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Keywords:

  • α(1,6)fucosyltransferase;
  • FUT8;
  • colorectal cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Changes in enzyme activity and the expression levels of α(1,6)fucosyltransferase [α(1,6)FT] have been reported in certain types of malignant transformations. To develop a better understanding of the role of α(1,6)FT in human colorectal carcinoma (CRC), we analysed the enzyme activity in healthy and tumour tissues. α(1,6)FT activity was considerably higher in tumour tissue than in healthy tissue and was related to gender, lymph node metastasis, type of growth and tumour stage. We also observed a significant increase in the α(1,6)FT expression in tumour tissues as compared to healthy and transitional tissues, inflammatory lesions and adenomas. The immunohistochemical expression in tumour tissues was correlated with the degree of infiltration through the intestinal wall. Finally, a statistical correlation was found between enzyme activity and expression obtained by Western blot in colorectal tumours when compared in the same patient. All these findings demonstrate an alteration of α(1,6)FT activity and expression in CRC. © 2008 Wiley-Liss, Inc.

It is well documented that N-linked oligosaccharides on glycoproteins are structurally altered during malignant transformation.1 An increase in fucosylated carbohydrates during pathological conditions has been reported by a number of researchers.2, 3 GDP-L-Fuc: N-acetyl-β-D-glucosaminide α(1,6)fucosyltransferase [α(1,6)FT, EC 2.4.1.68] catalyses the transfer of a fucose residue to the innermost GlcNAc on N-glycans.4 This enzyme is widely distributed in nature and its products, core fucosylated oligosaccharides, are found in many glycoproteins from a variety of tissues.5 α(1,6)FT is thought to play an important role in foetal development.6 Thus, it seems that the loss of the fucosylated core of N-glycoproteins could be related to the severe alterations of development observed in leukocyte adhesion deficiency (LAD II) or in the congenital disorder of glycosylation IIc (CDG IIc).7 Recent studies have demonstrated that the fucose α(1,6)-linked deficiency affects the normal function of glycoprotein receptors related to cellular growth, such as the EGF, PDGF and TGF-β1 receptors.8, 9 It has also been reported that the absence of core fucosylation from the IgG1 molecule enhances antibody- dependent cellular cytotoxicity.10–12

Moreover, several alterations of α(1,6)fucosylation during carcinogenesis have been reported. For example, the level of core fucosylation is elevated in both liver and serum from patients with hepatocarcinoma (HCC).13, 14 Hence, the presence of α(1,6)fucosylated α-fetoprotein, L3 fraction AFP, is a highly specific marker for HCC.15, 16 More recently, a glycoform of haptoglobin (Hp) with altered sialylation and fucosylation has been described in HCC and has been proposed as potential diagnostic biomarker.17 Moreover, the serum levels of fucosylated Hp are also increased in breast, ovarian and pancreatic cancer.17, 18 In human ovarian serous adenocarcinomas, both α(1,6)FT mRNA and protein levels show important and specific elevations.19 Likewise, in papillary carcinoma of the thyroid, an increase in α(1,6)FT levels has been demonstrated, as well as a relevant correlation with the biological aggressiveness and anaplastic transformation of the primary tumour.20

The behaviour of α(1,6)FT in human colorectal adenocarcinoma (CRC) remains almost unknown. Here we combined immunohistochemical, Western blot and enzymatic techniques to elucidate whether the activity and/or expression of α(1,6)FT are enhanced in CRC. With these, we evaluated the potential of α(1,6)FT activity and expression as indicators for the clinical outcome and monitoring of colorectal tumours. The results presented here reveal some functional changes to α(1,6)FT during CRC development in terms of enzyme activity and protein expression. Therefore, this enzyme could well play an important role in the progression of this malignant tumour.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tissue specimens

Colorectal tissue samples were obtained from patients with CRC undergoing surgery at the Cristal-Piñor Hospital (Ourense, Spain) and the Xeral Hospital (Vigo, Spain) after receiving approval from the appropriate local Institutional Review Board. Transitional tissue was defined as the tissue immediately adjacent to the edge of the tumour, which did not show any microscopic features of malignancy. Mucosa was considered healthy if distant by at least 10 cm from the tumour. The specimens used for immunohistochemical analysis (7 healthy tissues, 20 adenomas and 10 inflammatory lesions of free-CRC patients and 168 tumour, 29 transitional and 62 healthy tissues of CRC patients) were fixed in formalin (10%), embedded in paraffin (60°C) and subjected to haematoxylin and eosin staining (standard procedure) to evaluate their microscopic features. The specimens employed in both α(1,6)FT assays (98 tumour and healthy tissues) and Western Blot (20 tumour and healthy tissues) were washed with ice-cold saline buffer and stored frozen at −85°C until use.

