June 2007
Volume 48, Issue 6
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Retina  |   June 2007
Neurokinin-1 Receptors Located in Human Retinoblastoma Cell Lines: Antitumor Action of Its Antagonist, L-732,138
Author Affiliations
  • Miguel Muñoz
    From the Pediatric Intensive Care Unit, Virgen del Rocío University Children’s Hospital, Sevilla, Spain;
  • Marisa Rosso
    From the Pediatric Intensive Care Unit, Virgen del Rocío University Children’s Hospital, Sevilla, Spain;
  • Rafael Coveñas
    Laboratory of Neuroanatomy of the Peptidergic Systems, Institute of Neurosciences of Castilla y León, Salamanca, Spain; and
  • Ignacio Montero
    From the Pediatric Intensive Care Unit, Virgen del Rocío University Children’s Hospital, Sevilla, Spain;
  • Miguel Angel González-Moles
    University of Granada, School of Dentistry, Department of Oral Medicine, Granada, Spain.
  • María José Robles
    From the Pediatric Intensive Care Unit, Virgen del Rocío University Children’s Hospital, Sevilla, Spain;
Investigative Ophthalmology & Visual Science June 2007, Vol.48, 2775-2781. doi:10.1167/iovs.05-1591
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      Miguel Muñoz, Marisa Rosso, Rafael Coveñas, Ignacio Montero, Miguel Angel González-Moles, María José Robles; Neurokinin-1 Receptors Located in Human Retinoblastoma Cell Lines: Antitumor Action of Its Antagonist, L-732,138. Invest. Ophthalmol. Vis. Sci. 2007;48(6):2775-2781. doi: 10.1167/iovs.05-1591.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The authors have recently demonstrated that substance P and L-733,060 induce cell proliferation and cell inhibition, respectively, in human retinoblastoma cell lines. However, the presence of neurokinin-1 receptors has not been demonstrated in such cell lines, nor is it known whether other neurokinin-1 receptor antagonists exert antitumoral action against retinoblastoma cell lines. The purpose of this study was to demonstrate the presence of neurokinin-1 receptors in the human retinoblastoma cell lines WERI-Rb-1 and Y-79 and to study the growth inhibitory capacity of the neurokinin-1 receptor antagonist L-732,138 against those cell lines. The authors also sought to demonstrate that the administration of L-732,138 or L-733,060 induces apoptosis in retinoblastoma cells and that neurokinin-1 receptors and substance P are present in primary retinoblastoma.

methods. Immunoblot analysis was used to determine neurokinin-1 receptors, and a Coulter counter was used to determine viable cell numbers; this was followed by application of the tetrazolium compound WST-8, a colorimetric method, to evaluate cell viability. DAPI stain was applied to assess chromatin condensation, characteristic of apoptosis, and immunoperoxidase was used to demonstrate neurokinin-1 receptors and substance P in eyes with primary retinoblastoma.

results. Neurokinin-1 receptors were present in both retinoblastoma cell lines studied. Three identical bands (isoforms of approximately 33, 58, and 75 kDa) were observed in both cell lines. Moreover, L-732,138 inhibited the growth of both cell lines studied, with and without previous administration of substance P. This inhibition occurred in a dose-dependent manner, with the IC50 values of 60.47 μM for WERI-Rb1 and 56.78 μM for Y-79. Moreover, apoptosis was observed in both cell lines after the administration of L-732,138 or L-733,060. In fixed eyes with primary retinoblastoma, a high density of neurokinin-1 receptors was observed in tumor cells, whereas a very low number of such cells contained substance P.

conclusions. This study showed that the same isoforms of the neurokinin-1 receptor are present in human retinoblastoma cell lines WERI-Rb-1 and Y-79. Both L-732,138 and L-733,060 can induce apoptosis in these cell lines and therefore can act as antitumoral agents. Primary retinoblastoma specimens display neurokinin-1 receptor immunolabeling. These results suggest that the neurokinin-1 receptor may be a promising new target for the treatment of retinoblastoma.

