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Clinical Biochemistry 43 (2010) 725–731

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Clinical Biochemistry

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Alterations of insulin-like growth factor binding protein 3 (IGFBP-3) glycosylation inpatients with breast tumours

Ivona Baričević a,⁎, Romana Masnikosa a, Dragana Lagundžin a, Vera Golubović b, Olgica Nedić a

a Institute for the Application of Nuclear Energy (INEP), University of Belgrade, Banatska 31b, 11080 Belgrade, Serbiab Institute of Oncology and Radiology of Serbia, Belgrade, Serbia

⁎ Corresponding author. Tel.: +381 11 2 617 252; faxE-mail address: [email protected] (I. Baričević).

0009-9120/$ – see front matter © 2010 The Canadiandoi:10.1016/j.clinbiochem.2010.03.006

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 13 October 2009Received in revised form 3 March 2010Accepted 10 March 2010Available online 19 March 2010

Keywords:Insulin-like growth factor binding protein-3(IGFBP-3)Breast tumourGlycosylationLectin-affinity chromatography

Objectives: Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator ofdevelopment and progression of breast cancer as it regulates the amount of free, physiologically active IGF-Iand IGF-II. Changes in the glycosylation pattern within IGFBP-3 may affect its interaction with ligands. Theaim of this study was to investigate whether such changes occur during disease progression.

Design and methods: IGFBP-3 in serum samples from healthy women and from women with breasttumours was characterised in terms of its concentration (IRMA), glycosylation moiety (lectin-affinitychromatography) and distribution of molecular species (immunoblotting).

Results: In patients with benign tumours the concentration and carbohydrate content of IGFBP-3 wasunaltered compared to healthy women. In patients with malignant tumours in most cases these twoparameters were unchanged, but there were women whose concentration of IGFBP-3 was reduced and itsstructure was altered. In non-surviving cancer patients the concentration of IGFBP-3 was significantly

reduced and these molecules contained a greater amount of biantennary complex type N-glycans havingmore mannose, fucose, bisecting GlcNAc and terminal sialic acid residues.

Conclusion: Our results showed that breast cancer progression causes alterations of IGFBP-3glycosylation. The extent of changes increases with breast cancer severity.

© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

The growth hormone/insulin-like growth factor I (GH/IGF-I) axisis an essential modulator of growth and development of the breast byexerting potent mitogenic and anti-apoptotic effects [1]. The axis isalso a very important regulator of the metabolic activity within cellsand tissues. Imbalances within the axis lead to aberrant signallingnetworks and over-expression of growth factors that are implicated inthe development and progression of breast cancer. Insulin-likegrowth factor binding proteins (IGFBP-1 to -6) are multi-functionalregulatory proteins that bind IGFs [2]. They are differentiallyexpressed in cells, tissues and physiological fluids. IGFBP-1, -2, -3and -4 are the most abundant IGFBPs in blood. IGFBP-3 is by far themain circulating IGFBP that maintains a circulating reservoir of IGFs,has a role in their transport and prolongs their half-life [3]. Nativenon-glycosylated IGFBP-3 has a molecular weight of 29 kDa. In thecirculation IGFBP-3 exists as two glycoforms of 40 and 45 kDacontaining 3N-glycosylated sites (Asn-X-Ser/Thr) located at Asn89,Asn109 and Asn172 (4, 4.5 and 5 kDa, respectively). The 40 kDaglycoform has two occupied N-glycosylation sites whereas the

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45 kDa glycoform has all three [4]. The presence of N-glycosylationsites affects the ability of the IGFBPs to adhere to the cell surfacewhichcan modify their affinity for IGF. Both non-glycosylated andglycosylated forms of IGFBP-3 are functionally similar in their abilityto bind IGFs. However, in T47D breast cancer cells glycosylated IGFBP-3 binds less to the cell surface compared to its non-glycosylatedcounterpart. It would appear that post-translational modifications ofIGFBP-3 are important and are likely to affect IGFBP-3's regulatoryfunctions in breast tissue. Furthermore, IGFBPs can be cleaved byproteases into forms that possess either reduced or no affinity for IGFs.Prostate-specific antigen (PSA), cathepsin D and plasmin can cleaveIGFBP-3 and all of these proteases have been localised within breastcancer tissue [5].

