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Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck
Oral Oncology (2004) 40 28–35
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Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck Burkhard M. Helmkea,1, Christoph Reisserb,1, Mario Idzkoeb, Gerhard Dyckhoffb, Christel Herold-Mendeb,c,* a
Department of Pathology, University of Heidelberg, INF 220, 69120 Heidelberg, Germany Molecular Cell Biology Group, Department of Head and Neck Surgery, University of Heidelberg, INF 400, 69120 Heidelberg, Germany c Molecular Biology Laboratory, Department of Neurosurgery, University of Heidelberg, INF 400, 69120 Heidelberg, Germany b
Received 6 May 2003; accepted 27 May 2003
KEYWORDS HNSCC; SGLT-1; GLUT; Squamous cell carcinomas; Head and neck tumors; Glucose transport
Summary Tumors show an increased glucose uptake that is mediated by glucose transport proteins. We have analyzed the expression of the sodium-dependent glucose co-transporters SGLT-1 and-2 in short-term cultures of squamous cell carcinomas of the head and neck (HNSCC) by RT-PCR. Distribution of the SGLT-1 protein in HNSCC tissues was investigated by immunohistochemistry. While we observed in 17/36 HNSCC shortterm cultures the SGLT-1 mRNA, we found no SGLT-2 expression. SGLT-1 mRNA expression occurred preferentially in cultures originating from moderately and well differentiated HNSCC. In tumor tissues a heterogeneous SGLT-1 staining restricted to differentiated tumor cells was seen in 27/30 HNSCC analyzed. In normal mucosa SGLT-1 staining was also confined to differentiated compartments and lacked in dysplastic areas. Our data indicate a differentiation-dependent expression of SGLT-1 in HNSCC. This is important knowledge for the planning of glucose-targeting therapies and suggest SGLT-1 as a differentiation marker in head and neck tissues. # 2003 Elsevier Ltd. All rights reserved.
Introduction The incidence of malignant tumors of the head and neck (HNSCC) has increased all over the world during the last decades.1,2 Despite progress in surgical techniques and an improved application of * Corresponding author. Tel.: +49-6221-563-9504; fax: +496221-565362. E-mail address: [email protected] (C. Herold-Mende). 1 The first two authors have contributed equally to this paper.
radio- and chemotherapy, the mortality of these tumors is still high therefore demanding the development of new treatment modalities. In this respect therapeutical inhibition of the tumor metabolism has gained special interest since the metabolic activity provides the basis for tumor cell proliferation and thus tumor growth. As most important sources of energy the oxidative metabolism in mammalian cells needs glucose.3,4 Uptake of glucose into cells is mediated by two different processes, a facilitated saturable non-energy-dependent transport mechanism and
1368-8375/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1368-8375(03)00129-5
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck an active energy- and sodium-dependent transport mechanism. Specific transmembrane glucose transporter proteins have been identified that are involved in the facilitated transport (glucose transport proteins, GLUTs) or in the active transport (sodium-dependent glucose co-transporters, SGLTs). The gene family of proteins of the facilitated glucose transport mechanism (GLUTs) is comprised of different members, which are structurally related and exhibit considerable homology in their primary sequences (GLUT-1-75). The glucose transporter proteins have a marked tissue-specific pattern: in normal tissues GLUT-1 is mainly expressed in erythrocytes and vascular tissue, GLUT-2 in cells of liver, pancreas, kidney and intestine, GLUT-3 in brain tissues and in inflammatory cells.6 The insulin-responsive transporter GLUT-4 is expressed in muscular and fat tissue. GLUT-5 is present in human enterocytes and is largely a fructose transporter.7 Putative GLUT-7 is a hepatic intracellular glucose transporter.8 In previous studies on preneoplastic and neoplastic tissues of the head and neck we have shown a marked expression of GLUT-1 in squamous cell carcinomas of the head and neck as well as in preneoplastic lesions throughout the whole dysplastic compartment, while in normal mucosa GLUT-1 staining was weak and always confined to the basal cell layer.9 Compared to the facilitated glucose transport mechanism knowledge about the active energyand sodium-dependent transport mechanism and the involved SGLT proteins is rather incomplete. SGLT-1 was the first of a group of sodium-dependent transport proteins that have been postulated in 196010 and described and named ‘‘sodiumdependent d-glucose co-transporter 1 (SGLT-1)‘‘ in 1987.11 The human SGLT-1 is an integral membrane protein consisting of 664 amino acids and a molecular weight of 73 497 Da. There are 11 transmembranous parts with both the carboxy- and the amino-end within the cytoplasm. SGLT-1 has a high glucose affinity with a Na+/glucose-binding rate of 2:1. Physiologically, SGLT is localized in apical epithelial cells of the microvilli in the intestine and mediates glucose uptake from the food.12 In addition, SGLT-1 was shown in the brush border of renal cells and in vitro in the brush border of Caco-2 cells that originate from a colon carcinoma.12,13 In respect to squamous epithelia such as the mucosa of the head and neck region and therefrom derived tumors, expression of SGLTs has never been studied before. However, this information would be important since several inhibitors of SGLTs have been successfully developed.14 17 These include the dihydrochalcone flavonoid phloretin18 and
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phloridzin a polycyclic glucosid that has been shown to be a potent competitive inhibitor of the Na+/glucose-transport by reducing the glucose uptake in cultivated cells of a mammary adenocarcinoma of the rat.19 Therefore, the aim of our study was to analyze expression of the active glucose transport on the mRNA level in tumor cell cultures as well as on the protein level in normal mucosa in comparison to premalignant and malignant lesions of the head and neck (HNSCC).
Materials and methods Cell lines and cell culture Human colon carcinoma cells HT29 were obtained from American Type Culture Collection (Rockville, MD). Thirty-six short-term cell cultures derived from human HNSCC (maxillary sinus n=3, oral cavity n=8, oropharynx n=14, larynx n=11, hypopharynx n=5, CUP n=3, Table 1) have been established in our laboratory as described earlier.20 All short-term cultures were characterized for their epithelial origin by the immunohistochemical detection of tissue-specific markers using antibodies recognizing a broad spectrum of cytokeratins (clone MNF 116 recognizing cytokeratins 5, 6, 8, 17, and 19 and clone 34BE12, recognizing cytokeratins 1, 5, 10, and 14, both DAKO, Hamburg, Germany). Lack of endothelial cell contamination was determined by staining with endothelial cellspecific antibodies directed against factor VIII (DAKO, Hamburg, Germany) and PECAM-1 (PharMingen, Heidelberg, Germany). Only cultures showing a homogenous staining for the cytokeratin markers and no staining for both endothelial cellmarkers were selected for this study. Cells were cultured routinely in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics at 37 C, 5% CO2, and 95% air in a humidified incubator with medium changes twice a week. After reaching confluency, cells were harvested by a brief incubation with Trypsin IEDTA solution (Viralex; PAA, Linz, Austria) and seeded into a fresh 75 cm2 plastic tissue culture flask. Mycoplasma contamination of the cells was excluded by DAPI staining.