Retinoblastoma is the most common intraocular malignancy of infancy and childhood. 1 Its incidence is approximately 1 in 15,000 live births, but it occurs at increased frequency in families with a positive history for it. 2 3 Retinoblastoma arises from amacrine cell, horizontal cell, or Müller cell precursors, though the cell of origin of retinoblastoma remains controversial. 4 5 This malignancy leads to metastatic disease and death in 50% of affected children in undeveloped nations, but that rate falls to less than 5% of affected children in developed nations with advanced medical care. 6  
We have recently demonstrated that the undecapeptide substance P induces the growth of human retinoblastoma WERI-Rb-1 and Y-79 cell lines and that the piperidine neurokinin (NK)-1 receptor antagonist L-733,060 exerts antitumoral action against both cell lines. 7 In addition, the presence of substance P has been demonstrated in retinoblastoma cells. 8 NK-1 receptors have been described in the rabbit 9 and mouse retina, 10 and the expression of substance P and NK-1 receptors has been studied in the developing mouse retina and in the retinas of mice with genetic deletion of the NK-1 receptor. 11 It is also known that the NK-1 receptor shows preferential affinity for substance P and that, after binding to the NK-1 receptor, this peptide regulates many biological functions. 12 13 14 However, it is not yet known whether NK-1 receptors are present in the human retinoblastoma cell lines WERI-Rb-1 and Y-79 or whether NK-1 receptor antagonists other than L-733,060 have antitumoral action against these cell lines. The NK-1 receptor antagonist L-733,060 has antitumoral activity against several human cell lines (neuroblastoma, glioma, melanoma, and retinoblastoma). 7 15 16  
L-732,138 is a potent and highly selective competitive nonpeptide tachykinin NK-1 receptor antagonist (N-acetyl-l-tryptophan 3, 5-bis (trifluoromethyl) benzyl ester). It is approximately 1000-fold more potent in cloned human NK-1 receptors than in cloned human NK-2 and NK-3 receptors and approximately 200-fold more potent in human NK-1 receptors than in rat NK-1 receptors. 17 The IC50 for the human NK-1 receptor expressed in Chinese hamster ovary cells is approximately 2.3 nM. 18 It is known that the administration of L-732,138 leads to an attenuation of hyperalgesia 19 and that L-732,138 is able to antagonize H3 antagonist–induced skin vascular permeability. 20 Finally, we have recently demonstrated the antitumoral action of the tryptophan-based antagonist L-732,138 in the human glioma cell line GAMG. 21  
To our knowledge, no studies have been carried out on the presence of NK-1 receptors in the human retinoblastoma cell lines WERI-Rb-1 and Y-79, nor has any work been carried out on the effect of the NK-1 receptor antagonist L-732,138 on these human cancer cell lines. In addition, it is known that a substance P antagonist (other than L-732,138 or L-733,060) induces apoptosis in lung cancer, 22 but the induction of apoptosis in WERI-Rb-1 and Y-79 cell lines by L-732,138 or L-733,060 is unknown. Thus, the aims of this study were (1) to demonstrate in vitro the presence of NK-1 receptors in the human retinoblastoma WERI-Rb-1 and Y-79 cell lines; (2) to demonstrate in tumor cells the presence of NK-1 receptors in fixed eyes with primary retinoblastoma; (3) to demonstrate, using a colorimetric method to evaluate cell viability, the antitumoral action of the NK-1 receptor antagonist L-732,138 on both cell lines; (4) to determine whether NK-1 antagonists L-732,138 and L-733,060 induce apoptosis in WERI-Rb-1 and Y-79 cells; (5) to study the ability of substance P to reverse cell growth inhibition by L-732,138 of both retinoblastoma cell lines; and (6) to demonstrate in both cell lines the presence of substance P, given that it has been previously reported that retinoblastoma contained clusters of pleomorphic cells with an intense substance P immunoreaction. 8 A final goal was to compare the degree of antitumoral action of two molecules that are structurally very different—piperidine (L-733,060) and l-tryptophan (L-732,138)—on WERI-Rb-1 and Y-79 cells. 
Materials and Methods
Source of Cell Lines and Culture Conditions
The human retinoblastoma cell lines WERI-RB-1 and Y-79 were purchased by Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Cell lines were maintained in RPMI 1640 (Gibco, Barcelona, Spain) supplemented with 10% and 20% heat-inactivated fetal bovine serum, respectively. Both lines grew as cell clusters in suspension. Cell lines were subcultured in 75-cm2 tissue culture flasks (Falcon, Heidelberg, Germany), and the medium was renewed once a week. Cells were incubated at 37°C in a humidified atmosphere of 95% air/5% CO2. In these culture conditions, we observed that the doubling times for WERI-RB-1 and Y-79 were, respectively, approximately 49 hours and 40 hours. 