Several large prospective and cohort studies have sought toidentify relationships between IGF-I, IGFBP-3 and breast cancer risk. Apositive association between IGF-I and breast cancer risk exists in pre-menopausal patients [6–8]. No evidence supports a role for IGF-I inpredicting post-menopausal risk [9–13]. Evidence linking IGFBP-3 tobreast cancer risk is inconsistent. The consequence of high circulatinglevels of IGFBP-3 on breast cancer risk in some studies is associatedwith increased breast cancer risk in pre-menopausal woman whereasother studies report a protective role, concluding that high IGFBP-3concentrations are associated with decreased cancer risk [7,12,14].Recent meta-analyses indicate a positive association between

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circulating IGFBP-3 levels and pre-menopausal cancer risk [15]. Incontrast, women with high IGF-I and low IGFBP-3 tend to be at highrisk for carcinoma in situ [16]. Careful scrutiny of the circulating formsof IGFBP-3 has been suggested. Rinaldi et al. demonstrated that highlevels of functional IGFBP-3 (intact IGFBP-3 and some IGFBP-3fragments) could be associated with reduced breast cancer risk,whereas high levels of total IGFBP-3 (intact and fragmented) could beassociated with increased breast cancer risk [17]. However, Li et al.failed to find any association of intact, fragmented or total IGFBP-3with breast cancer risk [18]. Espelund et al. reported that in breastcancer patients total IGFBP-3 was unchanged, whereas intact IGFBP-3was increased due to reduced IGFBP-3 protease activity [19]. Contraryto most other studies, a recently performed large prospective studyrevealed an association between IGF-I, IGFBP-3 and breast cancer riskin post-menopausal woman [10]. The prospective Nurses' HealthStudy II noted that both IGF-I and IGFBP-3 were not associated withbreast cancer risk in pre-menopausal patients [20].

The results from several studies that have investigated therelationship between IGF-I, IGFBP-3 and breast cancer risk are,therefore, unclear and contradictory. Such inconsistencies warranteda re-examination of the role of the IGF system in breast tumours. Inour current study we have measured the concentrations of IGF-I andIGFBP-3 in patients diagnosed with breast cancer (benign ormalignant) and were indicated for surgery. Our goal was not tocompare the levels of IGF-I and IGFBP-3 between pre-menopausal andpost-menopausal woman, rather we sought to determine whetherchanges in the glycosylation pattern of IGFBP-3 were involved in theoverall alteration of the circulating IGF/IGFBP system and whetherthese changes were related to malignancy. As protein glycosylationinfluences stability and function of the IGF system components and aschanges in N-linked glycosylation occur during cancer development,we characterised the carbohydrate moiety of IGFBP-3 in breasttumour patient groups using eight lectin-affinity columns withdifferent carbohydrate specificities. This allowed us to identifysaccharide residues present in IGFBP-3 from patients and to comparethe glycomic profile with that characteristic for IGFBP-3 from healthywomen. Another approach was to investigate the presence andrelative abundance of different IGFBP-3 molecular species in patientswith breast tumour. Therefore, our aim was primarily focused onalterations of IGFBP-3 glycosylation during disease progression. Thesealterations may reflect IGFBP-3 stability, half-life, binding capacityand, as a consequence, they may regulate IGF bioavailability.

Methods

Study population

Serum samples were obtained from healthy adult women (n=20,age 50±10.2 years) and from women diagnosed with breast tumourindicated for surgery (n=53, age 57±11.3 years). All womencompleted a questionnaire at interview containing details of personalcharacteristics and reproductive history. Patients were treated withchemotherapy for several months prior to surgery in the Institute ofOncology and Radiology of Serbia, Belgrade, Serbia during 2007.Patients included in this study were those indicated for surgery in theperiod July–August 2008. After post-operative histopathologicalanalysis and follow up that lasted 1 year after the surgery the patientswere divided into three groups: group 1 — patients diagnosed withbenign breast disease, survivors (n=8, age 58±11.6 years), group2— patients diagnosed with breast cancer, survivors (n=36, age 56±11.6) and group 3 — non-surviving patients diagnosed with breastcancer (n=9, age 55±10.3). All the patients were also consideredclustered as pre-menopausal and post-menopausal women. Thesesubgroups, however, did not show any differences in the measuredparameters. Therefore, we decided not to analyse them further assuch. 13% of patients underwent short surgical interventions

(tumorectomia, up to 30 min) whereas 3% of the patients hadextensive surgery (resectio partialis mamme). 52% of patientsunderwent longer operations (Mastectomia radicalis that lastedfrom 50 to 175 min) whereas 32% of patients underwent Quad-rantectomia cum dissectioaxillae dex (surgery lasted from 70 to140 min).

The BMI of the volunteers and patients ranged between 24.5 and26.7 kg/m2. No statistical difference in the BMI between the studygroups was observed (pN0.05). Blood samples from the patients werecollected in the morning after a 12-hour fast and before the surgeryfrom the patients. The serumwas separated by centrifugation, dividedinto aliquots and stored at −20 °C until use. All volunteers andpatients agreed that their sera could be used in this study according tothe current ethical standards (Helsinski Declaration of 1975, asrevised in 1983).

Determination of serum IGF-I and IGFBP-3 concentration

The IGF-I concentration was measured by RIA-IGF-I (INEP-Belgrade, Serbia) using 125I-labelled IGF-I [21]. The assay wasstandardised against WHO reference material 87/518. The serumIGFBP-3 concentration was measured by an immunoradiometricassay IRMA-IGFBP-3 (INEP-Belgrade, Serbia) using affinity purified125I-labelled polyclonal goat anti-IGFBP-3 antibodies (DiagnosticSystems Laboratories Inc., Webster, TX, USA). The assay wasstandardised against the DSL-6600 IGFBP-3 IRMA kit [22].

