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Peptides 38 (2012) 318–325

Contents lists available at SciVerse ScienceDirect

Peptides

j our na l ho me p age : www.elsev ier .com/ locate /pept ides

he substance P/neurokinin-1 receptor system in lung cancer: Focus on thentitumor action of neurokinin-1 receptor antagonists

iguel Munoza,∗ , Ana González-Ortegaa , Marisa Rossoa , María José Robles-Friasb , Andres Carranzab ,anuel Vicente Salinas-Martínb, Rafael Covenasc

Research Laboratory on Neuropeptides, Virgen del Rocío University Hospital, Sevilla, SpainDeparment of Pathology, Virgen del Rocío University Hospital, Sevilla, SpainLaboratory of Neuroanatomy of the Peptidergic Systems, Institute of Neurosciences of Castilla y León (INCYL, Lab. 14), Salamanca, Spain

r t i c l e i n f o

rticle history:eceived 3 September 2012eceived in revised form0 September 2012ccepted 20 September 2012vailable online 28 September 2012

eywords:poptosis

a b s t r a c t

The last decades have seen no significant progress in extending the survival of lung cancer patients andthere is an urgent need to improve current therapies. The substance P (SP)/neurokinin-1 receptor (NK-1R) system plays an important role in the development of cancer: SP and NK-1R antagonists respectivelyinduce cell proliferation and inhibition in human cancer cell lines. No study of the involvement of thissystem in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells has been carried out indepth. Here, we demonstrate the involvement of the SP/NK-1R system in human H-69 (SCLC) and COR-L23(NSCLC) cell lines: (1) they express isoforms of the NK-1R and mRNA for the NK-1R; (2) they overexpressthe tachykinin 1 gene; (3) the NK-1R is involved in their viability; (4) SP induces their proliferation; (5)

prepitant-732,138-733,060on-small cell lung cancermall cell lung cancer

NK-1R antagonists (Aprepitant (Emend), L-733,060, L-732,138) inhibit the growth of both cell lines ina concentration-dependent manner; (6) the specific antitumor action of these antagonists against suchcells occurs through the NK-1R; and (7) lung cancer cell death is due to apoptosis. We also demonstratethe presence of NK-1Rs and SP in all the human SCLC and NSCLC samples studied. Our findings indicatethat the NK-1R may be a promising new target in the treatment of lung cancer and that NK-1R antagonists

ntitu

could be new candidate a

. Introduction

Lung cancer is the first cause of cancer-related death in west-rn world. Despite multiple trials with cytotoxic chemotherapeuticgents, anti-growth factor-signaling agents and/or radiotherapy,he last two decades have seen no significant progress in extendinghe survival of patients suffering the disease [10]. Cytostatic drugshow a low safety profile and severe side effects, since they areot specific to tumor cells. Research should focus on drugs withhe same or greater anti-tumor action but with fewer side effects.his can only be achieved if the drug is specific against tumor cellsnd researchers are therefore seeking to identify novel molecularargets for blocking tumor growth.

The expression and secretion of peptides by tumors has

ttracted increasing interest, and there are data suggesting thathe substance P (SP)/neurokinin-1 receptor (NK-1R) system playsn important role in cancer development [19,20,24]: the number

∗ Corresponding author at: Hospital Infantil Virgen del Rocío, Unidad de Cuidadosntensivos Pediátricos, Av. Manuel Siurot s/n, 41013 Sevilla, Spain.el.: +34 955012965; fax: +34 955012921.

E-mail address: mmunoz@cica.es (M. Munoz).

