Fax +41 61 306 12 34
`E-Mail karger@karger.ch
`www.karger.com
` Research Article
` Tumor Biol 2008;29:245–254
` DOI: 10.1159/000152942
` The NK-1 Receptor Is Expressed in Human Primary
`Gastric and Colon Adenocarcinomas and Is
`Involved in the Antitumor Action of L-733,060 and
`the Mitogenic Action of Substance P on Human
`Gastrointestinal Cancer Cell Lines
` Marisa Rosso a María Jose Robles-Frías b Rafael Coveñas c
`Manuel Vicente Salinas-Martín b Miguel Muñoz a
` a ‘Virgen del Rocío’ University Children’s Hospital and b Department of Pathology, ‘Virgen del Rocío’
`University Hospital, Sevilla , and c Laboratory of Neuroanatomy of the Peptidergic Systems, Institute of
`Neurosciences of Castilla y León (INCYL), Salamanca , Spain
`sults: We observed the presence of several NK-1 receptor
`isoforms in human gastric and colon adenocarcinomas.
`Nanomolar concentrations of SP increased the growth of
`both cell lines and micromolar concentrations of L - 733,060
`inhibited the growth of such cell lines, with and without pre-
`vious administration of SP . L-733,060 inhibited the growth of
`the 23132/87 and SW-403 cell lines in a dose-dependent
`manner. After administration of L-733,060, apoptosis was ob-
`served in both cell lines. In both human primary gastric and
`colon adenocarcinomas, a high density of NK-1 receptors
`was observed. Immunoreactivi ty, showing a diffuse cyto-
`plasmic staining, was observed in the epithelial cells of nor-
`mal and tumor glands and in numerous stromal elements.
` Conclusions: We demonstrated that NK-1 receptors were ex-
`pressed in 23132/37 and SW-403 cell lines and in human pri-
`mary gastric and colon adenocarcinomas, that SP is a mito-
`gen and that the antitumor action of L-733,060 on both
`human cell lines occurs through the NK-1 receptor. Data also
`indicate that the cell death observed is produced by apop-
`tosis. These data suggest that the NK-1 receptor is a new and
`promising target in the treatment of human gastrointestinal
`adenocarcinomas.
` Copyright © 2008 S. Karger AG, Basel
` Key Words
` NK-1 receptor antagonists /H11554 L-733,060 /H11554 NK-1 receptor
`isoforms /H11554 Apoptosis /H11554 Tachykinins /H11554 Substance P /H11554
`Gastrointestinal carcinoma /H11554 Human primary
`gastrointestinal adenocarcinoma
` Abstract
` Background/Aims: It has been demonstrated that sub-
`stance P (SP) and neurokinin-1 (NK-1) receptor antagonist L-
`733,060 induces cell proliferation and inhibition, respective-
`ly, in several human cancer cell lines . At present, it is unk nown
`whether such actions are exerted on human gastric and co-
`lon adenocarcinomas. We carried out an in vitro study of the
`growth-inhibitory capacity of L-733,060 against human gas-
`tric and colon adenocarcinomas. Methods: A coulter coun-
`ter was used to determine viable cell numbers followed by
`application of the tetrazolium compound MTS. Immunoblot
`analysis was used to determin e the NK-1 receptors and the
`DAPI method was applied to demonstrate apoptosis. Immu-
`nohistochemistry was used to demonstrate NK-1 receptors
`in primary human gastric and colon adenocarcinomas. Re-
` Received: January 11, 2008
` Accepted: June 5,2008
` Published online: September 9, 2008
` Dr. Miguel Muñoz
` Hospital Infantil Universitario Virgen del Rocío
` Unidad de Cuidados Intensivos Pediátricos, Av. Manuel Siurot s/n
` ES–41013 Sevilla (Spain)
` Tel. +34 955 012 965, Fax +34 955 012 921, E-Mail mmunoz@cica.es
` © 2008 S. Karger AG, Basel
`1010–4283/08/0294–0245$24.50/0
` Accessible online at:
`www.karger.com/tbi
`Downloaded from http://karger.com/tbi/article-pdf/29/4/245/3567675/000152942.pdf by Lauren Wortman on 28 August 2025
`HELSINN EXHIBIT 2052
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00945
`Page 1 of 10
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` Rosso /Robles-Frías /Coveñas /
`Salinas-Martín /Muñoz
`Tumor Biol 2008;29:245–254246
` Introduction
` Gastric cancer accounts for approximately 12% of all
`cancer deaths. It remains the world’s second leading cause
`of cancer mortality [1] and is predicted to be the eighth
`l
`eading cause of all deaths worldwide by 2010 [2] . Surgery
`i
`s the only potentially curative treatment for localized
`gastric cancer, but most cases present at an advanced
`stage. The prognosis for this disease is extremely poor,
`with overall 5-year survival rates ranging from 10 to 15%
`in the United States and most developed countries [3] .