Immunohistochemical study

Immunohistochemical study was performed as described previously Ito et al.20 Tissue sections (2–3 μm) were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was blocked with 0.5% (v/v) hydrogen peroxide in methanol. After a rinse in PBS, bovine serum was applied to block non-specific interactions. Sections were then incubated with the primary antibody (15C6, anti-human α(1,6)FT, Fujirebio Corp., Tokyo, Japan) at 4°C overnight. After a rinse in PBS, sections were incubated with the secondary antibody bound to peroxidase; the peroxidase reaction was visualized by incubating with DAB (3,3′-diaminobenzidine). Finally, after a wash in water, the sections were counterstained with haematoxylin, dehydrated in a graded ethanol series, washed in xylene and mounted on a glass slide. Negative controls were performed using PBS instead of primary antibody. The semiquantitative staining analysis was performed by expert pathologists and the expression pattern was classified as follows: 0, tissues without staining; 1, less than 10%; 2, 10–50%; and 3, more than 50% of the tissue stained.

Preparation of tissue extracts

Colorectal tissues were homogenized in 6 volumes of 0.01 M Tris-HCl buffer (pH 7.4), containing 0.25 M sucrose. The homogenate was centrifuged for 10 min at 500g at 4°C. The supernatant obtained was centrifuged at 33,000g for 60 min at 4°C. Subsequently, the pellet was re-suspended in 1.5 mL of 0.01 M Tris-HCl buffer (pH 7.4) and centrifuged at 145,000g for 45 min at 4°C. The final pellet containing the total cell membrane fraction was re-suspended in 300 μL of 0.01 M Tris-HCl buffer (pH 7.4) and stored at −20°C until used in the enzymatic assays and Western Blot. The protein content of the final membrane preparation was determined with the BCA protein assay, using bovine serum albumin as standard.

Enzyme activity assay for α(1,6)FT

α(1,6)FT activity was determined using a modification of the method described by Hartel-Schenk et al.,21 using asialo-agalacto-fetuin (ASAGF) as exogenous acceptor. The commercial fetuin (Sigma, St. Louis, MO) was desialylated and degalactosylated as described by Ko and Raguppathy22 to obtain the ASAGF.

In the standard assays, the reaction mixture for α(1,6)FT activity had the following final concentrations in a total volume of 100 μL: 0.15 M Tris-HCl buffer (pH 7.4), 40 mM MgCl2, 80 mM NaF, 3% (v/v) Triton X-100, 0.5 μM GDP-L-[14C]-Fucose, 99.5 μM GDP-L-Fucose, 0.32 mg ASAGF and 50 μg of enzyme solution. Assays were run in duplicate for 1 hr at 37°C. The reaction was stopped with 0.5 mL of 20% (w/v) TCA. The precipitate was collected on Whatman glass-fibre filters (GF/C) and washed with 10 mL of 0.01 M Tris-HCl buffer (pH 7.4). Filters were dried overnight at room temperature and radioactivity was measured in a Wallac 1409-12 Scintillator system, using Ecoscint H as scintillation counting mixture. Enzyme activity was expressed as μU/mg protein.

