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NIS – sodium/iodide symporter is expressed in human colon cancer. A therapeutical alternative? Dick S. Delbroa,b*, Cecilia Bengtssona, Gunnar Nylundc , Amir KhorramManeshc, Svante Nordgrena, and Torsten Grunditzd a Department of Surgery, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden c Department of Surgery, Kungälv Hospital, SE-442 83 Kungälv, Sweden d Department of Oto-Rhino-Laryngology, Sahlgrenska University Hospital, Göteborg, Sweden b * Corresponding author.: Tel.: +46 31 342 1235; fax: +46 31 4138 92. E-mail address: [email protected]. Abstract The sodium/iodide symporter (NIS) which transports iodide into the thyroid gland is expressed also in extrathyroid tissue. Notably, it is absent from normal breast tissue but appears in the lactating breast as well as in breast cancer. With respect to the large intestine, data concerning its expression are conflicting. In the current study, we demonstrate by immunocytochemistry NIS expression in the HT-29 human colon cancer cell line. Immunhistochemistry showed the localization of NIS in normal colon mucosa in immune cells of the lamina propria solely. In contrast, there was a very strong expression in colon cancer in the tumor cells while expression in adjacent, macroscopically tumor free colon tissue was restricted essentially to the surface epithelium. Preliminary studies showed that NIS is functionally active in the HT-29 cells, which may suggest novel therapies in colon cancer. Introduction The sodium/iodine symporter (NIS) is a cell membrane protein that accomplishes active iodide transport into the thyroid gland and also into a number of extrathyroidal tissues. Interestingly, while the non-lactating does not express NIS, this will be evident in the lactating mammary gland and also in breast cancer (Dohan et al., 2003; Kogai et al., 2006). Thyroid NIS is activated by TSH, which hormone, however, is inefficient in breast cancer, where instead lactogenic hormones and certain nuclear receptor ligands serve to activate the pump (Kogai et al., 2006). In both thyroid and breast cancer, retinoic acid elicits the expression of NIS. The up-regulation of NIS in breast cancer could have both diagnostic and therapeutic values. Thus, it has been proposed that breast cancer metastases may be treated with 131I-(Wapnir et al., 2004). With particular emphasis on the gastrointestinal tract, mRNA for NIS could be demonstrated in human stomach but not colon (Ajjar et al., 1998; Spitzweg et al., 1998). In contrast to these findings, Wapnir et al. (2003) reported the immunohistochemical expression of NIS in human colonocytes as well as colonic adenocarcinoma. The reason for this discrepancy is unknown. The aim of the current study was to examine possible NIS expression on the protein level in the HT-29 human colon cancer cell line, colon cancer tissue and adjacent, macroscopically tumor free colon tissue. Materials and methods This study was approved by the local Ethics Committee of the Göteborg University. Cell culture The HT-29 (ATCC HTB 38; a kind gift from the laboratory of Prof. K. Lundholm, Department of Surgery, Sahlgrenska University Hospital, Göteborg, Sweden) is a human colon cancer cell line derived from an adenocarcinoma. The cells were maintained in culture in McCoy’s 5a medium (Invitrogen, Stockholm, Sweden) supplemented with 1%L-glutamine (Bio Whittaker Europe, Verviers, Belgium) and 1% penicillin–streptomycin (Invitrogen) in the presence of 4% fetal calf serum (FCS, Invitrogen). A split ratio of 1:8 once weekly was chosen, with a change of medium in between (McCoy’s 5a plus 2% FCS). Cells were renewed after approximately 22 passages. For the experiments, the cells were seeded in chamber slides (2 ml; 100,000 cells/ml). On day 5 following seeding, the cells were prepared for immunocytochemistry. The experiments were repeated at least three times, unless specified otherwise. Immunocytochemistry Before fixing the cells with phosphate-buffered formaldehyde, pH 7.4 (Substratavdelningen, Sahlgrenska University Hospital, Göteborg, Sweden) for 25 min, the cells were washed twice with PBS. Fixation was followed by a rinse (3-5 min) in TBS. Endogenous peroxidase was blocked with 0.3% hydrogen peroxidase (VWR International, Stockholm, Sweden) in methanol (Merck, Stockholm, Sweden) for 30 min followed by the blocking of unspecific protein binding with 2% normal horse serum PK6200 (ImmunKemi, Järfälla, Sweden) for 1 h in