`C
`olorectal cancer remains one of the leading causes of
`cancer death in the Western world and was estimated to
`have affected 148,000 people in 2002 in the United States
`alone [4] . Despite advances in multimodality therapies,
`5-
`year survival in colorectal cancer is approximately 55%
`and has remained essentially unchanged over the last 40
`years. Because metastatic disease is the major cause of
`treatment failure, new therapeutic strategies are essential
` [5] .
`
` Substance P (SP) is an undecapeptide that belongs to
`th e ta c h y kinin f amil y o f p e p ti d e s . I t i s kn o wn th a t S P ,
`neurokinin (NK) A, neuropeptide K and neuropeptide /H9253
`(the 2 latter elongated forms of NKA) are derived from
`the preprotachykinin A gene (PPT-1), whereas NKB is de-
`rived from the preprotachykinin B gene (PPT-2). The bio-
`logical actions of SP, NKA and NKB are mediated by 3
`receptors, named NK-1, NK-2 and NK-3, the NK-1 recep-
`tor showing preferential affinity for SP. After binding to
`the NK-1 receptor, SP regulates many biological func-
`tions [6] , and this neuropeptide has also been implicated
`in
` neurogenic inflammation, pain and depression [7] .
`M
`oreover, SP is known to have a widespread distribution
`in both the central and peripheral nervous systems, and
`it is also known that the undecapeptide is released from
`primary sensory nerve fibers. Moreover, activation of the
`NK-1 receptor induces mitogenesis in several tumor cells
` [8–12] .
`
` L-733,060 is a selective, potent and long-acting central
`nonpeptide tachykinin NK-1 receptor antagonist show-
`ing high affinity for the human NK-1 receptor in vitro
` [13] . The administration of L-733,060 produces analgesia
` [14] and antidepressive effects [15] . Moreover, the com-
`po
`und has been used in the treatment of a broad range of
`anxiety and mood disorders [16] as well as in inflamma-
`to
`ry liver disease; its action is most likely due to an inhi-
`bition of the effects of SP [17] . In addition, in vitro and in
`vivo studies have demonstrated that SP antagonists in-
`hibit the growth of both small cell lung cancer and glioma
` [18, 19] . Recently, we have also demonstrated that L-
`7
`33,060 shows antitumor activity against human neuro-
`blastoma, glioma, melanoma, retinoblastoma and pan-
`creas carcinoma cell lines [10–12, 20] . However, to our
`k
`nowledge no study has been carried out on the antitu-
`mor effect of the potent and long-acting NK-1 receptor
`antagonist L-733,060 against human gastric 23132/87 and
`co l o n a d e n ocar cin o ma SW -40 3 cell lin es. I t is also un -
`known whether SP exerts a mitogenic action or not on
`both tumor cell lines, and the presence of NK-1 receptors
`in those cell lines is also unknown. Thus, the aims of this
`study were: (1) to demonstrate the presence of isoforms of
`the NK-1 receptor in the human gastric adenocarcinoma
`23132/87 and the human colon adenocarcinoma SW-403
`cell lines; (2) to study the role of SP and the NK-1 receptor
`in the induction of the proliferation of human gastric ad-
`enocarcinoma 23132/87 and the human colon adenocar-
`cinoma SW-403 cell lines; (3) to demonstrate, using an
`MTS colorimetric method to eval uate cell viability , the
`a n t i t u m o r a c t i o n o f t h e N K - 1 r e c e p t o r a n t a g o n i s t L -
`73 3,060 on both cancer cell lines and to show that this
`antitumor action occurs through the NK-1 receptor; (4)
`to know whether the antagonist produces, or not, apop-
`tosis in both gastrointestinal tumor cell lines; (5) to dem-
`onstrate, in several human primary human gastric and
`colon adenocarcinomas, the presence and distribution of
`the NK-1 receptor.
