Involvement of substance P and the NK-1 receptor in cancer progression

Peptides 48 (2013) 1–9

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Involvement of substance P and the NK-1 receptor in cancer progression ˜ a,∗ , Rafael Covenas ˜ b Miguel Munoz a

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

a r t i c l e

i n f o

Article history: Received 3 June 2013 Received in revised form 29 July 2013 Accepted 29 July 2013 Available online 7 August 2013 Keywords: Angiogenesis Cancer Metastasis NK-1 receptor NK-1 receptor antagonists Substance P

a b s t r a c t Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis. © 2013 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of the SP/NK-1 receptor system in cancer progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. SP and the NK-1 receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. SP is present in tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. The NK-1 receptor is expressed in tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. The NK-1 receptor is overexpressed in tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. The NK-1 receptor is involved in the viability of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. SP induces the proliferation of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. SP induces the migration of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. SP stimulates neoangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. The SP/NK-1 receptor system is altered in chronic inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10. The SP/NK-1 receptor system and stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NK-1 receptor antagonists as antitumor drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. NK-1 receptor antagonists inhibit tumor cell growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. NK-1 receptor antagonists inhibit the migratory activity of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. NK-1 receptor antagonists exert antiangiogenic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. NK-1 receptor antagonists exert antiinflammatory, antidepressant and anxiolytic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Hospital Infantil Universitario Virgen del Rocío, Unidad de Cuidados Intensivos Pediátricos, Av. Manuel Siurot s/n, 41013 - Sevilla, Spain. Tel.: +34 955012965; fax: +34 955012921. ˜ E-mail address: [email protected] (M. Munoz). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.07.024

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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Numerous studies have reported the involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer (see [60] for review). SP shows great affinity for the tachykinin NK-1 receptor, which is widespread throughout the body. SP/NK-1 receptors have been detected in tumor cells and in intra- and peri-tumoral blood vessels [31,57,59–61]. SP induces mitogenesis in normal and tumor cells, protecting the latter from apoptosis and controls the migration of tumor cells [13,21,59]. This is extremely important since the prevention of metastasis is a major goal in the treatment of tumors, because over 90% of cancer deaths are derived not from the primary tumor but from the development of metastases. Moreover, it has recently been reported that the extravasation of tumor cells into the brain to form cerebral metastases may be an SP-mediated process [43]. It is known that malignant tissues express more NK-1 receptors than benign tissues and that tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression [23,59,60] (Fig. 1). Moreover, the expression of the precursor of SP (preprotachykinin A) is increased in breast cancer in comparison with that found in normal mammary epithelial cells [80]. Thus, it seems that tumor cells depend strongly on the potent mitotic signal mediated by SP and that by means of the overexpression of the NK-1 receptor tumor cells neutralize their own pathways, leading to cell death [55–57,59–61,63,76]. This means that the NK-1 receptor could be a specific molecular target for the treatment of cancer, since tumor cells overexpress NK-1 receptors. This could be an excellent strategy for the treatment of cancer (using NK-1 receptor antagonists), because the cytostatic drugs currently used in clinical practice are not specific against tumor cells and hence elicit serious side effects. In agreement with the above, there are numerous data showing that non-peptide NK-1 receptor antagonists (e.g., L-733,060, L-732,138, the drug aprepitant. . .) exert antitumor, antiangiogenesis and antimetastasis actions (see [59,60] for review). These antagonists cause apoptosis in tumor cells, this antitumor action being dose-dependent [55–57,61,63,76]. Aprepitant (Emend, MK869, L-754,030) is an NK-1 receptor antagonist with no serious side-effects that is currently used in clinical practice for the treatment of chemotherapy-induced nausea and vomiting [54]. In in vitro experiments, this drug exerts a potent antitumor action against a broad number of different types of human tumor cells [55–57]. In sum, all the data reported above, taken from in vivo and in vitro experiments, suggest that novel possibilities for translational research are emerging to improve the diagnosis and treatment of cancer. Here, we review the involvement of the SP/NK1 receptor system in cancer progression and, specifically, the use of NK-1 receptor antagonists as antitumor drugs.

