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Laboratory Investigation

Oncology 2005;69:436–444 DOI: 10.1159/000089999

Elevation of ST6Gal I Activity in Malignant and Transitional Tissue in Human Colorectal Cancer

Cristina Vázquez-Martín Emilio Gil-Martín Almudena Fernández-Briera

Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, Vigo , Spain

ied with the histological grade of the tumor; however, we failed to fi nd a correlation with the AJCC tumor clas-sifi cation. Conclusions: This work reports enhanced ST6Gal I activity in tumor and transitional tissues from CRC patients. However, our overall results suggest that ST6Gal I activity is not indicative of the patient’s out-come.

Copyright © 2005 S. Karger AG, Basel

Introduction

The median survival time of colorectal cancer (CRC) patients mainly depends on the presence of metastasis [1, 2] . It is hypothesized that the progression of cancer to an advanced stage is due to highly malignant and metastatic tumor cells arising within the primary tumor [3–5] . Rec-ognition of such metastatic tumor cells should be useful for detecting the metastatic potential of CRC cell sub-populations [4, 5] , which has been attributed to various cell properties, including sialylated complex carbohy-drates [4–8] . CRC-related sialo-oligosaccharide structur-al changes are well documented [3–9] , although there is still little information available concerning the regulated expression of sialyltransferase activities in human colon cancer tissues implied in their synthesis [10] . These stud-ies are greatly hindered because sialyltransferase is weak-ly expressed and may have identical transfer activity to-wards the same acceptor [10, 11] . In fact, there are few

Key Words Colorectal cancer � ST6Gal I � Transitional tissue � Tumor progression

Abstract Objectives: The aim of the present study was to investi-gate the activity of CMP-NeuAc:Gal � (1,4)GlcNAc sialyl-transferase (ST6Gal I) in colorectal cancer (CRC). Meth-

ods: ST6Gal I activity was determined in healthy, transitional and tumor tissues from the same patient using asialotransferrin and N-acetyllactosamine as ac-ceptors. Results: ST6Gal I activities with asialotransfer-rin (n = 85) and N-acetyllactosamine (n = 40) as acceptors were statistically signifi cantly enhanced in CRC tissue compared with healthy mucosa from the same patient (p = 0.001). Using transitional tissue (n = 27), enhance-ment versus healthy tissue was observed (p ! 0.05). A positive correlation was found between ST6Gal I activity with N-acetyllactosamine and asialotransferrin in healthy (n = 32), tumorous (n = 32) and transitional tissue (n = 27), supporting the fact that the same enzyme was de-tected using both acceptors. Furthermore, we studied the relationship between some patients’ clinicopatho-logical features and ST6Gal I activity. Although the dif-ferences were not statistically signifi cant, the levels of ST6Gal I activity in tumorous and transitional tissues var-

Received: April 6, 2005 Accepted after revision: October 1, 2005 Published online: November 25, 2005

Oncology

Dr. Almudena Fernández-Briera Department of Biochemistry Genetics and Immunology, Faculty of Biology, University of Vigo ES–36310 Vigo (Spain) Tel. +34 86 812573, Fax +34 86 812556, E-Mail abriera@uvigo.es

© 2005 S. Karger AG, Basel 0030–2414/05/0695–0436$22.00/0

Accessible online at: www.karger.com/ocl

E.G.-M. and A.F.-B. contributed equally to this work.

ST6Gal I Activity in Human Colorectal Cancer

Oncology 2005;69:436–444 437

studies in which a tissue-specifi c sialyltransferase activity was correlated with tumor progression, and in such stud-ies, the number of samples included has usually been too low for achieving defi nite conclusions [12–14] .