Western Blot Analysis
As previously reported, 23 total protein was prepared from suspension cultures of WERI-RB-1 and Y-79 retinoblastoma cells in 125-cm2 culture flasks. Briefly, cells were harvested, washed with phosphate-buffered saline (PBS), pH 7.4, and resuspended in HEN buffer (50 mM HEPES, 5 mM EDTA, 250 mM NaCl, pH 7.3) containing 5 mM dithiothreitol (DTT), 1 mM Na3VO4, 0.2% IGEPAL CA-630 (Sigma-Aldrich, Madrid, Spain), and 1% protease inhibitor cocktail (Sigma-Aldrich). Once resuspended, cells were vortexed, incubated on ice for 5 minutes, and centrifuged for 15 minutes at high speed in a microcentrifuge, after which the protein-containing supernatant was collected. Protein concentrations were determined with a protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. 
From each sample, 50 μg protein was separated by electrophoresis on 10% SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were incubated in blocking solution (5% nonfat milk in PBS, 0.1% Tween-20 [PBS-T]), followed by overnight incubation with an antibody against the KTMTESSSFYSNMLA conserved domain, corresponding to the C terminus of the NK-1 receptor (Sigma-Aldrich) and diluted 1:4000. Membranes were then washed with PBS-T and incubated with a horseradish peroxidase–conjugated goat anti–rabbit IgG antibody for 2 hours at room temperature (1:10,000 dilution). Antibody detection was performed with an enhanced chemiluminescence reaction (ECL Western Blotting Detection; Amersham Life Science, UK). 
Drug Treatments
The NK-1 receptor antagonist N-acetyl-l-tryptophan 3, 5-bis (trifluoromethyl) benzyl ester, molecular weight 472.39 (L-732,138; Sigma-Aldrich), was dissolved in distilled water containing 2.5% dimethyl sulfoxide (DMSO) before sample treatment. To determine the 50% inhibition concentration (IC50), different concentrations (5, 10, 20, 40, 60, 80, and 100 μM for WERI-RB-1 and Y-79) of L-732,138 were evaluated. Substance P, acetate salt (Sigma-Aldrich) was dissolved in distilled water, and different concentrations of the compound (50 and 100 nM) were evaluated to determine substance P–induced cell proliferation. Finally, the most effective substance P concentration for each cell line was incubated for 1 hour before L-732,138 was added. 
Proliferation Assays
We evaluated cell proliferation using the tetrazolium compound WST-8 ([2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)]-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt), as described previously. 7 At the time of the assay, cells cultured for 4 to 5 days were harvested, cell viability was evaluated by trypan blue exclusion, and cell numbers were quantified with a Coulter counter. 
Cells were cultured in 96-well plates. Each well contained 104 cells in a total volume of 100 μL; each assay included one plate. The plate included blank wells, control wells (104 cells/0.1 mL), control wells treated with L-732,138, control wells with DMSO, and control wells treated with different concentrations of substance P (with or without L-732,138). The plates were inoculated with L-732,138 (5, 10, 20, 40, 60, 80, and 100 μM for WERI-RB-1 and Y-79) and were incubated for a period of 49 hours or 40 hours, respectively, for their first doubling times. The plates were also inoculated with substance P (50 and 100 nM), with (10 μM) or without L-732,138 for their first doubling times (49 hours or 40 hours). For the proliferation assays, 10 μL cell-counting kit-8 reagent was added to each well 90 minutes before samples were read on a multiscanner microplate reader (Multiskan Spectrum; Thermo Labsystem, Barcelona, Spain) at 450 nm. The quantity of product, as measured by optical density, 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-732,138 or substance P) was assayed in duplicate, and all experiments were performed at least three times. The IC50 of L-732,138 was calculated. 
IC50
To determine IC50, linear regression was used (Excel; Microsoft, Redmond, WA), and 50% inhibition was interpolated from the equation of the line. 
Statistical Analyses
ANOVA, the test of homogeneity of variances, and a post hoc test (Dunnett or Bonferroni test) were used. P ≤ 0.05 was considered significant. No significant differences were observed between the control and the control-DMSO groups (data not shown). 
DAPI Staining