Western blot analyses

For Western blotting of α(1,6)FT, 20 μg of protein from total cell membrane extracts was subjected to SDS-PAGE (12% polyacrylamide gel) under reducing conditions and then electrotransferred onto a polyvinylidene difluoride membrane (Hybond-P, Amersham Bioscience, Europe GMBH). Gels were run in duplicate, one for Coomassie Blue staining, and the other for the immunoassay. After blotting, the membrane was washed with TBS containing 0.05% Tween-20 (T-TBS) (v/v), blocked with 5% (w/v) dried skimmed milk in T-TBS overnight at 4°C and incubated with 1/1,000 diluted primary antibody (15C6, anti-human α(1,6)FT, Fujirebio Corp., Tokyo, Japan) for 2 hr at room temperature. Some blots were also probed with 1/7,000 diluted β-actin antibody (Sigma) and used as a protein loading control. After a wash with T-TBS, the blot was incubated with 1/1,500 diluted secondary antibody, a peroxidase-conjugated antibody against mouse IgG (DakoCytomation, Denmark), for 1 hr at room temperature. Finally, the blot was washed with T-TBS and colour was developed using BCIP/NBT Liquid Substrate System (Sigma).

The pattern and the intensity of bands were obtained by densitometric analysis (GS-800 Calibrated Densitometer, Bio-Rad laboratories), using Quantity One for PC software (Bio-Rad laboratories, Hercules, CA).

Statistical analyses

Statistical analyses were performed using SPSS v. 14.00 for WINDOWS XP. Univariate analysis for categorical data was carried out with the χ2 test or Fisher's exact probability test. For continuous data, we employed Wilcoxon's test, the Mann-Whitney U test and the Kruskall-Wallis test. Finally, the possible association between α(1,6)FT protein expression and α(1,6)FT activity was tested using the Spearman test. The results were considered significant when p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

α(1,6)FT activity

The enzyme α(1,6)FT is highly specific for its acceptor substrate, needing a biantennary oligosaccharide sequence non-sialylated and non-galactosylated such as that shown by ASAGF. To determine the level of fucose incorporation into endogenous acceptors, we carried out parallel assays without adding ASAGF. This endogenous incorporation, the result of the action of α(1,6)FT and other FTs, was fairly low and similar for both healthy and tumour tissues; additionally, according to Wilcoxon's test (data not shown) there were no statistically significant differences in endogenous activity between tumour and control samples.

The mean α(1,6)FT activity observed in healthy and tumour colorectal tissues from the same patient (n = 98) was quite different (Table I), a statistically significant increase in the tumour specimens being observed with respect to the healthy ones (p < 0.001, according to Wilcoxon's test).

Table I. α(1,6)FT Activity in Healthy and Tumour Colorectal Tissues
Tissuenα(1,6)FT activity [mean (μU/mg) ± SEM]P
  • SEM, standard error of the means.

  • *

    Statistically significant.

Healthy9828.66 ± 2.98(Wilcoxon's test) p < 0.001*
Tumour9842.64 ± 3.52

We also performed a correlation analysis between α(1,6)FT activity and the standard clinicopathological features employed in our study. The results revealed that α(1,6)FT activity was significantly higher in men than in women; higher in tumours without lymph node metastasis or with polypoid growth than in non-polypoid growth tumours and, finally, higher in early stages than in advanced ones (Table II).

Table II. Relationship between Patients' Clinicopathological Features and α(1,6)FT Activity in Tumour Tissues
Featurenα(1,6)FT activity [mean (μU/mg) ± SEM]p
  • SEM, standard error of the means.

  • *

    Statistically significant.

Gender   
 Men5849.67 ± 4.98(Mann-Whitney U)
 Women4032.44 ± 4.300.015*
Age (years)   
 >701529.51 ± 4.65(Kruskall-Wallis)
 70–772030.64 ± 6.010.733
 <771740.75 ± 9.65 
Tumour location   
 Right colon2042.24 ± 8.00 
 Left colon1338.20 ± 8.12(Kruskall-Wallis)
 Sigma2451.57 ± 7.860.49
 Rectum3339.43 ± 6.26 
Size (cm)   
 >43238.77 ± 5.32(Kruskall-Wallis)
 4–64443.45 ± 5.290.91
 <61945.94 ± 9.46 
Histologic type   
 Well differentiated228.63 ± 17.5(Kruskall-Wallis)
 Moderately differentiated4733.43 ± 4.470.67
 Poorly differentiated339.84 ± 12.7 
Growth type   
 Polypoid1851.92 ± 9.1(Mann-Whitney U)
 Non-polypoid3423.93 ± 3.020.002*
Dukes' stage   
 A1858.27 ± 9.93 
 B4542.01 ± 4.34(Kruskall-Wallis)
 C y D3535.41 ± 6.100.049*
Astler-Coller classification   
 A, B1 y B22637.10 ± 4.66(Mann-Whitney U)
 C1, C2 y D2630.13 ± 6.810.052*
TNM classification   
 T   
  Tis/T1/T2950.01 ± 10.4(Mann-Whitney U)
  T3/T44230.19 ± 4.450.036*
 N   
  N02630.58 ± 4.66(Mann-Whitney U)
  N1/N22622.42 ± 6.810.052*
 M   
  M05134.04 ± 4.25(Mann-Whitney U)
  M1123.03 ± 10.00.66
AJCC classification   
 IIa/IIb2637.67 ± 4.63(Mann-Whitney U)
 IIIa, IIIb, IIIc, IV2629.56 ± 6.810.02*

