DL262014 authorized by Minister of Health Italy) This studywas carried out in accordance with the recommendationsthat cover all scientific procedures involving the use of liveanimals

26 Generation of Heterotopic Mouse Model of Breast Cancerand Experimental Protocol MDAMB231 breast cancer cellswere harvested from subconfluent cultures after a briefexposure to 025 trypsin Trypsinization was stopped withmedium containing 10 FBS The cells were washed once inserum-freemediumand suspended in PBSOnly suspensionsconsisting of single cells with gt90 viability were used forthe injections A total of 16 female Foxn1nunu mice wereused in this experiment and maintained in a barrier facilityon HEPA-filtered racks Animals were individually identifiedusing numbered ear tags All experiments were conducted ina biological laminar flow hood and all surgical procedureswere conducted with strict adherence to aseptic techniqueThe mice were anesthetized with Avertin solution injectedintraperitoneally according to their weight A suspension of25 times 106 MBAMB231 cells in 25 120583L of PBS 1Xmouse wasinjected subcutaneously into the right-side flank area ofmiceWhen tumors reached sim30ndash60mm3 mice were randomizedinto the following treatment groups (119899 = 4) (a) normalsaline (control) and (b) morphine sulphate at 0714mgkgmouseday for first 15 days and then 143mgkg mouseday(equivalent to 50mg and 100mg morphine per day respfor a 70 kg human) Tumor volumes were monitored oncea week by using a digital caliper Therapy was continuedfor 4 weeks and animals were sacrificed 2 weeks later Thetumor size was measured using digital caliper and thetumor volumewas estimated by the following formula tumorvolume (mm3) = (119882 times 119871) 2 times 12 where 119871 is the lengthand119882 is the width of the tumor Normally distributed datawere represented as mean plusmn SEM Paired 119905-test one-tailedanalysis was used to examine the significance of differencesamong groups (GraphPad Prism 50) A probability valuewith lowast119875 lt 005 and lowastlowast119875 lt 001 was considered to bestatistically significant Fluorescein isothiocyanate- (FITC-)dextran (100 120583L) was injected into the tail vein of mice tovisualize microvessels within 150120583m (using single-photonmicroscopy) or sim600120583m (using multiphoton laser-scanningmicroscopy [MPLSM]) of a tumorwindow interface Half ofthe tumor tissue was formalin-fixed and paraffin-embeddedfor immunohistochemistry and routine HampE staining Theother half was snap-frozen in liquid nitrogen and stored atminus80∘C

27 Preparation of Nuclear Extract from Tumor SamplesBreast tumor tissues (75ndash100mgmouse) from control and

BioMed Research International 3

experimental mice were prepared according to standardprotocols The supernatant (nuclear extract) was collectedand stored at minus70∘C until use Protein concentration wasdetermined by the Bradford protein assay with BSA as thestandard

28 Immunohistochemical Analysis for CD31 in Tumor TissueBreast cancer tumor samples from controls and treated micewere embedded in paraffin and fixed with paraformalde-hyde After being washed in PBS the slides were blockedwith protein block solution (DakoCytomation) for 20minand then incubated overnight with polyclonal anti-goatPECAM-1 (1 100) After the incubation the slides werewashed and then incubated with biotinylated link universalantiserum followed by horseradish peroxidase-streptavidinconjugate (LSAB+ kit) The slides were rinsed and color wasdeveloped using 3 31015840-diaminobenzidine hydrochloride as achromogen Finally sections were rinsed in distilled watercounterstained with haematoxylin and mounted with DPXmounting medium for evaluation Pictures were capturedwith a Photometrics CoolSNAP CF colour camera (NikonLewisville TX) and MetaMorph version 465 software (Uni-versal Imaging Downingtown PA)

29 Western Blot Analysis Breast tumor tissues (75ndash100mgmouse) from control and experimental mice were mincedand incubated on ice for 1 h in 05mL of ice-cold LysisBuffer (10mM Tris ph80 130mM Nacl 1 Triton X-100 10mM NaF 10mM sodium phosphate 10mM sodiumpyrophosphate 2 120583gmL aprotinin 2120583gmL leupeptin and2 120583gmL pepstatin) The minced tissue was homogenizedusing a Dounce homogenizer and centrifuged at 16000timesgat 4∘C for 10min Western blotting analysis was performedaccording to standard protocols 120573-Actin was used as loadingcontrol

3 Results

31 Morphine Enhances the Proliferation of Triple NegativeBreast Cancer Cells by Performing In Vitro Assays on MBAMB231 breast cancer cells Wound-healing assay demon-strated that morphine enhances the migration of breastcancer cells at 48 h in dose dependent manner These resultswere also confirmed by MTT assay and flow cytometry(Figure 1(e) and data not shown) In order to assess ifmorphine enhances the apoptosis in breast cancer cellswe performed western blotting analysis of p53 expressionon cell lysate extracted from MBAMB231 cells not treatedand treated with morphine (Figure 1(g)) Taken togetherour data showed that morphine inhibits apoptosis andpromotes proliferation in dose dependent manner Thesame results were also obtained for MCF-7 cells (data notshown)

32 Morphine Promotes Tumor Growth and Microvessel For-mation in Heterotopic Mouse Model of Triple Negative BreastCancer In order to study the role of morphine in the tumorgrowth in vivo we generated a mouse model of breast

cancer by injection of MDAMB231 cells subcutaneously intothe right-side flank area of mice When tumors reachedsim30ndash60mm3 2 weeks after cell injection the mice wererandomized into three groups (a) normal saline (control)and (b) morphine sulphate at 0714mgkg mouseday forfirst 15 days and then 143mgkg mouseday (equivalentto 50mg and 100mg morphine per day resp for a 70 kghuman) Tumor volumes were monitored once a week byusing a digital caliper Therapy continued for 4 weeks andanimals were sacrificed 2 weeks later We also monitoredthe body weight of mice twice a week until the end oftreatment No difference was observed between the bodyweights of two groups of animals indicating that treatmentsof mice with drug are not associated with toxicity effectsMice were sacrificed at the end of treatment The finaltumor volumes on day 35 after the start of treatment showeda significant increase in the morphine group comparedwith control (Figure 2(a)) Interestingly administration ofmorphine enhanced tumor volumes and resulted in rapidgrowth of tumors with respect to controls In order to assessif morphine inhibits microvessel formation in breast tumorsfluorescein isothiocyanate- (FITC-) dextranwas injected intothe tail vein of mice Our data demonstrate that morphineenhancedmicrovessel formation inmice tumors with respectto controls (Figures 2(b)-2(c)) In order to confirm these datawe performed an immunohistochemical staining with CD31on tumor tissues from control and treated mice Our datademonstrate that morphine promotes microvessel formationin breast tumors of mice treated with respect to controls(Figures 3(a)-3(b))

4 Discussion

Several experimental studies performed in in vitro and in vivocancer cell lines and mouse models showed that morphineplays a role in regulation of cancer cell growth andmetastasisThe results obtained by these studies are still controversialsince many reports showed that morphine was able to inhibitthe growth of various human cancer cell lines [6ndash12] oranimal models [13ndash16] On the contrary other studies provedthat morphine increased tumor cell growth in in vivo [17 18]or in vitro [19]models To study cancer cell growth promotingor inhibiting effects of morphine several xenograft mousemodels were generated Tegeder et al [13] generated a mousemodel of breast cancer by subcutaneous injection of MCF-7 and MDA-MB231 cells in NMRI-nunu mice In thispaper it has been demonstrated that morphine significantlyreduced tumor growth through a p53-dependent mecha-nism Additionally in these mice naloxone increased thegrowth-inhibitory effects of morphine Similar results wereobtained in rat model of colon cancer in which subcutaneousadministration of morphine leads to significant decreasein the hepatic tumor burden On the contrary severalexperimental studies demonstrated that morphine increasedtumor growth Gupta et al in orthotropic mouse model ofbreast cancer obtained by injection of MCF-7 cells into themammary fat pad of nudemice demonstrated thatmorphinein clinically relevant doses increased tumor growth This

4 BioMed Research International

Prol

ifera

tion

()

Days

0

50

100

150

200

250

300

1 2 3 4 5

CTRM 1120583M

M 10120583MM 100120583M

(e)

Relat

ive t

o ac

tion

()

CTR M 1120583M M 10120583M M 100120583M0

051

152

253

354

CTR M 1120583M M 10120583M M 100120583M

P53

120573-Actin

(f)

(g)

(a) (b) (c) (d)

CTR M 1120583M M 10120583M M 100120583M

Figure 1 Morphine stimulates proliferation in MDAMB231 cell lines MDAMB231 cells were incubated in medium containing (a) mediumcontrol (b) 1 120583mmorphine (c) 10120583mmorphine and (d) 100 120583mmorphine Cell migration rates were quantitatively assessed by counting thenumber of cells in the denuded area at 0 24 and 48 h after wound induction At 48 h after wound induction there were clearly more cellsin the denuded area of morphine treated cells than untreated cells (e) MTT assay results show an enhancement of proliferation in breastcancer cells treated with morphine with respect to control cells Data are representative of three independent experiments (119875 value lt 005)(f-g) Western blot showing that morphine reduces the expression of p53 in MDAMB231 cells treated with morphine (lanes 2 3 and 4) withrespect to controls (lane 1) in dose dependent manner 120573-Actin was used as loading control

was associated with increased angiogenesis and inhibitionof apoptosis and promotion of cell cycle progression [20]In this study it was also reported that naloxone itself hadno significant effect on angiogenesis According to theseresults in another study it was demonstrated that morphinesubcutaneously administrated in mice increased the tumorgrowth in mouse model of leukaemia and sarcoma In thesemice morphine had also a general immunosuppressive effect[25]

These contrasting results are probably associated withdifferent concentration andor time of administration ofmorphine In fact in vitro and in vivo studies demonstratedthat tumor-enhancing effects with morphine occur afteradministration of low daily doses or single dose of morphine[22] while tumor suppression occurs after chronic high dosesof morphine [11 15 16]

Thus there is a dilemma about the effects of morphineon cancer cell growth and angiogenesis in various types ofcancer [23] The role of morphine in the regulation of tumorcell growth is not yet correctly established

It has also been demonstrated that the 120583-opioid recep-tor by which morphine exerts its action directly regu-lates tumor growth and metastasis On the basis of theseresults different mechanisms of opioid receptor-mediatedinfluence of morphine on tumor growth have been proposedMorphine as mentioned above after binding to the 120583-opioid receptor regulates cell cycle progression by stimulat-ing mitogen-activated protein kinase (MAPK)extracellulargrowth factor (Erk) pathways [20] Alternatively morphinecan mediate apoptosis by activating phosphatidylinositol 3-kinase (PI3K)protein kinase B (Akt) pathway [26] Addi-tionallymorphine by upregulation of urokinase plasminogen

BioMed Research International 5

0 7 14 21 28 35 420

500

1000

1500

Days after tumor injection

CTRMorphine

Tum

or v

olum

e (m

m3)

(a)

Control

(b)

Morphine

(c)

Figure 2 Morphine promotes the tumor growth in TBNC mouse model (a) Morphine promotes tumor growth in breast tumor xenograftmodel Breast tumor growth in mice treated with vehicle (∙) and morphine (119887119897119886119888119896119904119898119886119897119897119904119902119906119886119903119890) Tumor volumes increased after 28 days ofmorphine treatment until 35 days (119875 lt 005) as compared with control (vehicle-treated) Each point represents the mean of five separateexperiments (b-c) Measurements of fluorescence per second depicting microvessel tumor (FITC-DEXTRANE) using MacroFluo imagesshowed that morphine enhances the angiogenesis in tumor of mice (b) with respect to controls (c)

CTR

PECA

M-1

(a)

Morphine

(b)

Figure 3 Morphine promotes angiogenesis formation in heterotopic mouse model of breast cancer Immunohistochemical analysis forPECAM-1showed the enhancement of PECAM-1 expression inmorphine treated group (b) compared to controls (a) Arrows indicate positivestaining of microvessel staining

activator (uPA) expression induces metastasis formation[27] while by transactivation of VEGF receptor it inducesangiogenesis [28] Finally morphine affects also the functionof T lymphocytes leading to immunosuppression [29]

It has been proposed that morphine plays also a role intumor apoptosis Apoptosis is a form of cell death in whicha programmed sequence of events leads to the eliminationof cells without releasing harmful substances into the sur-rounding area It is noted that apoptosis is regulated by twopathways the mitochondrial-mediated pathway (intrinsic)[30] and death receptor-mediated pathway (extrinsic) [31] Itis noted that in cancer cells apoptosis is deregulated andthis leads to quick proliferation and tumor growth [32 33]Morphine was shown to induce apoptosis of macrophages T

lymphocytes and human endothelial cells [34 35] Experi-ments performed on human tumor cell lines demonstratedthat morphine in high concentration induces apoptosis andinhibits cancer cell growth by activation of different signalpathways involving caspase 39 and cytochrome c sigma-2 receptor Additionally in SH-SY5Y cells morphine hasantiapoptotic effect by antagonizing doxorubicin [36] Thesediscrepancies also in these cases are associatedwith differentcell line tumor type used andor in vivo dosetime of mor-phine administrated

Recent data demonstrated a role of morphine in angio-genesis Angiogenesis is required for invasive tumor growthand metastasis and represents an important point in thecontrol of cancer progression Proangiogenic activity of