To determine whether apoptosis is induced by NK-1 receptor antagonists, DAPI staining was performed. In short, cells were cultured on four-chamber slides. After treatment with NK-1 receptor antagonist L-732,138 for WERI-RB-1 and Y-79 first doubling times (49 hours and 40 hours) or L-733,060, cells were washed twice with phosphate-buffered saline (PBS) and fixed by incubation in 4% paraformaldehyde for 30 minutes. After a second washing in PBS, cells were incubated in 1:1000 dilution (1 μg/mL DAPI solution; Sigma-Aldrich) for 30 minutes in the dark, and the supporting slides were mounted with a mixture of PBS and glycerol (70%). Cells were then observed through a fluorescence microscope (Zeiss, Oberkochen, Germany). Apoptotic cells were defined by the condensation and fragmentation of nuclear chromatin. We counted the number of apoptotic cells observed in WERI-RB-1 or Y-79 cells not treated with the NK-1 receptor antagonists, WERI-RB-1 cells treated with L-733,060 (20 μM) or L-732,138 (100 μM), and Y-79 cells treated with L-733,060 (25 μM) or L-732,138 (100 μM). In each case, the count was repeated in three different slides. For each slide, we counted the total number of cells and the number of apoptotic cells in five different fields (40× objective) and calculated the percentage of apoptotic cells per field. Each field was captured using digital photography. 
Immunocytochemistry for NK-1 Receptors and Substance P
Single eyes with primary retinoblastoma were obtained from four children (2 to 4 years of age; Hospital Juan Ramón Jiménez, Huelva, Spain; Hospital Virgen del Rocío, Sevilla, Spain) after surgical intervention. After formaldehyde and ethanol fixation, eyes were embedded in paraffin. The experimental design, protocols, and procedures of this work were performed under the guidelines of the ethics and legal recommendations of Spanish and European law and in accordance with the Declaration of Helsinki. 
For the detection of substance P and NK-1 receptor, 3- to 5-μm sections were cut from the tissue block. Sections were deparaffinized with xylene and rehydrated through a series of ethanol. Endogenous peroxidase activity was blocked by incubating the slides in methanol with 1.5% H2O2 for 2 minutes. For antigen retrieval, sections were boiled in citrate buffer (2.94 g/L sodium citrate, pH 6.0) for 15 minutes and subsequently cooled to 30°C. After washing with PBS, sections were incubated with 10% nonimmune horse serum for 15 minutes. Subsequently, they were incubated for 60 minutes with 1:500 diluted anti–substance P (Sigma, St. Louis, MO) and with anti–NK-1 receptor (Sigma-Aldrich, Madrid, Spain) antibodies, followed by washing in PBS and incubation with 1:500 diluted biotinylated horse anti–mouse antibody (Vector Laboratories Inc., Burlington, VT) for 30 minutes with peroxidase-streptavidin conjugase (Immunotech; Beckman Coulter, Fullerton, CA) in a 1:400 dilution. Sections were washed in phosphate-citrate buffer (pH 5.8), and substance P and NK-1 receptors were visualized for light microscopy with DAB reagent (0.06%) and 0.03% H2O2 in phosphate-citrate buffer). Sections were counterstained with hematoxylin for 3 minutes Finally, as previously published, 24 to determine the specificity of the immunostaining, several histologic controls were included (primary and secondary antibodies omitted, preabsorption of the primary antibodies). In all cases, such controls indicated the specificity of the antibodies used in this study. Moreover, as a positive control, the immunocytochemical technique was applied on glioma cells to show NK-1 receptors. As previously reported, 25 we also observed that such cells expressed NK-1 receptors. 
In sections of retinoblastoma treated with the anti–NK-1 receptor antiserum, we counted the number of tumor cells showing immunoreactivity (brown staining) or not and the location of the staining. 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; when 10% to 40% were stained, the number was considered moderate; when greater than 40% were stained, the number was considered high. Tumor cells were recorded as positive when they showed moderate or strong labeling. In all cases studied, we observed a high number of immunoreactive tumor cells expressing NK-1 receptors, with immunoreactivity located on the tumor cells, plasma membranes, or both. 
Results
NK-1 Receptors