Lectin-affinity chromatography

Lectin-affinity chromatography was performed using eight columnswith the following agarose-immobilised lectins [23]: 1) Con A (lectinfrom Canavalia ensiformis binds strongly to high-mannose (Man) typeN-glycans), 2) succinylated WGA (succinylated wheat germ agglutininhas specificity towards GlcNAcβ1→4GlcNAc, but does not bind sialicacid (Sia) residues), 3) RCA I (Ricinus communis agglutinin I is a lectinspecific for N-glycans containing a bisecting GlcNAc in a sequenceGalβ1→4GlcNAc; high affinity binding indicates the presence of corefucosylation), 4) ECL (Erythrina cristagalli lectin has the highest affinitytowards Galβ1→4GlcNAc disaccharides, but sialylation of the latterabolishes lectin binding), 5) SNA (Sambucus nigra agglutinin is specificfor Siaα2→6Gal and Siaα2→6GalNAc sequences present in N-glycans),6) UEA (Ulex europaeus agglutinin is specific for terminal α1→2 linkedL-Fuc residues), 7) PHA-E (Phaseolus vulgaris erythroagglutinin recog-nises complex type oligosaccharides and binds with the highest affinityto biantennary chains that contain a bisecting GlcNAc residue which isß1→4-linked to the Man in the trimannosyl core) and 8) PHA-L(Phaseolus vulgaris leukoagglutinin also binds complex type oligosac-charides, but it prefers multiantennary chains with GlcNAcβ1→6residues). All agarose-immobilised plant lectins were from VectorLaboratories (Burlingame, CA, USA). All buffers and sugar solutionswereprepared following procedures recommended by the producer and thedetails are described in Table 1.

Serum samples (0.1 mL) were pre-incubated at 4 °C overnightwith 125I-IGF-I (3×105 cpm) to allow 125I-IGF-I binding to IGFBP-3and then applied to the lectin columns. 125I-IGF-I binds to all IGFBPs,but 125I-IGF-I–IGFBP-3 complexes are those that predominantlyinteract with lectins due to the fact that IGFBP-3 is the most abundantIGF-binding protein in postnatal circulation [24]. The samples wererecirculated through the columns at 25 °C for 1 h to ensure maximalbinding. The unbound material was washed away with 20 mL of thecorresponding buffer that was used for equilibration. The elution ofbound complexes was performed using two consecutive steps (all thedetails are in Table 1), the two specific elutions were at pH 7.5 and pH3.0. PHA-E- and PHA-L-agarose columnswere eluted in one step usingacetic acid. The radioactivity (cpm) within the collected fractions(1 mL) was determined in a γ-counter. The recovery of radioactivity

Table 1Lectin-affinity columns: equilibration and specific elution buffers (pH 7.5 and pH 3.0).

Lectin Equilibration buffer Specific elution at pH 7.5 Specific elution at pH 3.0

Con A 0.02 M HEPES pH 7.5, 0.5 M NaCl, 0.01 M NaN3a 0.2 M methyl-α-glucopyranoside, 0.2 M

methyl-α-mannopyranoside in 0.02 M HEPES0.2 M methyl-α-glucopyranoside, 0.2 Mmethyl-α-mannopyranoside in 0.2 M acetic acid

Succ. WGA 0.01 M HEPES pH 7.5, 0.15 M NaCl, 0.01 M NaN3 0.5 M GlcNAc in 0.01 M HEPES 0.5 M GlcNAc in 0.2 M acetic acidRCA I 0.01 M HEPES pH 7.5, 0.15 M NaCl, 0.01 M NaN3 0.2 M Lac in 0.01 M HEPES 0.2 M Lac in 0.2 M acetic acidECL 0.01 M HEPES pH 7.5, 0.15 M NaCl, 0.01 M NaN3

b 0.2 M Lac in 0.01 M HEPES 0.2 M Lac in 0.2 M acetic acidSNA 0.01 M HEPES pH 7.5, 0.15 M NaCl, 0.01 M NaN3

b 0.5 M Lac in 0.01 M HEPES 0.5 M Lac in 0.2 M acetic acidUEA 0.01 M HEPES pH 7.5, 0.15 M NaCl, 0.01 M NaN3

b 0.1 M Fuc in 0.01 M HEPES 0.1 M Fuc in 0.2 M acetic acidPHA-E 0.01 M HEPES pH 8.0, 0.15 M NaCl, 0.01 M NaN3

c – 0.1 M acetic acidPHA-L 0.01 M HEPES pH 7.5, 0.15 M NaCl, 0.01 M NaN3

c – 0.1 M acetic acid

All the buffers that were used for specific elution at pH 7.5 were supplemented with the same cations that were used for equilibration.a Con A equilibration buffer contained 1 mM CaCl2, 1 mM MgCl2 and 1 mM MnCl2.b HEPES buffer used for ECL-, SNA- and UEA-agarose contained 0.1 mM CaCl2.c HEPES buffer used for PHA-E and PHA-L-agarose contained 0.1 mM CaCl2 and 0.1 mM MnCl2.