The disaccharide Gal � (1,4)GlcNAc (N-acetyllactos-amine) is a common constituent of glycoconjugate N-linked oligosaccharide chains that could be sialylated by � (2,3)- or � (2,6)-sialyltransferases (ST3 or ST6) [10, 12, 13] . The � -galactoside � (2,6)-sialyltransferase (ST6Gal I, EC 2.4.99.1) has long been the only ST6 so far identifi ed as being able to catalyze the � (2,6)-sialylation of N-acetyl-lactosamine [10] , but very recently a second type of hu-man � -galactoside, � (2,6)-sialyltransferase (ST6Gal II), has been cloned which preferentially sialylates N-acetyl-lactosamine structures on oligosaccharides [15, 16] . Ad-ditionally, the ST6Gal II gene showed a very tissue-spe-cifi c pattern of expression because it was found essen-tially in brain tissue whereas the ST6Gal I gene was ubiquitously expressed [16] . The oligosaccharide struc-ture NeuAc � (2,6)Gal � (1,4)GlcNAc, known as CDw75 antigen, is a B lymphocyte surface antigen, a ligand for the cell-specifi c lectin CD22/siglec-2 which is important for B cell function [15] . This epitope is widely distributed in CRC tissues and was detected by means of Sambucus nigra agglutinin [17–20] and has further been shown to be positively associated with an invasive phenotype in some experimental studies [6, 17, 18] . The upregulation of ST6Gal I is probably at the origin of the increased � (2,6)-sialylation of cancer cells [20] , therefore, it would be very important to clarify the role of this enzyme in CRC.

In the current study, we focus on the comparison of ST6Gal I activity determined in healthy and CRC tissues from the same patient using two different acceptors: asialotransferrin (n = 85) and N-acetyllactosamine (n = 40), further checking the reaction products in order to as-sess the type of sialyltransferase activity measured. Tran-sitional tissue (n = 27) was also studied to detect the grad-ual changes related to neoplasic transformation. Since an enhancement of sialyltransferase activity with both ac-ceptors in transitional and tumorous tissues was observed, we analyzed the relationship between ST6Gal I activity in those tissues and clinicopathological features of the patient and the tumor to elucidate the clinical signifi cance of the altered enzyme activity. To our knowledge, it is the fi rst time that ST6Gal I activity is related to clinicopa-thological features in a consistent number of samples, wherein the transitional tissue is also included for a better understanding of the progressive alterations of the onco-genic process.

Methods

Materials Labeled cytidine-5 � -monophosphate-N-acetylneuraminic acid

(CMP-[ 14 C]-NeuAc, specifi c activity 10 GBq/mmol) was purchased from New England Nuclear (Boston, Mass.) and Ecoscint H from National Diagnostics (Atlanta, Ga.). Transferrin, acetonitrile, N-acetyllactosamine, oligosaccharides, N-acetylneuraminic acid (NeuAc), bicinchoninic acid solution, bovine serum albumin, thio-barbituric acid, CMP-NeuAc and a specifi c � (2,3)-neuraminidase were from Sigma (St. Louis, Mo.). All the other reagents used were of analytical grade.

Patients and Tissue Samples A total of 116 patients suffering from CRC were studied (59

women, 57 men). Their ages ranged from 46 to 90 years, with a me-dian age of 69 years. In each case, tumor tissue and healthy mucosa (distant by at least 10 cm from the tumor), were surgically obtained from the same patient. In some cases, we studied transitional tissue, i.e., the mucous membrane adjacent to the edge of a tumor, which did not show any microscopic features of malignancy. Clinical and pathological examinations were performed by two different anato-mopathologists stating that this was really colorectal and nonnecrot-ic tissue. All data, including gender, age, stage of disease according to the American Joint Committee on Cancer Classifi cation (AJCC) system [21] and pathological factors were prospectively recorded.

Tissue specimens from CRC patients were obtained immedi-ately following resection, then washed with ice-cold saline buffer and stored at –85 ° C until use. From these specimens, total cell membranes were obtained as described by López et al . [22] and employed as an enzyme source. All procedures were carried out between 0–4 ° C, and tumor, transitional and healthy tissues from the same patient were always processed in parallel. Protein concen-trations in membrane preparations were determined by the bicin-choninic acid solution method, using bovine serum albumin as standard, and run in triplicate.

Sialyltransferase Assays Sialyltransferase activity was determined as previously de-

scribed by Vázquez-Martín et al. [23] . Endogenous acceptor assays were carried out using the standard incubation system without add-ing exogenous acceptors. Enzyme activity was expressed as � U/mg of protein (U = international unit of enzyme activity). Assays were run in duplicate for 2 h at 37 ° C and the product was determined by one of the following methods depending on the acceptor sub-strate, asialotransferrin or N-acetyllactosamine.