6 BioMed Research International

morphine was demonstrated in the MCF-7 breast cancermodel In these mice morphine at clinically relevant con-centrations enhanced tumor neovascularization [20] In ananimal model of hormone-dependent breast cancer it hasalso been demonstrated that morphine promoted activationof vascular endothelial growth factor (VEGF) receptor andincreased metastasis [21 29] It has been proposed thatmorphine explains its proangiogenic activity by stimula-tion of mitogen-activated protein kinase (MAPK) signallingpathway via G protein-coupled receptors and nitric oxide(NO) Alternatively several in vivo studies provided evidencethat morphine can induce tumor growth by upregulationof cyclooxygenase-2 (COX-2) [37ndash40] and or prostaglandinE2-mediated stimulation of angiogenesis [41ndash44] On thecontrary several in vivo and in vitro studies demonstratedthat morphine can inhibit angiogenesis by regulation ofdifferent pathways [8 34 45 45ndash52] These different resultscan be due to different experimental conditions (cell linetumor type used andor dosetime of morphine) Morphineplays a role not only in tumor cell growth but also inmetastasis formation which is the main process related tomost cancer deaths and failure in cancer treatment [53 54]

For these reasons in order to elucidate the role ofmorphine in regulation of tumor growth and angiogenesis inbreast cancer we performed in vitro and in vivo studies on theMDAMB231 breast cancer cellsThese cells are triple negative(basal-like) breast cancer (TNBC) cells It is noted that TNBCdoes not express the estrogen receptor (ER) progesteronereceptor (PR) or human epidermal growth factor receptortype 2 (HER2) Due to lack of both hormone receptors andHER2 expression patients with this type of breast cancer haveno chance to benefit from the endocrine therapy and HER2targeted therapy In addition we used immunodeficient micethat are able to mimic the compromised immune systemof a patient with breast cancer So in these experimentalconditions it has become interesting for us to study therole of morphine in the regulation of cancer cell growthand angiogenesis Our data showed that morphine in TNBCat clinical relevant doses promotes tumor angiogenesis andincreases breast cancer progression For these reasons it isvery important for the management of severe pain associatedwith cancer to consider accurately the dose and route ofadministration of morphine in order to avoid severe effectof cancer progression Further studies are ongoing in ourlaboratory in order to dissect the molecular mechanismsunderlying the role of morphine in cancer development andmetastasis formation in breast cancer Specifically we willgenerate orthotropic mouse models of breast cancer by usingnot only MDAMB231 cells but also MCF-7 (human breastadenocarcinoma cell line) cells which represent estrogenreceptor (ER) positive control cell lines The results obtainedfrom these data will shed light on the role of morphine inregulation of breast cancer progression

Conflict of Interests

The authors have no other relevant affiliations or financialinterests with any organization or entity No writing assis-tance was used in the production of paper

Acknowledgments

The authors would like to specially thank MassimilianoSpinelli Data Manager of SSD Sperimentazione animaleIstituto Nazionale per lo Studio e la Cura dei TumorildquoFondazione G Pascalerdquo IRCCS Italy for kind help inproviding informatics assistance and Dr Di Napoli Danielefor supervising animal care This work was supported by the5x mille and current research programs of National Instituteof Tumors IRCCS ldquoFoundation G Pascalerdquo Naples (Italy)

References

[1] J N Lickiss ldquoApproaching cancer pain reliefrdquo European Journalof Pain vol 5 pp 5ndash14 2001

[2] R Schmitz ldquoFriedrich Wilhelm Serturner and the discovery ofmorphinerdquo Pharmacy in History vol 27 no 2 pp 61ndash74 1985

[3] G W Pasternak ldquoPharmacological mechanisms of opioidanalgesicsrdquo Clinical Neuropharmacology vol 16 no 1 pp 1ndash181993

[4] B L Kieffer and C Gaveriaux-Ruff ldquoExploring the opioidsystem by gene knockoutrdquo Progress in Neurobiology vol 66 no5 pp 285ndash306 2002

[5] P W Mantyh ldquoCancer pain and its impact on diagnosissurvival and quality of liferdquo Nature Reviews Neuroscience vol7 no 10 pp 797ndash809 2006

[6] N Sueoka E Sueoka S Okabe and H Fujiki ldquoAnti-cancereffects of morphine through inhibition of tumour necrosisfactor-120572 release and mRNA expressionrdquo Carcinogenesis vol 17no 11 pp 2337ndash2341 1996

[7] R Maneckjee and J D Minna ldquoOpioids induce while nicotinesuppresses apoptosis in human lung cancer cellsrdquo Cell Growthand Differentiation vol 5 no 10 pp 1033ndash1040 1994

[8] A Pasi B Qu R Steiner H-J Senn W Bar and F S MessihaldquoAngiogenesis modulation with opioidsrdquo General Pharmacol-ogy vol 22 no 6 pp 1077ndash1079 1991

[9] A Hatzoglou E Bakogeorgou E Papakonstanti C StournarasD S Emmanouel and E Castanas ldquoIdentification and char-acterization of opioid and somatostatin binding sites in theopossum kidney (OK) cell line and their effect on growthrdquoJournal of Cellular Biochemistry vol 63 no 4 pp 410ndash421 1996

[10] A Hatzoglou L Ouafik E Bakogeorgou K Thermos and ECastanas ldquoMorphine cross-reacts with somatostatin receptorSSTR2 in the T47D human breast cancer cell line and decreasescell growthrdquo Cancer Research vol 55 no 23 pp 5632ndash56361995

[11] R Maneckjee R Biswas and B K Vonderhaar ldquoBinding ofopioids to humanMCF-7 breast cancer cells and their effects ongrowthrdquo Cancer Research vol 50 no 8 pp 2234ndash2238 1990

[12] M Kampa E Bakogeorgou A Hatzoglou A Damianaki P-MMartin and E Castanas ldquoOpioid alkaloids and casomorphinpeptides decrease the proliferation of prostatic cancer cell lines(LNCaP PC3 and DU145) through a partial interaction withopioid receptorsrdquo European Journal of Pharmacology vol 335no 2-3 pp 255ndash265 1997

[13] I Tegeder S Grosch A Schmidtko et al ldquoG protein-independent G1 cell cycle block and apoptosis with morphinein adenocarcinoma cells involvement of p53 phosphorylationrdquoCancer Research vol 63 no 8 pp 1846ndash1852 2003

BioMed Research International 7

[14] M P Yeager and T A Colacchio ldquoEffect ofmorphine on growthof metastatic colon cancer in vivordquo Archives of Surgery vol 126no 4 pp 454ndash456 1991

[15] T Sasamura S Nakamura Y Iida et al ldquoMorphine analgesiasuppresses tumor growth and metastasis in a mouse modelof cancer pain produced by orthotopic tumor inoculationrdquoEuropean Journal of Pharmacology vol 441 no 3 pp 185ndash1912002

[16] YHarimaya K Koizumi T AndohHNojima Y Kuraishi andI Saiki ldquoPotential ability of morphine to inhibit the adhesioninvasion and metastasis of metastatic colon 26-L5 carcinomacellsrdquo Cancer Letters vol 187 no 1-2 pp 121ndash127 2002

[17] R H Simon and T E Arbo ldquoMorphine increases metastatictumor growthrdquo Brain Research Bulletin vol 16 no 3 pp 363ndash367 1986

[18] J W Lewis Y Shavit G W Terman L R Nelson R P Galeand J C Liebeskind ldquoApparent involvement of opioid peptidesin stress-induced enhancement of tumor growthrdquo Peptides vol4 no 5 pp 635ndash638 1983

[19] M G Sergeeva Z V Grishina and S D VarfolomeyevldquoMorphine effect on proliferation of normal and tumor cells ofimmune originrdquo Immunology Letters vol 36 no 2 pp 215ndash2181993

[20] K Gupta S Kshirsagar L Chang et al ldquoMorphine stimu-lates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growthrdquo Can-cer Research vol 62 no 15 pp 4491ndash4498 2002

[21] M Farooqui Y Li T Rogers et al ldquoCOX-2 inhibitor celecoxibprevents chronic morphine-induced promotion of angiogene-sis tumour growth metastasis and mortality without compro-mising analgesiardquo British Journal of Cancer vol 97 no 11 pp1523ndash1531 2007

[22] J Zong and G M Pollack ldquoMorphine antinociceptionis enhanced in mdr1a gene-deficient micerdquo PharmaceuticalResearch vol 17 no 6 pp 749ndash753 2000

[23] S Bimonte A Barbieri G Palma and C Arra ldquoThe role ofmorphine in animal models of human cancer does morphinepromote or inhibit the tumor growthrdquo BioMed Research Inter-national vol 2013 Article ID 258141 4 pages 2013

[24] J Nguyen ldquoMorphine stimulates cancer progression and mastcell activation and impairs survival in transgenic mice withbreast cancerrdquo British Journal of Anaesthesia vol 113 supple-ment 1 pp i4ndashi13 2014

[25] M Ishikawa K Tanno A Kamo Y Takayanagi and K-ISasaki ldquoEnhancement of tumor growth by morphine and itspossible mechanism in micerdquo Biological and PharmaceuticalBulletin vol 16 no 8 pp 762ndash766 1993

[26] M Iglesias M F Segura J X Comella and G Olmos ldquo120583-opioid receptor activation prevents apoptosis following serumwithdrawal in differentiated SH-SY5Y cells and cortical neuronsvia phosphatidylinositol 3-kinaserdquoNeuropharmacology vol 44no 4 pp 482ndash492 2003

[27] K Gach J Szemraj J Fichna M Piestrzeniewicz D S Delbroand A Janecka ldquoThe influence of opioids on urokinase plas-minogen activator on protein andmRNA level inMCF-7 breastcancer cell linerdquo Chemical Biology and Drug Design vol 74 no4 pp 390ndash396 2009

[28] P A Singleton and J Moss ldquoEffect of perioperative opioids oncancer recurrence a hypothesisrdquo Future Oncology vol 6 no 8pp 1237ndash1242 2010

[29] C Chen M Farooqui and K Gupta ldquoMorphine stimulatesvascular endothelial growth factor-like signaling in mouse

retinal endothelial cellsrdquo Current Neurovascular Research vol3 no 3 pp 171ndash180 2006

[30] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[31] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[32] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[33] M O Hengartner ldquoThe biochemistry of apoptosisrdquoNature vol407 no 6805 pp 770ndash776 2000

[34] P-N Hsiao M-C Chang W-F Cheng et al ldquoMorphineinduces apoptosis of human endothelial cells through nitricoxide and reactive oxygen species pathwaysrdquo Toxicology vol256 no 1-2 pp 83ndash91 2009

[35] A A Kapasi S A CosciaM P Pandya and P C Singhal ldquoMor-phine modulates HIV-1 gp160-induced murine macrophageand human monocyte apoptosis by disparate waysrdquo Journal ofNeuroimmunology vol 148 no 1-2 pp 86ndash96 2004

[36] X Lin Q Li Y-J Wang et al ldquoMorphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor120581B transcriptional activation in neuroblastoma SH-SY5Y cellsrdquoBiochemical Journal vol 406 no 2 pp 215ndash221 2007

[37] R Arerangaiah N Chalasani A M Udager et al ldquoOpioidsinduce renal abnormalities in tumor-bearing micerdquo NephronmdashExperimental Nephrology vol 105 no 3 pp e80ndashe89 2007

[38] D Salvemini T P Misko J L Masferrer K Seibert M G Cur-rie and P Needleman ldquoNitric oxide activates cyclooxygenaseenzymesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 15 pp 7240ndash7244 1993

[39] E Nedelec A Abid C Cipolletta et al ldquoStimulation ofcyclooxygenase-2-activity by nitric oxide-derived species inrat chondrocyte lack of contribution to loss of cartilageanabolismrdquo Biochemical Pharmacology vol 61 no 8 pp 965ndash978 2001

[40] M Farooqui ZHGeng E J StephensonN Zaveri D Yee andK Gupta ldquoNaloxone acts as an antagonist of estrogen receptoractivity in MCF-7 cellsrdquo Molecular Cancer Therapeutics vol 5no 3 pp 611ndash620 2006

[41] D Salvemini K Seibert J L Masferrer T P Misko M GCurrie and P Needleman ldquoEndogenous nitric oxide enhancesprostaglandin production in a model of renal inflammationrdquoJournal of Clinical Investigation vol 93 no 5 pp 1940ndash19471994

[42] R J Griffin BWWilliams RWild JM CherringtonH Parkand CW Song ldquoSimultaneous inhibition of the receptor kinaseactivity of vascular endothelial fibroblast and platelet-derivedgrowth factors suppresses tumor growth and enhances tumorradiation responserdquo Cancer Research vol 62 no 6 pp 1702ndash1706 2002

[43] K M Leahy R L Ornberg Y Wang B S Zweifel A T Kokiand J L Masferrer ldquoCyclooxygenase-2 inhibition by celecoxibreduces proliferation and induces apoptosis in angiogenicendothelial cells in vivordquoCancer Research vol 62 no 3 pp 625ndash631 2002

[44] S H Chang C H Liu and R Conway ldquoRole of prostaglandinE2-dependent angiogenic switch in cyclooxygenase 2-inducedbreast cancer progressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 2 pp 591ndash596 2004

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006

BioMed Research International 3

experimental mice were prepared according to standardprotocols The supernatant (nuclear extract) was collectedand stored at minus70∘C until use Protein concentration wasdetermined by the Bradford protein assay with BSA as thestandard

