Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas

Clinical Biochemistry 45 (2012) 1421–1431

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Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas Amel ben Anes a, 1, Hela ben Nasr b, 1, Philippe Hammann c, Lauriane Kuhn c, Mounir Trimeche d, Bechr Hamrita e, Iheb Bougmiza f, Anouar Chaieb g, Hedi Khairi g, Karim Chahed a, h,⁎ a

Laboratoire d'Immuno-Oncologie Moléculaire, Faculté de Médecine de Monastir, Tunisia Unité de recherche, adaptations cardio-circulatoires et hormonales à l'exercice musculaire, Faculté de Medecine, Sousse, Tunisia Plate Forme Protéomique, Institut de Biologie Moléculaire et Cellulaire, CNRS, 67084 Strasbourg, France d Département de Pathologie, CHU Farhat Hached, Sousse, Tunisia e Institut Préparatoire des écoles d'Ingénieurs, Sfax, Tunisia f Département de Médecine Communautaire, Faculté de Médecine de Sousse, Tunisia g Service d'obstétrique et des maladies féminines, Centre Hospitalo-Universitaire-Farhat-Hached, Sousse, Tunisia h Faculté des Sciences de Sfax, Département de Biochimie, Université de Sfax, Tunisia b c

a r t i c l e

i n f o

Article history: Received 3 February 2012 Received in revised form 28 May 2012 Accepted 15 July 2012 Available online 26 July 2012 Keywords: Breast cancer α1-proteinase inhibitor Metastasis Proteomics

a b s t r a c t Objectives: To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients. Design and methods: Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression. Results: A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found. Conclusions: Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

1. Introduction Tumor cells are characterized by their ability to invade normal tissues and to spread. This invasion is in part due to proteases, which are suspected to play a major role in extracellular matrix proteolysis and cell migration. While under normal physiological conditions a balance exists between proteases and their inhibitors, recent findings suggest that an imbalance between these two counterpart proteins may arise as either a causative or a responsive element in malignant diseases and could be related to tissue damage and disease progression [1]. Up to now, although increasing evidence suggests that proteolytic enzymes play crucial roles in cell proliferation and spread of cancer, the role of the body's natural inhibitors of these enzymes in such processes remains largely unknown.

⁎ Corresponding author at: Laboratoire d'Immuno-Oncologie Moléculaire, Faculté de Médecine de Monastir, Tunisia. E-mail address: [email protected] (K. Chahed). 1 Amel ben Anes and Hela ben Nasr are co-first authors and contributed equally to the study.

The α1-proteinase inhibitor (α1-Pi), also known as serpin A1 is among the protease inhibitors whose main function is to inhibit leukocyte elastase, trypsin and plasminogen [2]. This proteinase inhibitor has also been shown to block TGF-alpha release from its membrane-bound precursor on MCF-7 breast cancer cells, which may affect anchorage dependent growth thus acting as a tumor suppressor [3]. It was also found to stimulate fibroblast proliferation and procollagen synthesis and interact with enzymes involved in apoptosis [4, 5]. Additionally, native α1-Pi has been shown to increase insulin-induced mitogenesis, to inhibit cell growth in human plasma and decrease tendency towards metastasis [6]. α1-Pi is synthesized essentially in the liver but also in alveolar macrophages, circulating monocytes and in tumor cells [7]. This protein plays a crucial role in many processes and was found to be increased above normal values during the acute phase response and in chronic diseases such as in liver cirrhosis and hepatitis [8]. Up to now, several studies have revealed that serum levels of α1-Pi are significantly elevated in neoplastic diseases and may correlate with tumor progression [9]. In contrast, others have reported a significant correlation between α1-Pi down-regulation and increased risk of different types of cancer and may thus play a protective role in tumorogenesis [3].

0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.07.099

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The aim of the present study was therefore to examine the specificity of α1-Pi as a tumor marker in infiltrating ductal breast carcinomas and to assess the clinical significance of antigenic and functional levels of this proteinase inhibitor in disease progression. 2. Design and methods 2.1. Patients and controls Patients (80) and controls (95) were selected from the same population living in the middle coast of Tunisia. Sera were obtained at the time of diagnosis prior to any therapy from patients with histologically diagnosed breast cancer at the Department of Gynecology at Sousse Hospital after informed consent was given. Tumors classified as infiltrating ductal carcinomas were pathologically staged according to the tumor-node-metastasis classification system of UICC. Histological grade was assessed according to the system of Elston and Ellis. All patients were divided into four groups according to their disease stage (stages I–IV). Stage grouping was based on the TNM formula of the patient depending on tumor size, presence of regional metastatic lymph nodes (N) and presence of distant metastasis. This patient population consisted of 80 females with an age range of 29–85 years (median age, 48 years). Sera from 95 age-matched healthy volunteer women who visited the general health check-up division at Sousse Hospital (age range: 26–75 years, median age, 45 years) with no history of cancer or autoimmune disease were collected and used as controls. Aliquots of sera were immediately frozen at −80 °C until used and were never refrozen. CA15.3 levels were expressed as units/mL using a radio-immunometric assay. 2.2. Determination of immunoreactive levels of α1-Pi, tryptic inhibitory capacity and α1-Pi specific activity Determination of tryptic inhibitory capacity (TIC) against bovine trypsin in the serum was performed with N-benzoyl-DL-argininep-nitroaniline (BAPNA, Sigma) as substrate according to Dietz et al., [10]. Reduction in tryptic activity after the addition of plasma to a standard trypsin solution was determined. TIC was expressed in international units (μmol/min per mL or units/mL). The immunoreactive levels of α1-Pi were quantified by the single-radial immunodiffusion technique. Radial immunodiffusion (IDR) was performed by a minor modification of the method of Mancini et al. [11] with a final concentration of 1% agarose in barbital buffer pH 7.6 and 1:30 dilution of antiserum. The values were presented as mg/dL. We used a calibration set for serum proteins (SPQ TM Test system) for the quantitative determination of α1-proteinase inhibitor protein in human serum. The accuracy of the procedure was determined with control sera containing a predetermined concentration of α1-proteinase inhibitor. The immunoprecipitates were stained with Coomassie brilliant blue R-250. We performed preliminary experiments to ensure linearity of the measured α1-Pi protein concentration. For each experiment, the same set of diluted serum standards was used to generate the calibration curve on each plate and the diameter of each serum was estimated from the standard curves (supplementary file 1). The determined values by radial immunodiffusion and TIC were then used for the calculation of the specific inhibitory activity (inhibitory capacity/ antigen concentration ratio in μmol/min/mg of α1-Pi) [10]. Each experiment (IDR, TIC) was repeated at least twice and reproducibility was about 90% or more which reflected the small variations between experiments (Supplementary file 2). 2.3. Enzyme linked immunosorbent assay (ELISA) for the determination of circulating immune complexes Circulating immune complexes (CIC) were measured by an ELISA method employing the A0012 α1-Pi antibody (Dako). Immune complexes present in serum samples (CIC) were detected through a

