Download Harvesting of human material.

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 4C 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 50m.
Bar is 50m.
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.
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