28 Immunohistochemical Analysis for CD31 in Tumor TissueBreast cancer tumor samples from controls and treated micewere embedded in paraffin and fixed with paraformalde-hyde After being washed in PBS the slides were blockedwith protein block solution (DakoCytomation) for 20minand then incubated overnight with polyclonal anti-goatPECAM-1 (1 100) After the incubation the slides werewashed and then incubated with biotinylated link universalantiserum followed by horseradish peroxidase-streptavidinconjugate (LSAB+ kit) The slides were rinsed and color wasdeveloped using 3 31015840-diaminobenzidine hydrochloride as achromogen Finally sections were rinsed in distilled watercounterstained with haematoxylin and mounted with DPXmounting medium for evaluation Pictures were capturedwith a Photometrics CoolSNAP CF colour camera (NikonLewisville TX) and MetaMorph version 465 software (Uni-versal Imaging Downingtown PA)

29 Western Blot Analysis Breast tumor tissues (75ndash100mgmouse) from control and experimental mice were mincedand incubated on ice for 1 h in 05mL of ice-cold LysisBuffer (10mM Tris ph80 130mM Nacl 1 Triton X-100 10mM NaF 10mM sodium phosphate 10mM sodiumpyrophosphate 2 120583gmL aprotinin 2120583gmL leupeptin and2 120583gmL pepstatin) The minced tissue was homogenizedusing a Dounce homogenizer and centrifuged at 16000timesgat 4∘C for 10min Western blotting analysis was performedaccording to standard protocols 120573-Actin was used as loadingcontrol

3 Results

31 Morphine Enhances the Proliferation of Triple NegativeBreast Cancer Cells by Performing In Vitro Assays on MBAMB231 breast cancer cells Wound-healing assay demon-strated that morphine enhances the migration of breastcancer cells at 48 h in dose dependent manner These resultswere also confirmed by MTT assay and flow cytometry(Figure 1(e) and data not shown) In order to assess ifmorphine enhances the apoptosis in breast cancer cellswe performed western blotting analysis of p53 expressionon cell lysate extracted from MBAMB231 cells not treatedand treated with morphine (Figure 1(g)) Taken togetherour data showed that morphine inhibits apoptosis andpromotes proliferation in dose dependent manner Thesame results were also obtained for MCF-7 cells (data notshown)

32 Morphine Promotes Tumor Growth and Microvessel For-mation in Heterotopic Mouse Model of Triple Negative BreastCancer In order to study the role of morphine in the tumorgrowth in vivo we generated a mouse model of breast

cancer by injection of MDAMB231 cells subcutaneously intothe right-side flank area of mice When tumors reachedsim30ndash60mm3 2 weeks after cell injection the mice wererandomized into three groups (a) normal saline (control)and (b) morphine sulphate at 0714mgkg mouseday forfirst 15 days and then 143mgkg mouseday (equivalentto 50mg and 100mg morphine per day resp for a 70 kghuman) Tumor volumes were monitored once a week byusing a digital caliper Therapy continued for 4 weeks andanimals were sacrificed 2 weeks later We also monitoredthe body weight of mice twice a week until the end oftreatment No difference was observed between the bodyweights of two groups of animals indicating that treatmentsof mice with drug are not associated with toxicity effectsMice were sacrificed at the end of treatment The finaltumor volumes on day 35 after the start of treatment showeda significant increase in the morphine group comparedwith control (Figure 2(a)) Interestingly administration ofmorphine enhanced tumor volumes and resulted in rapidgrowth of tumors with respect to controls In order to assessif morphine inhibits microvessel formation in breast tumorsfluorescein isothiocyanate- (FITC-) dextranwas injected intothe tail vein of mice Our data demonstrate that morphineenhancedmicrovessel formation inmice tumors with respectto controls (Figures 2(b)-2(c)) In order to confirm these datawe performed an immunohistochemical staining with CD31on tumor tissues from control and treated mice Our datademonstrate that morphine promotes microvessel formationin breast tumors of mice treated with respect to controls(Figures 3(a)-3(b))

4 Discussion

Several experimental studies performed in in vitro and in vivocancer cell lines and mouse models showed that morphineplays a role in regulation of cancer cell growth andmetastasisThe results obtained by these studies are still controversialsince many reports showed that morphine was able to inhibitthe growth of various human cancer cell lines [6ndash12] oranimal models [13ndash16] On the contrary other studies provedthat morphine increased tumor cell growth in in vivo [17 18]or in vitro [19]models To study cancer cell growth promotingor inhibiting effects of morphine several xenograft mousemodels were generated Tegeder et al [13] generated a mousemodel of breast cancer by subcutaneous injection of MCF-7 and MDA-MB231 cells in NMRI-nunu mice In thispaper it has been demonstrated that morphine significantlyreduced tumor growth through a p53-dependent mecha-nism Additionally in these mice naloxone increased thegrowth-inhibitory effects of morphine Similar results wereobtained in rat model of colon cancer in which subcutaneousadministration of morphine leads to significant decreasein the hepatic tumor burden On the contrary severalexperimental studies demonstrated that morphine increasedtumor growth Gupta et al in orthotropic mouse model ofbreast cancer obtained by injection of MCF-7 cells into themammary fat pad of nudemice demonstrated thatmorphinein clinically relevant doses increased tumor growth This

4 BioMed Research International

Prol

ifera

tion

()

Days

0

50

100

150

200

250

300

1 2 3 4 5

CTRM 1120583M

M 10120583MM 100120583M

(e)

Relat

ive t

o ac

tion

()

CTR M 1120583M M 10120583M M 100120583M0

051

152

253

354

CTR M 1120583M M 10120583M M 100120583M

P53

120573-Actin

(f)

(g)

(a) (b) (c) (d)

CTR M 1120583M M 10120583M M 100120583M

Figure 1 Morphine stimulates proliferation in MDAMB231 cell lines MDAMB231 cells were incubated in medium containing (a) mediumcontrol (b) 1 120583mmorphine (c) 10120583mmorphine and (d) 100 120583mmorphine Cell migration rates were quantitatively assessed by counting thenumber of cells in the denuded area at 0 24 and 48 h after wound induction At 48 h after wound induction there were clearly more cellsin the denuded area of morphine treated cells than untreated cells (e) MTT assay results show an enhancement of proliferation in breastcancer cells treated with morphine with respect to control cells Data are representative of three independent experiments (119875 value lt 005)(f-g) Western blot showing that morphine reduces the expression of p53 in MDAMB231 cells treated with morphine (lanes 2 3 and 4) withrespect to controls (lane 1) in dose dependent manner 120573-Actin was used as loading control

was associated with increased angiogenesis and inhibitionof apoptosis and promotion of cell cycle progression [20]In this study it was also reported that naloxone itself hadno significant effect on angiogenesis According to theseresults in another study it was demonstrated that morphinesubcutaneously administrated in mice increased the tumorgrowth in mouse model of leukaemia and sarcoma In thesemice morphine had also a general immunosuppressive effect[25]

These contrasting results are probably associated withdifferent concentration andor time of administration ofmorphine In fact in vitro and in vivo studies demonstratedthat tumor-enhancing effects with morphine occur afteradministration of low daily doses or single dose of morphine[22] while tumor suppression occurs after chronic high dosesof morphine [11 15 16]

Thus there is a dilemma about the effects of morphineon cancer cell growth and angiogenesis in various types ofcancer [23] The role of morphine in the regulation of tumorcell growth is not yet correctly established

It has also been demonstrated that the 120583-opioid recep-tor by which morphine exerts its action directly regu-lates tumor growth and metastasis On the basis of theseresults different mechanisms of opioid receptor-mediatedinfluence of morphine on tumor growth have been proposedMorphine as mentioned above after binding to the 120583-opioid receptor regulates cell cycle progression by stimulat-ing mitogen-activated protein kinase (MAPK)extracellulargrowth factor (Erk) pathways [20] Alternatively morphinecan mediate apoptosis by activating phosphatidylinositol 3-kinase (PI3K)protein kinase B (Akt) pathway [26] Addi-tionallymorphine by upregulation of urokinase plasminogen

BioMed Research International 5

0 7 14 21 28 35 420

500

1000

1500

Days after tumor injection

CTRMorphine

Tum

or v

olum

e (m

m3)

(a)

Control

(b)

Morphine

(c)

Figure 2 Morphine promotes the tumor growth in TBNC mouse model (a) Morphine promotes tumor growth in breast tumor xenograftmodel Breast tumor growth in mice treated with vehicle (∙) and morphine (119887119897119886119888119896119904119898119886119897119897119904119902119906119886119903119890) Tumor volumes increased after 28 days ofmorphine treatment until 35 days (119875 lt 005) as compared with control (vehicle-treated) Each point represents the mean of five separateexperiments (b-c) Measurements of fluorescence per second depicting microvessel tumor (FITC-DEXTRANE) using MacroFluo imagesshowed that morphine enhances the angiogenesis in tumor of mice (b) with respect to controls (c)

CTR

PECA

M-1

(a)

Morphine

(b)

Figure 3 Morphine promotes angiogenesis formation in heterotopic mouse model of breast cancer Immunohistochemical analysis forPECAM-1showed the enhancement of PECAM-1 expression inmorphine treated group (b) compared to controls (a) Arrows indicate positivestaining of microvessel staining

activator (uPA) expression induces metastasis formation[27] while by transactivation of VEGF receptor it inducesangiogenesis [28] Finally morphine affects also the functionof T lymphocytes leading to immunosuppression [29]

It has been proposed that morphine plays also a role intumor apoptosis Apoptosis is a form of cell death in whicha programmed sequence of events leads to the eliminationof cells without releasing harmful substances into the sur-rounding area It is noted that apoptosis is regulated by twopathways the mitochondrial-mediated pathway (intrinsic)[30] and death receptor-mediated pathway (extrinsic) [31] Itis noted that in cancer cells apoptosis is deregulated andthis leads to quick proliferation and tumor growth [32 33]Morphine was shown to induce apoptosis of macrophages T

lymphocytes and human endothelial cells [34 35] Experi-ments performed on human tumor cell lines demonstratedthat morphine in high concentration induces apoptosis andinhibits cancer cell growth by activation of different signalpathways involving caspase 39 and cytochrome c sigma-2 receptor Additionally in SH-SY5Y cells morphine hasantiapoptotic effect by antagonizing doxorubicin [36] Thesediscrepancies also in these cases are associatedwith differentcell line tumor type used andor in vivo dosetime of mor-phine administrated

Recent data demonstrated a role of morphine in angio-genesis Angiogenesis is required for invasive tumor growthand metastasis and represents an important point in thecontrol of cancer progression Proangiogenic activity of

6 BioMed Research International

morphine was demonstrated in the MCF-7 breast cancermodel In these mice morphine at clinically relevant con-centrations enhanced tumor neovascularization [20] In ananimal model of hormone-dependent breast cancer it hasalso been demonstrated that morphine promoted activationof vascular endothelial growth factor (VEGF) receptor andincreased metastasis [21 29] It has been proposed thatmorphine explains its proangiogenic activity by stimula-tion of mitogen-activated protein kinase (MAPK) signallingpathway via G protein-coupled receptors and nitric oxide(NO) Alternatively several in vivo studies provided evidencethat morphine can induce tumor growth by upregulationof cyclooxygenase-2 (COX-2) [37ndash40] and or prostaglandinE2-mediated stimulation of angiogenesis [41ndash44] On thecontrary several in vivo and in vitro studies demonstratedthat morphine can inhibit angiogenesis by regulation ofdifferent pathways [8 34 45 45ndash52] These different resultscan be due to different experimental conditions (cell linetumor type used andor dosetime of morphine) Morphineplays a role not only in tumor cell growth but also inmetastasis formation which is the main process related tomost cancer deaths and failure in cancer treatment [53 54]

For these reasons in order to elucidate the role ofmorphine in regulation of tumor growth and angiogenesis inbreast cancer we performed in vitro and in vivo studies on theMDAMB231 breast cancer cellsThese cells are triple negative(basal-like) breast cancer (TNBC) cells It is noted that TNBCdoes not express the estrogen receptor (ER) progesteronereceptor (PR) or human epidermal growth factor receptortype 2 (HER2) Due to lack of both hormone receptors andHER2 expression patients with this type of breast cancer haveno chance to benefit from the endocrine therapy and HER2targeted therapy In addition we used immunodeficient micethat are able to mimic the compromised immune systemof a patient with breast cancer So in these experimentalconditions it has become interesting for us to study therole of morphine in the regulation of cancer cell growthand angiogenesis Our data showed that morphine in TNBCat clinical relevant doses promotes tumor angiogenesis andincreases breast cancer progression For these reasons it isvery important for the management of severe pain associatedwith cancer to consider accurately the dose and route ofadministration of morphine in order to avoid severe effectof cancer progression Further studies are ongoing in ourlaboratory in order to dissect the molecular mechanismsunderlying the role of morphine in cancer development andmetastasis formation in breast cancer Specifically we willgenerate orthotropic mouse models of breast cancer by usingnot only MDAMB231 cells but also MCF-7 (human breastadenocarcinoma cell line) cells which represent estrogenreceptor (ER) positive control cell lines The results obtainedfrom these data will shed light on the role of morphine inregulation of breast cancer progression

Conflict of Interests

The authors have no other relevant affiliations or financialinterests with any organization or entity No writing assis-tance was used in the production of paper

Acknowledgments

The authors would like to specially thank MassimilianoSpinelli Data Manager of SSD Sperimentazione animaleIstituto Nazionale per lo Studio e la Cura dei TumorildquoFondazione G Pascalerdquo IRCCS Italy for kind help inproviding informatics assistance and Dr Di Napoli Danielefor supervising animal care This work was supported by the5x mille and current research programs of National Instituteof Tumors IRCCS ldquoFoundation G Pascalerdquo Naples (Italy)