peroxidase-conjugated anti-human IgM or IgG. The reaction was revealed with ABTS as substrate and OD at 405 nm was measured. Briefly, 100 μL of 1:20 antibody diluted in 10 mM PBS buffer pH 7.2 was adsorbed in each well of 96‐well ELISA microplates. After overnight incubation at 4 °C, four washes with 10 mM sodium phosphate buffer, 0.1% Tween 20 (PBST) were done. In each well, 200 μL of blocking buffer (1% BSA in PBS) was added and plates were incubated at 37 °C for 3 h. One hundred microliter of 1:50 serum samples diluted in PBS was applied in triplicate and incubated overnight at 4 °C with the adsorbed antibody. After, plates were washed with PBST and 1% Triton-X100 in PBS; after that 1:1500 anti-human IgM (Sigma) or 1:2000 anti-human IgG horseradish peroxidase conjugates (Sigma, A8775) was added and incubated at 4 °C for 2 h. Freshly prepared 2,2′-azino-bis (3-ethylbenzothiazoline)-6-sulphonic acid (ABTS, Sigma) as substrate in sodium citrate buffer, pH 5.0 and 30% H2O2 were then added. Results were expressed as optical density (OD) units at 405 nm. Each experiment was repeated at least twice and reproducibility after assessment of intraclass correlation coefficients for relative reliability and coefficients of variation was about 90% or more which reflected the small variations between experiments (supplementary file 2). 2.4. Sensitivity, specificity and ROC analyses To determine the optimal cutoff values of α1-Pi levels, specific activity and circulating immune complexes as single markers, receiver operator characteristic (ROC) curves were drawn and the areas under the curves (AUC) were calculated for each test using SPSS 11.0. Their diagnostic values were evaluated by plotting sensitivity against 1-specificity in ROC space. Sensitivity (Se) was defined as the incidence of true positive results when the assays were applied to patients known to have cancer and was calculated using: Se = TP/TP + FN, where TP= true positive and FN= false negative. Specificity (Sp) was defined as the incidence of true negative results when the assays were applied to subjects known to be free of cancer and was determined using: Sp = TN/TN + FP, where TN= true negative and FP= false positive. The percentage of patients known to have cancer that were correctly classified as positive by the assay was: PPV (positive predictive value) = TP × 100 / TP + FP. The percentage of healthy donors correctly classified as negative by the assay was: NPV (negative predictive value) = TN × 100 / TN + FN. A similar procedure was followed in order to discriminate patients with node metastasis from node negative ones. Depending on the cutoff value, a range of sensitivity and corresponding specificity values were obtained for α1-Pi, specific activity and circulating immune complexes. 2.5. Total protein determination, SDS-PAGE and Western-blotting analyses The amount of total protein was determined with the Bio-Rad protein assay [12]. Bovine serum albumin was used as a standard. Equal amounts (2 μg) of protein samples from neoplastic or non‐tumor homogenates (serum, tissues) along with a standard mixture of proteins (Bio-Rad) were treated with SDS according to Laemmli [13]. SDSPAGE (10% resolving and 3% stacking gel) was performed at a constant current of 35 mA for 1 h, until the tracer dye (bromophenol blue) was within 2 mm of the lower edge. After electrophoresis, gels were either stained with Coomassie blue (Sigma) or were electrophoretically transferred to a nitrocellulose membrane (in 48 mM Tris–HCl pH 9.2, 39 mM glycine, 20% v/v methanol) for 1 h at 20 V using a semi-dry immunoblot transfer system (Bio-Rad). It was estimated after staining of the polyacrylamide gel that >90% of proteins transferred to the membrane. The membrane containing serum/tissue samples was then processed as follows: a, gently shake the membrane for 2 h in a 3% BSA solution in 10 mM PBS; b, wash three times for 10 min with 10 mM PBS, 0.1% Tween 20 (PBST); c, shake for 2 h in 1% BSA in 10 mM PBS containing