RNA isolation and RT-PCR analysis Total RNA was isolated from cell cultures grown on 75 cm2 plastic tissue culture flasks (Qiagen RNeasy total RNA preparation kit) according to the manufacturer’s instructions. RT-PCR reactions were performed using a reverse transcription system kit
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Table 1 Clinical data and SGLT expression of HNSCC short-term cultures
HNO35 p12 HNO41 p20 HNO64 p2 HNO66 p20 HNO67 p2, p5 HNO70 p9 HNO74 p7 HNO76 p15 HNO80 p5 HNO81 p5 HNO89 p14 HNO91 p34 HNO94 p3 HNO97 p58 HNO103 p5 HNO106 p3 HNO107 p7 HNO111 p5 HNO123 p4 HNO124 p13 HNO129 p4 HNO133 p4 HNO136 p88 HNO147 p20 HNO150 p15 HNO160 p18 HNO161 p12 HNO168 p4 HNO178 p11 HNO180 p7 HNO184 p15 HNO191 p36 HNO199 p30 HNO206 p9 HNO210 p12, p24 HNO212 p48
Localization
TNM
Grading
Oropharynx Oropharynx Larynx Oropharynx Oral cavity CUP Larynx Larynx Larynx Oral cavity Larynx Oral cavity CUP Oral cavity Oropharynx Larynx Larynx Hypopharynx Larynx Oral cavity CUP Maxillary sinus Maxillary sinus Hypopharynx Larynx Oral cavity Hypopharynx Maxillary sinus Hypopharynx Hypopharynx Larynx Oropharynx Oral cavity Oropharynx Larynx Oral cavity
T2N0M0 T3N3M0 T4N0M0 T2N1M0 T2N2M0 — T4N2M0 T2N2M0 T4N2M0 T1N0M0 T3N0M0 T1N1M0 — T2N2M0 T4N2Mo T2N0M0 T3N2M0 T3N2M0 T4N2M0 T4N2M0 — T3N2M0 T4N0M0 T4N2M0 T3N2M0 T2N2M0 T4N1M0 T2N2M0 T2N1M0 T2N0M0 T4N0M0 T2N2M0 T2N2M0 T2N0M0 T3N0M0 T4N2M0
G1 G2 G3 G3 G2 G3 G3 G1 G3 G1 G3 G3 G2 G3 G3 G2 G3 G2 G2 G3 G3 G3 G3 G3 G2 G1 G2 G3 G2 G3 G3 G3 G3 G3 G2 G2
(Sigma, Germany). Reverse transcription was done in a volume of 20 ml using 2 mg of total RNA, 1 U/ ml enhanced AMV reverse transcriptase, 0.5 mM of each dATP, dGTP, dCTP and dTTP and 2.5 mM random nonamers in 1PCR buffer II containing 5 mM MgCl2, and 1 U/ml RNase inhibitor. PCR (50 ml) reaction contained 3 ml of the RT-reaction, 8% DMSO, 0.2 mM of each dATP, dGTP, dCTP and dTTP, 2.5 mM MgCl2, 10 pM of each primer and 2.5 U AmpliTaq LA DNA Polymerase. Primer annealing temperature was optimized for each primer set: SGLT-1: 35 cycles of 95 C 1 min 30 s, 61 C 1 min, 72 C 1 min 30 s. SGLT-2: 35 cycles of 94 C 1 min 30 s, 62 C 1 min, 72 C 1 min 30 s.
SGLT-1
SGLT-2
+
+ + + + + + +
+
+ + + +
+ + + +
GAPDH: 30 cycles of 94 C 1 min 30 s, 62 C 1 min, 72 C 1 min 30 s. Forward and reverse primers were synthesized according to the sequences extracted from genbank. Primer sets used are listed in Table 2. All oligonucleotide primer pairs spanned intron—exon splice sites ensuring that PCR products did not generate from any DNA contamination present in the RNA preparations. PCR of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was done as control for the quality of the RNA preparation.
Tissue samples For further immunohistochemical analysis, biopsy specimens of 30 squamous cell carcinomas
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck
Table 2 SGLT and control primers used for RT-PCR SGLT-1(bp 201—221, forward) TGG CAG GCC GAA GTA TGG TGT (bp 507—528, reverse) ATG AAT ATG GCC CCC GAG AAG A SGLT-2(bp 615—637, forward) ACA CGG ACA CGG TAC AGA CCT T (bp 1599—1622, reverse) GAA CAG CAC AAT GGC GAA GTA GA GAPDH(bp 69—91, forward) GGT GGA GGT CGG AGT CAA CGG A (bp 286—308, reverse) GAG GGA TCT CGC TCC TGG AGG A
of the head and neck were obtained intra-operatively (maxillary sinus n=3, oral cavity n=5, oropharynx n=10, larynx n=8, hypopharynx n=4). Fresh tissues were snap-frozen immediately after surgery in liquid nitrogen and stored at 80 C until processing. Twenty-eight squamous epithelia in close vicinity to the tumor as well as from non-tumor patients were also cryopreserved.
Western blots and antibody production Proteins from cultured cells were extracted in 50 mM Tris (pH8), 150 mM NaCl, 0.1% SDS, 1% NP4O, 0.5% sodium deoxycholate, 0.02% NaN3, 1 mg/ml Pefabloc, 5 mg/ml Leupeptin, 1 mg/ml Pepstatin, 1 mg/ml Aprotinin, and 0.5 mg/ml EDTA. Total protein concentration was determined using the Bradford method (DC Protein Assay, Bio-Rad Laboratories, Muenchen). Equal amounts of protein were loaded on 5% SDS-PAGE mini-gels, analyzed and transferred to Hybond-P membranes (Amersham Pharmacia Biotech, Freiburg). Blocking was performed by incubating the PVDF membranes with 5% (w/v) non-fat dry milk in Trisbuffered saline (TBS) pH 7.5, for 1 h at room temperature and under continuous agitation. The membranes were then incubated with an affinitypurified rabbit polyclonal peptide antibody against peptide IETQVPEKKKGIFRR of SGLT-1, which represents residues 590—604 (diluted 1:300) for 18 h at 8 C under continuous agitation. After three washes with 20 mM Tris (pH 7.5), 500 mM glycine, and 0.05% (w/v) Tween the antibody binding was visualized by incubation with peroxidase-coupled anti-rabbit IgG antibody (diluted 1:4000) in 5% (w/v) non-fat dry milk in PBS for 45 mm at room temperature followed by chemiluminescence detection with the ECL system (Amersham Pharmacia Biotech, Freiburg) according to the manufacturer’s instructions.