a moist chamber. The primary antibody: a monoclonal mouse antibody to human NIS (Chemicon, Calif. USA; 1:20-1:100) diluted in horse serum was added and incubated in a moist chamber at 4C over-night. The following day, the slides were washed in TBS (2 x 5 min), incubated with the secondary antibody for 30 min (also in a moist chamber), followed by a second wash in TBS (2 x 5 min). For staining, the slides were incubated with the ABCreagents (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA) for 30 min in a moist chamber, again rinsed with TBS (2 x 5 min). Positive immunoreactivity was visualised with 3-diaminobenzidine tetrahydrochloride (DAB, DakoCytomation), resulting in brown staining. The color reaction was stopped after about 8 min by a rinse in water for 10 min. The cells were then counterstained with Mayer’s hematoxylin (Histolab, Göteborg, Sweden) for about 1–2 min, rinsed in water for 5 min before being mounted with Faramount Aqueous Mounting Medium (DakoCytomation); they were then photographed under a light microscope (Nikon Eclipse E400 & Nikon Digital Camera DXM 1200; Upplands Väsby, Sweden). Negative controls were performed by excluding the primary antibody and incubating the cells instead with horse serum, resulting in no immunoreactivity. Harvesting of human material. Fourteen patients, 6 women (mean age: 74, range 65-82 years, 3 Dukes B and C, respectively) and 8 men (mean age: 73, range 43-92 years, 4 Dukes B and C, respectively) underwent surgical resection for colon tumours. All patients included in this study had their surgical procedures performed at Kungälv Hospital, Kungälv, Sweden. They were treated on an elective basis. For all patients, full-thickness samples of both neoplastic and macroscopically tumour-free bowel wall, the latter resected 10 cm from the tumour, were dissected immediately upon colonic resection. The portion of the tumour analysed was obtained from the edge of the tumour, representing a viable area devoid of necrosis. Moreover, endoscopically harvested biopsies from normal colon were obtained from three patients. The specimens were immersed in 4% phosphate-buffered formaldehyde (pH 7.0, Apoteket AB, Sweden) for 24 h and were then embedded in paraffin and were processed for immunohistochemistry. Immunohistochemistry. Sections of 4 m thickness were cut. After de-paraffination, the tissue sections were immersed in 10mM citrate buffer pH 6, and were placed in a microwave oven at max power (2x5min). Next, endogenous peroxidase was blocked with 0.3% hydrogen peroxide (VWR) in methanol (Merck) for 30 min. Non-specific protein binding was blocked with 2% normal horse serum PK6200 (Immunokemi) for 60 min. The sections were incubated with the monoclonal, anti-NIS antibody (see above; 1:50) over-night in a moist chamber. Next, DAKO-Envision (DakoCytomation, Stockholm, Sweden) was applied for 30 min (also in a moist chamber), followed by a second wash in TBS (2-5 min). Positive immunoreactivity was visualised with DAB (DakoCytomation), as above. The tissues were counterstained with Mayer’s hematoxylin (Histolab). The tissue sections were mounted with Pertex Mounting Medium (Histolab) and were then photographed under a light microscope as above. Controls were performed as reported for immunocytochemistry (above). Results NIS is heterogenously expressed in HT-29 cells. The antibody to NIS produced, in a concentration dependent fashion, immunoreactivity in the HT-29 cells, albeit in a heterogenous manner (Fig. 1b). Fig. 1a. Control. HT-29 human colon Fig. 1b. Demonstration of NIS immuno- cancer cells treated with secondary reactivity. Antibody concentration: 1:25. antibody only. Bar is 50m. Bar is 50m. NIS is expressed in immune cells in normal colon Fig. 2 demonstrates NIS expression in a biopsy from normal colon, showing immunoreactivity solely in the mononuclear cells of the lamina propria. Fig. 2. Demonstration of NIS immunoreactivity in normal colon mucosa. Antibody concentration: 1:50. Bar is 50 m. NIS is expressed in surface epithelium of macroscopically tumor free, human colon tissue. In Fig. 3 it may be seen that NIS immunoreactivity is localized in a scattered fashion in the surface epithelium and also the crypt, and, weakly, in immune cells of the lamina propria. Fig.3. Demonstration of NIS immunoreactivity in macroscopically tumor free colon tissue. Antibody concentration: 1:50. Bar is 50 m. NIS is strongly expressed in the tumor cells but not tumor stroma, in human colon