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rabbit antiserum to human α1-Pi (A0012, Dako) at a dilution of 1:1000; d, wash three times for 15 min each with PBS containing 1% Tween 20; e, shake for 1 h in 1% BSA containing goat anti-rabbit IgG linked with horseradish peroxidase at a dilution of 1:5000; f, wash twice 15 min each with 10 mM PBS, 0.5% Tween 20; and g, 100 μL of substrate solution containing 3,3′-diaminodiazobenzidine in PBST and 30% H2O2 was added to each well. 2.6. Two-dimensional gel electrophoresis (2-DE) and 2-DE western blotting Analytical 2-DE was carried out in a Bio-Rad system (Miniprotean II) as described previously [14]. Equal amounts of plasmatic proteins issued from breast cancer patients with different tumor stages (n= 10) were applied to the first dimension and at least three IEF gels were run for each sample. IEF was performed on 7 cm IEF rod gels (pH 4.0– 8.0) at 200 V for 15 min, 300 V for 15 min and 400 V for 18 h. Focused strips were equilibrated in SDS equilibration buffer (125 mM Tris–HCl pH 6.8, 2.5% (w/v) SDS, 10% (w/v) glycerol, 0.025% (w/v) bromophenol blue) and were then loaded onto 12% SDS gel slabs for separation in the second dimension. For each experiment, IEF and SDS-PAGE were carried under similar conditions. Western blotting of 2-DE gels with α1-Pi antibody was performed as previously described (Section 2.5). 2.7. Spot picking, in-gel digestion and protein identification by MALDI-TOF-MS Picked spots were washed with 100 μL of 25 mM NH4HCO3 and dehydrated with 100 μL of acetonitrile (ACN). This operation was repeated twice and the pieces of gel were dried under vacuum for 10 min. Reduction was achieved by 1-hour treatment with 10 mM DTT in NH4HCO3 buffer (100 μL) at 56 °C. After discarding the DTT solution, alkylation reaction was performed by addition of 100 μL of 25 mM iodoacetamide in 25 mM NH4HCO3 buffer for 1 h at room temperature, protected from light. Finally, the excised gel pieces were again washed 3 times for 5 min with 25 mM NH4HCO3 and ACN alternately. Gel pieces were completely dried under vacuum before tryptic digestion. The dried gel volume was evaluated (about 1 to 2 μL) and three volumes of trypsin (Promega, V5111), 12.5 ng/μL, in 25 mM NH4HCO3 buffer (freshly diluted) were added. The digestion was performed at room temperature overnight. Afterwards 5 μL of 35% H2O/60% ACN/5% HCOOH was added and the mixture sonicated for 30 min and centrifuged in order to extract tryptic peptides. Mass measurements were carried out on a BIFLEX III TM MALDI-TOF (Bruker, Daltonics, Bremen, Ge) equipped with the SCOUT TM high resolution Optics with an X–Y multisample probe and gridless reflector. This instrument was used at a maximum accelerating potential of 19 kV (in positive mode) and was operated in reflector mode. A saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma, Saint Louis, MO) in acetone was used as a matrix. A first layer of fine matrix crystals was obtained by spreading and fast evaporation of 0.5 μL of the matrix solution. On this fine layer of crystals, a droplet of 0.5 μL of aqueous HCOOH (5%) solution was deposited. Afterwards, 0.5 μL tryptic digest was added and mixed to a second 0.3 μL droplet of saturated matrix solution (in 50% H2O/50% ACN). The preparation was dried under vacuum. The sample was washed once by applying 0.7 μL of aqueous HCOOH (5%) solution on the target and then flushed after a few seconds. In positive mode, internal calibration was performed with tryptic peptides coming from autodigestion of trypsin, with monoisotopic masses at m/z= 842.510 and m/z =2211.105. Monoisotopic peptide masses were assigned and used for database searches. For mass measurements, up to one missed tryptic cleavage and optional methionine oxidation were considered. In most cases the mass accuracy was less than 50 ppm, a value which is generally considered adequate for achieving statistically significant results for protein identification. These files were then fed into the search engine MASCOT (Matrix Science, London, UK). The data were searched against the Swiss-Prot and NCBI non-redundant (NCBInr) protein sequence databases.

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2.8. Immunohistochemistry (IHC) IHC analyses were performed on formalin-fixed, paraffin-embedded sections of infiltrating ductal breast tumors (n= 34). Among these, 14 cases were node negative (N−) whereas 20 cases were node positive (N+). Standard indirect immunoperoxidase procedures were used for the detection of α1-Pi immunoreactivity in tumor tissues and adjacent non‐neoplastic sections. Briefly, five-micrometer sections were deparaffinized in toluene and subsequently hydrated with graded ethanol, and rehydrated in water. Slides were microwaved in 0.01 M citrate buffer at pH 6.0 for 20 min at 750 W. Thereafter, sections were rinsed thoroughly with water and placed in a Tris‐buffered saline (TBS) solution (0.05 M Tris–HCl pH 7.6, 1.15 M NaCl). Endogenous peroxidase activity was blocked with hydrogen peroxide solution for 7 min and sections were rinsed gently with TBS. Sections were then incubated at 4 °C overnight with the antibody against α1-Pi (Dako, A0012) at a dilution of 1:600. After rinsing in TBS, sections were incubated with the secondary antibody, DakoCytomation EnVision + Dual Link System Peroxydase (DakoCytomation, Glostrup, Denmark), for 30 min. Sections were washed 2 times with TBS, followed by application of the diaminobenzidine substrate pack according to the manufacturer's instructions (DakoCytomation), yielding a brown-colored signal. Finally, tissue sections were counterstained with hematoxylin and mounted. IHC staining was evaluated by two independent pathologists for staining intensity. The staining of cytoplasm, plasma membrane and nucleus was evaluated. Cells were considered positive when at least one of these components was stained. α1-Pi staining in infiltrating ductal carcinomas was scored with regard to the approximate percentage of tumor cells and their relative immunostaining intensities (% positive tumor cells× intensity). After counting both immunoreactive cells and the total of tumor cells, the average percentages of immunoreactive cells were calculated. The percentage of positive tumor cells was graded as 0, b 10%, 1, 10–25%, 2, 26–50%, 3, 51–74% and 4, 75–100%. Immunostaining intensity in tumor cells was rated as 0, none, 1, weak, 2, moderate and 3, intense. Cases scored with values below 3 were considered as a group as negative expression levels whereas cases scored with 4 or more were considered as a group as positive. 2.9. Statistical analysis The obtained results were presented as a mean±S.D. The differences in the means of experimental results were analyzed for their statistical significance using unpaired Student's t-test. For assessment of reproducibility, intraclass correlation coefficients (ICC) for relative reliability and coefficients of variation (CV) were determined using SPSS.11.0. Correlations were analyzed with spearman rank correlation. The chi-square test was used to determine the differences between groups. Statistical significance was defined as a P-valueb 0.05. Determination of median values and interquartile ranges and comparison of the studied parameters were performed using SPSS.11.0. The ROC curves with 95% confidence interval (CI) were performed to determine cutoff values. Diagnostic criteria, such as sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV) and areas under the ROC curves were determined using SPSS.11.0 and Epiinfo 6.0. P-value was considered significant if b0.05 and highly significant if b0.005. 3. Results 3.1. Determination of immunoreactive levels of α1-Pi, tryptic inhibitory capacity and α1-Pi specific activity A study of immunoreactive values of α1-Pi and tryptic inhibitory capacity (TIC) in serum from patients with infiltrating ductal breast carcinomas and controls was performed. There was a significant