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Immunohistochemistry Immunohistochemical staining was performed on serial cryostat sections (5—7 mm) of the frozen biopsies, mounted on 3-aminopropyl-triethoxysilan coated slides. As fixative acetone was used at 20 C for 10 min. Incubation with the primary and secondary antibodies and detection was carried out as described elsewhere21 with Vectastain Laboratories Elite ABC Kit (Vector Laboratories, Burlingame, California). Primary antibody used was the before described affinity-purified rabbit polyclonal anti SGLT-1 antibody (diluted 1:100). Specificity of the SGLT-1 antibody reaction was confirmed in parallel sections using preimmune serum.
Results mRNA expression of SGLT-1 and SGLT-2 in HNSCC cultures In vitro expression of SGLT-1, as analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), was detected in 17 of 36 HNSCC cultures in different passages (between p3 and p88, Fig. 1, Table 1). Comparison of tumor localization with SGLT-1 expression revealed SGLT-1 mRNA in 1/3 maxillary sinus carcinomas, 5/8 oral cavity carcinomas, 2/6 oropharynx carcinomas, 6/11 larynx carcinomas, 1/5 hypopharynx carcinomas, and 2/3 CUP. Comparison with the grading of the respective tumor tissue showed SGLT-1 expression in 75% of well differentiated G1 tumors (3/4), 73% of moderately differentiated G2 tumors (8/11), and 29% of poorly differentiated G3 tumors (6/21) analyzed. The human colon carcinoma cell line HT29 served as positive control.22 In contrast to SGLT-1, we could not observe in any of the HNSCC cultures expression of SGLT-2 (Table 1). Amplification of GAPDH in all samples confirmed the integrity of the mRNA used.
Antibody production and specificity To further analyze localization of SGLT-1 protein in native tissues and to determine SGLT-1-expressing cell types and homogeneity of SGLT-1 expression we developed affinity-purified SGLT-1specific peptide antibodies in several animals (see A1 and A2 in Fig. 2). When proteins from SGLT-1expressing HT29 cells (see mRNA analysis) separated by SDS-polyacrylamide gel electrophoresis and blotted on PVDF membranes were developed with these peptide antibodies, the major reaction product was observed at 75 kDa (Fig. 2), as has
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Figure 1 mRNA Expression of SGLT-1 in HNSCC short-term cultures. Upper lane shows results for SGLT-1 expression as determined by RT-PCR. HT29 colon carcinoma cells served as positive control. Lower lane shows expression of the house keeping gene GAPDH as control for the RNA integrity. p=passage number of tumor cell cultures.
Figure 2 Western blot analysis of affinity-purified antiSGLT-1-specific peptide antibodies raised against residues 590—604 from human SGLT-1 in HT29 cells. The major reaction products of antisera of two different animals (A1 and A2) were observed at the expected size of 75 kDa.
been described in small intestine.23 Because reactivity of the antibody developed in animal 2 (A2) appeared to be stronger and because the antibody did not show any cross-reactivity, it was used for our further immunohistochemical analysis of native tissues.
immunohistochemistry including 20 cases, where corresponding tumor cell cultures had been analyzed by RT-PCR before. The lesions contained two well (G1), 12 moderately (G2), and 16 poorly (G3) differentiated squamous cell carcinomas. Only three G3 tumors were negative for SGLT-1, while we observed a hetereogeneous expression of SGLT1 in the remaining 27 tumors analyzed. Interestingly, in all tumors that were positive for SGLT-1, strong staining was confined to differentiated tumor cells localized in the center of tumor islands (Fig. 3E and F), and thus resembling the expression observed in the normal epithelium. Comparison of results obtained in tumor cell cultures by RT-PCR with protein expression in the native tumor tissue revealed no SGLT-1 detection in tumor cultures, where the tumor tissue failed to express SGLT-1. However, in 6/20 cases, where SGLT-1 staining was weak and restricted to few tumor cells in the tumor tissue, we found no mRNA message in the corresponding short-term culture.
Protein expression of SGLT-1 in squamous epithelia and squamous cell carcinomas of the head and neck
Discussion
Immunohistochemical staining in stratified squamous epithelium of the oral mucosa with the antiSGLT-1 antibody revealed a constitutive targeting of SGLT-1 in all epithelia (n=23). However, staining was strictly confined to differentiated cell layers starting with the prickle cell layer (Fig. 3A). In contrast, in the basal cell layers SGLT-1 was always absent. Also, non-epithelial structures like smooth and skeletal muscle cells were positive for SGLT-1 (Fig. 3B). In addition, in five epithelial specimen that contained dysplastic areas (including mild, moderate, and severe dysplasias) SGLT-1 expression was either reduced to non-dysplastic areas [Fig. 3C showing mild (area II) and moderate (area III) dyplastic foci] or lacked completely in severe dyplasias [Fig. 3D (area I)]. To investigate the expression of SGLT-1 in carcinomas arising from squamous epithelia, 30 cases of squamous cell carcinomas were analyzed by
Positron emission tomography (PET) studies on malignant HNSCC have shown that tumor growth is associated with an up to 10-fold increased uptake of the glucose analogue fluorodeoxyglucose (FDG) compared to normal epithelium and benign tumors.24 These observations together with similar results on other tumor types25,26 have brought up the idea to control tumor growth by inhibiting the glucose transport. Two well-known compounds in this respect are phloretin and phloridzin.18,19 Since both seem to target preferentially the active glucose transport mediated by SGLTs and since these proteins have not been studied before in head and neck tumors, our aim was to investigate the expression of SGLT-1 and-2 in HNSCC. We were able to identify the SGLT-1 mRNA in 17/ 36 HNSCC short-term cultures by RT-PCR. Most of the SGLT-1 positive HNSCC cultures originated from tumors exhibiting an advanced differentiation (3/4
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck
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Figure 3 Immunohistochemical staining for SGLT-1 in normal, preneoplastic and neoplastic tissues of the head and neck. Normal squamous epithelium (A) shows a SGLT-1 staining starting with the prickle cell layer. In the basal compartment SGLT-1 is negative. SGLT-1 expression in non-epithelial structures (B) like smooth and skeletal muscle cells. Squamous epithelia in (C) and (D) show dysplastic alterations including mild (II) and moderate (III) dyplastic foci beside non-dysplastic mucosa (I) in (C) and a severe dyplasia (I) beside a moderate dysplasia (II) in (D). In dysplastic areas with a disturbed differentiation SGLT-1 staining is absent. (E) and (F) demonstrate heterogeneous SGLT-1 staining in the moderately differentiated tumor HNO212 (E) and the poorly differentiated tumor HNO89 (F). SGLT-1 expression can only be found in differentiated tumor cells.