` Materials and Methods
` Cell Cultures
` We used the human gastric adenocarcinoma cell line 23132/87
`(DSMZ, Braunschweig, Germany) and the human colon adeno-
`carcinoma cell line SW-403 (DSMZ). These cell lines were main-
`tained in DMEM or RPMI 1640 (Gibco, Barcelona, Spain) supple-
`mented with 10% heat-inactivated fetal bovine serum according
`to the culture conditions suggested by
` the ATCC, the ICLC and
`the DSMZ. Cell lines were seeded in 75-cm 2 tissue culture flasks
`(Falcon, Heidelberg, Germany). The medium was renewed every
`2 days and the cells were harvested by treatment with trypsin
`(0.05 and 0.02% EDTA without Ca2
`+ and Mg 2+ ; Sigma-Aldrich,
`Madrid, Spain) on the sixth day after seeding. Cells were incu-
`bated at 37
` ° C in a humidified atmosphere of 95% air/5% CO 2 .
` D r u g T r e a t m e n t s
` The NK-1 receptor antagonist (2S, 3S) 3-([3,5-Bis(trifluoro-
`methyl)phenyl]methoxy)-2-phenylpiperidine, MW 438.9, (L-
`733,060; Sigma-Aldrich) was dissolved in distilled water contain-
`ing 0.2% dimethylsulphoxide (DMSO) before sample treatment.
`In order to determine the 50% inhibition concentration (IC
`50 ),
`different concentrations (5, 10, 15, 20, 25 and 30 /H9262 M ) of L-733,060
`were evaluated. SP, acetate salt (Sigma-Aldrich), was dissolved in
`distilled water. In order to determine SP-induced cell prolifera-
`tion, different concentrations of SP (5, 10, 50 and 100 n
`M ) were
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` Antitumor Action of L-733,060 on
`Gastrointestinal Cancer Cell Lines
`Tumor Biol 2008;29:245–254 247
`evaluated. The most effective SP concentration for each cell line
`was incubated 1 h before the addition of L-733,060.
` Proliferation Assays
` Cell proliferation was evaluated using the tetrazolium com-
`pound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
`nyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), accord-
`ing to the manufacturer’s instructions (CellTiter 96 Aqueous One
`Solution Cell Proliferation Assay; Promega Corp., Madison,
`Wisc., USA). At the time of the assay, cells cultured for 4–5 days
`were harvested by trypsinization and cell viability was evaluated
`by Trypan blue exclusion. Cell numbers were quantified using a
`Coulter counter. Cells were cultured in 96-well plates: each well
`contained 10
`4 cells in a total volume of 100 /H9262 l. Each assay includ-
`ed 1 plate. The plate included blank wells (0 cells/0.1 ml), control
`wells (10
`4 cells /0.1 ml), control wells with DMSO, control wells
`treated with L-733,060, control wells treated with exogenous SP at
`different concentrations and control wells treated with the most
`effective SP concentration and L-733,060. The plates were inocu-
`lated with L-733,060 (5, 10, 20 and 30 /H9262
`M for 23132/87 or 10, 15,
`20 and 25 /H9262 M for SW-403) and were incubated for a period of 50
`or 48 h, respectively. The plates were also inoculated with exoge-
`nous SP (5, 10 and 100 n
`M for 23132/87 or 5, 50 and 100 n M for
`SW-403) with (10 /H9262 M for 23132/87 or 15 /H9262 M for SW-403) and
`without L-733,060 for their first doubling times (50 or 48 h). For
`the proliferation assay, 20 /H9262 l of the MTS reagent was added to
`each well 90 min before reading the samples on a multiscanner
`microplate reader (Spectra Classic; TECAN, Barcelona, Spain) at
`492 nm. The quantity of the product, as measured by optical den-
`sity , is directly proportional to the number of living cells. Each
`experimental condition (blank wells, control wells and control
`wells treated with different concentrations of L-733,060 or SP) was
`assayed in duplicate and all experiments were performed at least
`3 times. The IC
`50 of L-733,060 was calculated using the regression
`straight-line function based on the least-squares technique.
` Statistical Analyses
` Data are expressed as means 8 SD. Statistical analysis
` was
`performed with SPSS statistical software for Microsoft Windows,
`release 13.0 (SPSS Inc., Chicago, Ill., USA). The homogeneity of
`the variance was tested using the Levene test. If the variances were
`homogeneous, the data were analyzed by using the one-way
`ANOV A test with Bonferroni’s correction for multiple compari-
`sons. For data sets with nonhomogeneous variances, the ANOVA
`test with T3 Dunnett post hoc
` analysis was applied. The criterion
`for significance was p ! 0.05 for all comparisons.