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Table 1 Values of α1-Pi levels, antitryptic activity and circulating immune complexes in serum of patients with breast cancer according to clinicopathological parameters of the disease. Clinical parameters

Age ≤45 >45 Tumor size T1–T2 T3–T4 Lymph node metastasis Negative (N−) Positive (N+) Tumor stage I–II III–IV SBR grade I II–III

Measured parameters N (%)

α1-Pi levels

43.1 56.9

a

b

P-value

α1-Pi specific activity

4.06 ± 1.15 3.42 ± 1.47

0.06

0.59

3.63 ± 1.37 3.88 ± 1.32

259 ± 14 208 ± 8

0.01

50 50

258 ± 12 259 ± 13

9.1 90.9

260 ± 10 258 ± 13

c

P-value

α1-Pi/IgG /CIC

1.58 ± 0.4 1.31 ± 0.56

0.038

0.47

1.4 ± 0.52 1.5 ± 0.48

3.37 ± 1.35 3.75 ± 1.28

0.07

0.92

3.51 ± 1.32 3.99 ± 1.33

0.8

3.03 ± 0.59 3.87 ± 1.36

P-value

TIC

256 ± 14 256 ± 11

0.23

54.1 45.9

258 ± 11 260 ± 14

39 61

d

d

P-value

α1-Pi/IgM/CIC

0.094 ± 0.044 0.09 ± 0.022

0.61

0.057 ± 0.015 0.052 ± 0.015

0.21

0.45

0.091 ± 0.04 0.092 ± 0.026

0.95

0.057 ± 0.017 0.048 ± 0.011

0.04

1.29 ± 0.5 1.75 ± 0.47

0.023

0.099 ± 0.043 0.084 ± 0.019

0.03

0.059 ± 0.014 0.05 ± 0.015

0.045

0.15

1.35 ± 0.49 1.55 ± 0.49

0.12

0.096 ± 0.042 0.086 ± 0.023

0.27

0.058 ± 0.016 0.049 ± 0.014

0.03

0.18

1.16 ± 0.2 1.5 ± 0.5

0.14

0.079 ± 0.022 0.090 ± 0.035

0.41

0.057 ± 0.016 0.053 ± 0.015

0.6

P-value

Results are expressed as mean ± S.D. Values in bold cases are significant (P b 0.05). a α1-Pi levels (immunoreactive values) in serum are expressed in mg/dL. b Trypsin inhibitory capacity (TIC) is expressed in international units/mL. c Specific α1-Pi activity is expressed in μmol/min.mg of α1-Pi. d Values of α1-Pi/IgG/CIC and α1-Pi/IgM/CIC are expressed as optical density values (OD units).

decrease in the concentration of α1-Pi in the serum of breast cancer patients, as compared to healthy controls (258 ± 12 vs. 304 ± 18, respectively, P-value = 0.04). Mean ± S.D of α1-Pi levels among different clinicopathological parameters of breast cancer including tumor size, lymph node metastasis (N), clinical tumor stage and SBR grade were also determined (Table 1). Box-plots representing median values and interquartile ranges are presented in Fig. 1. A significant association (P-value = 0.01) between mean values of α1-Pi levels and lymph node metastasis was retrieved (Table 1, Fig. 1A). As shown in Table 1, the mean value of serum titers of α1-Pi in lymph node negative (N−) patients (259 ± 14) was higher than values reported in the lymph node positive (N+) group (208 ± 8). These calculated values were significantly low as compared to the healthy group value. There was however, no difference in the mean values of immunoreactive α1-Pi levels with the other clinical parameters of breast cancer (clinical tumor size, P-value = 0.59; tumor stage, P-value = 0.92; SBR grade, P-value = 0.80). From our findings, no significant difference in the activity of trypsin inhibitor (TIC) in the serum among patients and controls was found (3.84 ± 1.39 vs. 3.9 ± 1.53, respectively, P-value = 0.54). When we tested the relationship between the tryptic inhibitory capacity and prognostic indicators, no significant association was found with tumor size (3.63 ± 1.37 (T1–T2) vs. 3.88 ± 1.32 (T3–T4)), tumor stage (3.51 ± 1.32 (I–II) vs. 3.99 ± 1.33 (III–IV)) and SBR grade (3.03 ± 0.59 (I) vs. 3.87 ± 1.36 (II–III)). Interestingly, the mean value of TIC in node positive patients (3.75 ± 1.28) was higher than the mean value found in the node negative group (3.37 ± 1.35), although no significant difference was determined between them at the 5% level (P-value = 0.07, Fig. 1C). Specific activity of α1-Pi was also investigated but no difference in the mean values of the explored parameter between different groups was found (healthy controls: 1.28 ± 0.66, breast cancer patients: 1.48 ± 0.52, P-value = 0.16). When specific activity was determined among different clinicopathological parameters, the values were significantly higher in N+ patients in comparison to N− patients (1.75 ± 0.47 vs. 1.29 ± 0.50, P-value = 0.023, Fig. 1D). No difference in the mean values of specific activity of α1-Pi was retrieved for the other clinical parameters investigated (tumor size, P-value = 0.45; tumor stage, P-value= 0.12; SBR grade, P-value = 0.14). In addition to these prognostic indicators, we stratified our patients according to age. Only a significant difference in specific α1-Pi activity according to age was retrieved (age ≤ 45 years, 1.58 ± 0.4; age> 45 years, 1.31 ± 0.56, P-value=0.038).