Asialotransferrin. Desialylated transferrin was prepared by treatment of transferrin with 0.1 N H 2 SO 4 for 1 h at 80 ° C, followed by dialysis and lyophilization. Desialylation was checked by means of the thiobarbituric acid method [24] . In the standard assay, the reaction mixture had the following fi nal concentrations in a total volume of 100 � l: 40 m M MES (pH 6.0), 4 m M NaF, 5 m M MnCl 2 , 0.2% (v/v) Triton X-100, 1 � M CMP-[ 14 C]-NeuAc, 199 � M CMP-NeuAc, 0.9 mg asialotransferrin and 50 � g of enzyme solution. The reaction was stopped with 0.5 ml of 20% (w/v) trichloroacetic acid. The precipitate was collected on Whatman glass fi ber fi lters (GF/C) and washed with 10 ml of 0.01 M MES buffer (pH 6.0). Filters were dried overnight at room temperature and radioactivity was mea-sured in a Wallac 1409-12 Scintillator system, using Ecoscint H as the liquid scintillation counting mixture.

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Oncology 2005;69:436–444 438

N-Acetyllactosamine. Assays were carried out under the same conditions as for asialotransferrin, using 20 m M N-acetyllactos-amine as acceptor substrate. Reaction mixtures were quenched with 0.4 ml of cold 5 m M potassium phosphate buffer (pH 5.5) and applied to a 3-ml column of Dowex 1X8-400 eluted with that buffer. Fractions of 1 ml were collected and aliquots of 200 � l were counted in 5 ml scintillation fl uid. Examination of the behavior of 14 C-labeled products showed that CMP-[ 14 C]-NeuAc was com-pletely adsorbed and that sialo-oligosaccharides were eluted in the second and third fraction (2–3 ml) so that they were collected for counting.

Determination of K m and V max Kinetic studies were carried out for the donor substrate (CMP-

NeuAc) and the acceptor (asialotransferrin) within the ranges 0.67–100 � M and 0.3–1.2 mg/100 � l of incubation, respectively, using the previously detailed standard mixture reaction. K m and V max were determined using the ‘Enzfi tter’ nonlinear regression data analysis program (Elsevier, Biosoft).

� (2,3)-Neuraminidase Treatment In order to cleave the potential NeuAc linked in � (2,3)-linkage

from the NeuAc-asialotransferrin, and to assess the � (2,6)-activity fraction evaluated, two standard reactions were undertaken using healthy and tumorous tissue as an enzyme source under the condi-tions of the above standard assay. The reaction was stopped by 1 ml of 1% (w/v) phosphotungstic acid in 0.5 M HCl. Samples were cen-trifuged at 2,000 g for 10 min and the resulting pellet was washed once with 1 ml of 1% (w/v) phosphotungstic acid in 0.5 M HCl and three times with methanol (1 ml). The fi nal pellet containing ([ 14 C]-NeuAc-asialotransferrin) and free of phosphotungstic acid was dried overnight at room temperature and was then incubated in the absence or presence of 25 mU of � (2,3)-neuraminidase. Digestion was performed at 37 ° C for 12 h in 200 � l of 0.1 M sodium acetate buffer (pH 5.5) containing 10 m M CaCl 2 . The reaction was stopped by the addition of 0.5 ml of 7.5% (w/v) trichloroacetic acid. Samples were centrifuged (2,000 g for 10 min) and NeuAc was obtained af-ter four washings of the pellets (1 ml), the fi rst with phosphotungstic acid, and the rest with methanol. [ 14 C]-NeuAc linked to asialotrans-ferrin in � (2,3) was recovered in the supernatant and in the pellet it remained the corresponding to the � (2,6)-linkage. In order to prove the effi ciency of the process, a Maackia amurensis lectin bound to a Sepharose chromatographic column (MAL-Sepharose) was used [25] to confi rm the complete elimination of the NeuAc � (2,3) linked to the protein after undertaking the exposed process.