` Western Blot Analyses
` W e f o ll o w e d a p r e vi o u s l y r e p o rt e d
` methodology [10] . Thus,
`tot
`al protein was prepared from subconfluent human gastric ad-
`enocarcinoma cell line and human colon adenocarcinoma cell
`cultures in 125-cm 2 culture flasks. As a control, we included a
`protein extract from rat pheochromocytoma cell line (PC12 cells).
`Briefly, cells were harvested by trypsin treatment, washed with
`phosphate-buffered saline (PBS), pH 7.4, and resuspended in
`HEN buffer (5 m
`M EDTA, 250 m M NaCl, 50 m M HEPES, pH 7.3)
`containing 5 m M DTT, 1 m M Na 3 VO 4 , 0.2% IGEPAL CA-630 (Sig-
`ma) and 1% protease inhibitor cocktail (Sigma). Once resuspend-
`ed, cells were vortexed, incubated on ice for 5 min, and centri-
`fuged for 15 min at high speed in a microcentrifuge, after which
`the protein-containing supernatant was collected. Protein con-
`centrations were determined with the protein assay kit from Bio-
`Rad according to the manufacturer’s instructions.
` Fifty micrograms of protein, from each sample, was separated
`by electrophoresis on 10% SDS-polyacrylamide gels and electro-
`blotted onto PVDF membranes. Blots were incubated in blocking
`s o l u t i o n ( 5 % n o n f a t m i l k i n P B S , 0 . 1 % T w e e n - 2 0 ) , f o l l o w e d b y
`overnight incubation with a polyclonal antibody (S8305; Sigma)
`developed in rabbit against the KTMTESSSFYSNMLA conserved
`domain, corresponding to the C terminus of the NK-1 receptor
`(Sigma), and diluted 1:
` 1,000. The membranes were then washed
`with PBS/Tween-20 and incubated with a horseradish peroxi-
`dase-conjugated goat anti-rabbit IgG antibody for 2 h at room
`temperature (1:
` 10,000 dilution). Antibody detection was per-
`formed with an enhanced chemiluminescence reaction (ECL
`Western blotting detection; Amersham Life Science, Piscataway,
`N.J., USA). As previously reported
` [10] , we included a protein ex-
`t
`ract from a glioma cell line as a control (data not shown). Several
`isoforms of the NK-1 receptor shown by this cell line are known
` [10] . In addition, no bands were detected when incubation was
`c
`arried out with the secondary antibody alone.
` D A P I S t a i n i n g
` In order to determine whether apoptosis was induced by the
`N K - 1 r e c e p t o r a n t a g o n i s t u s e d h e r e , D A P I s t a i n i n g w a s p e r -
`formed. Briefly, cells were cultured on 4-chamber slides. After
`treatment with L-733,060 for their first doubling times (48–50 h),
`the cells were washed twice with PBS and fixed by incubation in
`4% paraformaldehyde for 30 min. Following a second wash in
`PBS, cells were incubated in DAPI solution (Sigma-Aldrich) at a
`dilution of 1/1,000 (1 /H9262 g/ml) for 30 min in the dark and the sup-
`porting slides were mounted with a mixture of PBS and glycerol
`at 70%. The cells were then observed through a fluorescence mi-
`croscope (Zeiss, Oberkochen, Germany). Apoptotic cells were de-
`fined by the condensation and fragmentation of nuclear chroma-
`tin. We counted the number of apoptotic cells observed in the
`following cases: human gastric adenocarcinoma 23132/87 and
`human colon adenocarcinoma SW-403 cells not treated with the
`NK-1 receptor antagonists; 23132/87 cells treated with the NK-1
`receptor antagonist L-733,060 (30 /H9262
`M ) and SW-403 cells treated
`with the NK-1 receptor antagonist L-733,060 (25 /H9262 M ). In each
`case, the count was repeated in 3 different slides. Finally, in each
`slide, we counted the number of apoptotic cells located in 5 dif-
`ferent sequential fields (we considered a field as the image ob-
`served with the 40 ! lens). Images of such fields were captured by
`means of a microscope equipped with a digital camera.