3.2. Enzyme linked immunosorbent assay (ELISA) for the determination of circulating immune complexes An ELISA method was developed to detect α1-Pi circulating immune complexes among patients and controls but no significant differences were found (P >0.05). Differences among clinicopathological parameters (tumor size, tumor stage, SBR grade) were studied by SPSS on standardized data and no difference was found for α1-Pi/IgG/CIC (Table 1). Interestingly, a statistically significant difference among breast cancer lymph node status was found for both α1-Pi/IgM/CIC and α1-Pi /IgG/ CIC levels (Table 1, Fig. 1). Correlations between circulating immune complexes (α1-Pi/IgG/CIC, α1-Pi/IgM/CIC) and serum concentration of α1-Pi in breast cancer samples were investigated using the Spearman rank correlation coefficient. A poor but significant correlation of α1-Pi levels with α1-Pi/IgM/CIC and α1-Pi/IgG/CIC levels was found (R2 = 0.242, P-value= 0.035 and R2 =0.241, P-value= 0.033, respectively). In cancer samples, considering CA15.3 levels versus α1-Pi levels, α1-Pi/IgM/CIC and α1-Pi/IgG/CIC, no significant correlation was found (R 2 = − 0.007, P-value = 0.971; R 2 = − 0.24, P-value = 0.178; R 2 = − 0.218, P-value = 0.231, respectively). 3.3. Sensitivity, specificity and ROC analyses We used the results from our study at specific cutoff values to calculate sensitivity (incidence of true positive results when the assays were applied to breast cancer patients or node metastatic patients), specificity (incidence of true negative results when the assays were applied to healthy controls or N− patients) and predictive values. Depending on the cutoff value, a range of sensitivity and corresponding specificity values were obtained for each group investigated (Table 2). PPV and NPV were determined for various cutoff levels of the parameters studied (Table 2). The relationship between specificity and sensitivity was profiled by ROC curves and the areas under the curves estimated (Fig. 2). With the best diagnostic cutoff levels determined by ROC curves, the estimation of specific activity (μmol/min/mg α1-Pi) generates a greater area under curve (AUC: 0.69, P =0.0004) than areas determined for α1-Pi levels (mg/dL) or for circulating immune complexes (Fig. 2). At a cutoff value of 1.65, the estimation of specific activity yielded 50% sensitivity and 80% specificity for predicting node metastatic development among breast cancer patients. The PPV was 77.4% and the NPV was 52.9%. In the same context, α1-Pi levels presented at the best cutoff values (245.5 mg/dL and 253 mg/dL)

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Fig. 1. Box-plots representing median values and interquartile ranges of α1-Pi levels (panels A and B), tryptic inhibitory capacity (panel C), specific activity (panel D) and circulating immune complexes (α1-Pi/IgG/CIC. Panels E and F; α1-Pi/IgM/CIC, panels G and H) among different clinicopathological parameters (N− versus N+, panels A,C,D,E,G; T1–T2 versus T3–T4, panel B; stages I–II versus stages III–IV, panels F and H). P-value was considered significant if b0.05.

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Table 2 The sensitivity, specificity, PPV and NPV were determined at selected cutoff values for α1-Pi, specific activity and circulating immune complexes. Single tests were constructed in order to discriminate breast cancer patients from controls (A) and node positive patients from node negative patients (B–E). Marker

Cutoff value

Sensitivity Specificity Positive (%) (%) predictive value % (95% CI)

Negative predictive value % (95% CI)

267.5

21

60.7

34 (21.9–48.4)

44.3 (35.4–53.5)

245.5 253

65.4 65.4

26.5 30

57.6 (44.1–70.2) 58.6 (45–71.1)

33.3 (17.2–54.0) 35.7 (19.3–55.9)

A—α1-Pi

allowed the unambiguous identification of the protein components investigated (Fig. 4). Using this approach, the corresponding spectra of three protein spots were identified as α1-Pi precursor. Fig. 4 shows the MS spectra obtained from the proteins investigated. Peaks numbered in the figure are identified as the m/z of tryptic peptides from α1-Pi by their high score (Access no.P01009). As shown in Fig. 3, the isoforms corresponding to α1-Pi precursor exhibited on 2-DE gels as a chain of protein spots with slightly different isoelectric points and molecular mass suggesting post-translational alterations.

B—α1-Pi

C—α1-Pi specific activity 1.45 1.65 1.81

60 50 35.4

68 80 82

72.5 (55.9–84.9) 77.4 (58.5–89.7) 73.9 (51.3–88.9)

54.8 (38.8–69.8) 52.9 (38.6–66.8) 47.5 (34.5–60.8)

0.09 44.7 0.095 42 0.097 32

50 50 63

56.8 (39.6–72.5) 55.6 (38.3–71.7) 53.6 (34.2–72.0)

38.1 (24–54.4) 37.2 (23.4–53.3) 37.3 (24.5–51.9)

0.052 34 0.06 21.3

34 58

43.2 (27.5–60.4) 27.9 (15.8–43.9) 44 (25.0– 64.7) 34.5 (22.6–48.7)