Immunohistochemistry

After the immunohistochemistry assays, the presence of α(1,6)FT was shown up by a brown staining, clearly localized in the cytoplasm of epithelial cells (Fig. 1). Positive α(1,6)FT expression was detected in 7 of the 62 healthy specimens (11.29%); in 1 of the 29 transitional specimens (3.4%) and in 103 of the 168 tumour specimens (61.3%) evaluated in CRC patients (Fig. 2a). No positive expression was found in the healthy and inflammatory tissues and, only 1 of the 20 polyps analysed (0.5%) was positive for α(1,6)FT expression. The statistical analysis revealed significant differences (p < 0.001, according to Wilcoxon's test) for tumour vs. healthy and transitional comparisons (Fig. 2a) and for tumour vs. adenomas, inflammatory lesions and control healthy tissues (p < 0.01, according to Mann Whitney U).

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Figure 1. Healthy colon tissue without specific α(1,6)FT staining (a), CRC tissue with positive staining (b), adenoma (c) and inflammatory lesion (d) without positive α(1,6)FT staining. α(1,6)FT was expressed in the colon epithelial cells as a brown staining in the cytoplasm. Original magnification, a and b ×400, c ×100 and d ×200.

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thumbnail image

Figure 2. Percentage of specimens with positive immunohistochemical α(1,6)FT expression (a) and classification of positive specimens according to the intensity of expression (b). (n = 62 for healthy tissues, n = 29 for transitional tissues and n = 162 for tumour tissues). **p < 0.001 and *p < 0.01 for comparison of tumour vs. healthy and transitional tissues.

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Furthermore, taking into account the positive degree of the immunohistochemical staining, the tumour tissue displayed a higher α(1,6)FT expression than both healthy and transitional tissues (Fig. 2b). The Wilcoxon test showed that this difference was statistically significant (p < 0.01).

Finally, α(1,6)FT expression in CRC was independent of gender, tumour location, size, histological type, lymph node metastasis, type of tumour growth (polypoid or non-polypoid) or stage according to Dukes, Astler-Coller and AJCC classifications (data not shown). Nevertheless, a statistically significant increase in α(1,6)FT-positive expression was found in the T3 and T4 stages of the TNM classification when compared with the Tis, T1 and T2 stages (p = 0.03, according to Fisher's exact probability test).

Western blot analysis

The analysis of α(1,6)FT expression by Western blot was performed in 20 patients; in 15 of these, the levels of α(1,6)FT were elevated in tumour tissue as compared to neighbouring healthy tissue (Fig. 3). This difference was statistically significant (p = 0.002, according to Wilcoxon's test). The calculated molecular weight for α(1,6)FT from colorectal tissues was approximately 60 kDa, as expected (Fig. 3).

thumbnail image

Figure 3. Representative western blot of α(1,6)FT in colorectal tissues. Protein (20 μg) from tumour colorectal tissues (TT, lines 3, 5 and 7) and their neighbouring healthy tissues (HT, lines 2, 4 and 6) were analysed as described above. β-actin was used as loading control. MW standards in lane 1.

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Moreover, after applying the pertinent statistical test no correlation was found between the levels of enzyme expression in tumour tissues and the standard clinicopathological features (data not shown).