We performed Western blot analysis to test the presence of NK-1 receptors in WERI-Rb-1 and Y-79 cell lines. Cell protein extracts were loaded onto polyacrylamide gels, electrophoresed, and transferred to membranes. Incubation with an antibody against an epitope whose sequence is conserved in several species revealed the presence of different isoforms of the NK-1 receptor in WERI-Rb-1 and Y-79 cell lines (Fig. 1) . The relative amount of each isoform was similar in both cell lines. Three similar bands were observed in both retinoblastoma cell lines at approximately 33, 58, and 75 kDa. 
As previously reported, 23 protein from the rat PC12 pheochromocytoma cell line was included as a positive control (not shown). It has been demonstrated that this cell line showed several isoforms of the NK-1 receptor; the 46-kDa protein was the most abundant. 23 No bands were detected when incubation was carried out with the secondary antibody alone. 
Antitumor Action of L-732,138
Growth inhibition of WERI-Rb-1 and Y-79 retinoblastoma cell lines by L-732,138 was observed in concentration-dependent cytotoxicity assays after the addition of increasing concentrations of L-732,138 (Figs. 2A 3A) . Thus, a lower inhibition of the growth of both cell lines was observed in the presence of low doses of L-732,138 for each line, whereas at the first doubling time a strong decrease in the number cells in both lines was found at intermediate concentrations, and very low cell numbers were observed at the maximum concentrations of the NK-1 receptor antagonist. 
The IC50 observed in the controls treated with L-732,138 was 60.47 μM for 49 hours for WERI-Rb1 and 56.78 μM for 40 hours for Y-79, with maximum inhibition for both cell lines seen when L-732,138 was present at 100 μM. 
NK-1 Receptor Antagonist L-732,138 Blocks Substance P-Induced Mitogen Stimulation
Treatment with L-732,138 at 10 μM for WERI-Rb-1 and Y-79 partially inhibited the growth of both cell lines (Figs. 2B 3B) . Moreover, as previously reported, 21 both cell lines were observed to grow after the addition of substance P (Figs. 2B 3B) . Thus, substance P stimulation was evident at 50 nM, and the maximum level was reached in our study at 100 nM for WERI-Rb-1 and Y-79 (Figs. 2B 3B) . The percentage of cell proliferation in both cell lines increased from 20% to 50% in WERI-Rb-1 and from 28% to 35% in Y-79, depending on the dose of substance P administered (Figs. 2B 3B)
To determine whether L-732,138 inhibited cell proliferation through interaction with its own receptor, we used the specific NK-1 receptor agonist substance P in competition experiments. The cellular concentration at 10 μM L-732,138 and 100 nM substance P was higher than that observed with L-732,138 alone, indicating that L-732,138 blocks substance P-induced mitogen stimulation. 
Apoptosis
After the administration of both NK-1 receptor antagonists, apoptotic cells were observed in both cell lines (Figs. 4A 4B) . After the administration of L-733,060, we observed 40% ± 5.2% (SEM) and 39% ± 3.7% apoptotic cells in WERI-Rb-1 and Y-79 cell lines, respectively, whereas after the administration of L-732,138, we found 40% ± 6.7% and 25% ± 6.0% apoptotic cells in WERI-RB-1 and Y-79 cell lines, respectively. These apoptotic cells were not found in retinoblastoma cell cultures that were not with NK-1 receptor antagonists. 
NK-1 Receptors and Substance P in Eyes with Primary Retinoblastoma
With the use of IHC, we observed a high density of NK-1 receptors in the tumor mass (Figs. 4C 4D)but few tumor cells containing substance P. 
Discussion
NK-1 Receptors
In the mammalian retina, NK-1 receptor immunoreactivity has been observed in amacrine, displaced amacrine, bipolar cells, interplexiform, and some ganglion cells, 9 10 whereas substance P has been found in amacrine cells. 9 10 We have demonstrated the presence of NK-1 receptors in retinoblastoma cells (WERI-Rb-1 and Y-79 tumor cell lines) and in primary retinoblastoma. Several previous reports have shown that different isoforms of the NK-1 receptor can be found in human and rat tissues. 26 For instance, four distinct proteins with molecular weights of 33, 58, 78, and 116 kDa can be affinity-labeled using [125I]-substance P in human lymphocytes, 27 with a 58-kDa hydrophobic glycoprotein the principal substance P-binding protein on human lymphocyte cell membranes. 28 In rat tissues, several NK-1 receptor forms have been detected; sizes ranged from 46 kDa to 54 kDa. 29 30 Recently, we have demonstrated the presence of isoforms of the NK-1 receptor in SKN-BE 2 neuroblastoma and GAMG glioma cell lines. 23 Thus, in the SKN-BE 2 cell line, a major 54-kDa band was observed, whereas in GAMG cells, two additional and more abundant isoforms of approximately 33 and 38 kDa were detected. Our present data are in agreement with those of previous studies. We demonstrated the presence of several NK-1 receptor isoforms (33, 58, and 75 kDa) in both retinoblastoma cell lines, though the functional roles of these NK-1 receptor isoforms are unknown. 