` Immunohistochemistry for NK-1 Receptors in Human
`Primary Adenocarcinomas
` After surgical intervention, 3 human primary gastric adeno-
`carcinomas and 4 human primary colon adenocarcinomas were
`obtained from patients at the Department of Pathology, Hospital
`‘Virgen del Rocío’, Sevilla, Spain. The experimental design, pro-
`tocols and procedures were performed under the ethical guide-
`lines and legal recommendations of Spanish and European law, as
`well as in accordance with the Declaration of Helsinki.
` Formalin-fixed and paraffin-embedded tumors (gastric and
`colon adenocarcinomas) were cut at 5 /H9262 m and dried overnight at
`37
` ° C. The sections were deparaffinized with xylene, hydrated
`through a series of solutions containing decreasing concentra-
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`Tumor Biol 2008;29:245–254248
`tions of ethanol, and immersed in water. After pressure cooker
`antigen retrieval in 10 ! citrate buffer, pH 6.0, slides were cooled
`at room temperature for 10 min. Endogenous peroxidase activity
`was blocked with 3% hydrogen peroxide for 30 min at room tem-
`perature. After washing with 0.05
`M Tris, the sections were incu-
`bated with 10% nonimmune pig serum for 30 min at room tem-
`perature. Subsequently, they were incubated overnight at 4
` ° C
`with 1: 500 diluted anti-NK-1 receptor primary antibody (Sigma-
`Aldrich). The sections were then washed in 0.05 M Tris at room
`temperature. The next step was the addition of Envision System-
`HRP (Dako ) reagents during 30 min at room temperature. The
`slides were rinsed with 0.05
`M Tris, and immunoreactivity was
`visualized for light microscopy with 3,3 /H11541 -diaminobenzidine chro-
`mogen solution (DAB+; Dako). Cell nuclei were lightly counter-
`stained with hematoxylin. Finally, as previously published [21] , in
`o
`rder to determine the specificity of the immunostaining, pri-
`m ary r e tin o b l a s t o m a w a s u s e d a s p o s i ti v e c o n tr o l . A s n e g a ti v e
`control, the primary antibody was omitted, being replaced by
`nonimmune serum. In both cases, the results obtained confirmed
`the specificity of the NK-1 receptor antibody used here.
` All slides were evaluated by 2 independent investigators. In
`each slide, 10 representative microscopic high-power fields were
`evaluated using a 40 ! lens. The presence or absence of staining
`and the intensity of immunoreactivity were noted, as well as the
`number of cells showing a brown staining and whether or not the
`staining was localized on the cytoplasm and/or in the plasma
`membrane. The number of immunoreactive cells was scored as
`follows: when less than 10% of the total tumor cells were stained,
`the number of immunoreactive cells was considered low, it was
`considered moderate when 10–40% were stained and high when
`more than 40% were stained. Tumor cells were recorded as posi-
`tive when they showed a moderate or strong labeling.
` R e s u l t s
` NK-1 Receptors
` We performed Western blot analyses in order to test
`the presence of NK-1 receptor in both human gastric ad-
`enocarcinoma 23132/87 and colon adenocarcinoma SW-
`403 cell lines. Total cell protein extracts were loaded onto
`polyacrylamide gels, resolved, and transferred to mem-
`branes as described in Material and Methods. Incubation
`with an antibody against an epitope whose sequence is
`conserved in several species (see Materials and Methods)
`revealed the presence of different isoforms of the NK-1
`r e c e p t o r i n b o t h t h e 2 3 1 3 2 / 8 7 a n d S W - 4 0 3 c e l l l i n e s
`( fig. 1 ). The relative amount of each protein form was
`similar between both cell lines. Four similar bands (iso-
`forms of about 75, 58, 46 and 34 kDa) were observed in
`the human gastric adenocarcinoma 23132/87 cell line
`( fig. 1 ), whereas the colon adenocarcinoma SW-403 cell
`line expressed isoforms of 75, 58 and 34 kDa. An addi-
`tional band was observed in both cell lines. In addition,
`no bands were detected when incubation was performed
`with the secondary antibody alone.