196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.peptides.2012.09.024

mor drugs in the treatment of SCLC and NSCLC.© 2012 Elsevier Inc. All rights reserved.

of NK-1Rs expressed in normal human cells is lower than thatexpressed in human tumoral cells; the expression of NK-1Rs is cor-related with the degree of malignancy; the activation of NK-1Rs bySP induces mitogenesis in human tumor cell lines; the migrationof tumor cells, a crucial requirement for invasion and metastasis, isregulated by SP signals, and NK-1R antagonists exert an antitumoralaction against human cancer cell lines. Moreover, NK-1R antago-nists do not usually have serious side effects. One such antagonist,the drug aprepitant, is used in clinical practice as antiemetic [2].Thus, there are sufficient data suggesting that the NK-1R could be anew promising target in human cancer treatment and that NK-1Rantagonists should be designed as novel therapeutic agents.

Currently, a few studies implicating the SP/NK-1R system in lungcancer have been reported: synthetic analogs of SP (identified asantagonists of SP-mediated cellular effects) induce cell death byapoptosis in both small-cell lung cancer (SCLC) and non-small-celllung cancer (NSCLC) cells [12,26,28,32]. To our knowledge, thereare many unknown issues regarding the involvement of the SP/NK-1R system in SCLC and NSCLC. Thus, the aims of this study are to

demonstrate: (1) in vitro the presence of NK-1Rs and their isoformsin the H-69 SCLC and COR-L23 NSCLC cell lines; (2) that both celllines express mRNA for the NK-1R and overexpress the tachykinin1 gene; (3) that the NK-1R is involved in their viability; (4) that SP

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nduces their proliferation; (5) that NK-1R antagonists, other thanynthetic analogs of SP, such as L-733,060, L-732,138 and the drugprepitant, inhibit the growth of both cell lines; (6) that the specificntitumor action of such antagonists occurs through the NK-1R androduces apoptosis in both cancer cell lines; and (7) the presence ofK-1Rs and SP in human SCLC and NSCLC samples. The data suggest

hat the NK-1R is a promising new target in the treatment of lungancer.

. Materials and methods

.1. Cell cultures and drug treatments

We used H-69 SCLC and COR-L23 NSCLC lung cancer cell linesEuropean Collection of Cell Cultures). These cells were maintaineds previously reported [23].

Three NK-1R antagonists were used: L-733,060, L-732,138 andprepitant. In order to determine the IC50, different concentrations5–40 �M) of L-733,060; (10–100 �M) of L-732,138; (5–50 �M) ofprepitant were evaluated. Different concentrations of SP (1, 5, 10,00 and 500 nM) were used. The most mitogenic nanomolar SP con-entration for each cell line was added for 1 h before the additionf each NK-1R antagonist.

.2. Proliferation assays and statistical analyses

Cell proliferation was evaluated as previously reported [23].ach experimental condition (blank wells, control wells, and con-rol wells treated with different concentrations of each antagonistr SP) was assayed in duplicate and all experiments were performedt least three times.

Data were expressed as means ± SD. Statistical analysis waserformed with SPPS statistical software for Microsoft Windows,elease 14.0 (Professional Statistics, Chicago). The homogeneity ofhe variance was tested using the Levene test. If the variances wereomogeneous, the data were analyzed using the one-way ANOVAest with Bonferroni’s correction for multiple comparisons. For dataets with non-homogeneous variances, the ANOVA test with T3unnett post hoc analysis were applied. The criterion for signifi-ance was p < 0.05 for all comparisons.

.3. Western blot analyses and immunohistochemistry for NK-1eceptors and SP

Western blot analyses were carried out as previously reported22,23]. Fifteen large NSCLC and fifteen SCLC human carcinomapecimens were retrieved from the archives of the Departmentf Pathology of the Virgen del Rocío University Hospital (Sevilla,pain). Paraffin-embedded SCLC and NSCLC tissues were used. Themmunocytochemical protocols for the demonstration of NK-1Rsnd SP, as well as the immunocytochemical controls carried outere, have been reported previously [8,23]. Ten representativeigh-power microscopic fields (40×) were evaluated in each slide.he presence or absence of staining and the number of cells show-ng a brown staining were noted, and we assessed whether or nothe staining was localized in the cytoplasm of tumor cells and/or inhe nucleus.