Antitumor Action of L-732,138
We have demonstrated in vitro a potent growth inhibition in the human WERI-Rb-1 and Y-79 retinoblastoma cell lines after administration of the nonpeptide NK-1 receptor antagonist L-732,138, an l-tryptophan derivative. This is in agreement with the results of a study in which the antitumor activity of the antagonist was demonstrated against glioma cell lines. 21 Our findings are in agreement with recent reports showing that L-733,060, a piperidine derivative NK-1 receptor antagonist, also shows antitumor activity against human neuroblastoma, glioma, 16 melanoma, 15 and retinoblastoma cell lines 7 and with reports that NK-1 receptor antagonists other than L-732,138, such as substance P antagonists, inhibited the growth of small cell lung cancer and glioma 16 22 31 32 33 34 in vitro and in vivo. In sum, it has been demonstrated that both NK-1 receptor antagonists produce growth inhibition in many cancer cell lines. Given that these NK-1 receptor antagonists can inhibit growth in tumors in which substance P and NK-1 receptors are expressed, 19 20 21 35 36 they could be candidates as broad-spectrum antineoplastic drugs. 
Here we have demonstrated that treating retinoblastoma cell lines with L-732,138 produces growth inhibition and cell death by apoptosis. Similarly, it is known that another substance P antagonist induces apoptosis in lung cancer and causes a concentration-dependent loss of cell viability, 22 as we observed with the use of L-732,138. Moreover, the blockade of NK-1 receptors in both retinoblastoma cell lines by L-732,138 could inhibit DNA synthesis and cell proliferation through the mitogen-activated protein kinase (MAPK) pathway. 37  
NK-1 Receptor Antagonist L-732,138 Blocks Substance P Mitogen Stimulation
In this study and elsewhere, 7 we have demonstrated that substance P increases the proliferation of retinoblastoma cell lines. Our results are consistent with previous findings because it has been reported that the activation of NK-1 receptors by substance P induces mitogenesis in several cancer cell types. 15 23 36 38 39 In addition, our results indicate that L-732,138 blocks substance P mitogen stimulation because L-732,138-induced growth inhibition was partially reversed by the administration of exogenous substance P. This observation suggests that the NK-1 receptor plays an important role in the growth of the retinoblastoma cell lines studied (Figs. 2B 3B) . After treatment with substance P and L-732,138, an increase in cellular concentration (27.82% and 24.63%) was observed in the WERI-Rb-1 and Y-79 cell lines, respectively (Figs. 2B 3B)compared with when the NK-1 receptor antagonist was administered alone. These values were 27.56% and 9.65%, respectively, for WERI-Rb-1 and Y-79 cell lines after treatment with substance P and L-733,060. 7  
Finally, it should be noted that our results on the blocking action of L-732,138 against substance P-induced mitogen stimulation are in agreement with our previous findings using L-733,060 and substance P in the same retinoblastoma cell lines. 7 Substance P is expressed at very low levels in primary retinoblastoma. This is partially in agreement with data previously reported by Tarkkanen et al., 8 who reported that the tumor mass, though mainly substance P negative, contained clusters of pleomorphic cells with an intense substance P immunoreaction. 
Antitumor Action of the NK-1 Receptor Antagonists L-732,138 and L-733,060
We have demonstrated that the NK-1 receptor antagonist molecules L-733,060 and L-732,138, which are structurally very different, have antitumor action. Both antagonists have in common their specificity for the NK-1 receptor, with Ki values of 2.3 nM for L-732,138 and 0.8 nM for L-733,060. However, on comparing the results obtained with L-732,138 and with L-733,060 in the same human retinoblastoma cell lines, 7 we observed 3- to 5-fold lower antitumor action for L-732,138 than for L-733,060, which is probably related to the different affinities of these two antagonists for the NK-1 receptor (2.3 nM for L-732,138 and 0.8 nM for L-733,060). 
All the data presented here suggest that the action of both NK-1 receptor antagonists is specifically through the target NK-1 receptor 23 and that the antitumor action on cancer cell lines (e.g., retinoblastoma, glioma, neuroblastoma, and melanoma) of both molecules is related to their ability to block NK-1 receptors. 
 