Correlation between α(1,6)FT expression and α(1,6)FT activity

We also analysed whether the increase in α(1,6)FT activity was correlated with the increase in the α(1,6)FT expression of the tumour tissue from the same patient. In this sense, we evaluated the possible association between α(1,6)FT immunohistochemical expression and α(1,6)FT activity in 49 specimens of CRC. To this end, 3 groups of enzyme activity were established on the basis of percentiles 33 and 67 (<18 μU/mg, low activity; 18–35 μU/mg, moderate activity; and >35 μU/mg, high activity). Finally, these groups were compared with positive/negative and low (0, 1)/ high2, 3 α(1,6)FT immunohistochemical expression groups, and no correlation was found between them (data not shown).

However, study of the possible association between α(1,6)FT activity and α(1,6)FT expression obtained by Western blot revealed a significant correlation between the 2 when the variation in enzyme expression and activity in tumour tissues was compared with that seen in healthy tissues (n = 17) (c = 0.68, r = 0.02 according to the Spearman test).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

During malignant transformation the levels of glycosyltransferases activities are altered dramatically.23, 24 α(1,6)FT activity is no exception and is altered in several tumour processes. In this sense, specific increases in its expression and/or enzyme activity have been reported in human tumour processes such as HCC,5, 13 thyroid follicular cancer20 and ovarian serous adenocarcinoma.19

In accordance with these previous works focused on cancers of varying origins our results show that α(1,6)FT activity is considerably elevated in colorectal tumours as compared with the surrounding healthy tissues. Moreover, study of the association between tumour α(1,6)FT activity and standard clinicopathological features revealed significant correlations between enzyme activity and gender, lymph node infiltration, type of growth and tumour stage. An increase in the levels of α(1,6)FT activity in polypoid tumours, which are more localized and less invasive than non-polypoid tumours, was observed and corresponded to the increase observed in tumours without lymph node metastasis. Similarly, we observed a progressive decrease in α(1,6)FT activity as the degree of infiltration in the intestinal wall progressed. These differences were statistically significant when we grouped the stages of the Dukes, Astler-Coller, TNM and AJCC classifications according to the depth of infiltration and the presence of lymph node invasion.

These data are in line with the evidence provided by the determination of α(1,6)FT mRNA in CRC tissues and in healthy colon from people not suffering from CRC: the presence of α(1,6)FT mRNA is detected in tumour tissues whereas it is not detected in healthy samples.25 Moreover, tumours with less advanced stages, without node infiltration or metastasis, show higher levels of mRNA.25 However, in conflict with our results, higher levels of enzyme activity have been detected in the plasma of patients with HCC in advanced stages as compared with that of those in early stages.26

Recent studies have shown that the absence of the FUT8 gene leads to an alteration of the normal structure and function of cell growth glycoprotein receptors.8 With the inhibition of the intracellular signalling routes controlled by these receptors an important delay takes place in cellular differentiation and growth. If this occurs with a deficit of α(1,6)FT activity, the enhancement of such activity could act as an activating factor on cell growth. The initial steps of tumour formation are characterized by an uncontrolled division of the transformed cells. In our study, these initial stages of the colorectal tumour showed the highest levels of α(1,6)FT activity. It is likely that this increase in enzyme activity, in combination with many other cell alterations produced during the malignant transformation, would be involved in the initial development of the nascent tumour.

α(1,6)FT activity decreased in the last steps of CRC malignancy. Several studies have demonstrated the association between the α(1,6)fucosylation of different cell adhesion molecules during tumour development and the capacity of dissemination of these transformed cells.5, 27, 28 Therefore, taking into account this preliminary evidence, we hypothesized that the decrease in α(1,6)fucosylation in advanced stages could be associated with the acquisition of the invasive potential of tumour cells. In this sense, determination of the factor which induces the increase in α(1,6)FT activity and its subsequent decrease in CRC appears to be of great importance.