References

[1] J N Lickiss ldquoApproaching cancer pain reliefrdquo European Journalof Pain vol 5 pp 5ndash14 2001

[2] R Schmitz ldquoFriedrich Wilhelm Serturner and the discovery ofmorphinerdquo Pharmacy in History vol 27 no 2 pp 61ndash74 1985

[3] G W Pasternak ldquoPharmacological mechanisms of opioidanalgesicsrdquo Clinical Neuropharmacology vol 16 no 1 pp 1ndash181993

[4] B L Kieffer and C Gaveriaux-Ruff ldquoExploring the opioidsystem by gene knockoutrdquo Progress in Neurobiology vol 66 no5 pp 285ndash306 2002

[5] P W Mantyh ldquoCancer pain and its impact on diagnosissurvival and quality of liferdquo Nature Reviews Neuroscience vol7 no 10 pp 797ndash809 2006

[6] N Sueoka E Sueoka S Okabe and H Fujiki ldquoAnti-cancereffects of morphine through inhibition of tumour necrosisfactor-120572 release and mRNA expressionrdquo Carcinogenesis vol 17no 11 pp 2337ndash2341 1996

[7] R Maneckjee and J D Minna ldquoOpioids induce while nicotinesuppresses apoptosis in human lung cancer cellsrdquo Cell Growthand Differentiation vol 5 no 10 pp 1033ndash1040 1994

[8] A Pasi B Qu R Steiner H-J Senn W Bar and F S MessihaldquoAngiogenesis modulation with opioidsrdquo General Pharmacol-ogy vol 22 no 6 pp 1077ndash1079 1991

[9] A Hatzoglou E Bakogeorgou E Papakonstanti C StournarasD S Emmanouel and E Castanas ldquoIdentification and char-acterization of opioid and somatostatin binding sites in theopossum kidney (OK) cell line and their effect on growthrdquoJournal of Cellular Biochemistry vol 63 no 4 pp 410ndash421 1996

[10] A Hatzoglou L Ouafik E Bakogeorgou K Thermos and ECastanas ldquoMorphine cross-reacts with somatostatin receptorSSTR2 in the T47D human breast cancer cell line and decreasescell growthrdquo Cancer Research vol 55 no 23 pp 5632ndash56361995

[11] R Maneckjee R Biswas and B K Vonderhaar ldquoBinding ofopioids to humanMCF-7 breast cancer cells and their effects ongrowthrdquo Cancer Research vol 50 no 8 pp 2234ndash2238 1990

[12] M Kampa E Bakogeorgou A Hatzoglou A Damianaki P-MMartin and E Castanas ldquoOpioid alkaloids and casomorphinpeptides decrease the proliferation of prostatic cancer cell lines(LNCaP PC3 and DU145) through a partial interaction withopioid receptorsrdquo European Journal of Pharmacology vol 335no 2-3 pp 255ndash265 1997

[13] I Tegeder S Grosch A Schmidtko et al ldquoG protein-independent G1 cell cycle block and apoptosis with morphinein adenocarcinoma cells involvement of p53 phosphorylationrdquoCancer Research vol 63 no 8 pp 1846ndash1852 2003

BioMed Research International 7

[14] M P Yeager and T A Colacchio ldquoEffect ofmorphine on growthof metastatic colon cancer in vivordquo Archives of Surgery vol 126no 4 pp 454ndash456 1991

[15] T Sasamura S Nakamura Y Iida et al ldquoMorphine analgesiasuppresses tumor growth and metastasis in a mouse modelof cancer pain produced by orthotopic tumor inoculationrdquoEuropean Journal of Pharmacology vol 441 no 3 pp 185ndash1912002

[16] YHarimaya K Koizumi T AndohHNojima Y Kuraishi andI Saiki ldquoPotential ability of morphine to inhibit the adhesioninvasion and metastasis of metastatic colon 26-L5 carcinomacellsrdquo Cancer Letters vol 187 no 1-2 pp 121ndash127 2002

[17] R H Simon and T E Arbo ldquoMorphine increases metastatictumor growthrdquo Brain Research Bulletin vol 16 no 3 pp 363ndash367 1986

[18] J W Lewis Y Shavit G W Terman L R Nelson R P Galeand J C Liebeskind ldquoApparent involvement of opioid peptidesin stress-induced enhancement of tumor growthrdquo Peptides vol4 no 5 pp 635ndash638 1983

[19] M G Sergeeva Z V Grishina and S D VarfolomeyevldquoMorphine effect on proliferation of normal and tumor cells ofimmune originrdquo Immunology Letters vol 36 no 2 pp 215ndash2181993

[20] K Gupta S Kshirsagar L Chang et al ldquoMorphine stimu-lates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growthrdquo Can-cer Research vol 62 no 15 pp 4491ndash4498 2002

[21] M Farooqui Y Li T Rogers et al ldquoCOX-2 inhibitor celecoxibprevents chronic morphine-induced promotion of angiogene-sis tumour growth metastasis and mortality without compro-mising analgesiardquo British Journal of Cancer vol 97 no 11 pp1523ndash1531 2007

[22] J Zong and G M Pollack ldquoMorphine antinociceptionis enhanced in mdr1a gene-deficient micerdquo PharmaceuticalResearch vol 17 no 6 pp 749ndash753 2000

[23] S Bimonte A Barbieri G Palma and C Arra ldquoThe role ofmorphine in animal models of human cancer does morphinepromote or inhibit the tumor growthrdquo BioMed Research Inter-national vol 2013 Article ID 258141 4 pages 2013

[24] J Nguyen ldquoMorphine stimulates cancer progression and mastcell activation and impairs survival in transgenic mice withbreast cancerrdquo British Journal of Anaesthesia vol 113 supple-ment 1 pp i4ndashi13 2014

[25] M Ishikawa K Tanno A Kamo Y Takayanagi and K-ISasaki ldquoEnhancement of tumor growth by morphine and itspossible mechanism in micerdquo Biological and PharmaceuticalBulletin vol 16 no 8 pp 762ndash766 1993

[26] M Iglesias M F Segura J X Comella and G Olmos ldquo120583-opioid receptor activation prevents apoptosis following serumwithdrawal in differentiated SH-SY5Y cells and cortical neuronsvia phosphatidylinositol 3-kinaserdquoNeuropharmacology vol 44no 4 pp 482ndash492 2003

[27] K Gach J Szemraj J Fichna M Piestrzeniewicz D S Delbroand A Janecka ldquoThe influence of opioids on urokinase plas-minogen activator on protein andmRNA level inMCF-7 breastcancer cell linerdquo Chemical Biology and Drug Design vol 74 no4 pp 390ndash396 2009

[28] P A Singleton and J Moss ldquoEffect of perioperative opioids oncancer recurrence a hypothesisrdquo Future Oncology vol 6 no 8pp 1237ndash1242 2010

[29] C Chen M Farooqui and K Gupta ldquoMorphine stimulatesvascular endothelial growth factor-like signaling in mouse

retinal endothelial cellsrdquo Current Neurovascular Research vol3 no 3 pp 171ndash180 2006

[30] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[31] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[32] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[33] M O Hengartner ldquoThe biochemistry of apoptosisrdquoNature vol407 no 6805 pp 770ndash776 2000

[34] P-N Hsiao M-C Chang W-F Cheng et al ldquoMorphineinduces apoptosis of human endothelial cells through nitricoxide and reactive oxygen species pathwaysrdquo Toxicology vol256 no 1-2 pp 83ndash91 2009

[35] A A Kapasi S A CosciaM P Pandya and P C Singhal ldquoMor-phine modulates HIV-1 gp160-induced murine macrophageand human monocyte apoptosis by disparate waysrdquo Journal ofNeuroimmunology vol 148 no 1-2 pp 86ndash96 2004

[36] X Lin Q Li Y-J Wang et al ldquoMorphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor120581B transcriptional activation in neuroblastoma SH-SY5Y cellsrdquoBiochemical Journal vol 406 no 2 pp 215ndash221 2007

[37] R Arerangaiah N Chalasani A M Udager et al ldquoOpioidsinduce renal abnormalities in tumor-bearing micerdquo NephronmdashExperimental Nephrology vol 105 no 3 pp e80ndashe89 2007

[38] D Salvemini T P Misko J L Masferrer K Seibert M G Cur-rie and P Needleman ldquoNitric oxide activates cyclooxygenaseenzymesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 15 pp 7240ndash7244 1993

[39] E Nedelec A Abid C Cipolletta et al ldquoStimulation ofcyclooxygenase-2-activity by nitric oxide-derived species inrat chondrocyte lack of contribution to loss of cartilageanabolismrdquo Biochemical Pharmacology vol 61 no 8 pp 965ndash978 2001

[40] M Farooqui ZHGeng E J StephensonN Zaveri D Yee andK Gupta ldquoNaloxone acts as an antagonist of estrogen receptoractivity in MCF-7 cellsrdquo Molecular Cancer Therapeutics vol 5no 3 pp 611ndash620 2006

[41] D Salvemini K Seibert J L Masferrer T P Misko M GCurrie and P Needleman ldquoEndogenous nitric oxide enhancesprostaglandin production in a model of renal inflammationrdquoJournal of Clinical Investigation vol 93 no 5 pp 1940ndash19471994

[42] R J Griffin BWWilliams RWild JM CherringtonH Parkand CW Song ldquoSimultaneous inhibition of the receptor kinaseactivity of vascular endothelial fibroblast and platelet-derivedgrowth factors suppresses tumor growth and enhances tumorradiation responserdquo Cancer Research vol 62 no 6 pp 1702ndash1706 2002

[43] K M Leahy R L Ornberg Y Wang B S Zweifel A T Kokiand J L Masferrer ldquoCyclooxygenase-2 inhibition by celecoxibreduces proliferation and induces apoptosis in angiogenicendothelial cells in vivordquoCancer Research vol 62 no 3 pp 625ndash631 2002

[44] S H Chang C H Liu and R Conway ldquoRole of prostaglandinE2-dependent angiogenic switch in cyclooxygenase 2-inducedbreast cancer progressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 2 pp 591ndash596 2004

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006

4 BioMed Research International

Prol

ifera

tion

()

Days

0

50

100

150

200

250

300

1 2 3 4 5

CTRM 1120583M

M 10120583MM 100120583M

(e)

Relat

ive t

o ac

tion

()

CTR M 1120583M M 10120583M M 100120583M0

051

152

253

354

CTR M 1120583M M 10120583M M 100120583M

P53

120573-Actin

(f)

(g)

(a) (b) (c) (d)

CTR M 1120583M M 10120583M M 100120583M

Figure 1 Morphine stimulates proliferation in MDAMB231 cell lines MDAMB231 cells were incubated in medium containing (a) mediumcontrol (b) 1 120583mmorphine (c) 10120583mmorphine and (d) 100 120583mmorphine Cell migration rates were quantitatively assessed by counting thenumber of cells in the denuded area at 0 24 and 48 h after wound induction At 48 h after wound induction there were clearly more cellsin the denuded area of morphine treated cells than untreated cells (e) MTT assay results show an enhancement of proliferation in breastcancer cells treated with morphine with respect to control cells Data are representative of three independent experiments (119875 value lt 005)(f-g) Western blot showing that morphine reduces the expression of p53 in MDAMB231 cells treated with morphine (lanes 2 3 and 4) withrespect to controls (lane 1) in dose dependent manner 120573-Actin was used as loading control

was associated with increased angiogenesis and inhibitionof apoptosis and promotion of cell cycle progression [20]In this study it was also reported that naloxone itself hadno significant effect on angiogenesis According to theseresults in another study it was demonstrated that morphinesubcutaneously administrated in mice increased the tumorgrowth in mouse model of leukaemia and sarcoma In thesemice morphine had also a general immunosuppressive effect[25]

These contrasting results are probably associated withdifferent concentration andor time of administration ofmorphine In fact in vitro and in vivo studies demonstratedthat tumor-enhancing effects with morphine occur afteradministration of low daily doses or single dose of morphine[22] while tumor suppression occurs after chronic high dosesof morphine [11 15 16]

Thus there is a dilemma about the effects of morphineon cancer cell growth and angiogenesis in various types ofcancer [23] The role of morphine in the regulation of tumorcell growth is not yet correctly established

It has also been demonstrated that the 120583-opioid recep-tor by which morphine exerts its action directly regu-lates tumor growth and metastasis On the basis of theseresults different mechanisms of opioid receptor-mediatedinfluence of morphine on tumor growth have been proposedMorphine as mentioned above after binding to the 120583-opioid receptor regulates cell cycle progression by stimulat-ing mitogen-activated protein kinase (MAPK)extracellulargrowth factor (Erk) pathways [20] Alternatively morphinecan mediate apoptosis by activating phosphatidylinositol 3-kinase (PI3K)protein kinase B (Akt) pathway [26] Addi-tionallymorphine by upregulation of urokinase plasminogen

BioMed Research International 5

0 7 14 21 28 35 420

500

1000

1500

Days after tumor injection

CTRMorphine

Tum

or v

olum

e (m

m3)

(a)

Control

(b)

Morphine

(c)