Table 2The serum concentrations of IGF-I, IGFBP-3 and the IGF-I/IGFBP-3 ratio in healthywomen and patients diagnosed with breast tumour: group 1 — patients with benigndisease, group 2 — patients with malignant disease (survivors) and group 3 — patientswith malignant disease (non-survivors).

Healthywomen

Group 1 Group 2 Group 3

IGF-I (nmol/L)mean±SD

27.2±5.24 15.7±4.45a 15.6±6.46a 5.8±2.30a,c,d

IGFBP-3 (nmol/L)mean±SD

90.8±27.90 79.4±26.38 77.3±29.60 56.7±21.56a

IGF-I/IGFBP-3mean±SD

0.31±0.067 0.21±0.051a 0.14±0.075a 0.10±0.037a,c

Statistically significant differences (pb0.05) between healthy women and patientsare indicated as following: a: comparing healthy subjects with each patient group;b: comparing patients diagnosed with benign disease (group 1) with patientsdiagnosed with breast cancer (survivors, group 2); c: comparing patients diagnosedwith benign disease (group 1) with patients diagnosed with breast cancer (non-survivors, group 3) and d: comparing surviving patients diagnosed with breast cancer(group 2) with non-surviving patients (group 3).

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from the columns in all cases ranged from 97% to 100%. All theindividual samples used in this study were analysed using all eightlectin columns.

To determine the presence of different forms of IGFBP-3 withrespect to molecular mass, four separate pools of sera were prepared(one for each of the four study groups) by mixing equal volumes ofindividual sera. All these pools were separately applied to the eightcolumns (without prior incubation with 125I-IGF-I) as describedabove. The fractions (both from pH 7.5 and from pH 3.0 elutions) thatcorresponded to those with the highest binding of 125I-IGF-I(determined previously) were collected from each column, pooledand immediately neutralised using 2 M Tris–HCl pH 8.9. The poolswere dialysed first against distilled water for 3 h at room temperatureand then against 0.9% NaCl overnight at 4 °C. After being concentratedto approximately 1 mL using Microcon centrifugal filter devices(10 kDa cut-off membrane, Millipore, Billerica, MA, USA) the sampleswere subjected to SDS-PAGE and immunoblotting.

IGFBP-3 detection by immunoblotting

Immunoblotting with an anti-IGFBP-3 antibody was used todetermine the IGFBP-3 profile in pooled sera prior to and afterlectin-affinity chromatography (peak fractions at both pH elutions). Apolyclonal affinity purified goat anti-IGFBP-3 antibody (DiagnosticSystems Laboratories Inc., Webster, TX, USA) and a HRP-conjugatedswine anti-goat antibody (Biosource, Camarillo, CA, USA) were used[25]. IGFBP-3 was detected using enhanced chemiluminescence (GEHealthcare, Buckinghamshire, UK).

Statistical analysis

All statistical comparisons were performed using SPSS 16.0software. Data for each measured parameter within all the examinedgroups were subjected to the Kolmogorov–Smirnov test. As onlynormal distributions were found, the results were expressed as themean value and the standard deviation. The non-parametric Kruskal–Wallis test was performed to evaluate differences between studygroups. In order to determine differences between specific groups theTukey HSD post hoc test was employed. The minimal statisticalsignificance was set at pb0.05.

Results

Serum concentrations of IGF-I and IGFBP-3 were measured inhealthy women and in patients diagnosed with breast tumoursindicated for surgery. The obtained results are presented in Table 2.The concentration of IGF-I was lower in groups 1 and group 2 whencompared to healthy women. However, no statistical differencebetween groups 1 and 2 was observed. In group 3 (non-survivingcancer patients) the IGF-I concentration was decreased to a greater

extent compared to healthy women, group 1 and group 2 patients.The IGFBP-3 concentration was significantly reduced only in group 3patients compared to that in healthy women, whereas no differencesbetween patient groups were found. The IGF-I/IGFBP-3 ratio wassignificantly decreased in all patients compared to healthy women.Non-surviving patients had significantly lower levels of IGF-I and,consequently, a lower IGF-I/IGFBP-3 ratio than the rest of the patients.

Lectin-affinity chromatography was used to characterise thecarbohydrate content of IGFBP-3. The reactivity of IGFBP-3 towardsthe immobilised lectins was monitored by measuring radioactivitythat represented the specific binding of 125I-IGF-I–IGFBP-3 complexesto a specific lectin. Most of the lectin-affinity columns (except PHA-Eand PHA-L) separated IGFBP-3 glycoforms into two peaks. Theradioactivity in the first peak (lower affinity binding) was elutedwith specific sugar solution at pH 7.5 whereas the radioactivity in thesecond peak (higher affinity binding) was eluted with specific sugarsolution at pH 3.0. In the case of PHA-E and PHA-L columns specificallybound complexes were eluted with acetic acid solution in one peak.