` Using an immunohistochemistry technique, a high
`density of NK-1 receptors was localized in all human pri-
`mary adenocarcinomas studied ( fig. 2 b, c, e, f). In both
`gastric and colon adenocarcinomas, a high number of tu-
`mor cells ( 1 75%) expressing the NK-1 receptor was ob-
`served. In all adenocarcinomas studied, the immunore-
`activity was observed in the epithelial cells of normal and
`tumor glands and in numerous stromal elements. This
`immunoreactivity was homogeneously expressed and
`showed a diffuse cytoplasmic staining. Moreover, in hu-
`man colon adenocarcinomas, a uniform granular cyto-
`plasmic staining was visualized in the apical areas of the
`epithelial cells of normal glands, whereas the most in-
`tense granular cytoplasmic staining was observed in the
`apical areas and other areas of the cytoplasm of the epi-
`thelial cells of the tumor glands.
` Antitumor Action of L-733,060
` Growth inhibition of the 23132/87 and SW-403 cell
`lines by L-733,060 was observed after the addition of in-
`creasing concentrations of L-733,060 ( fig. 3 a, 4 a). More-
`over, treatment of both cell lines with L-733,060 resulted
`in a concentration-dependent cytotoxicity ( fig. 3 a, 4 a).
`These figures also show the IC
`50 at the first doubling times.
`Thus, the concentrations required for a 50% reduction in
`optical density (IC
`50 ) observed in the controls treated with
`L-733,060 were 14.29 /H9262 M for 50 h for 23132/87 and 14.45
` /H9262 M for 48 h for SW-403 ( fig. 3 a, 4 a). Maximum inhibition
`Gastric
`23132/87
`Colon
`SW-403
`MW NK1-R
`75 kDa
`58 kDa
`46 kDa
`34 kDa
` Fig. 1. Western blot analysis of NK-1 receptors in human gastric
`adenocarcinoma 23132/87 and colon adenocarcinoma SW-403
`cell lines showing the presence of different NK-1 receptor com-
`plex isoforms. Dots indicate bands with molecular weights simi-
`lar to those previously reported.
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` Antitumor Action of L-733,060 on
`Gastrointestinal Cancer Cell Lines
`Tumor Biol 2008;29:245–254 249
`was observed when the drug was present at a concentra-
`tion of 30 /H9262 M (23132/87) and 25 /H9262 M (SW-403) during the
`culture periods. At the first doubling time, a strong de-
`crease in the number of the 2 cell lines studied was found
`at intermediate concentrations and no remaining living
`cells were observed at the maximal concentration. A low-
`er inhibition of growth of the 2 lines was observed in the
`presence of low doses of L-733,060. The figures also show
`that the SD values for the 2 cell lines studied were small,
`pointing to total agreement among the values obtained at
`the 3 times the experiments were carried out.
` The NK-1 Receptor Antagonist L-733,060 Blocks
`SP-Induced Mitogen Stimulation
` Growth of the 23132/87 and SW-403 cell lines was not-
`ed after the addition of SP and we observed that certain
`nanomolar concentrations of SP induced cell prolifera-
`tion compared to the controls ( fig. 3 b, 4 b). SP stimulation
`was evident at 5 n
`M and the maximum level was reached
`at 10 n M for 23132/87 and 50 n M for SW-403 ( fig. 3 b, 4 b).
`This indicates that the activation of SP receptors leads to
`mitogenesis in the 23132/87 and SW-403 human cancer
`cell lines. Thus, the percentage of cell proliferation of
`both cell lines increased from 10 to 20% in 23132/87 and
`from 10 to 25% in SW-403, depending on the dose of SP
`administered ( fig. 3 b, 4 b).
` Treatment with L-733,060, at 10 /H9262
`M for 23132/87 and
`15 /H9262 M for SW-403, partially inhibited the growth of both
`cell lines ( fig. 3 b, 4 b). In order to examine whether the
`NK-1 receptor antagonist L-733,060 inhibited cell prolif-
`eration via an interaction with its receptor, we used the
`specific NK-1 receptor agonist SP in competition experi-
`ments. Thus, the cellular concentration at 10 /H9262
`M
`(23132/87) and 15 /H9262 M (SW-403) of L-733,060 as well as 10
`n M (23132/87) and 50 n M (SW-403) of SP was higher than
`that observed with L-733,060 alone for 23132/87 ( fig. 3 b)
`and SW-403 ( fig. 4 b). These results indicate that L-
`733,060 blocks SP mitogen stimulation, since L-733,060-
`induced growth inhibition was partially reversed by the
`administration of a nanomolar dose of exogenous SP.
`a b c
`d e f
` Fig. 2. Hematoxylin-eosin-stained sections of human primary gastric ( a ) and colon ( d ) adenocarcinomas. ! 25.