.4. DAPI staining

In order to determine whether the NK-1R antagonists stud-ed induced apoptosis, DAPI staining was performed as previously

eported [23]. The numbers of apoptotic cells were counted, repeat-ng the counts on three different slides. On each slide, the numbersf apoptotic cells located in five different sequential fields wereounted.

38 (2012) 318–325 319

2.5. PCR (polymerase chain reaction) and real-time quantitativeRT-PCR

As previously reported [23], total RNA isolation from culturedcells was accomplished using the NucleoSpin RNA II Kit, allowingthe purification of cultured cells. Reverse transcription was per-formed with the elimination of genomic DNA. The amplificationproducts were visualized by electrophoresis.

Real-time quantitative RT-PCR was performed as previouslydescribed [3,16,29]. The sequences of primers for the human NK-1Rwere based on the common sequence of the TAC1R human isoforms(NM 001058, NM 015727), TAC1R-Foward (CTG CTG GTG ATT GGCTAT GC) and TACR1-Reverse (AGG AGG AAG AAG ATG TGG AAGG), which yielded a 186 bp fragment and for beta-actin gene theforward primer CGGCATCGTCACCAACTG, and the reverse primerCACGCAGCTCATTGTAGAAGGT were used, yielding a 70 bp frag-ment. The data were analyzed using the relative standard curvemethod, as reported previously [3,16].

Finally, BLAST analysis was conducted to confirm that theprimers did not show homology with any other DNA segment. Thus,we were able to rule out the possibility that the band observed inour study might have been due to DNA contamination.

2.6. Small interfering RNA gene silencing method

We followed the previously published protocol [15,23]. H69and COR-L23 human lung cancer cell lines were incubated, thenormal growth medium was removed, and antibiotic-free growthmedium was added. After 1 h, the latter medium was removedand the transfection mixture, containing the small interfering RNA(siRNA) TAC1R (Tachykinin receptor 1 gene) (Invitrogen) dilutedwith Transfection Reagent medium, was added. The technique wasapplied to a siRNA-negative control solution in the same way.

3. Results

3.1. NK-1 receptors

Three similar bands (isoforms of the NK-1R of about 34, 58and 75 kDa) were observed in the H-69 SCLC and COR-L23 NSCLCcell lines and an additional 46-kDa band was also observed in theformer cell line (Fig. 1A).

With PCR analyses we observed that the H-69 SCLC and COR-L23NSCLC tumor cell lines expressed mRNA for the tachykinin NK-1R(Fig. 1B). Real-time quantitative RT-PCR was performed to analyzeNK-1R expression using beta-actin as an internal control. The meanNK-1R/beta-actin ratio was 0.6 ± 3.1 for the H69 tumor cell lineand 0.37 ± 1.2 for the COR-L23 tumor cell line. However, the valueobtained was 0.01 ± 3.9 for the HEK293 normal cell line. The NK-1R mRNA level was approximately 30–60 folds higher in the lungcarcinoma cell lines than in the normal cell line.

3.2. Antitumor action of NK-1R antagonists

The NK-1R antagonists inhibited the growth of both the H-69SCLC and COR-L23 NSCLC cell lines in a concentration-dependentmanner (Fig. 2). The concentration required for a 50% reductionin optical density (IC50) observed with L-733,060 was 18.7 �M forH69, and 22 �M for COR-L23; with L-732,138 it was 51.9 �M forH69, and 87 �M for COR-L23, and with aprepitant it was 21.8 �M for

H69, and 30.04 �M for COR-L23. Maximum inhibition was observedwhen the drug was present at a concentration of 39.2 �M of L-733,060; 109 �M of L-732,138, and 45.2 �M of aprepitant (for H69),and 41.5 �M of L-733,060; 102.3 �M of L-732,138, and 60 �M of