High-Pressure Liquid Chromatography Studies Identifi cation of sialo-oligosaccharides was performed by HPLC

using a Beckman system gold liquid chromatograph, equipped with a Rheodyne 7725 injector, essentially as described by Joziasse et al . [26] . An analytical column (4.6 mm ! 250 mm) of Lichrosolv-NH 2 (particle size 4 � m, Merck) was used at a temperature of 20 ° C and a pressure of 11 MPa. The column was run isocratically with a mixture of acetonitrile/15 m M potassium phosphate buffer (pH 5.2) (83: 17, v/v) for 35 min, whereupon a linear gradient was start-ed in order to decrease the acetonitrile content by 0.5%/2 min. The fl ow rate was 2 ml/min. The reference compounds were injected together and were detected by UV absorption at 195 nm. The sialo-oligosaccharides from the reaction products were separated by ion

exchange chromatography as previously described; the second and third fractions were pooled, frozen and lyophilized. The elution time of the injected radioactive reaction products was determined by collecting an aliquot of 200 � l from 2 ml effl uent fractions in counting vials, followed by liquid scintillation counting.

Statistical Analysis Statistical analyses were performed using SPSS v. 9.0.1 (Inc.,

1989–1999) for Windows 95. The differences of sialyltransferase activity in different tissues or the relationship with different clini-copathological variables were statistically determined by means of the Wilcoxon test, Mann-Whitney U test, Kruskal-Wallis test and Dunn test. Correlation between sialyltransferase activity using asialotransferrin and N-acetyllactosamine as acceptors was evalu-ated by means of Pearson’s correlation test. A signifi cant difference was established when a p ! 0.05 was obtained.

Results

ST6Gal I Activity in Healthy, Transitional and Tumorous Human Colorectal Specimens CRC tissue and adjacent or distant uninvolved tissue

(transitional and healthy tissues) were homogenized and ST6Gal I activity was tested with two different acceptor substrates: asialotransferrin and N-acetyllactosamine. Given that endogenous acceptors could be present in the enzyme preparation and could alter the activity results from exogenous acceptors, 19 assays corresponding to 19 different patients were carried out without adding exog-enous acceptors. The incorporation into endogenous ac-ceptors (as means 8 standard error means) was very low: 4.56 8 0.81 � U/mg (0.00–11.53 � U/mg) and 4.40 8 0.76 � U/mg (0.00–13.28 � U/mg) in healthy and tumor-ous tissues, respectively; no statistically signifi cant differ-ences were found between these mean values (according to Wilcoxon’s test), therefore they were not considered in later assays.

Table 1 shows sialyltransferase activity with asialo-transferrin or N-acetyllactosamine determined in healthy and tumorous colorectal tissues from the same patients. Statistically signifi cant differences in sialyltransferase ac-tivities between healthy and tumorous tissues for both acceptors (p = 0.001) were observed. In addition, transi-tional tissue was included in the study ( table 2 ), and sta-tistically signifi cant differences were obtained among the three tissues, with both asialotransferrin (p ! 0.05) and N-acetyllactosamine (p = 0.001) as acceptors. As regards asialotransferrin, when comparing tissues considered in pairs, Wilcoxon’s test revealed statistically signifi cant dif-ferences between healthy and tumorous tissues (p ! 0.01) and healthy and transitional tissues (p ! 0.05), but not

ST6Gal I Activity in Human Colorectal Cancer

Oncology 2005;69:436–444 439

between transitional and tumorous tissues. With respect to N-acetyllactosamine, statistically signifi cant differ-ences were obtained again when comparing healthy and tumorous tissues (p ! 0.01) and healthy and transitional tissues (p ! 0.05) using Wilcoxon´s test; we also failed to fi nd differences between transitional and tumorous tis-sues. Note that the activity levels shown in tables 1 and 2 were compared for the same patients in order to avoid possible variability among different patients.

Enzyme Kinetics The increased ST6Gal I activity in colorectal tumors

could either be due to an alteration in the catalysis mech-anism or to an increased affi nity for the substrates. To investigate this point, the apparent K m and V max for do-nor and acceptor substrates of healthy and tumorous ST6Gal I were measured. Asialotransferrin was chosen as the acceptor because this glycoprotein refl ects the in vivo sialylation conditions better than the disaccharide N-acetyllactosamine. We failed to fi nd statistically signifi -cant differences in the V max values for healthy and tumor enzymes for any of the substrates (despite observations

showing that for asialotransferrin this parameter was in-creased 2.6-fold in tumorous tissue) ( table 3 ). On the oth-er hand, we did not fi nd any differences in K m values for asialotransferrin; but we found differences for CMP-NeuAc (p ! 0.05) as shown in table 3 .