Figure 2 Morphine promotes the tumor growth in TBNC mouse model (a) Morphine promotes tumor growth in breast tumor xenograftmodel Breast tumor growth in mice treated with vehicle (∙) and morphine (119887119897119886119888119896119904119898119886119897119897119904119902119906119886119903119890) Tumor volumes increased after 28 days ofmorphine treatment until 35 days (119875 lt 005) as compared with control (vehicle-treated) Each point represents the mean of five separateexperiments (b-c) Measurements of fluorescence per second depicting microvessel tumor (FITC-DEXTRANE) using MacroFluo imagesshowed that morphine enhances the angiogenesis in tumor of mice (b) with respect to controls (c)

CTR

PECA

M-1

(a)

Morphine

(b)

Figure 3 Morphine promotes angiogenesis formation in heterotopic mouse model of breast cancer Immunohistochemical analysis forPECAM-1showed the enhancement of PECAM-1 expression inmorphine treated group (b) compared to controls (a) Arrows indicate positivestaining of microvessel staining

activator (uPA) expression induces metastasis formation[27] while by transactivation of VEGF receptor it inducesangiogenesis [28] Finally morphine affects also the functionof T lymphocytes leading to immunosuppression [29]

It has been proposed that morphine plays also a role intumor apoptosis Apoptosis is a form of cell death in whicha programmed sequence of events leads to the eliminationof cells without releasing harmful substances into the sur-rounding area It is noted that apoptosis is regulated by twopathways the mitochondrial-mediated pathway (intrinsic)[30] and death receptor-mediated pathway (extrinsic) [31] Itis noted that in cancer cells apoptosis is deregulated andthis leads to quick proliferation and tumor growth [32 33]Morphine was shown to induce apoptosis of macrophages T

lymphocytes and human endothelial cells [34 35] Experi-ments performed on human tumor cell lines demonstratedthat morphine in high concentration induces apoptosis andinhibits cancer cell growth by activation of different signalpathways involving caspase 39 and cytochrome c sigma-2 receptor Additionally in SH-SY5Y cells morphine hasantiapoptotic effect by antagonizing doxorubicin [36] Thesediscrepancies also in these cases are associatedwith differentcell line tumor type used andor in vivo dosetime of mor-phine administrated

Recent data demonstrated a role of morphine in angio-genesis Angiogenesis is required for invasive tumor growthand metastasis and represents an important point in thecontrol of cancer progression Proangiogenic activity of

6 BioMed Research International

morphine was demonstrated in the MCF-7 breast cancermodel In these mice morphine at clinically relevant con-centrations enhanced tumor neovascularization [20] In ananimal model of hormone-dependent breast cancer it hasalso been demonstrated that morphine promoted activationof vascular endothelial growth factor (VEGF) receptor andincreased metastasis [21 29] It has been proposed thatmorphine explains its proangiogenic activity by stimula-tion of mitogen-activated protein kinase (MAPK) signallingpathway via G protein-coupled receptors and nitric oxide(NO) Alternatively several in vivo studies provided evidencethat morphine can induce tumor growth by upregulationof cyclooxygenase-2 (COX-2) [37ndash40] and or prostaglandinE2-mediated stimulation of angiogenesis [41ndash44] On thecontrary several in vivo and in vitro studies demonstratedthat morphine can inhibit angiogenesis by regulation ofdifferent pathways [8 34 45 45ndash52] These different resultscan be due to different experimental conditions (cell linetumor type used andor dosetime of morphine) Morphineplays a role not only in tumor cell growth but also inmetastasis formation which is the main process related tomost cancer deaths and failure in cancer treatment [53 54]

For these reasons in order to elucidate the role ofmorphine in regulation of tumor growth and angiogenesis inbreast cancer we performed in vitro and in vivo studies on theMDAMB231 breast cancer cellsThese cells are triple negative(basal-like) breast cancer (TNBC) cells It is noted that TNBCdoes not express the estrogen receptor (ER) progesteronereceptor (PR) or human epidermal growth factor receptortype 2 (HER2) Due to lack of both hormone receptors andHER2 expression patients with this type of breast cancer haveno chance to benefit from the endocrine therapy and HER2targeted therapy In addition we used immunodeficient micethat are able to mimic the compromised immune systemof a patient with breast cancer So in these experimentalconditions it has become interesting for us to study therole of morphine in the regulation of cancer cell growthand angiogenesis Our data showed that morphine in TNBCat clinical relevant doses promotes tumor angiogenesis andincreases breast cancer progression For these reasons it isvery important for the management of severe pain associatedwith cancer to consider accurately the dose and route ofadministration of morphine in order to avoid severe effectof cancer progression Further studies are ongoing in ourlaboratory in order to dissect the molecular mechanismsunderlying the role of morphine in cancer development andmetastasis formation in breast cancer Specifically we willgenerate orthotropic mouse models of breast cancer by usingnot only MDAMB231 cells but also MCF-7 (human breastadenocarcinoma cell line) cells which represent estrogenreceptor (ER) positive control cell lines The results obtainedfrom these data will shed light on the role of morphine inregulation of breast cancer progression

Conflict of Interests

The authors have no other relevant affiliations or financialinterests with any organization or entity No writing assis-tance was used in the production of paper

Acknowledgments

The authors would like to specially thank MassimilianoSpinelli Data Manager of SSD Sperimentazione animaleIstituto Nazionale per lo Studio e la Cura dei TumorildquoFondazione G Pascalerdquo IRCCS Italy for kind help inproviding informatics assistance and Dr Di Napoli Danielefor supervising animal care This work was supported by the5x mille and current research programs of National Instituteof Tumors IRCCS ldquoFoundation G Pascalerdquo Naples (Italy)

References

[1] J N Lickiss ldquoApproaching cancer pain reliefrdquo European Journalof Pain vol 5 pp 5ndash14 2001

[2] R Schmitz ldquoFriedrich Wilhelm Serturner and the discovery ofmorphinerdquo Pharmacy in History vol 27 no 2 pp 61ndash74 1985

[3] G W Pasternak ldquoPharmacological mechanisms of opioidanalgesicsrdquo Clinical Neuropharmacology vol 16 no 1 pp 1ndash181993

[4] B L Kieffer and C Gaveriaux-Ruff ldquoExploring the opioidsystem by gene knockoutrdquo Progress in Neurobiology vol 66 no5 pp 285ndash306 2002

[5] P W Mantyh ldquoCancer pain and its impact on diagnosissurvival and quality of liferdquo Nature Reviews Neuroscience vol7 no 10 pp 797ndash809 2006

[6] N Sueoka E Sueoka S Okabe and H Fujiki ldquoAnti-cancereffects of morphine through inhibition of tumour necrosisfactor-120572 release and mRNA expressionrdquo Carcinogenesis vol 17no 11 pp 2337ndash2341 1996

[7] R Maneckjee and J D Minna ldquoOpioids induce while nicotinesuppresses apoptosis in human lung cancer cellsrdquo Cell Growthand Differentiation vol 5 no 10 pp 1033ndash1040 1994

[8] A Pasi B Qu R Steiner H-J Senn W Bar and F S MessihaldquoAngiogenesis modulation with opioidsrdquo General Pharmacol-ogy vol 22 no 6 pp 1077ndash1079 1991

[9] A Hatzoglou E Bakogeorgou E Papakonstanti C StournarasD S Emmanouel and E Castanas ldquoIdentification and char-acterization of opioid and somatostatin binding sites in theopossum kidney (OK) cell line and their effect on growthrdquoJournal of Cellular Biochemistry vol 63 no 4 pp 410ndash421 1996

[10] A Hatzoglou L Ouafik E Bakogeorgou K Thermos and ECastanas ldquoMorphine cross-reacts with somatostatin receptorSSTR2 in the T47D human breast cancer cell line and decreasescell growthrdquo Cancer Research vol 55 no 23 pp 5632ndash56361995

[11] R Maneckjee R Biswas and B K Vonderhaar ldquoBinding ofopioids to humanMCF-7 breast cancer cells and their effects ongrowthrdquo Cancer Research vol 50 no 8 pp 2234ndash2238 1990

[12] M Kampa E Bakogeorgou A Hatzoglou A Damianaki P-MMartin and E Castanas ldquoOpioid alkaloids and casomorphinpeptides decrease the proliferation of prostatic cancer cell lines(LNCaP PC3 and DU145) through a partial interaction withopioid receptorsrdquo European Journal of Pharmacology vol 335no 2-3 pp 255ndash265 1997

[13] I Tegeder S Grosch A Schmidtko et al ldquoG protein-independent G1 cell cycle block and apoptosis with morphinein adenocarcinoma cells involvement of p53 phosphorylationrdquoCancer Research vol 63 no 8 pp 1846ndash1852 2003

BioMed Research International 7

[14] M P Yeager and T A Colacchio ldquoEffect ofmorphine on growthof metastatic colon cancer in vivordquo Archives of Surgery vol 126no 4 pp 454ndash456 1991

[15] T Sasamura S Nakamura Y Iida et al ldquoMorphine analgesiasuppresses tumor growth and metastasis in a mouse modelof cancer pain produced by orthotopic tumor inoculationrdquoEuropean Journal of Pharmacology vol 441 no 3 pp 185ndash1912002

[16] YHarimaya K Koizumi T AndohHNojima Y Kuraishi andI Saiki ldquoPotential ability of morphine to inhibit the adhesioninvasion and metastasis of metastatic colon 26-L5 carcinomacellsrdquo Cancer Letters vol 187 no 1-2 pp 121ndash127 2002

[17] R H Simon and T E Arbo ldquoMorphine increases metastatictumor growthrdquo Brain Research Bulletin vol 16 no 3 pp 363ndash367 1986

[18] J W Lewis Y Shavit G W Terman L R Nelson R P Galeand J C Liebeskind ldquoApparent involvement of opioid peptidesin stress-induced enhancement of tumor growthrdquo Peptides vol4 no 5 pp 635ndash638 1983

[19] M G Sergeeva Z V Grishina and S D VarfolomeyevldquoMorphine effect on proliferation of normal and tumor cells ofimmune originrdquo Immunology Letters vol 36 no 2 pp 215ndash2181993

[20] K Gupta S Kshirsagar L Chang et al ldquoMorphine stimu-lates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growthrdquo Can-cer Research vol 62 no 15 pp 4491ndash4498 2002

[21] M Farooqui Y Li T Rogers et al ldquoCOX-2 inhibitor celecoxibprevents chronic morphine-induced promotion of angiogene-sis tumour growth metastasis and mortality without compro-mising analgesiardquo British Journal of Cancer vol 97 no 11 pp1523ndash1531 2007

[22] J Zong and G M Pollack ldquoMorphine antinociceptionis enhanced in mdr1a gene-deficient micerdquo PharmaceuticalResearch vol 17 no 6 pp 749ndash753 2000

[23] S Bimonte A Barbieri G Palma and C Arra ldquoThe role ofmorphine in animal models of human cancer does morphinepromote or inhibit the tumor growthrdquo BioMed Research Inter-national vol 2013 Article ID 258141 4 pages 2013

[24] J Nguyen ldquoMorphine stimulates cancer progression and mastcell activation and impairs survival in transgenic mice withbreast cancerrdquo British Journal of Anaesthesia vol 113 supple-ment 1 pp i4ndashi13 2014

[25] M Ishikawa K Tanno A Kamo Y Takayanagi and K-ISasaki ldquoEnhancement of tumor growth by morphine and itspossible mechanism in micerdquo Biological and PharmaceuticalBulletin vol 16 no 8 pp 762ndash766 1993

[26] M Iglesias M F Segura J X Comella and G Olmos ldquo120583-opioid receptor activation prevents apoptosis following serumwithdrawal in differentiated SH-SY5Y cells and cortical neuronsvia phosphatidylinositol 3-kinaserdquoNeuropharmacology vol 44no 4 pp 482ndash492 2003

[27] K Gach J Szemraj J Fichna M Piestrzeniewicz D S Delbroand A Janecka ldquoThe influence of opioids on urokinase plas-minogen activator on protein andmRNA level inMCF-7 breastcancer cell linerdquo Chemical Biology and Drug Design vol 74 no4 pp 390ndash396 2009

[28] P A Singleton and J Moss ldquoEffect of perioperative opioids oncancer recurrence a hypothesisrdquo Future Oncology vol 6 no 8pp 1237ndash1242 2010

[29] C Chen M Farooqui and K Gupta ldquoMorphine stimulatesvascular endothelial growth factor-like signaling in mouse

retinal endothelial cellsrdquo Current Neurovascular Research vol3 no 3 pp 171ndash180 2006

[30] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[31] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[32] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[33] M O Hengartner ldquoThe biochemistry of apoptosisrdquoNature vol407 no 6805 pp 770ndash776 2000

[34] P-N Hsiao M-C Chang W-F Cheng et al ldquoMorphineinduces apoptosis of human endothelial cells through nitricoxide and reactive oxygen species pathwaysrdquo Toxicology vol256 no 1-2 pp 83ndash91 2009

[35] A A Kapasi S A CosciaM P Pandya and P C Singhal ldquoMor-phine modulates HIV-1 gp160-induced murine macrophageand human monocyte apoptosis by disparate waysrdquo Journal ofNeuroimmunology vol 148 no 1-2 pp 86ndash96 2004

[36] X Lin Q Li Y-J Wang et al ldquoMorphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor120581B transcriptional activation in neuroblastoma SH-SY5Y cellsrdquoBiochemical Journal vol 406 no 2 pp 215ndash221 2007

[37] R Arerangaiah N Chalasani A M Udager et al ldquoOpioidsinduce renal abnormalities in tumor-bearing micerdquo NephronmdashExperimental Nephrology vol 105 no 3 pp e80ndashe89 2007