D—α1Pi/ IgG/ CIC

E—α1Pi/ IgM/ CIC

α1-Pi levels (immunoreactive values) in serum are expressed in mg/dL. Specific α1-Pi activity is expressed in μmol/min/mg of α1-Pi. Values of α1-Pi/IgG/CIC and α1-Pi/IgM/CIC are expressed as optical density values (OD units).

specificities that are low (26.5% and 30%, respectively) for a possible clinical discrimination between N− and N+ patients, although P-value was statistically significant (P = 0.032, Fig. 2B). On the other hand, α1-Pi/IgG/CIC immune complexes presented at different cutoff values a poor diagnostic potential and the P-value was not statistically significant (P =0.063, Fig. 2D). 3.4. SDS-PAGE, 2-DE analyses, western blotting and MALDI-TOF mass spectrometry In the current study, the detection of α1-Pi was investigated in serum samples by immunoblotting. α1-Pi was detected in control or tumor samples as a single diffuse band with a molecular weight of approximately 47 kDa. Human plasma α1-Pi was used as a positive control and was detected as a single band with a similar molecular weight (data not shown). Due to the high α1-Pi level in serum samples and high background staining we were unable to decipher whether there are additional faint bands of lower or higher molecular weights corresponding to α1-Pi through SDS-PAGE experiments. Subsequently, we have used a proteomics approach that combines two-dimensional gel electrophoresis (2-DE), Western blotting and MALDI-TOF mass spectrometry to examine α1-Pi protein alteration in the serum of patients with different tumor stages (n = 10). As shown in Fig. 3, several large and diffuse protein spots with a similar molecular weight of approximately 47 kDa and different isoelectric points (pI:5–6) were detected. Additionally, a faint band of approximately 42 kDa was more pronounced in less advanced stages and became hardly detectable in breast cancer sera when disease progresses. Proteins showing immunoreactivity with α1-Pi antibody (spots 1–3) were subsequently excised and subjected to in-gel tryptic digestion and MALDI-TOF analyses. Protein identification was repeated at least twice using spots from different gels. The acquired peptide mass fingerprints (PMF) were used to search through the Swiss-Prot and National Center for Biotechnology Information non-redundant (NCBInr) databases by the Mascot search engine. This procedure

3.5. Immunoblotting and immunohistochemistry Given the differential mean expression of α1-Pi in serum among breast cancer patients (N− vs N+), subsequent immunoblots using protein extracts issued from tumor and adjacent non‐tumor tissues and immunohistochemical (IHC) staining of α1-Pi in infiltrating ductal breast carcinomas were conducted (Fig. 5). As shown in Fig. 5A, immunoblots revealed the increased expression of α1-Pi in 70% of tumors investigated indicating that tumor cells are able to synthesize the α1-Pi inhibitor. Immunohistochemical expression of α1-Pi was noted in all cases investigated and positivity retrieved in 75 to 80% of tumor cells. The signals detected in neoplastic cells were strong with little variation in intensity, whereas epithelial cells of the normal duct express lower levels of this protein (Fig. 5B). In all positive cases, immnoreactivity of α1-Pi was retrieved in the cytoplasm. No difference in α1-Pi expression was found, however, in immunoblots or IHC investigations among different tumor tissues with regard to node metastatic development (P-value = 0.68). 4. Discussion α1-Pi is an acute phase glycoprotein abundantly present in serum and extracellular fluids that is viewed as a major effector in the host response to trauma and inflammation [7]. It is one of the most important and abundant extracellular protein inhibitors of the serpin family which possesses a broad spectrum inhibitor activity against various proteases in several biological pathways including trypsin, chymotrypsin and elastase-like enzymes [15]. Albeit, cumulative evidence suggests that the liver is the most important source of α1-Pi, additional studies have demonstrated that epithelial cells and blood cells including neutrophils and cells of monocyte/macrophage lineage also synthesize this protease inhibitor [16]. Furthermore, α1-Pi expression has been found in neoplastic cells of certain types of tumors such as hepatoma, ovarian carcinoma and lung carcinomas [17]. Up to now, although tumor cell derived proteolytic enzymes are thought to play multiple roles in the spread of cancer including degradation of the basement membrane and stimulation of cell proliferation, the role of the body's natural inhibitors of these enzymes in these processes is largely unknown [18]. Therefore, the aim of the present study was to examine the specificity of α1-Pi as a breast tumor marker and its clinical significance in the prognosis of infiltrating ductal breast carcinomas. In the current study, we report a significantly decreased immunoreactive serum mean level of α1-Pi in breast cancer patients relative to healthy controls, which may indicate a tendency to proteolysis rather than tissue protection. This result was independent of α1-Pi genotypic-related deficiency since in most subjects, serum levels of α1-Pi ranged above 150 mg/dL (150 to 350 mg/dL). Our findings were similar to what was reported by Doustjalali et al. [19] showing that the mean expression of α1-Pi in breast cancer patients was lower than that of healthy controls. Low serum levels of α1-Pi were also linked to a higher risk of developing cancer of the liver, bladder, gall bladder and malignant lymphomas [20]. Some biochemical studies have shown, however, higher α1-Pi levels in breast cancer, while others did not show a significant change in these levels and are hence contrasting with our results [21, 22]. Our findings are also inconsistent with some previous studies that

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Fig. 2. Receiver operating characteristic curve (ROC) of α1-Pi levels (A,B), specific activity (C) and circulating immune complexes (α1-Pi/IgG/CIC (D), α1-Pi/IgM/CIC (E)) as a single test were constructed in order to discriminate breast cancer patients from controls (panel A) and node positive from node negative patients (panels B–E). For specific activity, a shorter distance to the upper left corner of the ROC space (panel C) indicates a higher diagnostic value. For circulating immune complexes and radial immunodiffusion, a shorter distance to the lower corner of the ROC space indicates rather a higher diagnostic value (panels A,B,D,E). The areas under the curve (AUC) are indicated. The AUC for a perfect discriminatory test would be 1.0 in the case of specific activity (panel C) and 0.0 for radial immunodiffusion (panels A and B) and circulating immune complexes (panels D and E). The diagonal line marks values of sensitivity and (1-specificity) for which no discrimination can be made by the test. P-value was considered significant if b0.05 and highly significant if b0.005.