G1, 8/11 G2, 6/21 G3). In contrast to SGLT-1, we failed to detect SGLT-2 in the same tumor cell cultures. Therefore we have developed an antiSGLT-1 peptide antibody to further analyze expression and distribution of the SGLT-1 protein in native head and neck tissues. We found a heterogeneous SGLT-1 staining in 27/30 tumors analyzed that was always restricted to differentiated tumor areas. In some tumor tissues, where only single
tumor cells were positive for SGLT-1 by immunohistochemistry, we could not detect the SGLT-1 mRNA in the corresponding tumor culture. This discrepancy might be explained by the fact that under in vitro conditions mainly less differentiated tumor cells are expanded. Immunohistochemical analysis of normal and preneoplastic mucosa revealed a similar differentiation-dependent SGLT-1 distribution. In the
34 normal mucosa SGLT-1 was never found in the basal compartment, where proliferation occurs, but was always detectable starting with the prickle cell layer. Whenever we located dysplastic alterations in squamous epithelia, SGLT-1 staining was completely absent in the lesioned area. This distribution pattern together with the observed expression of SGLT-1 in normal and neoplastic tissues as well as the preferential mRNA expression in tumor cultures originating from more differentiated tumors strongly supports the idea that SGLT-1 is expressed in a differentiation-dependent manner in tissues of the head and neck. We could strengthen this hypothesis by the finding that glycogen, the storage form of glucose only occurring in differentiated epithelial cells,9 could be identified by PAS-staining in the same tumor and epithelial compartments as SGLT-1 (data not shown). Further our observations are in line with data of Baron-Delage and co-workers27 describing a downregulation of SGLT-1 expression in colon carcinoma cells after oncogenic activation of p21ras or pp60csrc, while GLUT-1 was upregulated at the same time. Reports on SGLT expression in tumorigenesis are few. So far, SGLT-1 expression was found in several intestinal tumor cell lines13,22 and RT-PCR analysis on primary adenocarcinomas of the lung and corresponding metastases has shown that although expression of SGLT-1 can be found in these tumors, it is not increased in metastases.28 Altogether, our data on the expression and distribution of SGLT-1 in normal, preneoplastic and neoplastic head and neck tissues give new insights on the expression of glucose transport proteins in different compartments of squamous epithelia and therefrom derived tumors. This is important knowledge for the planning of glucose-targeting therapies on the one hand and on the other hand indicates that SGLT-1 may serve as a differentiation marker in head and neck tissues.
Acknowledgements We gratefully acknowledge the help of all medical colleagues in gathering the tumor biopsies. We thank Mrs. Melanie Bobko, and Mrs. Renate Steinle for excellent technical support, and Mr. Philip Benjamin for photographic work. References 1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics. CA Cancer J Clin 2002;52:23—47. 2. Parkin DM, Pisani P, Ferlay J. Global cancer statistics. CA Cancer J Clin 1999;49:33—64.
B.M. Helmke et al. ¨ ber den Stoffwechsel von 3. Warburg O, Wind F, Negelein E. U Tumoren. Klin Wochenschr 1926;5:829—832. 4. Tirone TA, Brunicardi FC. Overview of glucose regulation. World J Surg 2001;25:461—467. 5. Gould GW, Holman GD. The glucose transporter family: structure, function and tissue-specific expression. Biochem J 1993;295:329—341. 6. Mantych GJ, James DE, Chung HD, Devaskar SU. Cellular localization and characterization of Glut 3 glucose transporter isoform in human brain. Endocrinology 1992;131: 1270—1278. 7. Burant CF, Takeda J, Brot-Laroche E, Bell GI, Davidson NO. Fructose transporter in human spermatozoa and small intestine is GLUT5. J Biol Chem 1992;267:14523—14526. 8. Waddell ID, Zomerschoe AG, Voice MW, Burchell A. Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucosetransport protein GLUT 2. Biochem J 1992;286:173—177. 9. Reisser C, Eichhorn K, Herold-Mende C, Born AI, Bannasch P. Expression of facilitative glucose transport proteins during development of squamous cell carcinomas of the head and neck. Int J Cancer 1999;80:194—198. 10. Crone C. The diffusion of some non-electrolytes from blood to brain tissue. Acta Physiol Scand Suppl 1960;175:33—34. 11. Hediger MA, Coady MJ, Ikeda TS, Wright EM. Expression cloning and cDNA sequencing of the Na+/glucose co-transporter. Nature 1987;330:379—381. 12. Takata K. Glucose transporters in the transepithelial transport of glucose. J Electron Microsc (Tokyo) 1996;45:275— 284. 13. Bissonette P, Gagne H, Coady MJ, Benabdallah K, Lapointe JY, Berteloot A. Kinetic separation and characterization of three sugar transport modes in Caco-2 cells. Am J Physiol 1996;270:G833—G843. 14. Ader P, Block M, Pietzsch S, Wolffram S. Interaction of quercetin glucosides with the intestinal sodium/glucose cotransporter (SGLT-1). Cancer Lett 2001;26(162):175—180. 15. Crespy V, Morand C, Besson C, Manach C, Demigne C, Remesy C. Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats. J Nutr 2001; 131:2109—2114. 16. Oku A, Ueta K, Arakawa K, Kano-Ishihara T, Matsumoto M, Adachi T, Yasuda K, Tsuda K, Saito A. Antihyperglycemic effect of T-1095 via inhibition of renal Na+-glucose cotransporters in streptozotocin-induced diabetic rats. Biol Pharm Bull 2000;23:1434—1437. 17. Veyhl M, Wagner K, Volk C, Gorboulev V, Baumgarten K, Weber WM, Schaper M, Bertram B, Wiessler M, Koepsell H. Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1. Proc Natl Acad Sci USA 1998;95:2914—2919. 18. Malo C, Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na(+)-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 1991;122:127—141. 19. Nelson JA, Falk RE. The efficacy of phloridzin and phloretin on tumor cell growth. Anticancer Res 1993;13:2287—2292. 20. Herold-Mende C, Andl T, Steiner HH, Riede D, Buttler A, Reisser C, Fusenig NE, Mueller MM. Expression and functional significance of vascular endothelial growth factor receptors in human tumor cells. Lab Invest 1999;79:1573—1582. 21. Herold-Mende C, Seiter S, Born AI, Patzelt E, Schupp M, Zo ¨ller J, Bosch FX, Zo ¨ller M. Expression of CD44 splice variants in squamous epithelia and squamous cell carcinomas of the head and neck. J Pathol 1996;179:66—73.