� (2,3)-Neuraminidase Treatment In order to characterize the products generated in the

sialylation reaction using asialotransferrin as acceptor, a precipitation technique was developed for checking the NeuAc � (2,3) or NeuAc � (2,6) incorporation into asialo-transferrin. To demonstrate the complete NeuAc � (2,3) elimination arising from the action of a specifi c � (2,3)-neuraminidase, a MAL-Sepharose chromatographic col-umn was employed based on the ability of the immobi-lized lectin to interact in a highly specifi c way with the trisaccharide sequence NeuAc � (2,3)Gal � (1,4)GlcNAc [25] . Thus, fetuin, which contains NeuAc linked in � (2,3)-linkage, was treated with the enzyme in the same way as NeuAc-asialotransferrin, as exposed in Methods. After di-gestion with the enzyme, the column did not retain fetuin, therefore the effi cacy of the NeuAc � (2,3)-linked elimina-

Acceptor Tissue Means 8 SE Range n p

Asialotransferrin healthy 32.4982.69 1.68–123.27 85 0.001tumorous 55.8384.89 1.04–200.96

N-acetyllactosamine healthy 56.4485.66 1.37–150.30 40 0.001tumorous 157.95826.37 18.47–831.00

Data are expressed in �U/mg protein and represent means 8 standard error (SE) of n assays run independently and in duplicate. Statistical analysis was performed by Wilcox-on’s test using individual determinations.

Table 1. ST6Gal I activity in human co-lon adenocarcinoma and healthy mucosa from the same patient (at least 10 cm from the tumor)

Acceptor Tissue Means 8 SE Range n p

Asialotransferrin healthy 27.9983.76 2.73–68.28tumorous 57.4488.68 6.33–200.96 27 0.024transitional 45.6986.80 9.06–164.04

N-acetyllactosamine healthy 47.1486.17 1.37–122.09tumorous 114.03814.79 18.47–268.93 27 0.001transitional 84.81812.74 21.77–12.74

Data are expressed in �U/mg protein and represent means 8 standard error (SE) of n assays run independently and in duplicate. Statistical analysis was performed by the Kruskal-Wallis test using individual determinations.

Table 2. ST6Gal I activity in human co-lon adenocarcinoma, transitional tissue and healthy mucosa (at least 10 cm from the tumor) from the same patient

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Oncology 2005;69:436–444 440

tion was assessed. When the process was carried out with [ 14 C]NeuAc-asialotransferrin, the results revealed that the 100% NeuAc incorporation into asialotransferrin was due to the � (2,6)-linkage, since no radioactivity was found in-dicative of the NeuAc � (2,3) linked to asialotransferrin.

Identifi cation of the Sialo-Oligosaccharide Formed From the reaction carried out with N-acetyllactos-

amine, the two possible sialo-oligosaccharide isomeric products formed were separated by means of HPLC, as described in Methods. First, both the separation and spectrophotometric detection of the standards was under-

taken ( fi g. 1 a). Then, the standard assay was performed using N-acetyllactosamine as acceptor and using ion-ex-change chromatography to separate the sialo-oligosaccha-rides as explained earlier. The oligosaccharide products migrated as a single component identical to the reference tetrasaccharide NeuAc � (2,6)Gal � (1,4)GlcNAc on the HPLC ( fi g. 1 b). So, the sialo-oligosaccharides had a mean elution time of 53 min based on of 14 independent assays carried out with 7 healthy and 7 tumorous specimens. No traces of radioactivity were found in the elution time cor-responding to the NeuAc � (2,3) isomer in any of the 14 assays performed.

Substrates n Vmax Km

normal tumorous normal tumorous

CMP-NeuAc 5 35.0487.28 46.4084.13 21.4984.31 44.0685.05*Asialotransferrin 3 17.5285.13 45.17833.26 0.3380.09 0.4680.25

Vmax and Km of ST6Gal I from human colon adenocarcinoma and healthy mucosa were obtained as described in Methods. Values represent means 8 standard error (SE) of n de-terminations, performed independently and in duplicate. Vmax is expressed as �U/mg protein and Km as �M for CMP-NeuAc and as mg/100 �l/incubation for asialotransferrin. * p < 0.05 according to the Wilcoxon test.