320 M. Munoz et al. / Peptides 38 (2012) 318–325

Fig. 1. (A) Western blot analysis of NK-1Rs in human H-69 SCLC and COR-L23 NSCLCcell lines showing the presence of different NK-1R complex isoforms. Dots indicatebands with molecular weights similar to those reported previously. The analysiswas conducted three times. (B) Agarose gel showing the expression of TAC1R cDNAiPt

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Fig. 2. (A) Percentage of growth inhibition of human H69 SCLC cells at 24 h in in vitrocultures following the addition of increasing concentrations (5–40 �M) of L-733,060,(10–100 �M) of L-732,138, and (5–50 �M) of aprepitant. The percentage of inhibi-tion for the first doubling time of incubation is plotted on a linear graph. Levelof significance: *p ≤ 0.05. Values are means ± SD (bars). (B) Percentage of growthinhibition of human COR-L23 NSCLC cells at 24 h in in vitro cultures following theaddition of increasing concentrations (10–40 �M) of L-733,060, (40–100 �M) ofL-732,138 or (5–50 �M) of aprepitant. The percentage of inhibition for the first dou-bling time of incubation is plotted on a linear graph. Level of significance: *p ≤ 0.05.Values are means ± SD (bars). The experiment was repeated three times in duplicate.

Table 1Apoptosis results on half-inhibition (IC50) and full inhibition (IC100) experiments inSCLC and NSCLC cells after the administration of the NK-1R antagonist.

H69 Means (%) Standard deviation

L-733,060Control 3.2 3.150% 26 4.6100% 41.3 4.6

L-732,138Control 3.2 3.1100% 27 3.5

AprepitantControl 3.2 3.150% 41.1 2.2100% 45.1 3.1

COR-L23 Means (%) Standard deviation

L-733,060Control 3.1 1.8100% 52.1 2.9

L-732,138Control 3.1 1.8100% 34.7 2.4

Aprepitant

n human lung carcinoma cell lines. Equal aliquots of each cDNA were amplified byCR for 40 cycles (target genes) with specific primers. PCR was carried out threeimes.

prepitant (for COR-L23) during the culture periods. No remainingiving cells were observed at the maximal concentration.

.3. Apoptosis

After administration of the NK-1R antagonists many apoptoticells were found in both the human H-69 SCLC and COR-L23 NSCLCell lines (Fig. 3). In DAPI-stained cultures, we observed 41.3% ± 4.6D and 52.1% ± 2.9 SD of apoptotic cells in the H69 and COR-L23 cellines respectively after the administration of IC100 L-733,060, and7% ± 3.1 SD and 34.7% ± 2.4 SD of apoptotic cells in the H69 andOR-L23 cell lines respectively after the administration of IC100 L-32,138. 45.1% ± 3.1 SD and 54.3% ± 5.1 SD of apoptotic cells in H69nd COR-L23 cell lines were found respectively after the adminis-ration of IC100 aprepitant (Table 1).

.4. The NK-1R antagonists block substance P-induced mitogentimulation

Growth of the H-69 SCLC and COR-L23 NSCLC cell lines wasoted after the addition of SP, and certain nanomolar concentra-ions of SP induced cell proliferation (Fig. 4). SP stimulation wasvident at 1 and 5 nM respectively, and the maximum level waseached at 10 nM in both the H69 and COR-L23 cell lines. The per-entage of cell proliferation of both cell lines increased from 15% to5% in H69 and from 27% to 37% in COR-L23, depending on theose of SP administered. In order to examine whether the NK-R antagonists inhibited cell proliferation via an interaction withheir receptor, the specific NK-1R agonist SP was used in competi-ion experiments: the cellular concentration at 15 �M, 20 �M (H69nd COR-L23 respectively) of L-733,060; 40 �M, 80 �M (H69 andOR-L23 respectively) of L-732,138 and 20 �M, 30 �M (H69 andOR-L23 respectively) of aprepitant with 10 nM (both cell lines)

f SP was higher than that observed with the NK-1R antagonistlone for H69 and COR-L23 (Fig. 4). Thus, NK-1R antagonist-inducedrowth inhibition was partially reversed by the administration of

nanomolar dose of exogenous SP.