Figure 1.
 
Western blot analysis of NK-1 receptors (NK-1-R) in the WERI-RB-1 and Y-79 retinoblastoma cell lines showing the presence of different NK-1 receptor complex isoforms. Dots indicate bands with molecular weights similar to those previously reported.
Figure 1.
 
Western blot analysis of NK-1 receptors (NK-1-R) in the WERI-RB-1 and Y-79 retinoblastoma cell lines showing the presence of different NK-1 receptor complex isoforms. Dots indicate bands with molecular weights similar to those previously reported.
Figure 2.
 
(A) Percentage of growth inhibition of WERI-Rb-1 cells at 49 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80 and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values represent mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 49 hours. (B) Induction of cell proliferation of WERI-Rb-1 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist L-732,138 was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 49 hours. In both cases, L-732,138 inhibited WERI-Rb-1 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 2.
 
(A) Percentage of growth inhibition of WERI-Rb-1 cells at 49 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80 and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values represent mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 49 hours. (B) Induction of cell proliferation of WERI-Rb-1 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist L-732,138 was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 49 hours. In both cases, L-732,138 inhibited WERI-Rb-1 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 3.
 
(A) Percentage of growth inhibition of Y-79 cells at 40 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80, and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values are mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 40 hours. (B) Induction of cell proliferation of Y-79 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 40 hours. In both cases, L-732,138 inhibited Y-79 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 3.
 
(A) Percentage of growth inhibition of Y-79 cells at 40 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80, and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values are mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 40 hours. (B) Induction of cell proliferation of Y-79 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 40 hours. In both cases, L-732,138 inhibited Y-79 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 4.
 
(A) DAPI staining of Y-79 retinoblastoma cell line treated with NK-1 receptor antagonist L-733,060. Nuclear condensation and fragmentation are observed. (B) High-power image of the boxed area in (A). (C, D) After use of an immunocytochemical technique, note, in an eye with primary retinoblastoma, a high expression of NK-1 receptors (brown) in retinoblastoma cells. Cell nuclei were counterstained with hematoxylin. (C, inset) Region from which the photograph shown in (D) was taken. Original magnifications: (A) ×40; (B) ×200; (C) ×100; (D) ×150.
Figure 4.
 