2. Involvement of the SP/NK-1 receptor system in cancer progression There is a large body of evidence demonstrating that peptides are involved in cancer development. In particular, many authors have focused on the involvement of SP in cancer. The results obtained from such work suggest that a common mechanism for

Fig. 1. Involvement of the SP/NK-1 receptor system in cancer progression. Tumor cells express SP and the undecapeptide is located in the nucleus and in the cytoplasm of these cells. NK-1 receptors are overexpressed and are involved in the viability of tumor cells. After binding to the NK-1 receptor located in the plasma membrane, SP and NK-1 receptor antagonists exert opposite effects in tumor cells. SP could induce mitogenesis via autocrine (SP is secreted from tumor cells), paracrine (SP exert a mitogenic action in endothelial cells) and/or endocrine (SP is secreted from the tumor mass into the blood vessels) mechanisms. SP is also released from nerve terminals and/or the peptide reaches the whole body through the bloodstream (this is regulated by the limbic system).

cancer cell proliferation mediated by SP and the NK-1 receptor is activated (Fig. 1). Summarizing the data, the following key-points are relevant: 2.1. SP and the NK-1 receptor SP is an undecapeptide widely distributed throughout the body. It is derived from the preprotachykinin A gene and belongs to the tachykinin family of peptides. The biological actions of tachykinins (SP, neurokinin A, neurokinin B. . .) are mediated through the NK1, NK-2 and NK-3 receptors. SP shows the highest affinity for the NK-1 receptor, which means that the biological actions (e.g., pain, neurogenic inflammation, regulation of the cardiovascular system, mitogenesis. . .) exerted by the undecapeptide are mainly mediated by the NK-1 receptor [59,60], although other NK receptors could be also involved (e.g., NK-2). After the binding of SP to the NK-1 receptor, both are internalized into endosomes; the undecapeptide induces a clathrin-dependent internalization of the receptor, after which SP is degraded and the NK-1 receptor is recycled to the cell surface [9,24,28,48]. SP-NK-1 receptor binding can generate second messengers (cAMP accumulation via stimulation of adenylate cyclase; stimulation, via phospholipase C, of phosphatidyl inositol turnover, leading to calcium mobilization; arachidonic acid mobilization via phospholipase A2), triggering numerous effector mechanisms involved in cellular excitability and in the regulation of the cell function [51,59,60]. However, the effects of other tachykinins (e.g., hemokinin-1 (HK-1), neurokinin A) should be studied in depth since HK-1 is a potent ligand for the NK-1 receptor expressed in non-neural

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tissues. HK-1 enhances B cell proliferation and antibody production and activates the MAPK pathway [90], promotes the survival of dendritic cells [34], enhances the proliferation of T-cell precursors, increases the number of thymocytes [93] and decreases blood pressure [5]. Moreover, it has been demonstrated that HK-1, in contrast to SP, induces the proliferation of human pre-B lymphocytes [29], that HK-1 and the NK-1 receptor are expressed in human B-lymphocytes, but that these cells do not express SP [29], and that hemokinins promote angiogenesis through the NK-1 receptor [81]. It is also known that SP and HK-1 exert an antiapoptotic effect on bone-marrow-derived dendritic cells (which are the preferred targets for immunotherapy protocols). This effect carried out via the NK-1 receptor enhances the survival of the dendritic cells both in vitro and in vivo and induces a potent immunestimulatory activation in such cells, which promoted a robust cellular immunity [34]. A co-regulated decrease of NK-1 receptor and HK-1 gene expression has been demonstrated in monocytes and macrophages after activation with pro-inflammatory cytokinines (e.g., interferon-gamma, interleukin-1 beta) [6]. Thus, there are many data available concerning the involvement of HK1 in non-solid tumors but in solid tumors our knowledge is fairly limited. This should be studied in the near future. The exact endogenous agonists for the NK-1 receptor must be clearly established since it has been suggested that SP may be the main agonist in the central nervous system and HK-1 in peripheral tissues. Moreover, the antiproliferative effect of neurokinin A on hematopoietic progenitor cells has been reported, this effect being explained through the synthesis of transforming growth factor beta 1 and macrophage inflammatory protein-1 alpha [88]. This finding suggests that the stimulation of NK-2 receptors could be a beneficial strategy in cancer treatment, in addition to treatment with NK1 receptor antagonists (see below). It is also known that the inhibitory effect of neurokinin A could be reversed by SP and that the antiproliferative action of the peptide is partly mediated by p53 [88].