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck 22. Delezay O, Verrier B, Mabrouk K, van Rietschoten J, Fantini J, Mauchamp J, Gerard C. Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line. J Cell Physiol 1995;163:120—128. 23. Lescale-Matys L, Dyer J, Scott D, Freeman TC, Wright EM, Shirazi-Beechey SP. Regulation of the ovine intestinal Na+/ glucose co-transporter (SGLT1) is dissociated from mRNA abundance. Biochem J 1993;291:435—440. 24. Reisser C, Haberkorn U, Strauss LG. The relevance of positron emission tomography for the diagnosis and treatment of head and neck tumors. J Otolaryngol 1993;22:231—238. 25. Lin WC, Hung YC, Yeh LS, Kao CH, Yen RF, Shen YY. Usefulness of (18)F-fluorodeoxyglucose positron emission tomography to detect para-aortic lymph nodal metastasis in
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advanced cervical cancer with negative computed tomography findings. Gynecol Oncol 2003;89:73—76. 26. Higashi K, Matsunari I, Ueda Y, Ikeda R, Guo J, Oguchi M, Tonami H, Yamamoto I. Value of whole-body FDG PET in management of lung cancer. Ann Nucl Med 2003;17: 1—14. 27. Baron-Delage S, Mahraoui L, Cadoret A, Veissiere D, Taillemite JL, Chastre E, Gespach C, Zweibaum A, Capeau J, Brot-Laroche E, Cherqui G. Deregulation of hexose transporter expression in Caco-2 cells by ras and polyoma middle T oncogenes. Am J Physiol 1996;270:G314—G323. 28. Ishikawa N, Oguri T, Isobe T, Fujitaka K, Kohno N. SGLT gene expression in primary lung cancers and their metastatic lesions. Jpn J Cancer Res 2001;92:874—879.
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Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck Burkhard M. Helmkea,1, Christoph Reisserb,1, Mario Idzkoeb, Gerhard Dyckhoffb, Christel Herold-Mendeb,c,* a
Department of Pathology, University of Heidelberg, INF 220, 69120 Heidelberg, Germany Molecular Cell Biology Group, Department of Head and Neck Surgery, University of Heidelberg, INF 400, 69120 Heidelberg, Germany c Molecular Biology Laboratory, Department of Neurosurgery, University of Heidelberg, INF 400, 69120 Heidelberg, Germany b
Received 6 May 2003; accepted 27 May 2003
KEYWORDS HNSCC; SGLT-1; GLUT; Squamous cell carcinomas; Head and neck tumors; Glucose transport
Summary Tumors show an increased glucose uptake that is mediated by glucose transport proteins. We have analyzed the expression of the sodium-dependent glucose co-transporters SGLT-1 and-2 in short-term cultures of squamous cell carcinomas of the head and neck (HNSCC) by RT-PCR. Distribution of the SGLT-1 protein in HNSCC tissues was investigated by immunohistochemistry. While we observed in 17/36 HNSCC shortterm cultures the SGLT-1 mRNA, we found no SGLT-2 expression. SGLT-1 mRNA expression occurred preferentially in cultures originating from moderately and well differentiated HNSCC. In tumor tissues a heterogeneous SGLT-1 staining restricted to differentiated tumor cells was seen in 27/30 HNSCC analyzed. In normal mucosa SGLT-1 staining was also confined to differentiated compartments and lacked in dysplastic areas. Our data indicate a differentiation-dependent expression of SGLT-1 in HNSCC. This is important knowledge for the planning of glucose-targeting therapies and suggest SGLT-1 as a differentiation marker in head and neck tissues. # 2003 Elsevier Ltd. All rights reserved.
Introduction The incidence of malignant tumors of the head and neck (HNSCC) has increased all over the world during the last decades.1,2 Despite progress in surgical techniques and an improved application of * Corresponding author. Tel.: +49-6221-563-9504; fax: +496221-565362. E-mail address: [email protected] (C. Herold-Mende). 1 The first two authors have contributed equally to this paper.
radio- and chemotherapy, the mortality of these tumors is still high therefore demanding the development of new treatment modalities. In this respect therapeutical inhibition of the tumor metabolism has gained special interest since the metabolic activity provides the basis for tumor cell proliferation and thus tumor growth. As most important sources of energy the oxidative metabolism in mammalian cells needs glucose.3,4 Uptake of glucose into cells is mediated by two different processes, a facilitated saturable non-energy-dependent transport mechanism and
1368-8375/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1368-8375(03)00129-5
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck an active energy- and sodium-dependent transport mechanism. Specific transmembrane glucose transporter proteins have been identified that are involved in the facilitated transport (glucose transport proteins, GLUTs) or in the active transport (sodium-dependent glucose co-transporters, SGLTs). The gene family of proteins of the facilitated glucose transport mechanism (GLUTs) is comprised of different members, which are structurally related and exhibit considerable homology in their primary sequences (GLUT-1-75). The glucose transporter proteins have a marked tissue-specific pattern: in normal tissues GLUT-1 is mainly expressed in erythrocytes and vascular tissue, GLUT-2 in cells of liver, pancreas, kidney and intestine, GLUT-3 in brain tissues and in inflammatory cells.6 The insulin-responsive transporter GLUT-4 is expressed in muscular and fat tissue. GLUT-5 is present in human enterocytes and is largely a fructose transporter.7 Putative GLUT-7 is a hepatic intracellular glucose transporter.8 In previous studies on preneoplastic and neoplastic tissues of the head and neck we have shown a marked expression of GLUT-1 in squamous cell carcinomas of the head and neck as well as in preneoplastic lesions throughout the whole dysplastic compartment, while in normal mucosa GLUT-1 staining was weak and always confined to the basal cell layer.9 Compared to the facilitated glucose transport mechanism knowledge about the active energyand sodium-dependent transport mechanism and the involved SGLT proteins is rather incomplete. SGLT-1 was the first of a group of sodium-dependent transport proteins that have been postulated in 196010 and described and named ‘‘sodiumdependent d-glucose co-transporter 1 (SGLT-1)‘‘ in 1987.11 The human SGLT-1 is an integral membrane protein consisting of 664 amino acids and a molecular weight of 73 497 Da. There are 11 transmembranous parts with both the carboxy- and the amino-end within the cytoplasm. SGLT-1 has a high glucose affinity with a Na+/glucose-binding rate of 2:1. Physiologically, SGLT is localized in apical epithelial cells of the microvilli in the intestine and mediates glucose uptake from the food.12 In addition, SGLT-1 was shown in the brush border of renal cells and in vitro in the brush border of Caco-2 cells that originate from a colon carcinoma.12,13 In respect to squamous epithelia such as the mucosa of the head and neck region and therefrom derived tumors, expression of SGLTs has never been studied before. However, this information would be important since several inhibitors of SGLTs have been successfully developed.14 17 These include the dihydrochalcone flavonoid phloretin18 and
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phloridzin a polycyclic glucosid that has been shown to be a potent competitive inhibitor of the Na+/glucose-transport by reducing the glucose uptake in cultivated cells of a mammary adenocarcinoma of the rat.19 Therefore, the aim of our study was to analyze expression of the active glucose transport on the mRNA level in tumor cell cultures as well as on the protein level in normal mucosa in comparison to premalignant and malignant lesions of the head and neck (HNSCC).