Table 3. Kinetic parameters of ST6Gal I for the donor (CMP-NeuAc) and the acceptor (asialotransferrin) substrates

Fig. 1. Isolation and fractionation of NeuAc and sialylated N-acetyllactosamines. a HPLC separation and spectrophotometric detection of the standards was performed as described in Methods. They were composed of a mixture of (1) NeuAc, (2) Neu Ac � (2,3) Gal � (1,4) GlcNAc and (3) NeuAc � (2,6)Gal � (1,4)Glc Ac. b HPLC isolation and radioactive identifi -cation of the NeuAc isomers from the sialylation reaction of N-acetyllactosamine using healthy and tumorous colorectal tissues as enzyme source. The represented data are the means of 14 independent assays carried out with 7 healthy ( S ) and 7 tumorous specimens ( o ). No traces of radioactivity were found in the elution time corresponding to the NeuAc � (2,3)Gal � (1,4)GlcNAc isomer in any of the 14 assays performed.

ST6Gal I Activity in Human Colorectal Cancer

Oncology 2005;69:436–444 441

Correlation of ST6Gal I Activity Using Asialotransferrin and N-Acetyllactosamine as Acceptors A statistically signifi cant enhancement in ST6Gal I ac-

tivity was obtained using asialotransferrin and N-acetyl-lactosamine as acceptors in tumorous and transitional tissues upon comparison with healthy mucosa from the same patient. Therefore, we tried to fi nd if there was any correlation between the enhancement of activity for both acceptors in the same patients. To this end, we compared 32 healthy specimens, 32 tumorous specimens and 27 transitional specimens. A statistically signifi cant correla-tion was observed for healthy and tumorous tissue (p ! 0.01), and in transitional tissue (p = 0.001) ( table 4 ). Con-sequently, the altered sialyltransferase activity could pos-sibly belong to the same enzyme.

Table 5. Comparison of clinicopathological features of patients and ST6Gal I activity in tumorous and transitional colorectal tissues using asialotransferrin as acceptor

Table 4. Correlation between ST6Gal I ac-tivity to asialotransferrin and N-acetyllac-tosamine in healthy, tumorous and transi-tional tissues from the same patients with colorectal cancer (n)

Tissue n p

Healthy 32 0.002Tumorous 32 0.006Transitional 27 0.001

Statistical signifi cance (p) was based on Pearson’s correlation test.

Variable Tumorous tissue Transitional tissue

means 8 SE n p means 8 SE n p

Sex Women 52.9286.75 41 0.680 44.49811.00 13 0.115Men 59.8487.48 41 46.9888.17 14

Age, years <62 56.9589.68 19 37.9785.73 8<62–75 52.86810.87 21 0.250 40.48811.18 8 0.433>75 67.9589.90 21 61.59816.38 9

Tumor localizationRight colon 54.3389.54 22 42.4988.76 12Left colon 48.5786.44 38 0.190 27.2189.09 2 0.397Rectum 72.07811.88 21 56.14813.08 11

Size, cm<4 60.97811.82 22 65.36823.80 6<4–5.5 60.1988.88 29 0.834 34.0485.33 9 0.360>5.5 48.6086.84 27 53.63811.21 8

Histologic typeWell differentiated 63.06835.81 2 – 0Moderately differentiated 58.2985.86 65 0.705 51.5188.21 21 0.081Poorly differentiated 49.14811.79 12 25.0785.89 4

AJCC classifi cationA 47.96812.19 10 70.55831.54 4B 59.5287.91 39 0.918 50.79811.54 10 0.420C 55.2888.01 31 35.6285.53 11

Data are means 8 standard error (SE) expressed in �U/mg protein from n indepen-dent assays carried out in duplicate. Statistical signifi cance (p) was obtained by the Mann-Whitney test (for the sex and histologic type in transitional tissue) or the Kruskal-Wallis test (for the other comparisons).