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2.3. The NK-1 receptor is expressed in tumor cells This receptor has been demonstrated in human cancer cell lines and/or in primary tumors (e.g., glioma, astrocytoma, retinoblastoma, ganglioneuroblastoma, leukemia, neuroblastoma, carcinomas (pancreatic, larynx, gastric, colon, medullary thyroid, breast, oral). . .) [10,15,16,23,27,31,53,56,57,59–63] (Fig. 1). In addition, in most of the tumors investigated NK-1 receptors have been found in intra- and peri-tumoral blood vessels. This is quite important regarding the involvement of the NK-1 receptor in angiogenesis [31]. NK-1 receptors have been located in both the plasma membrane and the cytoplasm of tumor cells and, occasionally, in the nucleus of these cells [27,57,76]. Several isoforms (33–38, 46, 54–58 and 75 kDa) of the NK-1 receptor have been reported in human cancer cells (e.g., neuroblastoma, retinoblastoma, larynx carcinoma, gastric adenocarcinoma, leukemia...) [56,57,61,62,76]. However, in order to clarify the functional roles of these isoforms, further research is needed. In humans, the presence of two subtypes of the NK-1 receptor has been reported: the full-length and the truncated ones. It is known that the former mediates a slow growth of tumor cells and the second enhances the growth of these cells to a considerable extent and stimulates the production of cytokines with growth-promoting functions [71]. It seems that these cytokines activate a transcription factor (NF-␬B) that upregulates the truncated NK-1 receptor form and slightly increases the full-length form [52,74]. It is also known that the truncated form, an oncogenic isoform of the NK-1 receptor, mediates malignancy in tumor cells [71] and that the truncated NK-1 receptor is increased in colonic epithelial cells from patients with colitis-associated cancer [25]. At the present, it is unknown whether the NK-1 receptor antagonists exert or not a similar blockade of both long and short isoforms. Moreover, it has been demonstrated that tumor cells express mRNA for the tachykinin NK-1 receptor and that increased mRNA NK-1 receptor expression occurs in malignant tissues (e.g., breast biopsies), but not in benign ones (normal mammary epithelial cells, benign breast biopsies) [56,80].

2.2. SP is present in tumor cells The undecapeptide has been found in keratocystic odontogenic tumors, oral squamous cell carcinoma, larynx carcinoma, blast cells, melanoma, glioma, retinoblastoma, neuroblastoma, atypical nevi and in spindle and epithelioid nevi [10,16,27,63,65,66,69]. SP has been detected in the cytoplasm and in the nucleus of tumor cells [10,16,27,57] (Fig. 1). The location of SP inside the nucleus suggests that the peptide could act as a genetic modulator [58]. Like other peptides, in normal cells SP is synthesized in the cytoplasm and is located in this part of the cell, but in tumor cells it is more strongly expressed in the nuclei than in the cytoplasm of these cells. What are the implications of this observation? What are the implications in cancer? The location of a peptide in a certain place indicates, a priori, that it could be involved in functions in which the structure containing the peptide is involved [58]. Thus, the high presence of SP within the nucleus of tumor cells means that the peptide could regulate the nuclear activity of tumor cells, and in fact it is known that SP regulates mitogenesis in these cells. Moreover, SP could modify the oncogenetic mechanisms of tumor cells. In the future, this should be investigated in depth. The expression of SP by tumor cells also suggests that the peptide could be secreted by primary tumors (Fig. 1). Moreover, it is known that SP is present in tumor and in peritumoral tissues [57], that the levels of preprotachykinin A (the precursor of SP) and NK-1 receptors are higher in breast cancer cells in comparison with that found in normal mammary epithelial cells, and that breast cancer cells have high levels of SP whereas non tumorigenic cells show very low levels of the undecapeptide [80].