`The presence of NK-1 receptors (arrows) in human primary gastric adenocarcinoma ( b , c ) and in human pri-
`mary colon adenocarcinoma ( e , f ) is shown. b , e NK-1 receptors (arrows) can be observed on the cytoplasm of
`the epithelial cells tumor glands and in numerous stromal elements. ! 25. c Magnification of the right-middle
`region observed in b . ! 50. e , f NK-1 receptors (arrows) showing an intense granular cytoplasmic staining in
`the tumor glands of human primary colon adenocarcinoma. ! 25 ( e ) and ! 50 ( f ).
`Color version available online
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`Tumor Biol 2008;29:245–254250
`This indicates the specificity of tachykinin NK-1 recep-
`tor activation in the growth of the human gastric adeno-
`carcinoma ( fig. 3 b) and human colon adenocarcinoma
`cell lines ( fig. 4 b), since an increase in the cellular con-
`centration (44.8 and 32.45%) was observed in the 23132/87
`and SW-403 cell lines, respectively, ( fig. 3 b, 4 b) with re-
`spect to the values found when the antagonist was ad-
`ministered alone. There were no significant differences
`between the control and the control-DMSO groups (data
`not shown).
`0
`20
`40
`60
`80
`100
`120
`Inhibition (%)
`0 5 10 15 20 25 30
`L-733,060 concentration (µ )M
`SW-403
`y = 4.3866x – 13.399
`R = 0.88132
`a
`0
`20
`40
`60
`80
`100
`140
`Cell concentration (%)
`none
`none
`b
`120
`5
`none
`50
`none
`100
`none
`none
`15
`50
`15
`SW-403
`** * **
`*
`*
`+
`SP (µ )
`L-733,060 (µ )
`M
`M
` Fig. 4. a Percentage of growth inhibition of human colon adeno-
`carcinoma SW-403 cells at 48 h in in vitro cultures following the
`a d d i t i o n o f i n c r e a s i n g c o n c e n t r a t i o n s ( 1 0 , 1 5 , 2 0 a n d 2 5 /H9262 M) of
`L-733,060. The percentage of inhibition for the first doubling time
`of incubation is plotted on a linear graph. * p ^ 0.05. Values are
`m e a n s 8 SD (bars). The regression line is indicated, as well as the
`equation to obtain the IC 50 . b Induction of cell proliferation of
`human colon adenocarcinoma SW-403 cells by SP at several
`nanomolar concentrations (5, 50 and 100 n M ). The NK-1 receptor
`antagonist L-733,060 was added (15 /H9262 M ) in the presence (50 n M )
`or absence (none) of SP for 48 h. In both cases, L-733,060 inhib-
`ited SW-403 cell proliferation. Using the ANOVA test, a signifi-
`cant difference between each group and the control group (none-
`none) was found. * p ^ 0.01; * * p ^ 0.05;
`+ p ^ 0.05, 50-none vs.
`50–15; ° p ^ 0 . 0 5 , 5 0 – 1 5 v s . n o n e - 1 5 . V e r t i c a l b a r s i n d i c a t e S D .
`0
`20
`40
`60
`80
`100
`120
`Inhibition (%)
`0 5 10 15 20 25 30 35
`L-733,060 concentration (µ )M
`23132/87
`y = 3.2701x + 3.2673
`R = 0.96152
`a
`0
`20
`40
`60
`80
`100
`140
`Cell concentration (%)
`none
`none
`120
`5
`none
`10
`none
`100
`none
`none
`10
`10
`10
`23132/87
`* * **
`*
`*
`+
`b
`SP (µ )
`L-733,060 (µ )
`M
`M
` Fig. 3. a Percentage of growth inhibition of human gastric adeno-
`carcinoma 23132/87 cells at 50 h in in vitro cultures following the
`addition of increasing concentrations (5, 10, 20 and 30 /H9262 M ) of L-
`733,060. The percentage of inhibition for the first doubling time
`of incubation is plotted on a linear graph. * p ^ 0.05. Values are
`means 8 SD (bars). The regression line is indicated, as well as the
`equation to obtain the IC
`50 . b Induction of cell proliferation of
`human gastric adenocarcinoma 23132/87 cells by SP at several
`nanomolar concentrations (5, 10 and 100 n M ). The NK-1 receptor
`antagonist L-733,060 was added (10 /H9262 M ) in the presence (10 n M )
`or absence (none) of SP for 50 h. In both cases, L-733,060 inhib-
`ited 23132/87 cell proliferation. Using the ANOVA test, a signifi-
`cant difference between each group and the control group (none-
`none) was found. * p ^ 0.01; * * p ^ 0.05;
`+ p ! 0.05, 10-none vs.