Control 3.1 1.850% 42 4.9100% 54.3 5.1

M. Munoz et al. / Peptides 38 (2012) 318–325 321

Fig. 3. Human COR-L23 NSCLC cell line. (A) Cultured cells not treated with the NK-1R antagonist (20×). (B) Cultured cells treated with aprepitant (20×) showed a slowerproliferation profile. (C) Higher-power magnifications of the regions delimited in B (40×). Many nuclei of the treated cells show apoptotic figures: chromatin condensationand nuclear fragmentation can be observed. The experiment was conducted three times.

Fig. 4. (A–C) Induction of cell proliferation of human H69 SCLC cells by SP at several nanomolar concentrations (5, 10, 100 and 500 nM). NK-1R antagonists L-733,060,L-732,138 or aprepitant were added (15 �M, 40 �M and 20 �M respectively) in the presence (10 nM) or absence (none) of SP for their first doubling time. In both cases, eachNK-1R antagonist inhibited H69 SCLC cell proliferation. (D–F) Induction of cell proliferation of human COR-L23 NSCLC cells by SP at several nanomolar concentrations (1, 5,10 and 100 nM). The NK-1R antagonists L-733,060, L-732,138 or aprepitant were added (20 �M, 80 �M and 30 �M respectively) in the presence (10 nM) or absence (none)of SP for their first doubling time. In both cases, each NK-1R antagonist inhibited COR-L23 NSCLC cell proliferation. Using the ANOVA test, a significant difference betweeneach group and the control group (none-none) was found. Level of significance: *p ≤ 0.01. **p ≤ 0.05. #The value of significance of IC50-most mitogenic SP concentration vs.IC50-none p < 0.05, and x indicates IC50-most mitogenic SP concentration vs. none-most mitogenic SP concentration, p ≤ 0.01. Vertical bars indicate SD. The experiment wasconducted at least three times in duplicate.

322 M. Munoz et al. / Peptides

Fig. 5. Viability of H69 SCLC (A) and COR-L23 NSCLC (B) cell lines. This experimentwas repeated three times in duplicate. The number of siRNA-negative control cells iscompared with the number of siRNA TAC1R cells. At time 0, 100,000 H69 and 100,000Csc

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OR-L23 cells were seeded. In both cases, at 72 h the decrease in the number ofiRNA TAC1R cells was significant in comparison with the number of siRNA-negativeontrol cells.

.5. Small interfering RNA gene silencing method

The knockdown method was used. After 72 h we found 4.4 × 105

iRNA-negative control H69 cells and 2 × 105 siRNA TAC1R cells,.04 × 106 siRNA-negative control COR-L23 cells and 4.8 × 105

iRNA TAC1R cells (Fig. 5). After the administration of siRNA TAC1Ro both lines of cultured cells, many apoptotic cells were found inll wells studied. In DAPI-stained cultures we observed means of6.2 ± 1.15 (SD) % of apoptotic H69 cells, whereas in the siRNA-egative control cells we observed means of 2.2 ± 0.6 (SD) %.egarding the COR-L23 cells, after 72 h we observed means of5 ± 0.9 (SD) % after the administration of siRNA TAC1R and 8 ± 0.8SD) % siRNA-negative control cells.