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Correlation of ST6Gal I Activity with Clinicopathological Features of Patients The enhanced ST6Gal I activity observed in tumorous

and transitional tissues using asialotransferrin and N-acetyllactosamine as acceptors was then correlated in those tissues with clinicopathological parameters of pa-tients such as sex, age, tumor localization, size, histologi-cal type and tumor stage (according to AJCC). The impor-tance of this study relies on the possibility of determining relevant information for patient progress. We applied ad-equate statistical tests and obtained the results shown in tables 5 and 6 for asialotransferrin and N-acetyllactos-amine, respectively. No signifi cant differences were found in ST6Gal I activity in tumorous or transitional tissues for any of the clinicopathological variables studied, with the

exception of N-acetyllactosamine activity ( table 6 ) in tu-morous tissue between moderately and poorly differenti-ated specimens, in which the post-test for Kruskal-Wallis analysis (Dunn test) revealed statistically signifi cant dif-ferences in their respective enzyme activities.

Discussion

A number of studies have demonstrated an enhance-ment of � (2,6)-sialyltransferase activity in colorectal car-cinomas [6, 12–14, 17, 18] . In this work, asialotransferrin and N-acetyllactosamine bearing the common sequence Gal � (1,4)GlcNAc were employed as acceptors. This oli-gosaccharide structure could be sialylated by ST3 and

Variable Tumorous tissue Transitional tissue

means 8 SE n p means 8 SE n p

SexWomen 175.37847.60 17 0.561 99.55822.02 10 0.261Men 135.43818.90 21 77.75817.78 15

Age, years <62 102.63817.38 10 73.57814.03 8

62–75 94.69823.72 10 0.457 79.07816.89 10 0.457>75 150.27834.49 10 111.80840.77 7

Tumor localizationRight colon 94.56816.54 10 109.23825.61 8Left colon 190.57859.46 13 0.259 66.06822.31 5 0.431Rectum 217.14867.99 9 109.64832.42 7

Size, cm<4 211.39859.74 13 108.54838.08 8

4–5.5 165.39865.32 10 0.388 46.34812.80 4 0.270>5.5 105.53814.48 15 85.25811.15 13

Histologic typeWell differentiated 162.51898.66 3 87.12 1Moderately

differentiated 167.97831.13 32 0.1381 90.70815.29 22 0.513Poorly differentiated 40.8383.55 3 39.6887.53 2

AJCC classifi cationA 162.32859.93 11 80.26830.71 8B 145.19826.52 15 0.896 101.27829.43 8 0.647C 168.47862.59 12 78.86811.19 9

Data are means 8 standard error (SE) expressed in �U/mg protein from n indepen-dent assays carried out in duplicate. Statistical signifi cance (p) was obtained by the Mann-Whitney test (for sex) or the Kruskal-Wallis test (for the other comparisons).

1The post-test for Kruskal-Wallis analysis (Dunn test) revealed statistically signifi cant differences when ST6Gal I activities of moderately and poorly differentiated specimens from tumorous tissue.

Table 6. Comparison of clinicopathological features of patients and ST6Gal I activity in tumorous and transitional colorectal tissues using N-acetyllactosamine as acceptor

ST6Gal I Activity in Human Colorectal Cancer

Oncology 2005;69:436–444 443

ST6, whose presence in colorectal tissues has been estab-lished [11, 19] . It has been demonstrated that each group of enzymes (ST3 and ST6) displays preferences for glyco-protein acceptors and for the type of oligosaccharides rec-ognized [27] . Therefore, to precisely determine which en-zyme is involved in the sialylation, it is necessary to per-form tests to check the reaction products. Thus, by means of a specifi c � (2,3)-neuraminidase, we demonstrated that there is no � (2,3)-linkage to asialotransferrin chains. Next, by using HPLC it was shown that only � (2,6)-link-ages were present in N-acetyllactosamine unit chains. Therefore, we can state that ST6 activity was measured using both kinds of acceptor. The enzyme that accounted for this sialylated structure is ST6Gal I: ST6Gal II was not expressed or less so than ST6Gal I and only acted in isolated oligosaccharide chains [15, 16] .