[38] D Salvemini T P Misko J L Masferrer K Seibert M G Cur-rie and P Needleman ldquoNitric oxide activates cyclooxygenaseenzymesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 15 pp 7240ndash7244 1993

[39] E Nedelec A Abid C Cipolletta et al ldquoStimulation ofcyclooxygenase-2-activity by nitric oxide-derived species inrat chondrocyte lack of contribution to loss of cartilageanabolismrdquo Biochemical Pharmacology vol 61 no 8 pp 965ndash978 2001

[40] M Farooqui ZHGeng E J StephensonN Zaveri D Yee andK Gupta ldquoNaloxone acts as an antagonist of estrogen receptoractivity in MCF-7 cellsrdquo Molecular Cancer Therapeutics vol 5no 3 pp 611ndash620 2006

[41] D Salvemini K Seibert J L Masferrer T P Misko M GCurrie and P Needleman ldquoEndogenous nitric oxide enhancesprostaglandin production in a model of renal inflammationrdquoJournal of Clinical Investigation vol 93 no 5 pp 1940ndash19471994

[42] R J Griffin BWWilliams RWild JM CherringtonH Parkand CW Song ldquoSimultaneous inhibition of the receptor kinaseactivity of vascular endothelial fibroblast and platelet-derivedgrowth factors suppresses tumor growth and enhances tumorradiation responserdquo Cancer Research vol 62 no 6 pp 1702ndash1706 2002

[43] K M Leahy R L Ornberg Y Wang B S Zweifel A T Kokiand J L Masferrer ldquoCyclooxygenase-2 inhibition by celecoxibreduces proliferation and induces apoptosis in angiogenicendothelial cells in vivordquoCancer Research vol 62 no 3 pp 625ndash631 2002

[44] S H Chang C H Liu and R Conway ldquoRole of prostaglandinE2-dependent angiogenic switch in cyclooxygenase 2-inducedbreast cancer progressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 2 pp 591ndash596 2004

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006

BioMed Research International 5

0 7 14 21 28 35 420

500

1000

1500

Days after tumor injection

CTRMorphine

Tum

or v

olum

e (m

m3)

(a)

Control

(b)

Morphine

(c)

Figure 2 Morphine promotes the tumor growth in TBNC mouse model (a) Morphine promotes tumor growth in breast tumor xenograftmodel Breast tumor growth in mice treated with vehicle (∙) and morphine (119887119897119886119888119896119904119898119886119897119897119904119902119906119886119903119890) Tumor volumes increased after 28 days ofmorphine treatment until 35 days (119875 lt 005) as compared with control (vehicle-treated) Each point represents the mean of five separateexperiments (b-c) Measurements of fluorescence per second depicting microvessel tumor (FITC-DEXTRANE) using MacroFluo imagesshowed that morphine enhances the angiogenesis in tumor of mice (b) with respect to controls (c)

CTR

PECA

M-1

(a)

Morphine

(b)

Figure 3 Morphine promotes angiogenesis formation in heterotopic mouse model of breast cancer Immunohistochemical analysis forPECAM-1showed the enhancement of PECAM-1 expression inmorphine treated group (b) compared to controls (a) Arrows indicate positivestaining of microvessel staining

activator (uPA) expression induces metastasis formation[27] while by transactivation of VEGF receptor it inducesangiogenesis [28] Finally morphine affects also the functionof T lymphocytes leading to immunosuppression [29]

It has been proposed that morphine plays also a role intumor apoptosis Apoptosis is a form of cell death in whicha programmed sequence of events leads to the eliminationof cells without releasing harmful substances into the sur-rounding area It is noted that apoptosis is regulated by twopathways the mitochondrial-mediated pathway (intrinsic)[30] and death receptor-mediated pathway (extrinsic) [31] Itis noted that in cancer cells apoptosis is deregulated andthis leads to quick proliferation and tumor growth [32 33]Morphine was shown to induce apoptosis of macrophages T

lymphocytes and human endothelial cells [34 35] Experi-ments performed on human tumor cell lines demonstratedthat morphine in high concentration induces apoptosis andinhibits cancer cell growth by activation of different signalpathways involving caspase 39 and cytochrome c sigma-2 receptor Additionally in SH-SY5Y cells morphine hasantiapoptotic effect by antagonizing doxorubicin [36] Thesediscrepancies also in these cases are associatedwith differentcell line tumor type used andor in vivo dosetime of mor-phine administrated

Recent data demonstrated a role of morphine in angio-genesis Angiogenesis is required for invasive tumor growthand metastasis and represents an important point in thecontrol of cancer progression Proangiogenic activity of

6 BioMed Research International

morphine was demonstrated in the MCF-7 breast cancermodel In these mice morphine at clinically relevant con-centrations enhanced tumor neovascularization [20] In ananimal model of hormone-dependent breast cancer it hasalso been demonstrated that morphine promoted activationof vascular endothelial growth factor (VEGF) receptor andincreased metastasis [21 29] It has been proposed thatmorphine explains its proangiogenic activity by stimula-tion of mitogen-activated protein kinase (MAPK) signallingpathway via G protein-coupled receptors and nitric oxide(NO) Alternatively several in vivo studies provided evidencethat morphine can induce tumor growth by upregulationof cyclooxygenase-2 (COX-2) [37ndash40] and or prostaglandinE2-mediated stimulation of angiogenesis [41ndash44] On thecontrary several in vivo and in vitro studies demonstratedthat morphine can inhibit angiogenesis by regulation ofdifferent pathways [8 34 45 45ndash52] These different resultscan be due to different experimental conditions (cell linetumor type used andor dosetime of morphine) Morphineplays a role not only in tumor cell growth but also inmetastasis formation which is the main process related tomost cancer deaths and failure in cancer treatment [53 54]

For these reasons in order to elucidate the role ofmorphine in regulation of tumor growth and angiogenesis inbreast cancer we performed in vitro and in vivo studies on theMDAMB231 breast cancer cellsThese cells are triple negative(basal-like) breast cancer (TNBC) cells It is noted that TNBCdoes not express the estrogen receptor (ER) progesteronereceptor (PR) or human epidermal growth factor receptortype 2 (HER2) Due to lack of both hormone receptors andHER2 expression patients with this type of breast cancer haveno chance to benefit from the endocrine therapy and HER2targeted therapy In addition we used immunodeficient micethat are able to mimic the compromised immune systemof a patient with breast cancer So in these experimentalconditions it has become interesting for us to study therole of morphine in the regulation of cancer cell growthand angiogenesis Our data showed that morphine in TNBCat clinical relevant doses promotes tumor angiogenesis andincreases breast cancer progression For these reasons it isvery important for the management of severe pain associatedwith cancer to consider accurately the dose and route ofadministration of morphine in order to avoid severe effectof cancer progression Further studies are ongoing in ourlaboratory in order to dissect the molecular mechanismsunderlying the role of morphine in cancer development andmetastasis formation in breast cancer Specifically we willgenerate orthotropic mouse models of breast cancer by usingnot only MDAMB231 cells but also MCF-7 (human breastadenocarcinoma cell line) cells which represent estrogenreceptor (ER) positive control cell lines The results obtainedfrom these data will shed light on the role of morphine inregulation of breast cancer progression

Conflict of Interests

The authors have no other relevant affiliations or financialinterests with any organization or entity No writing assis-tance was used in the production of paper

Acknowledgments

The authors would like to specially thank MassimilianoSpinelli Data Manager of SSD Sperimentazione animaleIstituto Nazionale per lo Studio e la Cura dei TumorildquoFondazione G Pascalerdquo IRCCS Italy for kind help inproviding informatics assistance and Dr Di Napoli Danielefor supervising animal care This work was supported by the5x mille and current research programs of National Instituteof Tumors IRCCS ldquoFoundation G Pascalerdquo Naples (Italy)

References

[1] J N Lickiss ldquoApproaching cancer pain reliefrdquo European Journalof Pain vol 5 pp 5ndash14 2001

[2] R Schmitz ldquoFriedrich Wilhelm Serturner and the discovery ofmorphinerdquo Pharmacy in History vol 27 no 2 pp 61ndash74 1985

[3] G W Pasternak ldquoPharmacological mechanisms of opioidanalgesicsrdquo Clinical Neuropharmacology vol 16 no 1 pp 1ndash181993

[4] B L Kieffer and C Gaveriaux-Ruff ldquoExploring the opioidsystem by gene knockoutrdquo Progress in Neurobiology vol 66 no5 pp 285ndash306 2002

[5] P W Mantyh ldquoCancer pain and its impact on diagnosissurvival and quality of liferdquo Nature Reviews Neuroscience vol7 no 10 pp 797ndash809 2006

[6] N Sueoka E Sueoka S Okabe and H Fujiki ldquoAnti-cancereffects of morphine through inhibition of tumour necrosisfactor-120572 release and mRNA expressionrdquo Carcinogenesis vol 17no 11 pp 2337ndash2341 1996

[7] R Maneckjee and J D Minna ldquoOpioids induce while nicotinesuppresses apoptosis in human lung cancer cellsrdquo Cell Growthand Differentiation vol 5 no 10 pp 1033ndash1040 1994

[8] A Pasi B Qu R Steiner H-J Senn W Bar and F S MessihaldquoAngiogenesis modulation with opioidsrdquo General Pharmacol-ogy vol 22 no 6 pp 1077ndash1079 1991

[9] A Hatzoglou E Bakogeorgou E Papakonstanti C StournarasD S Emmanouel and E Castanas ldquoIdentification and char-acterization of opioid and somatostatin binding sites in theopossum kidney (OK) cell line and their effect on growthrdquoJournal of Cellular Biochemistry vol 63 no 4 pp 410ndash421 1996

[10] A Hatzoglou L Ouafik E Bakogeorgou K Thermos and ECastanas ldquoMorphine cross-reacts with somatostatin receptorSSTR2 in the T47D human breast cancer cell line and decreasescell growthrdquo Cancer Research vol 55 no 23 pp 5632ndash56361995

[11] R Maneckjee R Biswas and B K Vonderhaar ldquoBinding ofopioids to humanMCF-7 breast cancer cells and their effects ongrowthrdquo Cancer Research vol 50 no 8 pp 2234ndash2238 1990

[12] M Kampa E Bakogeorgou A Hatzoglou A Damianaki P-MMartin and E Castanas ldquoOpioid alkaloids and casomorphinpeptides decrease the proliferation of prostatic cancer cell lines(LNCaP PC3 and DU145) through a partial interaction withopioid receptorsrdquo European Journal of Pharmacology vol 335no 2-3 pp 255ndash265 1997

[13] I Tegeder S Grosch A Schmidtko et al ldquoG protein-independent G1 cell cycle block and apoptosis with morphinein adenocarcinoma cells involvement of p53 phosphorylationrdquoCancer Research vol 63 no 8 pp 1846ndash1852 2003

BioMed Research International 7

[14] M P Yeager and T A Colacchio ldquoEffect ofmorphine on growthof metastatic colon cancer in vivordquo Archives of Surgery vol 126no 4 pp 454ndash456 1991

[15] T Sasamura S Nakamura Y Iida et al ldquoMorphine analgesiasuppresses tumor growth and metastasis in a mouse modelof cancer pain produced by orthotopic tumor inoculationrdquoEuropean Journal of Pharmacology vol 441 no 3 pp 185ndash1912002

[16] YHarimaya K Koizumi T AndohHNojima Y Kuraishi andI Saiki ldquoPotential ability of morphine to inhibit the adhesioninvasion and metastasis of metastatic colon 26-L5 carcinomacellsrdquo Cancer Letters vol 187 no 1-2 pp 121ndash127 2002

[17] R H Simon and T E Arbo ldquoMorphine increases metastatictumor growthrdquo Brain Research Bulletin vol 16 no 3 pp 363ndash367 1986

[18] J W Lewis Y Shavit G W Terman L R Nelson R P Galeand J C Liebeskind ldquoApparent involvement of opioid peptidesin stress-induced enhancement of tumor growthrdquo Peptides vol4 no 5 pp 635ndash638 1983

[19] M G Sergeeva Z V Grishina and S D VarfolomeyevldquoMorphine effect on proliferation of normal and tumor cells ofimmune originrdquo Immunology Letters vol 36 no 2 pp 215ndash2181993

[20] K Gupta S Kshirsagar L Chang et al ldquoMorphine stimu-lates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growthrdquo Can-cer Research vol 62 no 15 pp 4491ndash4498 2002

[21] M Farooqui Y Li T Rogers et al ldquoCOX-2 inhibitor celecoxibprevents chronic morphine-induced promotion of angiogene-sis tumour growth metastasis and mortality without compro-mising analgesiardquo British Journal of Cancer vol 97 no 11 pp1523ndash1531 2007

[22] J Zong and G M Pollack ldquoMorphine antinociceptionis enhanced in mdr1a gene-deficient micerdquo PharmaceuticalResearch vol 17 no 6 pp 749ndash753 2000

[23] S Bimonte A Barbieri G Palma and C Arra ldquoThe role ofmorphine in animal models of human cancer does morphinepromote or inhibit the tumor growthrdquo BioMed Research Inter-national vol 2013 Article ID 258141 4 pages 2013

[24] J Nguyen ldquoMorphine stimulates cancer progression and mastcell activation and impairs survival in transgenic mice withbreast cancerrdquo British Journal of Anaesthesia vol 113 supple-ment 1 pp i4ndashi13 2014