.6. NK-1R/SP immunohistochemistry in NSCLC and SCLC samples

Using an immunohistochemical technique, fifteen large-cell andfteen small-cell lung carcinomas were examined for the presencef NK-1Rs and SP (Fig. 6; Table 2). The expression of NK-1Rs and SPas observed in all samples studied (30/30). In both tumor types,

he immunoreactivity for NK-1Rs was found in the cytoplasm and,

ccasionally, in the nucleus of tumors cells, whereas immunoreac-ivity for SP was predominantly located in the nucleus, the nuclear

embrane showing a strong staining. Moreover, the immunore-ctivity for NK-1Rs was observed in smooth muscle cells and in

38 (2012) 318–325

the endothelial cells of the small- and medium-caliber blood ves-sels, located in the peritumoral area, and immunoreactivity wasalso observed in macrophages and plasma cells. Immunoreactiv-ity for SP was also observed in the nucleus of smooth muscle cellsand in the endothelial cells of the small- and medium-caliber bloodvessels, as well as in macrophages located in the peritumoral areaand in both the nucleus and cytoplasm of plasma cells. It should benoted that in the same human SCLC or NSCLC histological sectionwe observed cells expressing or not expressing NK-1Rs and SP.

4. Discussion

The data described here are in agreement with those of previousstudies, in which the presence of isoforms and the overexpressionof the NK-1R have been reported in several human cancer cell linesother than SCLC/NSCLC [16,21–23]. NK-1R expression is known tobe increased 25–36-fold in human pancreatic cancer cell lines incomparison with normal controls; in glioma, NK-1Rs are overex-pressed in comparison with normal cells, and tumors samples frompatients with advanced tumor stages exhibit significantly higherNK-1R levels [7,20]. Here, we found that NK-1R expression wasincreased approximately 30–60 fold in lung cancer cell lines incomparison with a normal cell line. Moreover, our results are inagreement with studies reporting that human melanoma and acutelymphoblastic leukemia cell lines express mRNA for the NK-1R andthat in such cell lines, as in the SCLC and NSCLC cell lines stud-ied here, the NK-1R is involved in tumor cell viability [16,23]. Theactivation of the NK-1R by SP is known to induce mitogenesis inseveral cancer cell lines [13,17,18,22,23,25]. This is also in agree-ment with the findings reported here. The presence of NK-1Rs andSP in human primary SCLC and NSCLC samples is in agreement withprevious works carried out on primary melanoma and keratocys-tic odontogenic tumors [8,23]. The presence of SP in the nuclei ofthe tumor cells suggests that the peptide could act as a geneticmodulator. However, our results are not in agreement with thoseof a previous study in which, upon using an autoradiography tech-nique, SP receptors were rarely found in non-small-cell carcinomasof the lung [9]. Here, using an immunocytochemical technique wefound NK-1 receptors in all the lung samples studied. It seems thatthe autoradiography analysis is not sufficiently sensitive to revealSP receptors in most NSCL cells. This could be due to the fact thatthe number of SP receptors expressed in NSCL cells is insufficientto be detected by autoradiography. By contrast, the NK-1 receptorantibody used here might not be specific. This possibility can beruled out because the immunocytochemical controls carried out inthis and previous studies (omission of the primary antibody, preab-sorption of the primary antibody with the corresponding syntheticpeptide, the use of human gliomas as positive controls) revealed thespecificity of the anti-NK-1 receptor antibody used here [see 8,23].Moreover, it should be noted that in the same human lung histolog-ical section we observed cells expressing NK-1 receptors and cellsnot expressing them; that after eliminating NK-1 receptors in SCLCand NSCLC cells by a knockdown method we demonstrated that theNK-1 receptor is involved in the viability of these cells, and that thelung cancer cells studied here express mRNA for the NK-1 receptor.

The results found here are also in agreement with previous stud-ies on lung cancer, since synthetic analogs of SP and the three NK-1Rantagonists tested in this study inhibited growth and induced celldeath by apoptosis in both SCLC and NSCLC cells [12,26,28,32].Previous studies have also demonstrated that NK-1R antagonistsexert an apoptotic effect on other human cancer cell lines [19,20].