Materials and methods Cell lines and cell culture Human colon carcinoma cells HT29 were obtained from American Type Culture Collection (Rockville, MD). Thirty-six short-term cell cultures derived from human HNSCC (maxillary sinus n=3, oral cavity n=8, oropharynx n=14, larynx n=11, hypopharynx n=5, CUP n=3, Table 1) have been established in our laboratory as described earlier.20 All short-term cultures were characterized for their epithelial origin by the immunohistochemical detection of tissue-specific markers using antibodies recognizing a broad spectrum of cytokeratins (clone MNF 116 recognizing cytokeratins 5, 6, 8, 17, and 19 and clone 34BE12, recognizing cytokeratins 1, 5, 10, and 14, both DAKO, Hamburg, Germany). Lack of endothelial cell contamination was determined by staining with endothelial cellspecific antibodies directed against factor VIII (DAKO, Hamburg, Germany) and PECAM-1 (PharMingen, Heidelberg, Germany). Only cultures showing a homogenous staining for the cytokeratin markers and no staining for both endothelial cellmarkers were selected for this study. Cells were cultured routinely in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics at 37 C, 5% CO2, and 95% air in a humidified incubator with medium changes twice a week. After reaching confluency, cells were harvested by a brief incubation with Trypsin IEDTA solution (Viralex; PAA, Linz, Austria) and seeded into a fresh 75 cm2 plastic tissue culture flask. Mycoplasma contamination of the cells was excluded by DAPI staining.
RNA isolation and RT-PCR analysis Total RNA was isolated from cell cultures grown on 75 cm2 plastic tissue culture flasks (Qiagen RNeasy total RNA preparation kit) according to the manufacturer’s instructions. RT-PCR reactions were performed using a reverse transcription system kit
30
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Table 1 Clinical data and SGLT expression of HNSCC short-term cultures
HNO35 p12 HNO41 p20 HNO64 p2 HNO66 p20 HNO67 p2, p5 HNO70 p9 HNO74 p7 HNO76 p15 HNO80 p5 HNO81 p5 HNO89 p14 HNO91 p34 HNO94 p3 HNO97 p58 HNO103 p5 HNO106 p3 HNO107 p7 HNO111 p5 HNO123 p4 HNO124 p13 HNO129 p4 HNO133 p4 HNO136 p88 HNO147 p20 HNO150 p15 HNO160 p18 HNO161 p12 HNO168 p4 HNO178 p11 HNO180 p7 HNO184 p15 HNO191 p36 HNO199 p30 HNO206 p9 HNO210 p12, p24 HNO212 p48
Localization
TNM
Grading
Oropharynx Oropharynx Larynx Oropharynx Oral cavity CUP Larynx Larynx Larynx Oral cavity Larynx Oral cavity CUP Oral cavity Oropharynx Larynx Larynx Hypopharynx Larynx Oral cavity CUP Maxillary sinus Maxillary sinus Hypopharynx Larynx Oral cavity Hypopharynx Maxillary sinus Hypopharynx Hypopharynx Larynx Oropharynx Oral cavity Oropharynx Larynx Oral cavity
T2N0M0 T3N3M0 T4N0M0 T2N1M0 T2N2M0 — T4N2M0 T2N2M0 T4N2M0 T1N0M0 T3N0M0 T1N1M0 — T2N2M0 T4N2Mo T2N0M0 T3N2M0 T3N2M0 T4N2M0 T4N2M0 — T3N2M0 T4N0M0 T4N2M0 T3N2M0 T2N2M0 T4N1M0 T2N2M0 T2N1M0 T2N0M0 T4N0M0 T2N2M0 T2N2M0 T2N0M0 T3N0M0 T4N2M0
G1 G2 G3 G3 G2 G3 G3 G1 G3 G1 G3 G3 G2 G3 G3 G2 G3 G2 G2 G3 G3 G3 G3 G3 G2 G1 G2 G3 G2 G3 G3 G3 G3 G3 G2 G2
(Sigma, Germany). Reverse transcription was done in a volume of 20 ml using 2 mg of total RNA, 1 U/ ml enhanced AMV reverse transcriptase, 0.5 mM of each dATP, dGTP, dCTP and dTTP and 2.5 mM random nonamers in 1PCR buffer II containing 5 mM MgCl2, and 1 U/ml RNase inhibitor. PCR (50 ml) reaction contained 3 ml of the RT-reaction, 8% DMSO, 0.2 mM of each dATP, dGTP, dCTP and dTTP, 2.5 mM MgCl2, 10 pM of each primer and 2.5 U AmpliTaq LA DNA Polymerase. Primer annealing temperature was optimized for each primer set: SGLT-1: 35 cycles of 95 C 1 min 30 s, 61 C 1 min, 72 C 1 min 30 s. SGLT-2: 35 cycles of 94 C 1 min 30 s, 62 C 1 min, 72 C 1 min 30 s.
SGLT-1
SGLT-2
+
+ + + + + + +
+
+ + + +
+ + + +
GAPDH: 30 cycles of 94 C 1 min 30 s, 62 C 1 min, 72 C 1 min 30 s. Forward and reverse primers were synthesized according to the sequences extracted from genbank. Primer sets used are listed in Table 2. All oligonucleotide primer pairs spanned intron—exon splice sites ensuring that PCR products did not generate from any DNA contamination present in the RNA preparations. PCR of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was done as control for the quality of the RNA preparation.
Tissue samples For further immunohistochemical analysis, biopsy specimens of 30 squamous cell carcinomas
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck
Table 2 SGLT and control primers used for RT-PCR SGLT-1(bp 201—221, forward) TGG CAG GCC GAA GTA TGG TGT (bp 507—528, reverse) ATG AAT ATG GCC CCC GAG AAG A SGLT-2(bp 615—637, forward) ACA CGG ACA CGG TAC AGA CCT T (bp 1599—1622, reverse) GAA CAG CAC AAT GGC GAA GTA GA GAPDH(bp 69—91, forward) GGT GGA GGT CGG AGT CAA CGG A (bp 286—308, reverse) GAG GGA TCT CGC TCC TGG AGG A
of the head and neck were obtained intra-operatively (maxillary sinus n=3, oral cavity n=5, oropharynx n=10, larynx n=8, hypopharynx n=4). Fresh tissues were snap-frozen immediately after surgery in liquid nitrogen and stored at 80 C until processing. Twenty-eight squamous epithelia in close vicinity to the tumor as well as from non-tumor patients were also cryopreserved.