cancer. Fig. 4 demonstrates that NIS is expressed in the adenocarcinoma cells while the expression in the stroma is absent. Compared with the adjacent, macroscopically tumor free colon tissues, NIS is clearly over expressed in the tumor. Fig. 3. Demonstration of NIS immunoreactivity in human colon cancer tissue. Antibody concentration: 1:50. Bar is 50 m. Discussion In the current study, we found a very marked discrepancy with regard to NIS expression between normal colon mucosa, macroscopically tumor free colon tissue, being adjacent to colon cancer, and to colon cancer itself. In normal tissue, NIS was only expressed in immune cells in the lamina propria. Such localization was sparse in tumor free colon tissue. In the latter, instead, expression was scattered predominantly in the surface epithelium. In colon cancer, the tumor cells expressed strong immunoreactivity for NIS while such was more or less absent in the tumor stroma. Our data support those of Wapnir et al. (2003). Our finding with regard to NIS expression in immune cells only in the normal colon is interesting and may suggest that although macro- and microscopically tumor free, the tissue being adjacent to colon cancer could exhibit a premalignant phenotype. Recent data from our laboratory (unpublished) strongly indicate that the HT-29 cells take up 125 - I , strongly suggesting that NIS is functionally active in these cells. Experiments are ongoing, analysing whether this pump may be activated by paracrine mediators. Interestingly, in breast cancer cells, NIS expression was stimulated by addition of adenosine-5´-triphosphate (ATP) via purinergic, cell membrane receptors (Dohán et al., 2006). It is tempting to speculate that a similar mechanism may operate in the HT-29 cells, since these also express purinergic receptors (Delbro et al., 2005). The current results may point at a novel therapeutic strategy in colon cancer, namely the aministration of radioiodide, as suggested for the treatment of certain breast cancer metastases (Wapnir et al., 2003). In fact, animal experiments already suggest that radioiodide therapy is beneficial in colon cancer (Mitrofanova et al., 2005). Acknowledgements The present study was supported by the Anna-Lisa and Bror Björnsson Foundation (D. Delbro), the Assar Gabrielsson Foundation (D. Delbro), the Göteborg Medical Society (A. Khorram-Manesh, G. Nylund), the King Gustaf V Jubilee Clinic Foundation (D. Delbro), the LUA-ALF agreement (S. Nordgren), the University of Kalmar (D. Delbro) and the Västra Götalandregionen (G. Nylund). The authors are grateful to Dr. F. Aldenborg for invaluable advice and suggestions, to Prof. K. Lundholm for the gift of the HT-29 cells, and to Ms. Lilian Karlsson for expert technical assistance. References Ajjan, R.A., Kamaruddin, N.A., Crisp, M., Watson, P.F., Ludgate, M., Weetman, A.P. (1998) Regulation and tissue distribution of the human sodium iodide symporter gene, Clin Endocrinol, 49:517-523 Delbro DS, Nylund G, Nordgren S. (2005) Demonstration of P2Y4 purinergic receptors in the HT-29 human colon cancer cell line, Auton Autacoid Pharmacol, 25:163-166. Dohán, O., De la Vieja, A., Paroder, V., Riedel, C., Artani, M., Reed, M., Ginter, C.S., Carrasco, N. (2003) The sodium/iodide Symporter (NIS): characterization, regulation, and medical significance, Endocr Rev, 24:47-88 Dohán, O., De la Vieja, A., Carrasco, N. (2006) Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells, Mol Endocrinol, 20:1121-1137. Kogai, T., Taki, K., Brent, G.A. (2006) Enhancement of sodium/iodide symporter expression in thyroid and breast cancer, Endocr Relat Cancer, 13:797-826 Mitrofanova, E., Unfer, R., Vahanian, N., Kane, S., Carvour, M., Link, C. (2005) Effective growth arrest of human colon cancer in mice, using rat sodium iodide symporter and radioiodine therapy, Human Gene Ther, 16:1333-1337. Spitzweg, C., Joba, W., Eisenmenger, W., Heufelder, A.E. (1998) Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa, J Clin Endocrinol Metab, 83:1746-1751 Wapnir, I.L., van de Rijn, M., Nowels, K., Amenta, P.S., Walton, K., Montgomery, K., Greco, R.S., Dohan, O., Carrasco, N. (2003) Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections, J Clin Endocrinol Metab,88:1880-1888. Wapnir, I.L., Goris, M., Yudd, A., Dohan, O., Adelman, D., Nowels, K., Carrasco, N. (2004) The NaC/IK symporter mediates iodide uptake in breast cancer metastases and can be selectively downregulated in the thyroid, Clin Cancer Res, 10:42944302