(A) DAPI staining of Y-79 retinoblastoma cell line treated with NK-1 receptor antagonist L-733,060. Nuclear condensation and fragmentation are observed. (B) High-power image of the boxed area in (A). (C, D) After use of an immunocytochemical technique, note, in an eye with primary retinoblastoma, a high expression of NK-1 receptors (brown) in retinoblastoma cells. Cell nuclei were counterstained with hematoxylin. (C, inset) Region from which the photograph shown in (D) was taken. Original magnifications: (A) ×40; (B) ×200; (C) ×100; (D) ×150.
The authors thank Nicholas Skinner for stylistic revision of the English text; Arturo Mangas and Manuel Sánchez for technical assistance; and Rocio Cabrera (Anatomic Pathology Service, Hospital Virgen del Rocío, Sevilla, Spain) and Juan José Borrero (Anatomic Pathology Service, Hospital Juan Ramón Jiménez, Huelva, Spain) for retinoblastoma diagnostics and for providing eyes with primary retinoblastoma. 
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Figure 1.
 
Western blot analysis of NK-1 receptors (NK-1-R) in the WERI-RB-1 and Y-79 retinoblastoma cell lines showing the presence of different NK-1 receptor complex isoforms. Dots indicate bands with molecular weights similar to those previously reported.
Figure 1.
 
Western blot analysis of NK-1 receptors (NK-1-R) in the WERI-RB-1 and Y-79 retinoblastoma cell lines showing the presence of different NK-1 receptor complex isoforms. Dots indicate bands with molecular weights similar to those previously reported.
Figure 2.
 
(A) Percentage of growth inhibition of WERI-Rb-1 cells at 49 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80 and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values represent mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 49 hours. (B) Induction of cell proliferation of WERI-Rb-1 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist L-732,138 was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 49 hours. In both cases, L-732,138 inhibited WERI-Rb-1 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 2.
 
(A) Percentage of growth inhibition of WERI-Rb-1 cells at 49 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80 and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values represent mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 49 hours. (B) Induction of cell proliferation of WERI-Rb-1 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist L-732,138 was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 49 hours. In both cases, L-732,138 inhibited WERI-Rb-1 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 3.
 
(A) Percentage of growth inhibition of Y-79 cells at 40 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80, and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values are mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 40 hours. (B) Induction of cell proliferation of Y-79 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 40 hours. In both cases, L-732,138 inhibited Y-79 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 3.
 
(A) Percentage of growth inhibition of Y-79 cells at 40 hours in in vitro cultures after the addition of increasing concentrations (5, 10, 20, 40, 60, 80, and 100 μM) of L-732,138. The percentage of inhibition for the first doubling time of incubation is plotted on a linear graph. *P ≤ 0.05. Values are mean ± SD (bars). Regression line is indicated, as is the equation to obtain the IC50. Dashed lines: IC50 at 40 hours. (B) Induction of cell proliferation of Y-79 cells by substance P at two nanomolar concentrations (50 and 100 nM). The NK-1 receptor antagonist was added (10 μM) in the presence (100 nM) or absence (none) of substance P for 40 hours. In both cases, L-732,138 inhibited Y-79 cell proliferation. With the use of ANOVA, a significant difference between each group and the control group (none-none) was found. *P ≤ 0.01. **P ≤ 0.05. #Value of significance of 100 to 10 versus none to 10. #P ≤ 0.05. Vertical bars: SD.
Figure 4.
 
(A) DAPI staining of Y-79 retinoblastoma cell line treated with NK-1 receptor antagonist L-733,060. Nuclear condensation and fragmentation are observed. (B) High-power image of the boxed area in (A). (C, D) After use of an immunocytochemical technique, note, in an eye with primary retinoblastoma, a high expression of NK-1 receptors (brown) in retinoblastoma cells. Cell nuclei were counterstained with hematoxylin. (C, inset) Region from which the photograph shown in (D) was taken. Original magnifications: (A) ×40; (B) ×200; (C) ×100; (D) ×150.
Figure 4.
 
(A) DAPI staining of Y-79 retinoblastoma cell line treated with NK-1 receptor antagonist L-733,060. Nuclear condensation and fragmentation are observed. (B) High-power image of the boxed area in (A). (C, D) After use of an immunocytochemical technique, note, in an eye with primary retinoblastoma, a high expression of NK-1 receptors (brown) in retinoblastoma cells. Cell nuclei were counterstained with hematoxylin. (C, inset) Region from which the photograph shown in (D) was taken. Original magnifications: (A) ×40; (B) ×200; (C) ×100; (D) ×150.
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