IGFBP-3 in sera from healthy subjects and patients diagnosed withbreast tumours showed specific binding to lectins that decreased inthe following order: SNANCon ANPHA-ENRCA INsuccinylatedWGANPHA-LNECLNUEA (Table 3 and Fig. 1). The Kruskal–Wallistest revealed significant differences between the study groups withrespect to the total specific binding to the following columns: Con A,RCA I, SNA, PHA-E and PHA-L (pb0.05). The Tukey post hoc testdemonstrated that the total binding to Con A-, SNA- and PHA-L-agarose was lower in non-surviving cancer patients (group 3) than inall other groups whereas a decrease in binding to RCA I was observedonly in non-surviving cancer patients compared to healthy women.Between patient groups significant differences were revealed only forPHA-E eluates: IGFBP-3 from non-surviving patients interacted to a

Table 3Total specific binding of IGFBP-3 complexes (cpm) to the Con A, succinylated WGA, RCA I, ECL, SNA, UEA, PHA-E and PHA-L columns, the ratio of the total amount of bound IGFBP-3complexes (cpm) and the concentration of IGFBP-3 (nmol/L) and the ratio of the amounts of IGFBP-3 complexes eluted with specific sugar solutions at pH 7.5 and at pH 3.0.

Lectin Binding characteristics Healthy women Group 1 Group 2 Group 3 Significant difference

Con A Total bound (cpm) 24437±4577.8 19329±2125.6 22640±5245.1 11290±2267.5 *Total bound (cpm)/conc. (nmol/L) 253±48.7 293±55.8 233±62.1 215±31.4Ratio pH 7.5/pH 3.0 6.1±0.66 4.4±0.51 4.6±0.84 2.4±0.51 *

Succ. WGA Total bound (cpm) 3859±1052.6 4081±649.6 3214±742.4 3445±901.5Total bound (cpm)/conc. (nmol/L) 38±12.9 66±17.9 32±7.9 78±34.6 *Ratio pH 7.5/pH 3.0 1.6±0.59 0.6±0.27 1.2±0.32 0.6±0.42 *

RCA I Total bound (cpm) 12570±2562.9 9705±2251.6 10699±1386.3 7023±2831.6 *Total bound (cpm)/conc. (nmol/L) 124±32.9 155±61.5 116±17.2 111±15.6Ratio pH 7.5/pH 3.0 10.0±6.07 5.3±1.83 7.0±0.42 3.9±0.94 *

ECL Total bound (cpm) 2646±748.6 2105±198.8 2481±297.9 2659±812.3Total bound (cpm)/conc. (nmol/L) 28±9.1 33±6.9 25±2.0 45±7.2 *Ratio pH 7.5/pH 3.0 2.9±1.34 1.8±0.45 2.5±0.82 2.4±0.79

SNA Total bound (cpm) 38586±5713.6 38742±6887.5 42456±6206.7 22619±3461.2 *Total bound (cpm)/conc. (nmol/L) 396±107.9 505±50.8 515±187.4 421±53.0Ratio pH 7.5/pH 3.0 1.6±0.32 1.4±0.29 1.4±0.18 0.93±0.15 *

UEA Total bound (cpm) 1336±267.6 1647±431.7 1222±296.8 1155±184.5Total bound (cpm)/conc. (nmol/L) 15±1.6 23±10.5 14±3.5 21±2.6Ratio pH 7.5/pH 3.0 0.2±0.10 0.3±0.13 0.3±0.15 0.3±0.21

PHA-E Total bound (cpm) 13282±2087.6 17540±1452.7 17580±4377.8 10339±2205.2 *Total bound (cpm)/conc. (nmol/L) 191±67.8 277±51.3 208±65.0 197±58.2

PHA-L Total bound (cpm) 2768±364.6 3236±549.2 2790±473.2 1868±231.4 *Total bound (cpm)/conc. (nmol/L) 32±8.6 48±18.9 32±7.1 35±5.3

Statistical analysis was performed using the non-parametric Kruskal–Wallis test and significant differences between the groups (pb0.05) are shown with *.

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lesser extent than IGFBP-3 from the rest of the patients. Specificelution at pH 7.5 from Con A and SNA columns was significantly lowerin group 3 patients than in all other groups whereas pH 7.5 specificelution from succinylated WGA and RCA I was lower only in non-surviving cancer patients when compared to healthy women.Significant differences in specific elution at pH 3.0 were obtainedjust for two lectins: succinylated WGA (healthy women/group1,group 1/group 2) and SNA (group 1/group 2, group 2/group 3).