Western blots and antibody production Proteins from cultured cells were extracted in 50 mM Tris (pH8), 150 mM NaCl, 0.1% SDS, 1% NP4O, 0.5% sodium deoxycholate, 0.02% NaN3, 1 mg/ml Pefabloc, 5 mg/ml Leupeptin, 1 mg/ml Pepstatin, 1 mg/ml Aprotinin, and 0.5 mg/ml EDTA. Total protein concentration was determined using the Bradford method (DC Protein Assay, Bio-Rad Laboratories, Muenchen). Equal amounts of protein were loaded on 5% SDS-PAGE mini-gels, analyzed and transferred to Hybond-P membranes (Amersham Pharmacia Biotech, Freiburg). Blocking was performed by incubating the PVDF membranes with 5% (w/v) non-fat dry milk in Trisbuffered saline (TBS) pH 7.5, for 1 h at room temperature and under continuous agitation. The membranes were then incubated with an affinitypurified rabbit polyclonal peptide antibody against peptide IETQVPEKKKGIFRR of SGLT-1, which represents residues 590—604 (diluted 1:300) for 18 h at 8 C under continuous agitation. After three washes with 20 mM Tris (pH 7.5), 500 mM glycine, and 0.05% (w/v) Tween the antibody binding was visualized by incubation with peroxidase-coupled anti-rabbit IgG antibody (diluted 1:4000) in 5% (w/v) non-fat dry milk in PBS for 45 mm at room temperature followed by chemiluminescence detection with the ECL system (Amersham Pharmacia Biotech, Freiburg) according to the manufacturer’s instructions.
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Immunohistochemistry Immunohistochemical staining was performed on serial cryostat sections (5—7 mm) of the frozen biopsies, mounted on 3-aminopropyl-triethoxysilan coated slides. As fixative acetone was used at 20 C for 10 min. Incubation with the primary and secondary antibodies and detection was carried out as described elsewhere21 with Vectastain Laboratories Elite ABC Kit (Vector Laboratories, Burlingame, California). Primary antibody used was the before described affinity-purified rabbit polyclonal anti SGLT-1 antibody (diluted 1:100). Specificity of the SGLT-1 antibody reaction was confirmed in parallel sections using preimmune serum.
Results mRNA expression of SGLT-1 and SGLT-2 in HNSCC cultures In vitro expression of SGLT-1, as analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), was detected in 17 of 36 HNSCC cultures in different passages (between p3 and p88, Fig. 1, Table 1). Comparison of tumor localization with SGLT-1 expression revealed SGLT-1 mRNA in 1/3 maxillary sinus carcinomas, 5/8 oral cavity carcinomas, 2/6 oropharynx carcinomas, 6/11 larynx carcinomas, 1/5 hypopharynx carcinomas, and 2/3 CUP. Comparison with the grading of the respective tumor tissue showed SGLT-1 expression in 75% of well differentiated G1 tumors (3/4), 73% of moderately differentiated G2 tumors (8/11), and 29% of poorly differentiated G3 tumors (6/21) analyzed. The human colon carcinoma cell line HT29 served as positive control.22 In contrast to SGLT-1, we could not observe in any of the HNSCC cultures expression of SGLT-2 (Table 1). Amplification of GAPDH in all samples confirmed the integrity of the mRNA used.
Antibody production and specificity To further analyze localization of SGLT-1 protein in native tissues and to determine SGLT-1-expressing cell types and homogeneity of SGLT-1 expression we developed affinity-purified SGLT-1specific peptide antibodies in several animals (see A1 and A2 in Fig. 2). When proteins from SGLT-1expressing HT29 cells (see mRNA analysis) separated by SDS-polyacrylamide gel electrophoresis and blotted on PVDF membranes were developed with these peptide antibodies, the major reaction product was observed at 75 kDa (Fig. 2), as has
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Figure 1 mRNA Expression of SGLT-1 in HNSCC short-term cultures. Upper lane shows results for SGLT-1 expression as determined by RT-PCR. HT29 colon carcinoma cells served as positive control. Lower lane shows expression of the house keeping gene GAPDH as control for the RNA integrity. p=passage number of tumor cell cultures.
Figure 2 Western blot analysis of affinity-purified antiSGLT-1-specific peptide antibodies raised against residues 590—604 from human SGLT-1 in HT29 cells. The major reaction products of antisera of two different animals (A1 and A2) were observed at the expected size of 75 kDa.
been described in small intestine.23 Because reactivity of the antibody developed in animal 2 (A2) appeared to be stronger and because the antibody did not show any cross-reactivity, it was used for our further immunohistochemical analysis of native tissues.
immunohistochemistry including 20 cases, where corresponding tumor cell cultures had been analyzed by RT-PCR before. The lesions contained two well (G1), 12 moderately (G2), and 16 poorly (G3) differentiated squamous cell carcinomas. Only three G3 tumors were negative for SGLT-1, while we observed a hetereogeneous expression of SGLT1 in the remaining 27 tumors analyzed. Interestingly, in all tumors that were positive for SGLT-1, strong staining was confined to differentiated tumor cells localized in the center of tumor islands (Fig. 3E and F), and thus resembling the expression observed in the normal epithelium. Comparison of results obtained in tumor cell cultures by RT-PCR with protein expression in the native tumor tissue revealed no SGLT-1 detection in tumor cultures, where the tumor tissue failed to express SGLT-1. However, in 6/20 cases, where SGLT-1 staining was weak and restricted to few tumor cells in the tumor tissue, we found no mRNA message in the corresponding short-term culture.