The enhancement of α(1,6)FT enzyme activity in tumour tissues could be the result of an increase in the protein levels of the enzyme. The immunohistochemical analysis performed revealed a significant increase in α(1,6)FT expression in tumour tissues with respect to healthy and transitional tissues (p < 0.001). The rate of the enzyme expression was also significantly higher in tumour tissue (p < 0.001). Similar results have been obtained on studying the tissue protein content in follicular thyroid and serous ovarian tumours,19, 20 as well as in tumour colon tissue,25 in tumour hepatic tissue29 and in tumour cell lines (MKN45, Colo201 and Colo205) in which mRNA levels were determined.5

Likewise, we must emphasize the absence of α(1,6)FT immunohistochemical expression in the inflammatory and healthy tissues of patients without CRC which suggests that the alteration of the enzyme expression is specific of the malignant transformation and is not related to the inflammatory process normally associated to the cancer. Besides, the very low rate of positive expression (0.5%) in pre-cancerous lesions, similar to the one obtained in transitional tissues, confirms that the increase in the tissue enzyme levels is strongly associated with a total transformation of the colorectal tumour.

On the other hand, in papillary carcinomas the increase in α(1,6)FT expression is significantly related to clinicopathological parameters such as tumour size, lymph node metastasis, an advanced stage, and the presence of poorly differentiated lesions, indicating that the expression of the enzyme is required for the progression of the carcinoma.20 In our immunohistochemical study, we found a statistically significant increase in the α(1,6)FT-positive expression in advanced stages (T4 and T3) of the TNM classification in comparison with early stages (Tis, T1 and T2), while enzyme activity was higher in early stages than in advanced stages. Accordingly, in spite of enzyme activity and immunohistochemical expression being elevated in colorectal tumour tissues in our experimental study, when we compared them in the same patient we found no statistical correlation. To interpret these results we must bear in mind that the immunohistochemical results, in spite of the painstaking protocol developed, originate from a semiquantitative determination of the enzyme levels, whereas the enzyme activity assays allow a quantitative determination of the catalytic potential of the enzyme.

Western blot studies revealed an elevated expression of α(1,6)FT in tumour tissue in comparison with the control tissue (p = 0.021), and we also detected a significant correlation between α(1,6)FT activity and expression obtained with Western blot when we compared the variation in enzyme expression and enzyme activity in tumour tissue with regard to healthy tissue (c = 0.68, r = 0.02). In spite of the existence of this correlation in some patients, major increases in enzyme activity were found to be associated with minor increases in enzyme expression. Several authors have described similar results after the assessment of mRNA and enzyme activity levels,5 together with FUT8 gene expression levels and the amount of core fucosylated glycans.30 Two hypothesis have been proposed for this discrepancy. The least credible is the existence of another fucosyltransferase that can catalyze the same reaction that α(1,6)FT. The existence of some mechanism of post-translational modification or alternative mechanisms for enzyme activation that would modulate the levels of active catalytic protein5, 30 seems to be the most suitable reason and could likely explain our results if it is accepted that the tissue levels of enzyme detected by us did not necessarily correspond to the levels of active enzyme for catalysis. Thus, in the later stage of the colorectal tumour these mechanisms of activation could be inhibited by a biological process which is at present unknown. Likewise, the differences between the results obtained with Western blot and immunohistochemical analysis could be due to their different sensitivities or to the semiquantitative nature of immunohistochemistry, as we commented previously.

In conclusion, we have demonstrated the alteration of α(1,6)fucosylation in colorectal human cancer as a result of increases in both enzyme activity and protein expression following malignant transformation. Moreover, we have observed a close association between the levels of α(1,6)FT activity and tumour aggressiveness. Nevertheless, further studies regarding the factors that determine the increase of the enzyme expression and activity, including experiments with FUT8 knock out colorectal cancer cells, will be necessary to clarify the role played by this enzyme in CRC. Finally, another very important research field is the identification of core fucosylated glycoproteins increased in CRC which could be related to the development and dissemination of the tumour.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This research would not have been possible without the collaboration of the Pathology Service of the Cristal-Piñor Hospital (Ourense, Spain) and the Xeral Hospital (Vigo, Spain). We also would like to thank Professor Carlos Villaverde and Molecular Biomarkers investigation group (Faculty of Biology, University of Vigo, Spain) as well as the predoctoral students Ms. Cloti Costa Nogueira and Ms. Iria García Parceiro for their help to develop the present work. Ms. L. Muinelo-Romay and Ms. S. Villar-Portela were supported by a predoctoral fellowship from the “Xunta de Galicia.”

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  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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