To analyse whether changes in specific binding between immo-bilised lectins and the 125I-IGF-I–IGFBP-3 complexes were a conse-quence of altered carbohydrate structure caused by tumours or aconsequence of decreased concentration of IGFBP-3, the ratiobetween total binding of IGFBP-3 complexes (cpm) and IGFBP-3concentration was calculated for each sample in all the study groups.The results are presented in Table 3. When the study groups werecompared only binding to succinylated WGA and ECL exhibitedstatistical difference. For both lectins a difference existed betweenhealthy women and non-surviving cancer patients. Specific IGFBP-3binding to these lectins was low and poised a technical limitationwhen considering the validity of the result.

The ratio of pH 7.5 and pH 3.0 elutions was calculated for eachcolumn and for all the subjects included in this study, as well.Significant differences in the ratio were observed for Con A,

Fig. 1. Bound radioactivity (cpm) corresponding to specific elution at pH 7.5 (grey bars) from(black bars) from the Con A, succinylatedWGA, RCA I, SNA, PHA-E and PHA-L columns. Study(patients with malignant disease, survivors) and G3 — group 3 (malignant disease patients

succinylated WGA, RCA I and SNA columns (Table 3). The Tukeypost hoc test showed that in all cases the ratio decreased in the group 3patients when compared with healthy women indicating thatincreased radioactivity was eluted with specific sugar solution at pH3.0. Other differences were the following: Con A column (healthywomen/patients with benign tumour, healthy women/survivingcancer patients, patients with benign tumour/non-surviving cancerpatients, surviving cancer patients/non-surviving cancer patients);succinylated WGA column (healthy women/patients with benigntumour) and SNA (patients with benign tumour/non-survivingcancer patients, surviving cancer patients/non-surviving cancerpatients).

Immunoblotting was used to analyse the IGFBP-3 profile from thepooled sera and from the collected peak fractions from all lectin-affinity columns. Fig. 2 indicates representative immunoblots of bothpH 7.5- and pH 3.0-eluates from SNA, Con A, RCA I and PHA-E columnsfor all four study groups. Immunoblotting was also performed usingeluates from the other lectin columns (those that bound significantlyless IGFBP-3). However, in such samples IGFBP-3 bands were notvisible (data not shown).

Immunoblotting demonstrated that the pooled sera from all studygroups contained the IGFBP-3 doublet at 40–45 kDa (intact IGFBP-3),a visible IGFBP-3 form at 29 kDa and poorly visible IGFBP-3 forms at

the Con A, succinylated WGA, RCA I and SNA columns and to specific elution at pH 3.0groups: H— healthy women, G1— group 1 (patients with benign disease), G2— group 2, non-survivors).

Fig. 2. Detection of IGFBP-3 immunoreactivity in the following samples: healthysubjects (panel a), patients diagnosed with benign breast disease (panel b), survivingbreast cancer patients (panel c) and non-surviving breast cancer patients (panel d). Inall the immunoblots the order of the samples is the same. Lane 1: “pooled” serumsample for the appropriate group (mix of all the samples that belong to that group).Lane 2: pH 7.5 elution from the SNA column. Lane 3: pH 3.0 elution from the SNAcolumn. Lane 4: pH 7.5 elution from the Con A column. Lane 5: pH 3.0 elution from theCon A column. Lane 6: pH 7.5 elution from the RCA I column. Lane 7: pH 3.0 elution fromthe RCA I column. Lane 8: pH 3.0 elution from the PHA-E column. The positions of themolecular weight markers are indicated on the right hand side.

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18 and 15 kDa (fragmented IGFBP-3). The intensity of the intact andfragmented IGFBP-3 was decreased in all patients compared tohealthy women. However, in non-surviving breast cancer patientsthe protein bands of both intact and fragmented IGFBP-3 were hardlyvisible. Furthermore, an additional band above 45 kDa was evident.These results firmly correlated with the results obtained in the IRMA-IGFBP-3 assay (Table 2). The collected and pooled chromatographicfractions were dialysed to remove sugars and concentrated in order toachieve a similar dilution as that of the initial pooled sera forelectrophoresis. Depending on the column the number of peakfractions that were collected varied. For example, in the case of theSNA and Con A columns, in which high specific binding at pH 7.5 wasfound, five fractions were collected. In contrast, in the case of the UEAand PHA-L columns the peak consisted of just one fraction. The finalconcentration step adjusted the sample volumes to approximately,but not exactly, 1 mL. Accordingly the “concentration factor” wasslightly different for different lectin columns and study groups.Therefore, the immunoblot results cannot be used for a directquantitative comparison but they are, on the other hand, valuablefor a qualitative comparison of the IGFBP-3 molecular forms presentin the eluates.

The IGFBP-3 doublet and the 29 kDa form were clearly visible in apool of sera obtained from healthy women, in pH 7.5 eluates fromSNA, Con A and RCA I columns and in the pH 3.0 eluate from the PHA-Ecolumn (Fig. 2, panel a). The minor IGFBP-3 forms at 18 and 15 kDawere present as very weak bands in most of the profiles. The pH 3.0eluate from the SNA column (lane 3) contained all anti-IGFBP-3immunoreactive forms, but to a significantly reduced degree (45, 40,

29, 18 and 15 kDa). A unique band with a molecular weight between30 and 40 kDa was also present in this sample. This immunoreactivespecies was not created by acidic pH as it was not present in the otherpH 3.0-eluates. IGFBP-3 species in pH 3.0-eluates from Con A (lane 5)and RCA I (lane 7) columns were barely detectable.