Protein expression of SGLT-1 in squamous epithelia and squamous cell carcinomas of the head and neck
Discussion
Immunohistochemical staining in stratified squamous epithelium of the oral mucosa with the antiSGLT-1 antibody revealed a constitutive targeting of SGLT-1 in all epithelia (n=23). However, staining was strictly confined to differentiated cell layers starting with the prickle cell layer (Fig. 3A). In contrast, in the basal cell layers SGLT-1 was always absent. Also, non-epithelial structures like smooth and skeletal muscle cells were positive for SGLT-1 (Fig. 3B). In addition, in five epithelial specimen that contained dysplastic areas (including mild, moderate, and severe dysplasias) SGLT-1 expression was either reduced to non-dysplastic areas [Fig. 3C showing mild (area II) and moderate (area III) dyplastic foci] or lacked completely in severe dyplasias [Fig. 3D (area I)]. To investigate the expression of SGLT-1 in carcinomas arising from squamous epithelia, 30 cases of squamous cell carcinomas were analyzed by
Positron emission tomography (PET) studies on malignant HNSCC have shown that tumor growth is associated with an up to 10-fold increased uptake of the glucose analogue fluorodeoxyglucose (FDG) compared to normal epithelium and benign tumors.24 These observations together with similar results on other tumor types25,26 have brought up the idea to control tumor growth by inhibiting the glucose transport. Two well-known compounds in this respect are phloretin and phloridzin.18,19 Since both seem to target preferentially the active glucose transport mediated by SGLTs and since these proteins have not been studied before in head and neck tumors, our aim was to investigate the expression of SGLT-1 and-2 in HNSCC. We were able to identify the SGLT-1 mRNA in 17/ 36 HNSCC short-term cultures by RT-PCR. Most of the SGLT-1 positive HNSCC cultures originated from tumors exhibiting an advanced differentiation (3/4
Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck
33
Figure 3 Immunohistochemical staining for SGLT-1 in normal, preneoplastic and neoplastic tissues of the head and neck. Normal squamous epithelium (A) shows a SGLT-1 staining starting with the prickle cell layer. In the basal compartment SGLT-1 is negative. SGLT-1 expression in non-epithelial structures (B) like smooth and skeletal muscle cells. Squamous epithelia in (C) and (D) show dysplastic alterations including mild (II) and moderate (III) dyplastic foci beside non-dysplastic mucosa (I) in (C) and a severe dyplasia (I) beside a moderate dysplasia (II) in (D). In dysplastic areas with a disturbed differentiation SGLT-1 staining is absent. (E) and (F) demonstrate heterogeneous SGLT-1 staining in the moderately differentiated tumor HNO212 (E) and the poorly differentiated tumor HNO89 (F). SGLT-1 expression can only be found in differentiated tumor cells.
G1, 8/11 G2, 6/21 G3). In contrast to SGLT-1, we failed to detect SGLT-2 in the same tumor cell cultures. Therefore we have developed an antiSGLT-1 peptide antibody to further analyze expression and distribution of the SGLT-1 protein in native head and neck tissues. We found a heterogeneous SGLT-1 staining in 27/30 tumors analyzed that was always restricted to differentiated tumor areas. In some tumor tissues, where only single
tumor cells were positive for SGLT-1 by immunohistochemistry, we could not detect the SGLT-1 mRNA in the corresponding tumor culture. This discrepancy might be explained by the fact that under in vitro conditions mainly less differentiated tumor cells are expanded. Immunohistochemical analysis of normal and preneoplastic mucosa revealed a similar differentiation-dependent SGLT-1 distribution. In the
34 normal mucosa SGLT-1 was never found in the basal compartment, where proliferation occurs, but was always detectable starting with the prickle cell layer. Whenever we located dysplastic alterations in squamous epithelia, SGLT-1 staining was completely absent in the lesioned area. This distribution pattern together with the observed expression of SGLT-1 in normal and neoplastic tissues as well as the preferential mRNA expression in tumor cultures originating from more differentiated tumors strongly supports the idea that SGLT-1 is expressed in a differentiation-dependent manner in tissues of the head and neck. We could strengthen this hypothesis by the finding that glycogen, the storage form of glucose only occurring in differentiated epithelial cells,9 could be identified by PAS-staining in the same tumor and epithelial compartments as SGLT-1 (data not shown). Further our observations are in line with data of Baron-Delage and co-workers27 describing a downregulation of SGLT-1 expression in colon carcinoma cells after oncogenic activation of p21ras or pp60csrc, while GLUT-1 was upregulated at the same time. Reports on SGLT expression in tumorigenesis are few. So far, SGLT-1 expression was found in several intestinal tumor cell lines13,22 and RT-PCR analysis on primary adenocarcinomas of the lung and corresponding metastases has shown that although expression of SGLT-1 can be found in these tumors, it is not increased in metastases.28 Altogether, our data on the expression and distribution of SGLT-1 in normal, preneoplastic and neoplastic head and neck tissues give new insights on the expression of glucose transport proteins in different compartments of squamous epithelia and therefrom derived tumors. This is important knowledge for the planning of glucose-targeting therapies on the one hand and on the other hand indicates that SGLT-1 may serve as a differentiation marker in head and neck tissues.
Acknowledgements We gratefully acknowledge the help of all medical colleagues in gathering the tumor biopsies. We thank Mrs. Melanie Bobko, and Mrs. Renate Steinle for excellent technical support, and Mr. Philip Benjamin for photographic work. References 1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics. CA Cancer J Clin 2002;52:23—47. 2. Parkin DM, Pisani P, Ferlay J. Global cancer statistics. CA Cancer J Clin 1999;49:33—64.
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Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck 22. Delezay O, Verrier B, Mabrouk K, van Rietschoten J, Fantini J, Mauchamp J, Gerard C. Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line. J Cell Physiol 1995;163:120—128. 23. Lescale-Matys L, Dyer J, Scott D, Freeman TC, Wright EM, Shirazi-Beechey SP. Regulation of the ovine intestinal Na+/ glucose co-transporter (SGLT1) is dissociated from mRNA abundance. Biochem J 1993;291:435—440. 24. Reisser C, Haberkorn U, Strauss LG. The relevance of positron emission tomography for the diagnosis and treatment of head and neck tumors. J Otolaryngol 1993;22:231—238. 25. Lin WC, Hung YC, Yeh LS, Kao CH, Yen RF, Shen YY. Usefulness of (18)F-fluorodeoxyglucose positron emission tomography to detect para-aortic lymph nodal metastasis in
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advanced cervical cancer with negative computed tomography findings. Gynecol Oncol 2003;89:73—76. 26. Higashi K, Matsunari I, Ueda Y, Ikeda R, Guo J, Oguchi M, Tonami H, Yamamoto I. Value of whole-body FDG PET in management of lung cancer. Ann Nucl Med 2003;17: 1—14. 27. Baron-Delage S, Mahraoui L, Cadoret A, Veissiere D, Taillemite JL, Chastre E, Gespach C, Zweibaum A, Capeau J, Brot-Laroche E, Cherqui G. Deregulation of hexose transporter expression in Caco-2 cells by ras and polyoma middle T oncogenes. Am J Physiol 1996;270:G314—G323. 28. Ishikawa N, Oguri T, Isobe T, Fujitaka K, Kohno N. SGLT gene expression in primary lung cancers and their metastatic lesions. Jpn J Cancer Res 2001;92:874—879.