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Fig. 3. Close-up sections of 2-DE Western blots in the location of α1-Pi are shown. A differential expression of specific isoforms of α1-Pi precursor (spots 1–3) identified by MALDI-TOF mass spectrometry occurs with the progression in the TNM stage of breast cancer.

demonstrated increased circulating levels of serine proteinase inhibitors in the course of various cancers such as brain, colorectal and gastric carcinomas [23]. This controversy with our findings may depend on several factors including the proteinase activity in plasma and the profiles and/or molecular forms of the inhibitors released. In the current investigation, when clinicopathological parameters were considered, significantly higher mean levels of serum α1-Pi were found in cases with local tumor compared to patients with tumor node metastasis. We have found, however, no relation between α1-Pi levels and the other clinicopathological parameters of breast cancer investigated including tumor stage, SBR grade and tumor size (Table 1). Interestingly, our findings are supported by recent data showing that α1-Pi is downregulated in metastatic axillary lymph nodes compared to node-negative breast carcinoma tissues which may highlight that similar alteration pathways may occur in these two compartments [24]. In addition, in agreement with our data, aprotinin, which is another serine proteinase inhibitor, has been reported to reduce invasion and metastasis when administered to hamsters with highly invasive fibrosarcomas or mice with mammary carcinoma [25]. Although molecular mechanisms associated to axillary lymph node metastasis are not well understood, increased levels of α1-Pi reported herein could be a part of a specific protective physiological mechanism among N− patients [26]. Reduced levels of α1-Pi in node positive patients may at least be linked to enhanced proteolytic tissue damage and thus increased risk of the occurrence of metastasis in the lymph nodes [27]. Based on literature, a decreased level of α1-Pi, as reported herein, may exert direct pro-inflammatory effects, whereas an increased expression appears rather to be an anti-inflammatory event, which may constitute an additional protective factor against the dissemination of tumor cells and spread of cancer [28]. This assumption agrees well with a previous study showing that

a negative correlation exists between α1-Pi levels and anchorageindependent growth of MCF-7 human breast cancer cells [29]. Our findings may also depend on the role of α1-Pi as an anti-angiogenic factor that is able to limit conversion of a small cluster of tumor cells into progressively growing tumor. This effect may be triggered by α1-Pi through the reduction of tumor capillary density by inducing apoptosis and inhibiting chemotaxis of endothelial cells [30]. In the current investigation, and contrary to what one would expect, the mean values of TIC and specific activity among patients were similar to those reported for healthy controls (P > 0.05). α1-Pi specific activity was found, however, to increase as a result of metastatic formation in the lymph nodes. No other significant association was found with the other clinicopathological parameters of the disease. In order to discriminate node positive patients from node negative ones, optimal cutoff conditions, determined by ROC analyses for specific activity yielded a sensitivity of 50% and a specificity of 80%. The PPV was 77.4% and the NPV was 52.9%. The diagnostic specificity of the α1-Pi specific activity needs, however, to be ascertained in a larger clinically set of patients among which node metastatic status is known. Up to now, circulating immune complexes are thought to play a crucial role as modulators of both cellular and humoral immune responses and are considered as markers for tumor burden and prognosis in patients with cancers [31]. Interestingly, our study revealed that immune complexes carrying α1-Pi are increased in node negative patients when IgM and IgG isotype immune complexes were measured (P-values= 0.045 and 0.03, respectively). One explanation is that the up-regulated α1-Pi protein may have triggered a specific humoral immune response in node negative patients versus N+ patients and caused binding of the antibodies to the serum α1-Pi protein. Whether such humoral immune response affects disease spread in breast cancer patients by eliminating blood-borne disseminated tumor cells (micrometastases), as suggested

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Fig. 4. MALDI-TOF peptide mass fingerprints of the tryptic digests corresponding to α1-Pi precursor (spots 1–3, upper panel). The matched peptide sequences are shown (lower panel).

previously for Muc 1, warrants, however, further studies [32]. Although the mechanisms by which proteins elicit autoantibody formation in cancer remain largely unknown, the level of expression of a protein in

tumors, post-translational modifications and an abnormal processing have been suggested to trigger such humoral autoimmune responses in cancer.

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Fig. 5. Expression of α1-Pi in infiltrating ductal breast carcinomas and adjacent non‐neoplastic tissues. Upper panel (A): immunoblots revealing an increased expression of α1-Pi in 70% of tumors investigated indicating that tumor cells are able to synthesize α1-Pi inhibitor (T: tumor tissue, N: non‐tumor tissue). Lower panel (B): Immunohistochemical investigation of α1-Pi expression in infiltrating ductal breast carcinomas. Immunoreactivity was restricted to the cytoplasm. The intensity of signals was greater in tumor cells whereas epithelial cells of the normal ducts express lower levels of this protein.