In patients with benign breast disease (Fig. 2, panel b) the IGFBP-3immunoreactivity found in lectin-affinity column eluates was similarto that found in healthy women except for the SNA pH 3.0 eluate (lane3) and the RCA I pH 7.5 eluate (lane 6), in which the IGFBP-3 formswere almost undetectable. In surviving breast cancer patients (Fig. 2,panel c) all of the IGFBP-3 bands were comparable to those present inhealthy women, in terms of their presence, but in a lesser amount,except those in the pH 7.5-eluate from immobilised RCA I (almostundetectable). In samples from non-surviving breast cancer patients(Fig. 2, panel d) all IGFBP-3 bands were weaker compared to bothhealthy women and the other two groups of patients.

Discussion

Breast cancer is among the most prevalent cancers causing one ofthe highest numbers of cancer-related deaths among womenworldwide [26]. IGF-I has been reported to have both mitogenic andanti-apoptotic effects, and is considered to be a risk factor forcarcinogenic transformation. The GH/IGF axis stimulates proliferationof normal breast epithelial cells as well as breast hyperplastic cells andbreast cancer cells (in vitro and in vivo). There is substantial datalinking IGF-I and IGFBP-3 to the assessment of risk and prevention ofcancer. However, the results from most of the published studies arenot consistent. Studies on differentially-aged cancer patient popula-tions bearing different tumour locations using different experimentaldesigns have hampered understanding their conclusions. Suchheterogeneous results warrant a re-examination of the involvementof IGF-I/IGFBP axis in breast cancer pathology.

In this current work we havemeasured concentrations of IGF-I andIGFBP-3 in healthy women and in patients that were previouslydiagnosed with breast tumour. Our results indicated that in all patientgroups the IGF-I concentration was significantly decreased comparedto healthy people. Furthermore, an extremely low IGF-I concentrationwas measured in non-surviving cancer patients. A significantlydecreased concentration of IGFBP-3 was also determined in non-surviving patients.

There are scarce data in the literature about the disturbances in theIGF system when breast cancer is diagnosed. Most of the publisheddata has concentrated on the connection between the IGF system andbreast cancer risk. Our results are in agreement with Kaulsay [27] whoreported that patients with breast cancer (compared to those withbenign disease) had low IGFBP-3. In vivo studies have suggested that ahigh local tumour level of IGFBP-3 is associated with a poor prognosis.In contrast, epidemiological studies regarding the circulating IGFBP-3concentration revealed both positive and inverse associations ofIGFBP-3 with breast cancer [28]. There is a reasonable evidence thathigh circulating IGF-I levels are related to increased cancer riskwhereas advanced cancers are often associated with a very low IGF-Ilevel causing a decrease in the IGF-I/IGFBP-3 ratio [29]. In this studywe found that all the patient groups had a decreased IGF-I/IGFBP-3ratio compared to healthy women and that non-surviving patientshad the lowest ratio. Espelund et al. did not observe any change in theratio between healthy subjects and early-stage breast cancer inwomen [19]. Yet, all the patients involved in our study had anadvanced stage of tumour development, they were treated bychemotherapy and were scheduled for surgery.

During carcinogenesis cells may undergo a dramatic transforma-tion of the glycosylation mechanism and, as a consequence, manycellular proteins show an altered glycosylation pattern. This phenom-enon is considered to be a hallmark of malignancy and is mainlycharacterised by enhanced synthesis of highly branched and sialylated

730 I. Baričević et al. / Clinical Biochemistry 43 (2010) 725–731

N-glycans [30,31]. In the case of IGFBP-3, altered glycosylation maychange its affinity towards IGFs, although it is not essential for IGF-Ibinding [32]. Glycosylation can, on the other hand, modulate the half-life of the molecule in the circulation and its clearance rate. In the caseof IGFBP-3 increased proteolysis liberates increased amounts of free,physiologically active IGFs.

In the literature there are scarce data on IGFBP-3 glycosylation andnone in conditions of malignancy. Our results suggest that IGFBP-3present in human serum contains mainly biantennary complex typeN-glycans with a bisecting GlcNAc residue that is ß1→4-linked to theß-linked Man in the trimannosyl core. These N-glycans have a highcontent of terminal Sia that is bound byα2→6 bonds to Gal or GalNAc.A bisecting GlcNAc residue is possibly core fucosylated.