We observed that the cell death noted here was due to a spe-cific toxic effect of NK-1R antagonists and not to a non-specificaction of these drugs, demonstrating for the first time in lung can-cer cell lines the specificity of NK-1R blockade by antagonists. The

M. Munoz et al. / Peptides 38 (2012) 318–325 323

Fig. 6. (A) a–d: Large cell lung carcinoma (case 13, Table 2). a: Hematoxylin–eosine-stained 40×; b: NK-1R 20×; c: NK-1R 40×; d: negative control 20×. e–h: Large cellneuroendocrine carcinoma (case 15, Table 2). e: Hematoxylin–eosine-stained 40×; f: NK-1R 20×; g: NK-1R 40×; h: negative control 20×. i–l: Basaloid carcinoma (case 8,T trol 20H l 20×.

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able 2). i: Hematoxylin–eosine-stained 40×; j: SP 20×; k: SP 40×; l: negative conematoxylin–eosine-stained 40×; b: NK-1R 20×; c: NK-1R 40×; d: negative controhe immunocytochemical technique was repeated at least three times.

lockade by NK-1R antagonists could inhibit both DNA synthesisnd cell proliferation through the mitogen-activated protein kinaseMAPK) pathway [13], and such antagonists could also inhibithe formation of a �-arrestin-containing complex that allows theuclear translocation of ERK1/2, inhibiting proliferation and induc-

ng apoptosis [4]. It has been reported that NK-1R antagonistsecrease the basal phosphorylation of Akt, indicating the presencef a constitutively active form of NK-1R for eliciting apoptosis inumor cells and also causing the cleavage of caspase-3 and the pro-eolysis of poly(ADP-ribose) polymerase [1]. The death of tumorells occurs after activation of the apoptotic machinery and thiseans that the induction of apoptosis represents one of the most

ppropriate methods for cancer treatment. Tumor cells need to

et up strategies to neutralize the multiple pathways leading toell death, and it may be proposed that at least one of the mostmportant is the activation and/or an increase in the phenotypicxpression of the NK-1R [5]. Increased NK-1R expression renders

×. (B) a–d: Small cells lung carcinoma (case 12, Table 2; e–h: case 14, Table 2). a:e: Hematoxylin–eosine-stained 40×; f: SP 20×; g: SP 40×; h: negative control 20×.

tumor cells highly dependent on the SP stimulus, a potent mitoticsignal. The increased SP-mediated mitogenic signal could counter-act the different death signal pathways activated in each tumor celldue to its own genetic damage, oncogene activation, etc. Lack of thismitogenic signal after the receptor has been blocked with the NK-1R antagonist could make the balance inside the cell favorable toapoptotic/death signals, leading to cell death. Thus, a number ofdifferent death signals are overriden by the SP-mediated mitoticstimulus; by cutting the potent mitotic signal induced by SP, NK-1R antagonists leave the cell alone with its death load or at leastrender the balance between life and death signals favorable to thelatter [5].

Active tumor cell migration is a crucial requirement for the

development of metastasis and cancer progression, and neoan-giogenesis is a hallmark of tumor development. SP inducesthe migration of tumor cells by binding to NK-1Rs in cancercells, the growth of capillary vessels and the proliferation of

324 M. Munoz et al. / Peptides 38 (2012) 318–325

Table 2Clinical findings. Evaluation of the expression of NK-1R/SP and its immunolocalization in tumor cells of lung cancer samples: (A) NSCLC and (B) SCLC.