[25] M Ishikawa K Tanno A Kamo Y Takayanagi and K-ISasaki ldquoEnhancement of tumor growth by morphine and itspossible mechanism in micerdquo Biological and PharmaceuticalBulletin vol 16 no 8 pp 762ndash766 1993

[26] M Iglesias M F Segura J X Comella and G Olmos ldquo120583-opioid receptor activation prevents apoptosis following serumwithdrawal in differentiated SH-SY5Y cells and cortical neuronsvia phosphatidylinositol 3-kinaserdquoNeuropharmacology vol 44no 4 pp 482ndash492 2003

[27] K Gach J Szemraj J Fichna M Piestrzeniewicz D S Delbroand A Janecka ldquoThe influence of opioids on urokinase plas-minogen activator on protein andmRNA level inMCF-7 breastcancer cell linerdquo Chemical Biology and Drug Design vol 74 no4 pp 390ndash396 2009

[28] P A Singleton and J Moss ldquoEffect of perioperative opioids oncancer recurrence a hypothesisrdquo Future Oncology vol 6 no 8pp 1237ndash1242 2010

[29] C Chen M Farooqui and K Gupta ldquoMorphine stimulatesvascular endothelial growth factor-like signaling in mouse

retinal endothelial cellsrdquo Current Neurovascular Research vol3 no 3 pp 171ndash180 2006

[30] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[31] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[32] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[33] M O Hengartner ldquoThe biochemistry of apoptosisrdquoNature vol407 no 6805 pp 770ndash776 2000

[34] P-N Hsiao M-C Chang W-F Cheng et al ldquoMorphineinduces apoptosis of human endothelial cells through nitricoxide and reactive oxygen species pathwaysrdquo Toxicology vol256 no 1-2 pp 83ndash91 2009

[35] A A Kapasi S A CosciaM P Pandya and P C Singhal ldquoMor-phine modulates HIV-1 gp160-induced murine macrophageand human monocyte apoptosis by disparate waysrdquo Journal ofNeuroimmunology vol 148 no 1-2 pp 86ndash96 2004

[36] X Lin Q Li Y-J Wang et al ldquoMorphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor120581B transcriptional activation in neuroblastoma SH-SY5Y cellsrdquoBiochemical Journal vol 406 no 2 pp 215ndash221 2007

[37] R Arerangaiah N Chalasani A M Udager et al ldquoOpioidsinduce renal abnormalities in tumor-bearing micerdquo NephronmdashExperimental Nephrology vol 105 no 3 pp e80ndashe89 2007

[38] D Salvemini T P Misko J L Masferrer K Seibert M G Cur-rie and P Needleman ldquoNitric oxide activates cyclooxygenaseenzymesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 15 pp 7240ndash7244 1993

[39] E Nedelec A Abid C Cipolletta et al ldquoStimulation ofcyclooxygenase-2-activity by nitric oxide-derived species inrat chondrocyte lack of contribution to loss of cartilageanabolismrdquo Biochemical Pharmacology vol 61 no 8 pp 965ndash978 2001

[40] M Farooqui ZHGeng E J StephensonN Zaveri D Yee andK Gupta ldquoNaloxone acts as an antagonist of estrogen receptoractivity in MCF-7 cellsrdquo Molecular Cancer Therapeutics vol 5no 3 pp 611ndash620 2006

[41] D Salvemini K Seibert J L Masferrer T P Misko M GCurrie and P Needleman ldquoEndogenous nitric oxide enhancesprostaglandin production in a model of renal inflammationrdquoJournal of Clinical Investigation vol 93 no 5 pp 1940ndash19471994

[42] R J Griffin BWWilliams RWild JM CherringtonH Parkand CW Song ldquoSimultaneous inhibition of the receptor kinaseactivity of vascular endothelial fibroblast and platelet-derivedgrowth factors suppresses tumor growth and enhances tumorradiation responserdquo Cancer Research vol 62 no 6 pp 1702ndash1706 2002

[43] K M Leahy R L Ornberg Y Wang B S Zweifel A T Kokiand J L Masferrer ldquoCyclooxygenase-2 inhibition by celecoxibreduces proliferation and induces apoptosis in angiogenicendothelial cells in vivordquoCancer Research vol 62 no 3 pp 625ndash631 2002

[44] S H Chang C H Liu and R Conway ldquoRole of prostaglandinE2-dependent angiogenic switch in cyclooxygenase 2-inducedbreast cancer progressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 2 pp 591ndash596 2004

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006

6 BioMed Research International

morphine was demonstrated in the MCF-7 breast cancermodel In these mice morphine at clinically relevant con-centrations enhanced tumor neovascularization [20] In ananimal model of hormone-dependent breast cancer it hasalso been demonstrated that morphine promoted activationof vascular endothelial growth factor (VEGF) receptor andincreased metastasis [21 29] It has been proposed thatmorphine explains its proangiogenic activity by stimula-tion of mitogen-activated protein kinase (MAPK) signallingpathway via G protein-coupled receptors and nitric oxide(NO) Alternatively several in vivo studies provided evidencethat morphine can induce tumor growth by upregulationof cyclooxygenase-2 (COX-2) [37ndash40] and or prostaglandinE2-mediated stimulation of angiogenesis [41ndash44] On thecontrary several in vivo and in vitro studies demonstratedthat morphine can inhibit angiogenesis by regulation ofdifferent pathways [8 34 45 45ndash52] These different resultscan be due to different experimental conditions (cell linetumor type used andor dosetime of morphine) Morphineplays a role not only in tumor cell growth but also inmetastasis formation which is the main process related tomost cancer deaths and failure in cancer treatment [53 54]

For these reasons in order to elucidate the role ofmorphine in regulation of tumor growth and angiogenesis inbreast cancer we performed in vitro and in vivo studies on theMDAMB231 breast cancer cellsThese cells are triple negative(basal-like) breast cancer (TNBC) cells It is noted that TNBCdoes not express the estrogen receptor (ER) progesteronereceptor (PR) or human epidermal growth factor receptortype 2 (HER2) Due to lack of both hormone receptors andHER2 expression patients with this type of breast cancer haveno chance to benefit from the endocrine therapy and HER2targeted therapy In addition we used immunodeficient micethat are able to mimic the compromised immune systemof a patient with breast cancer So in these experimentalconditions it has become interesting for us to study therole of morphine in the regulation of cancer cell growthand angiogenesis Our data showed that morphine in TNBCat clinical relevant doses promotes tumor angiogenesis andincreases breast cancer progression For these reasons it isvery important for the management of severe pain associatedwith cancer to consider accurately the dose and route ofadministration of morphine in order to avoid severe effectof cancer progression Further studies are ongoing in ourlaboratory in order to dissect the molecular mechanismsunderlying the role of morphine in cancer development andmetastasis formation in breast cancer Specifically we willgenerate orthotropic mouse models of breast cancer by usingnot only MDAMB231 cells but also MCF-7 (human breastadenocarcinoma cell line) cells which represent estrogenreceptor (ER) positive control cell lines The results obtainedfrom these data will shed light on the role of morphine inregulation of breast cancer progression

Conflict of Interests

The authors have no other relevant affiliations or financialinterests with any organization or entity No writing assis-tance was used in the production of paper

Acknowledgments

The authors would like to specially thank MassimilianoSpinelli Data Manager of SSD Sperimentazione animaleIstituto Nazionale per lo Studio e la Cura dei TumorildquoFondazione G Pascalerdquo IRCCS Italy for kind help inproviding informatics assistance and Dr Di Napoli Danielefor supervising animal care This work was supported by the5x mille and current research programs of National Instituteof Tumors IRCCS ldquoFoundation G Pascalerdquo Naples (Italy)

References

[1] J N Lickiss ldquoApproaching cancer pain reliefrdquo European Journalof Pain vol 5 pp 5ndash14 2001

[2] R Schmitz ldquoFriedrich Wilhelm Serturner and the discovery ofmorphinerdquo Pharmacy in History vol 27 no 2 pp 61ndash74 1985

[3] G W Pasternak ldquoPharmacological mechanisms of opioidanalgesicsrdquo Clinical Neuropharmacology vol 16 no 1 pp 1ndash181993

[4] B L Kieffer and C Gaveriaux-Ruff ldquoExploring the opioidsystem by gene knockoutrdquo Progress in Neurobiology vol 66 no5 pp 285ndash306 2002

[5] P W Mantyh ldquoCancer pain and its impact on diagnosissurvival and quality of liferdquo Nature Reviews Neuroscience vol7 no 10 pp 797ndash809 2006

[6] N Sueoka E Sueoka S Okabe and H Fujiki ldquoAnti-cancereffects of morphine through inhibition of tumour necrosisfactor-120572 release and mRNA expressionrdquo Carcinogenesis vol 17no 11 pp 2337ndash2341 1996

[7] R Maneckjee and J D Minna ldquoOpioids induce while nicotinesuppresses apoptosis in human lung cancer cellsrdquo Cell Growthand Differentiation vol 5 no 10 pp 1033ndash1040 1994

[8] A Pasi B Qu R Steiner H-J Senn W Bar and F S MessihaldquoAngiogenesis modulation with opioidsrdquo General Pharmacol-ogy vol 22 no 6 pp 1077ndash1079 1991

[9] A Hatzoglou E Bakogeorgou E Papakonstanti C StournarasD S Emmanouel and E Castanas ldquoIdentification and char-acterization of opioid and somatostatin binding sites in theopossum kidney (OK) cell line and their effect on growthrdquoJournal of Cellular Biochemistry vol 63 no 4 pp 410ndash421 1996

[10] A Hatzoglou L Ouafik E Bakogeorgou K Thermos and ECastanas ldquoMorphine cross-reacts with somatostatin receptorSSTR2 in the T47D human breast cancer cell line and decreasescell growthrdquo Cancer Research vol 55 no 23 pp 5632ndash56361995

[11] R Maneckjee R Biswas and B K Vonderhaar ldquoBinding ofopioids to humanMCF-7 breast cancer cells and their effects ongrowthrdquo Cancer Research vol 50 no 8 pp 2234ndash2238 1990

[12] M Kampa E Bakogeorgou A Hatzoglou A Damianaki P-MMartin and E Castanas ldquoOpioid alkaloids and casomorphinpeptides decrease the proliferation of prostatic cancer cell lines(LNCaP PC3 and DU145) through a partial interaction withopioid receptorsrdquo European Journal of Pharmacology vol 335no 2-3 pp 255ndash265 1997

[13] I Tegeder S Grosch A Schmidtko et al ldquoG protein-independent G1 cell cycle block and apoptosis with morphinein adenocarcinoma cells involvement of p53 phosphorylationrdquoCancer Research vol 63 no 8 pp 1846ndash1852 2003

BioMed Research International 7

[14] M P Yeager and T A Colacchio ldquoEffect ofmorphine on growthof metastatic colon cancer in vivordquo Archives of Surgery vol 126no 4 pp 454ndash456 1991

[15] T Sasamura S Nakamura Y Iida et al ldquoMorphine analgesiasuppresses tumor growth and metastasis in a mouse modelof cancer pain produced by orthotopic tumor inoculationrdquoEuropean Journal of Pharmacology vol 441 no 3 pp 185ndash1912002

[16] YHarimaya K Koizumi T AndohHNojima Y Kuraishi andI Saiki ldquoPotential ability of morphine to inhibit the adhesioninvasion and metastasis of metastatic colon 26-L5 carcinomacellsrdquo Cancer Letters vol 187 no 1-2 pp 121ndash127 2002

[17] R H Simon and T E Arbo ldquoMorphine increases metastatictumor growthrdquo Brain Research Bulletin vol 16 no 3 pp 363ndash367 1986

[18] J W Lewis Y Shavit G W Terman L R Nelson R P Galeand J C Liebeskind ldquoApparent involvement of opioid peptidesin stress-induced enhancement of tumor growthrdquo Peptides vol4 no 5 pp 635ndash638 1983

[19] M G Sergeeva Z V Grishina and S D VarfolomeyevldquoMorphine effect on proliferation of normal and tumor cells ofimmune originrdquo Immunology Letters vol 36 no 2 pp 215ndash2181993

[20] K Gupta S Kshirsagar L Chang et al ldquoMorphine stimu-lates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growthrdquo Can-cer Research vol 62 no 15 pp 4491ndash4498 2002

[21] M Farooqui Y Li T Rogers et al ldquoCOX-2 inhibitor celecoxibprevents chronic morphine-induced promotion of angiogene-sis tumour growth metastasis and mortality without compro-mising analgesiardquo British Journal of Cancer vol 97 no 11 pp1523ndash1531 2007

[22] J Zong and G M Pollack ldquoMorphine antinociceptionis enhanced in mdr1a gene-deficient micerdquo PharmaceuticalResearch vol 17 no 6 pp 749ndash753 2000

[23] S Bimonte A Barbieri G Palma and C Arra ldquoThe role ofmorphine in animal models of human cancer does morphinepromote or inhibit the tumor growthrdquo BioMed Research Inter-national vol 2013 Article ID 258141 4 pages 2013

[24] J Nguyen ldquoMorphine stimulates cancer progression and mastcell activation and impairs survival in transgenic mice withbreast cancerrdquo British Journal of Anaesthesia vol 113 supple-ment 1 pp i4ndashi13 2014

[25] M Ishikawa K Tanno A Kamo Y Takayanagi and K-ISasaki ldquoEnhancement of tumor growth by morphine and itspossible mechanism in micerdquo Biological and PharmaceuticalBulletin vol 16 no 8 pp 762ndash766 1993