In non-surviving breast cancer patients a decrease in total bindingtowards Con A, RCA I, SNA, PHA-E and PHA-L may be considered to bea consequence of decreased concentration of IGFBP-3 in thecirculation. The ratios between the total amount of bound IGFBP-3complexes and the concentration of IGFBP-3 were similar in allgroups. However, the calculated ratio between the radioactivityeluted at pH 7.5 and pH 3.0 indicated that specific alterations incarbohydrate content occurred in non-surviving breast cancerpatients. On average double the amount of IGFBP-3 complexes wereeluted at pH 3.0 from the samples of these patients than from thesamples of healthy women and other patients from the followinglectin columns: Con A, succinylated WGA, RCA I and SNA. IGFBP-3from non-surviving breast cancer patients had a significantly greateramount of biantennary complex and/or hybrid type N-glycans withmore terminal Siaα2→6Gal residues, abundance of Man residues,presence of bisecting GlcNAc and core fucosylation. Altered saccharidestructure of IGFBP-3 is most likely a consequence of activatedglycosyltransferases due to increased levels of glucocorticoides,cytokines and other factors [33]. Alteration of the glycan structuremay be part of the adaptive physiological mechanism that protectsIGFBP-3 from further degradation andmaintains its function as amaincarrier of IGFs.

Krzeslak et al. investigated differences in glycosylation of cytosolicproteins isolated from benign and malignant human thyroid neo-plasms using plant lectin ECL and RCA I [34]. They showed that lesslectin binding in the case of malignant carcinomas corresponded to alower degree of glycosylation of some cytosolic proteins and toincreased sialylation of terminal Gal residues. Tajiri et al. comparedthe glycan profiles of free and complexed prostate-specific antigen(PSA) from cancer patients [35]. The results indicated that serum PSAcontains fucosylated biantennary oligosaccharides that are abundant-ly sialylated (mainlyα2→3 bonds). In seminal plasma, high-mannoseand hybrid types of oligosaccharides predominate in PSA with Siabound by α2→6-links to biantennary oligosaccharides. Peracaula etal. analysed PSA N-glycans from seminal fluid and prostate cancercells (LNCaP cell line) [36]. Normal PSA from seminal plasma hadsialylated biantennary complex structure. PSA from LNCaP cells,however, showed a significantly different glycan pattern, especially inthe outer ends of the biantennary complex structures that were notsialylated and contained a high amount of Fuc. The level of GalNAcincreased in LNCaP. These carbohydrate differences allow differentialdiagnosis between benign prostate hyperplasia and prostate cancer.

Ovarian cancer is also characterised by changes in glycosylation, inparticular with increased levels of core fucosylation and an increasedpresence of agalactosyl biantennary glycans and sialil Lewis x (SLex)[37]. Hamid et al. found changes in fucosylation and sialylation on thetri- and tetraantennary structures in the advanced breast cancerpatients [38]. The increase was obtained in α1→3Fuc, that forms apart of the SLex epitope. The main differences in the structure of alpha-foetoprotein frompatientswith liver cancer and benign diseases are theincreased core fucosylation and bisecting GlcNAc antennary structures.According toQiu et al., possiblemarkers that coulddistinguish colorectalcancer from adenomas and normal tissue include elevated sialylation

and fucosylation of complement C3, histidine-rich glycoprotein andkininogen-1 [39]. In patientswith pancreatic cancer Zhao et al. observedincreased sialylation and fucosylation of hemopexin, kininogen-1,antithrombin-III and haptoglobin-related proteins while decreasedsialylation on plasma protease C1 inhibitor [40].

Our results have shown that benign transformation of breasttissue, in general, does not cause significant reduction in theconcentration of circulating IGFBP-3 or a significant change in thestructure of IGFBP-3 either in its carbohydrate moiety (determined bya lectin-affinity method) or in its protein core (determined byelectrophoresis and immunoblotting). In patients with malignantbreast tissue transformation the concentration of IGFBP-3 remainswithin the reference range or it may be reduced. The structure ofIGFBP-3, however, exhibits changes. The changes are more pro-nounced as the disease progresses particularly in non-survivingpatients. In patients that died within a year following the surgery (inmost cases within 6 months), an increased amount of IGFBP-3having a higher content of biantennary complex and/or hybrid typeN-glycans with more terminal Siaα2→6Gal residues, abundance ofMan residues, presence of bisecting GlcNAc and core fucosylation wasevident. These transformed IGFBP-3 molecules may correspond to animmunoreactive protein band having amolecular weight greater than45 kDa in immunoblot.

Alteration in the primary structure of IGFBP-3 may affect theconformation of this molecule which, in turn, may affect its affinity forligands (resulting in a different relative ratio of IGFs to IGFBP-3 and,thus, bioavailability of the peptides) and the affinity of proteases, andreceptors for IGFBP-3 (in the physiological milieu) or antibodies (inimmunochemical assays). We are aware that further studies employ-ing a higher number of cases and, possibly, grouping according todisease stage would elaborate our current results. The data reportedhere clearly showed that alterations in IGFBP-3 glycosylation occurredduring breast cancer progression as determined using a panel of plantlectins. This methodology maybe a useful tool to identify variations inprotein glycosylation in different stages of diseases.

Acknowledgment

This work was financed by the Serbian Ministry of Science andTechnological Development (grant number 143019).

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