From our study, 2-DE Western-blot analyses revealed the occurrence of α1-Pi isoform-specific alterations in sera from advanced breast cancer patients and there was a relative correlation with the progression in the TNM stage of this disease. These mass variants may correspond to proteolyzed fragments or result from glycosylation of α1-Pi which is thought to confer resistance to degradation by proteases and hence enhance the half-life of circulating α1-Pi [33, 34]. As reported herein, several studies indicated the occurrence of different molecular forms of α1-Pi including inhibitory and non-inhibitory forms that may express different biological effects on tumor growth and invasiveness [35, 36]. In the current study, as the number of specimens in each group is currently insufficient for a sound statistical conclusion, the truthfulness of our 2-DE findings with regard to CIC generation and alterations of α1-Pi activity awaits further investigations. We may speculate however, that in less advanced patients, one of the protein isoforms (spot3, Fig. 3) may be a minor and perhaps functionally inert form of α1-Pi and that in advanced stages this low molecular weight form may not predominate. In the current study, we have also performed immunoblots and immunohistochemical staining of α1-Pi in infiltrating ductal breast carcinoma tissues. Our findings revealed, however, no difference in α1-Pi expression according to node metastatic status. Taken together, our investigation of antigenic and functional levels of α1-Pi highlights that development of breast cancer and metastatic potential might not only be ascribed to a deficiency in circulating α1-Pi but also to alterations in its molecular and physical properties. As reported in 2-DE analyses, these alterations may affect its activity and probably its native structure. Our observations may also indicate that α1-Pi retains its antigenic behavior but lacks partially its tryptic inhibitory capacity among N− patients. This result underscores the occurrence of inactive forms in this group which might have other biological activities linked with a decreased tissue destruction and node metastasis occurrence [37]. This assumption

agrees well with a previous study showing that the antiproteolytic activity is only an indicator of the available level of α1-Pi that is able to inhibit proteases and that non-inhibitory forms may also play a role in affecting tumor development and invasion [38]. Likewise, others showed in a similar context that both native or inactive forms of α1-Pi are among the anti-angiogenic serpins whose angio-inhibitory activities are independent of their anti-proteolytic role [30]. As reported herein, biologically inactive and immunologically active forms of α1-Pi resulting from the oxidation at the active methionine site, abnormalities in its genetic synthesis or processing and polymerization, have been described in several malignant diseases and inflammatory conditions [39, 40]. Functionally inert forms of α1-Pi with a TIC that does not increase proportionally with circulating α1-Pi have also been reported in lung cancer and in leukemias [41]. Likewise, antiprotease activity does not increase after a cytokinininduced expression of α1-Pi, which may suggest the formation of inactive complexes with proteinases [42]. In conclusion, our data indicate that determination of both α1-Pi levels and its related specific activity could be the source of major complementary clinical information. Our findings suggest also a role for this plasmatic protein in node metastatic development. The alterations reported herein in both antigenic levels and specific activity of α1-Pi with regard to disease progression prove at least in part the participation of this proteinase inhibitor in the pathways connected with the maintenance of protease/antiprotease balance. Our results provide also additional evidence that malignant breast tumor tissues possess the ability to produce α1-Pi which may regulate proteolytic activities in the tumor microenvironment but do not support the involvement of tissular α1-Pi in node metastatic development. The present study does not identify, however, the contribution of tumor cells on α1-Pi levels and related circulating immune complexes. Our findings may indicate separate and distinctive regulatory pathways of α1-Pi in tissues and serum. We also present for the first time the evidence

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that the expression of different isoforms or post-translationally modified forms of precursors corresponding to α1-Pi is affected with progression of the disease. Further quantitative evaluation of the concentration and biological function of the different isoforms on a larger clinical set with different degrees of malignancy is required and is in progress. Acknowledgments This work was supported by le Ministère de l'Enseignement Supérieur et de la Recherche Scientifique, le Ministère de la Santé Publique de la République Tunisienne and by the Centre National de Recherche Scientifique (Strasbourg, France). Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.clinbiochem.2012.07.099. References [1] Liotta LA, Stetler-Stevenson WG. Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res 1991;51:5054-9. [2] Huber R, Carrell RW. Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry 1989;28:8951-66. [3] Yavelow J, Tuccillo A, Kadner SS, Katz J, Finlay TH. Alpha 1-antitrypsin blocks the release of transforming growth factor-alpha from MCF-7 human breast cancer cells. J Clin Endocrinol Metab 1997;82:745-52. [4] Dabbagh K, Laurent GJ, Shock A, Leoni P, Papakrivopoulou J, Chambers RC. Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways. J Cell Physiol 2001;186:73-81. [5] Ikari Y, Mulvihill E, Schwartz SM. Alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells. J Biol Chem 2001;276:11798-803. [6] Yao J, Baecher-Allan CM, Sharon J. Serpins identified as cell growth inhibitors in human plasma. Mol Cell Biol Res Commun 2000;3:76-81. [7] Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983;52:655-709. [8] Dabrowska M, Mantur M, Panasiuk A, Prokopowicz J. Does the concentration of alpha 1-proteinase inhibitor reflect the transformation of liver cirrhosis to liver carcinoma? Neoplasma 1997;44:305-7. [9] Thompson DK, Haddow JE, Smith DE, Ritchie RF. Elevated serum acute phase protein levels as predictors of disseminated breast cancer. Cancer 1983;51:2100-4. [10] Dietz A, Rubinstein HM, Hodges LV. Measurement of alpha 1-antitrypsin in serum by immunodiffusion and by enzymatic assay. Clin Chem 1974;20:396-9. [11] Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965;2:235-54. [12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;7:248-54. [13] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5. [14] Chahed K, Kabbage M, Ehret-Sabatier L, Lemaitre-Guillier C, Remadi S, Hoebeke J, et al. Expression of fibrinogen E-fragment and fibrin E-fragment is inhibited in the human infiltrating ductal carcinoma of the breast: the two-dimensional electrophoresis and MALDI-TOF-mass spectrometry analyses. Int J Oncol 2005;27:1425-31. [15] Potempa J, Korzus E, Travis J. The serpin superfamily of proteinase inhibitors: structure, function and regulation. J Biol Chem 1994;269:15957-60. [16] Bagdasarian A, Colman RW. Subcellular localization and purification of platelet alpha1-antitrypsin. Blood 1978;51:139-56. [17] Kataoka H, Seguchi K, Inoue T, Koono M. Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines. FEBS Lett 1993;328:291-5. [18] Pemberton PA. The role of serpin superfamily members in cancer. Cancer J 1997;10:1–10.

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