Patients Clinical data Percentage of tumor cellsshowing immunoreactivityfor NK-1 receptors

Percentage of tumor cellsshowing immunoreactivityfor SP

Age Sex TNM at diagnosis Comments Nucleus Cytoplasm Nucleus Cytoplasm

A: Non-small cell lung cancer1 59 Male T2, N2 10 90 90 902 66 Male T2, N1 10 80 80 603 73 Male T2, N0 Large cell neuroendocrine carcinoma 10 70 70 504 67 Male T2, N0 20 80 70 405 51 Female T2, N0 10 100 60 506 71 Male T2, N0 Large cell neuroendocrine carcinoma 20 70 60 607 69 Male T2, N0 5 80 60 608 57 Male T2, N2 Basaloid carcinoma 10 70 80 509 56 Male T1, N0 Large cell neuroendocrine carcinoma 30 70 60 4010 46 Male T2, N0 5 60 70 5011 55 Male T1, N0 Large cell neuroendocrine carcinoma 20 70 70 6012 74 Male T2, N0 10 70 70 6013 62 Male T2, N0 20 50 80 4014 77 Male T1, N0 30 90 10 3015 82 Male T2, N2 Large cell neuroendocrine carcinoma 10 60 70 50

Patients Clinical data Percentage of tumor cells showingimmunoreactivity for NK-1 receptors

Percentage of tumor cells showingimmunoreactivity for SP

Age Sex TNM at diagnosis Nucleus Cytoplasm Nucleus Cytoplasm

B: Small cell lung cancer1 60 Male T2, N2 10 60 80 202 71 Male T3, N0 10 50 30 503 71 Male T1, N0 10 60 50 404 79 Male T3, N2 20 50 70 205 76 Male T3, N2 10 70 20 106 51 Male T3, N2 80 80 80 307 69 Female T2, N1 20 80 20 208 77 Male T3, N1 10 60 60 309 71 Male T3, N1 10 90 50 3010 57 Male T2, N0 10 30 40 1011 63 Male T2, N1 10 30 30 3012 68 Male T2, N1 40 90 30 10

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13 60 Male T2, N0

14 71 Male T2, N0

15 76 Male T2, N2

ndothelial cells, these actions being blocked by NK-1R antago-ists [6,11,14,27,31,33]. Vessel neoformation has been associatedith increased tissue innervation and expression of NK-1Rs [30].

n fact, in a large majority of the tumors investigated NK-1Rs haveeen found in intra- and peritumoral blood vessels [9,23]. This is ingreement with the results reported here, since we observed NK-Rs in smooth muscle cells and in endothelial cells of the small-nd medium-caliber blood vessels, located in the peritumoral andn the intratumoral regions of the lung cancer samples.

. Conclusions

In sum, we have performed an in-depth study of the involve-ent of the SP/NK-1R system in lung cancer. Our findings suggest

hat the NK-1R could be a new candidate target in the treatmentf SCLC and NSCLC and that NK-1R antagonists (e.g., the drugprepitant (Emend)) could be a novel, promising antitumor drug inung cancer therapy, since they could exert an antitumoral actionhrough three mechanisms: (1) an antiproliferative effect, due tohe inhibition of tumor cell growth, inducing cell death by apopto-is; (2) an inhibition of angiogenesis in the tumor mass; and (3)n inhibition of tumor cell migration (invasion and metastasis). It

hould be noted that the findings described are not exclusive toung cancer cells, since there are sufficient data to suggest that aommon mechanism for cancer cell proliferation mediated by SPnd the NK-1 receptor occurs, and that NK-1 receptor antagonists

10 90 60 5020 70 50 1010 60 60 20

might be considered as broad-spectrum antineoplastic drugs. Thisshould be confirmed definitively in the coming years.

Conflicts of interest

USPTO Application no. 20090012086 “Use of non-peptidic NK-1receptor antagonists for the production of apoptosis in tumor cells”(Miguel Munoz).

Acknowledgements

This work was supported by the Consejería de Innovacion, Cien-cia y Empresa of the Regional Government of Andalucía (CTS-2247,Spain). The authors wish to thank Dr. José Palacios for providinglung cancer samples, and Mr. Manuel Sánchez and Mr. FranciscoJesus Fuentes for technical assistance and Mr. Nicholas Skinner forsupervising the English text.

References

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