[26] M Iglesias M F Segura J X Comella and G Olmos ldquo120583-opioid receptor activation prevents apoptosis following serumwithdrawal in differentiated SH-SY5Y cells and cortical neuronsvia phosphatidylinositol 3-kinaserdquoNeuropharmacology vol 44no 4 pp 482ndash492 2003

[27] K Gach J Szemraj J Fichna M Piestrzeniewicz D S Delbroand A Janecka ldquoThe influence of opioids on urokinase plas-minogen activator on protein andmRNA level inMCF-7 breastcancer cell linerdquo Chemical Biology and Drug Design vol 74 no4 pp 390ndash396 2009

[28] P A Singleton and J Moss ldquoEffect of perioperative opioids oncancer recurrence a hypothesisrdquo Future Oncology vol 6 no 8pp 1237ndash1242 2010

[29] C Chen M Farooqui and K Gupta ldquoMorphine stimulatesvascular endothelial growth factor-like signaling in mouse

retinal endothelial cellsrdquo Current Neurovascular Research vol3 no 3 pp 171ndash180 2006

[30] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[31] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[32] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[33] M O Hengartner ldquoThe biochemistry of apoptosisrdquoNature vol407 no 6805 pp 770ndash776 2000

[34] P-N Hsiao M-C Chang W-F Cheng et al ldquoMorphineinduces apoptosis of human endothelial cells through nitricoxide and reactive oxygen species pathwaysrdquo Toxicology vol256 no 1-2 pp 83ndash91 2009

[35] A A Kapasi S A CosciaM P Pandya and P C Singhal ldquoMor-phine modulates HIV-1 gp160-induced murine macrophageand human monocyte apoptosis by disparate waysrdquo Journal ofNeuroimmunology vol 148 no 1-2 pp 86ndash96 2004

[36] X Lin Q Li Y-J Wang et al ldquoMorphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor120581B transcriptional activation in neuroblastoma SH-SY5Y cellsrdquoBiochemical Journal vol 406 no 2 pp 215ndash221 2007

[37] R Arerangaiah N Chalasani A M Udager et al ldquoOpioidsinduce renal abnormalities in tumor-bearing micerdquo NephronmdashExperimental Nephrology vol 105 no 3 pp e80ndashe89 2007

[38] D Salvemini T P Misko J L Masferrer K Seibert M G Cur-rie and P Needleman ldquoNitric oxide activates cyclooxygenaseenzymesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 15 pp 7240ndash7244 1993

[39] E Nedelec A Abid C Cipolletta et al ldquoStimulation ofcyclooxygenase-2-activity by nitric oxide-derived species inrat chondrocyte lack of contribution to loss of cartilageanabolismrdquo Biochemical Pharmacology vol 61 no 8 pp 965ndash978 2001

[40] M Farooqui ZHGeng E J StephensonN Zaveri D Yee andK Gupta ldquoNaloxone acts as an antagonist of estrogen receptoractivity in MCF-7 cellsrdquo Molecular Cancer Therapeutics vol 5no 3 pp 611ndash620 2006

[41] D Salvemini K Seibert J L Masferrer T P Misko M GCurrie and P Needleman ldquoEndogenous nitric oxide enhancesprostaglandin production in a model of renal inflammationrdquoJournal of Clinical Investigation vol 93 no 5 pp 1940ndash19471994

[42] R J Griffin BWWilliams RWild JM CherringtonH Parkand CW Song ldquoSimultaneous inhibition of the receptor kinaseactivity of vascular endothelial fibroblast and platelet-derivedgrowth factors suppresses tumor growth and enhances tumorradiation responserdquo Cancer Research vol 62 no 6 pp 1702ndash1706 2002

[43] K M Leahy R L Ornberg Y Wang B S Zweifel A T Kokiand J L Masferrer ldquoCyclooxygenase-2 inhibition by celecoxibreduces proliferation and induces apoptosis in angiogenicendothelial cells in vivordquoCancer Research vol 62 no 3 pp 625ndash631 2002

[44] S H Chang C H Liu and R Conway ldquoRole of prostaglandinE2-dependent angiogenic switch in cyclooxygenase 2-inducedbreast cancer progressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 2 pp 591ndash596 2004

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006

BioMed Research International 7

[14] M P Yeager and T A Colacchio ldquoEffect ofmorphine on growthof metastatic colon cancer in vivordquo Archives of Surgery vol 126no 4 pp 454ndash456 1991

[15] T Sasamura S Nakamura Y Iida et al ldquoMorphine analgesiasuppresses tumor growth and metastasis in a mouse modelof cancer pain produced by orthotopic tumor inoculationrdquoEuropean Journal of Pharmacology vol 441 no 3 pp 185ndash1912002

[16] YHarimaya K Koizumi T AndohHNojima Y Kuraishi andI Saiki ldquoPotential ability of morphine to inhibit the adhesioninvasion and metastasis of metastatic colon 26-L5 carcinomacellsrdquo Cancer Letters vol 187 no 1-2 pp 121ndash127 2002

[17] R H Simon and T E Arbo ldquoMorphine increases metastatictumor growthrdquo Brain Research Bulletin vol 16 no 3 pp 363ndash367 1986

[18] J W Lewis Y Shavit G W Terman L R Nelson R P Galeand J C Liebeskind ldquoApparent involvement of opioid peptidesin stress-induced enhancement of tumor growthrdquo Peptides vol4 no 5 pp 635ndash638 1983

[19] M G Sergeeva Z V Grishina and S D VarfolomeyevldquoMorphine effect on proliferation of normal and tumor cells ofimmune originrdquo Immunology Letters vol 36 no 2 pp 215ndash2181993

[20] K Gupta S Kshirsagar L Chang et al ldquoMorphine stimu-lates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growthrdquo Can-cer Research vol 62 no 15 pp 4491ndash4498 2002

[21] M Farooqui Y Li T Rogers et al ldquoCOX-2 inhibitor celecoxibprevents chronic morphine-induced promotion of angiogene-sis tumour growth metastasis and mortality without compro-mising analgesiardquo British Journal of Cancer vol 97 no 11 pp1523ndash1531 2007

[22] J Zong and G M Pollack ldquoMorphine antinociceptionis enhanced in mdr1a gene-deficient micerdquo PharmaceuticalResearch vol 17 no 6 pp 749ndash753 2000

[23] S Bimonte A Barbieri G Palma and C Arra ldquoThe role ofmorphine in animal models of human cancer does morphinepromote or inhibit the tumor growthrdquo BioMed Research Inter-national vol 2013 Article ID 258141 4 pages 2013

[24] J Nguyen ldquoMorphine stimulates cancer progression and mastcell activation and impairs survival in transgenic mice withbreast cancerrdquo British Journal of Anaesthesia vol 113 supple-ment 1 pp i4ndashi13 2014

[25] M Ishikawa K Tanno A Kamo Y Takayanagi and K-ISasaki ldquoEnhancement of tumor growth by morphine and itspossible mechanism in micerdquo Biological and PharmaceuticalBulletin vol 16 no 8 pp 762ndash766 1993

[26] M Iglesias M F Segura J X Comella and G Olmos ldquo120583-opioid receptor activation prevents apoptosis following serumwithdrawal in differentiated SH-SY5Y cells and cortical neuronsvia phosphatidylinositol 3-kinaserdquoNeuropharmacology vol 44no 4 pp 482ndash492 2003

[27] K Gach J Szemraj J Fichna M Piestrzeniewicz D S Delbroand A Janecka ldquoThe influence of opioids on urokinase plas-minogen activator on protein andmRNA level inMCF-7 breastcancer cell linerdquo Chemical Biology and Drug Design vol 74 no4 pp 390ndash396 2009

[28] P A Singleton and J Moss ldquoEffect of perioperative opioids oncancer recurrence a hypothesisrdquo Future Oncology vol 6 no 8pp 1237ndash1242 2010

[29] C Chen M Farooqui and K Gupta ldquoMorphine stimulatesvascular endothelial growth factor-like signaling in mouse

retinal endothelial cellsrdquo Current Neurovascular Research vol3 no 3 pp 171ndash180 2006

[30] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[31] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[32] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[33] M O Hengartner ldquoThe biochemistry of apoptosisrdquoNature vol407 no 6805 pp 770ndash776 2000

[34] P-N Hsiao M-C Chang W-F Cheng et al ldquoMorphineinduces apoptosis of human endothelial cells through nitricoxide and reactive oxygen species pathwaysrdquo Toxicology vol256 no 1-2 pp 83ndash91 2009

[35] A A Kapasi S A CosciaM P Pandya and P C Singhal ldquoMor-phine modulates HIV-1 gp160-induced murine macrophageand human monocyte apoptosis by disparate waysrdquo Journal ofNeuroimmunology vol 148 no 1-2 pp 86ndash96 2004

[36] X Lin Q Li Y-J Wang et al ldquoMorphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor120581B transcriptional activation in neuroblastoma SH-SY5Y cellsrdquoBiochemical Journal vol 406 no 2 pp 215ndash221 2007

[37] R Arerangaiah N Chalasani A M Udager et al ldquoOpioidsinduce renal abnormalities in tumor-bearing micerdquo NephronmdashExperimental Nephrology vol 105 no 3 pp e80ndashe89 2007

[38] D Salvemini T P Misko J L Masferrer K Seibert M G Cur-rie and P Needleman ldquoNitric oxide activates cyclooxygenaseenzymesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 15 pp 7240ndash7244 1993

[39] E Nedelec A Abid C Cipolletta et al ldquoStimulation ofcyclooxygenase-2-activity by nitric oxide-derived species inrat chondrocyte lack of contribution to loss of cartilageanabolismrdquo Biochemical Pharmacology vol 61 no 8 pp 965ndash978 2001

[40] M Farooqui ZHGeng E J StephensonN Zaveri D Yee andK Gupta ldquoNaloxone acts as an antagonist of estrogen receptoractivity in MCF-7 cellsrdquo Molecular Cancer Therapeutics vol 5no 3 pp 611ndash620 2006

[41] D Salvemini K Seibert J L Masferrer T P Misko M GCurrie and P Needleman ldquoEndogenous nitric oxide enhancesprostaglandin production in a model of renal inflammationrdquoJournal of Clinical Investigation vol 93 no 5 pp 1940ndash19471994

[42] R J Griffin BWWilliams RWild JM CherringtonH Parkand CW Song ldquoSimultaneous inhibition of the receptor kinaseactivity of vascular endothelial fibroblast and platelet-derivedgrowth factors suppresses tumor growth and enhances tumorradiation responserdquo Cancer Research vol 62 no 6 pp 1702ndash1706 2002

[43] K M Leahy R L Ornberg Y Wang B S Zweifel A T Kokiand J L Masferrer ldquoCyclooxygenase-2 inhibition by celecoxibreduces proliferation and induces apoptosis in angiogenicendothelial cells in vivordquoCancer Research vol 62 no 3 pp 625ndash631 2002

[44] S H Chang C H Liu and R Conway ldquoRole of prostaglandinE2-dependent angiogenic switch in cyclooxygenase 2-inducedbreast cancer progressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 2 pp 591ndash596 2004

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006

8 BioMed Research International

[45] C-F Lam Y-C Liu F-L Tseng et al ldquoHigh-dose morphineimpairs vascular endothelial function by increased productionof superoxide anionsrdquo Anesthesiology vol 106 no 3 pp 532ndash537 2007

[46] J Blebea J E Mazo T K Kihara et al ldquoOpioid growth factormodulates angiogenesisrdquo Journal of Vascular Surgery vol 32 no2 pp 364ndash373 2000

[47] C-F Lam P-J Chang Y-S Huang et al ldquoProlonged use ofhigh-dose morphine impairs angiogenesis and mobilization ofendothelial progenitor cells in micerdquo Anesthesia and Analgesiavol 107 no 2 pp 686ndash692 2008

[48] J L Martin R Charboneau R A Barke and S Roy ldquoChronicmorphine treatment inhibits LPS-induced angiogenesis impli-cations in wound healingrdquo Cellular Immunology vol 265 no 2pp 139ndash145 2010

[49] S Roy S Balasubramanian J Wang Y Chandrashekhar RCharboneau and R Barke ldquoMorphine inhibits VEGF expres-sion in myocardial ischemiardquo Surgery vol 134 no 2 pp 336ndash344 2003

[50] L Koodie S Ramakrishnan and S Roy ldquoMorphine suppressestumor angiogenesis through aHIF-1120572p38MAPK pathwayrdquoTheAmerican Journal of Pathology vol 177 no 2 pp 984ndash997 2010

[51] N Faramarzi A Abbasi S M Tavangar M Mazouchi and AR Dehpour ldquoOpioid receptor antagonist promotes angiogen-esis in bile duct ligated ratsrdquo Journal of Gastroenterology andHepatology vol 24 no 7 pp 1226ndash1229 2009

[52] R L Shapiro J G Duquette D F Roses et al ldquoInductionof primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient andwild-typemicecellular blue nevi invade but do not progress to malignantmelanoma in uPA-deficient animalsrdquo Cancer Research vol 56no 15 pp 3597ndash3604 1996

[53] J A Engbring and H K Kleinman ldquoThe basement membranematrix in malignancyrdquo Journal of Pathology vol 200 no 4 pp465ndash470 2003

[54] M SWidel ldquoMechanisms of metastasis andmolecular markersof malignant tumor progression I Colorectal cancerrdquo PostępyHigieny i Medycyny Doswiadczalnej vol 60 pp 453ndash470 2006