`10-10; °p ! 0 . 0 5 , 1 0 - 1 0 v s . n o n e - 1 0 . V e r t i c a l b a r s i n d i c a t e S D .
`Downloaded from http://karger.com/tbi/article-pdf/29/4/245/3567675/000152942.pdf by Lauren Wortman on 28 August 2025
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`
` Antitumor Action of L-733,060 on
`Gastrointestinal Cancer Cell Lines
`Tumor Biol 2008;29:245–254 251
` A p o p t o s i s
` After administration of the NK-1 receptor antagonist
`L-733,060, a great number of apoptotic cells were found
`in both 23132/87 and SW -403 cell lines ( fig. 5 ). In fact,
`w e o b s e r v e d i n D A P I - s t a i n e d c u l t u r e s a 5 0 . 3 9 a n d
`45.83% increase in apoptotic cells in 23132/87 and SW-
`403 cell lines, respectively, after administration of L-
`7 3 3 , 0 6 0 ; S D v a l u e s w e r e 6 . 4 5 a n d 8 . 3 3 , r e s p e c t i v e l y .
`These apoptotic cells were not found in gastrointestinal
`carcinoma cell cultures not treated with NK-1 receptor
`antagonists.
` Discussion
` We have demonstrated the presence of isoforms of
`NK-1 receptors in gastrointestinal carcinoma cell lines.
`Several previous reports have shown that different iso-
`forms of the NK-1 receptor can be found in both human
`and rat tissues [22] . For instance, 4 different proteins with
`molecular weights of 33, 58, 78 and 116 kDa can be spe-
`cifically affinity labeled using [
`125 I]SP in human lympho-
`cytes [23] , the main SP-binding protein present on hu-
`man lymphocyte cell membranes being a 58-kDa hydro-
`phobic glycoprotein [24] . In rat tissues, several NK-1
`receptor forms have been detected, with sizes ranging
`a b
`c d
` Fig. 5. Apoptosis features induced by L-733,060 in human gastric adenocarcinoma 23132/87 ( a , b ) and colon
`adenocarcinoma SW-403 ( c , d ) cell lines. b , d Higher-power magnification of the regions delimited in the rect-
`angles shown in a and c , r e s p e c t i v e l y . ! 40 ( a , c ) a n d ! 100 ( b , d ). Nuclei of treated cells show chromatin con-
`d e n s a t i o n a n d n u c l e a r f r a g m e n t a t i o n .
`Color version available online
`Downloaded from http://karger.com/tbi/article-pdf/29/4/245/3567675/000152942.pdf by Lauren Wortman on 28 August 2025
`Page 7 of 10
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` Rosso /Robles-Frías /Coveñas /
`Salinas-Martín /Muñoz
`Tumor Biol 2008;29:245–254252
`from 46 to 54 kDa [25] . Recently, we have demonstrated
`t
`he presence of isoforms of the NK-1 receptor in both
`SKN-BE(2) neuroblastoma and GAMG glioma cell lines
` [10] . Thus, in the SKN-BE(2) cell line, a major 54-kDa
`ba
`nd was observed, whereas in GAMG cells, 2 additional
`and more abundant isoforms of about 33 and 38 kDa were
`detected. In addition, in human pancreas carcinoma
` CAPAN-1 and PA-TU 8902 cell lines, appearance of 75-,
`58-, 46- and 34-kDa bands was reported [12, 26] . Thus,
`th
`e present data are in agreement with those of previous
`studies, since we have demonstrated for the first time the
`presence of several NK-1 receptor isoforms in human
`gastric adenocarcinoma 23132/87 (34, 46, 58 and 75 kDa)
`and human colon adenocarcinoma SW-403 (34, 58 and
`75 kDa) cell lines, although the functional roles of these
`NK - 1 r ecep t o r iso f o rms in s u ch ce ll lin es ar e curr e n tl y
`unknown.
` It is known that NK-1 receptors are overexpressed in
`primary glioblastomas, breast and pancreatic carcino-
`mas as well as retinoblastoma. However