ST6Gal I activity was enhanced in tumorous tissue compared with healthy tissue (p = 0.001). These results are in accordance with others previously reported [12, 13, 17] . We failed to fi nd an explanation for the observed signifi cant differences based on values of the kinetic pa-rameters K m and V max as determined for asialotransferrin and CMP-NeuAc, since those were higher in tumorous than in normal tissue; we only fi nd a statistically signifi -cant increase in the K m of CMP-NeuAc substrate. How-ever, Dall’Olio et al. [12] , using asialotransferrin as accep-tor, described a 6-fold decrease in the K m of ST6Gal I for CMP-NeuAc in colon adenocarcinoma with respect to healthy tissue, although based on the kinetic test of only 1 patient, thus variability among subjects could explain this discrepancy. The activity levels of transitional tissue were observed to be intermediate between healthy and tumor levels and the statistical test revealed signifi cant differences compared with healthy tissue for both accep-tors (p ! 0.05). In the light of our results, the altered ac-tivity in transitional tissue could be considered as a proof of its preneoplasic stage in spite of its microscopically healthy appearance. This information could be very use-ful as an indicator of a malignancy potential and as a tar-get for novel therapeutic approaches. On the other hand, we determined the degree of correlation between activity levels obtained for each acceptor in every tissue from the same patient. As expected, we found a positive correla-tion in the three tissues studied with both acceptors.

In an attempt to fi nd clinical applicability for the in-crease of ST6Gal I activity, we tested the existence of a correlation between the level of ST6Gal I activity in tu-morous and transitional tissues and several clinicopatho-logical features of the patient. Although the differences by the Mann-Whitney or Kruskal-Wallis test were not

statistically signifi cant, our study suggests that the levels of ST6Gal I activity in tumorous and transitional tissues varied with the histological grade of the tumor: well to moderately differentiated tumors had higher enzyme ac-tivity levels than poorly differentiated tumors. In this sense, the test performed after the Kruskal-Wallis analy-sis revealed a statistically signifi cant decrease in enzyme activity for N-acetyllactosamine in poorly differentiated specimens as compared to moderately differentiated ones. These results are in accordance with other immu-nohistochemical studies in which the enzyme was detect-ed using poorly differentiated tissue [28] and human CRC cell lines [29] . Although altered glycoconjugates correlate with an increased propensity to liver metastasis in animal models [30] , we failed to fi nd a correlation with AJCC tumor classifi cation. In some studies, � (2,6)-sialylation is negatively correlated with a good clinical outcome in CRC patients [18, 20, 28] . But in those stud-ies the most widely used tool for the investigation of the sialyl- � (2,6)-linkage was Sambucus nigra agglutinin and this lectin not only recognizes the CDw75 antigen, but also recognizes the sTn epitope, another glycan structure highly expressed in colon cancer tissues that has also been related to patient survival [31] . As the sTn antigen is con-sidered to be sialylated by ST6GalNAc I (EC 2.4.99.3) [10] , the results of these works should be reconsidered.

In any case, a large percentage of primary and meta-static gastrointestinal carcinomas show increased activity with anti-CDw75 antibodies [32, 33] and elevation of ST6Gal I mRNA in colorectal tissues [34, 35] and in colorectal cancer cells [20] have also been related to the metastasis process. One possible explanation is connect-ed with the nonadhesiveness of the cells [28] , because it has been proposed that a process of selection for meta-static cells by hypersialylation of specifi c tumor cell sur-face glycoconjugates would promote the release of single cells from the primary tumor mass [20, 30] .

This study reports enhanced ST6Gal I activity in tu-morous and transitional tissues from CRC patients. Fur-ther studies must be carried out on more samples and at different levels, such as mRNA or the CDw75 expression using monoclonal antibodies. All results should be com-pared in an attempt to understand the complex regula-tory factors in colon cancer, notably, by including transi-tional tissue in the study, since our results suggest the need for a more detailed study.

Vázquez-Martín/Gil-Martín/Fernández-Briera

Oncology 2005;69:436–444 444

Acknowledgements

We are very grateful to Dr. M. Butrón (Hospital Xeral, Vigo, Spain) and Drs. E. Cuevas and C. Miranda (Complejo Hospitala rio Cristal-Piñor, Ourense, Spain) for providing us with specimens

and patient data for this study and Dr. C. Villaverde for thestatistical analysis. This work was supported by grants from the ‘Xunta de Galicia’ (Projects PGIDT00PXI30103PR and PGIDIT02BTF30101PR).

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