2.4. The NK-1 receptor is overexpressed in tumor cells It is known not only that the NK-1 receptor is expressed in tumor cells, but also that those receptors are overexpressed in such cells (e.g., glioblastoma, breast cancer, retinoblastoma, larynx, pancreatic, gastric and colon carcinomas. . .) [16,23,56,57,59,60,63] (Fig. 1). This is important, since the visualization of NK-1 receptors by immunohistochemistry would facilitate the identification of tumors overexpressing this receptor for diagnostic or therapeutic purposes [78]. It is known that normal cells express a lower number of NK-1 receptors than tumor cells (e.g., human pancreatic cancer cell lines express more NK-1 receptors than control cells) [23]; that tumor samples from patients with advanced tumor stages exhibit significantly higher NK-1 receptor levels [23]; that TACR1 mRNA is present in human acute lymphoblastic leukemia cell lines, with the highest levels in these cells and the lowest ones in normal cells [56]; that astrocytoma/glioma cell lines in culture shows a lower number of NK-1 receptors than astrocytoma/glioma primary tumors; that glioblastomas express more NK-1 receptors than astrocytomas, and that the most malignant phenotypes of tumors show a higher rate of NK-1 receptor expression and were associated with advanced tumor stages and poorer prognosis [23,31,45]. The data suggest that the number of NK-1 receptors could be correlated with the degree of malignancy. Thus, the overexpression of the NK1 receptor in tumor cells (Fig. 1) suggests the possibility of finding a specific treatment against cancer using NK-1 receptor antagonists (see below) and, in this way, the side-effects of the treatment would

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be decreased considerably. This strategy opens up new approaches for cancer treatment. 2.5. The NK-1 receptor is involved in the viability of tumor cells After a knockdown gene-silencing method (siRNA), it has been reported that the NK-1 receptor is involved in the viability of the tumor cells [56,57,63] (Fig. 1). Following the administration of the siRNA TACR1 (tachykinin 1 receptor gene) to cultured tumor cells, more apoptotic cells were found in siRNA cells than in cells not transfected and hence the number of siRNA tumor cells was significantly decreased in comparison with the number of nontransfected cells [56,57,63]. 2.6. SP induces the proliferation of tumor cells It has been demonstrated that SP acts as a mitogen in normal and tumor (e.g., neuroblastoma, astrocytoma, melanoma, retinoblastoma, pancreas carcinoma, glioma, melanoma, larynx carcinoma, gastric and colon carcinoma, lynphoblastic leukemia) cells [56,57,59–63,70,76]. The mitogenic action is carried out via the NK-1 receptor, since the growth inhibition of many human tumor cells after the administration of NK-1 receptor antagonists is partially reversed by the administration of SP [56,57,61–63,76] (Fig. 1). For example, in these competition experiments the cellular concentration at 30 ␮M of aprepitant with 10 nM concentration of SP was higher than that observed with aprepitant alone [57]. The data indicates that SP in a universal mitogen in NK-1 receptorexpressing tumor cells. SP can be synthesized and secreted by tumor and non-tumor cells; the peptide can be released from nerve terminal, and/or it can be released into the blood vessels [59,60]. Through these ways, the peptide can exert a mitogenic action in tumor cells. The regulation of local tumor activity through sensory nerves containing SP is quite important, since the undecapeptide could modulate the growth of tumor cells, exerting a direct interaction between the nervous system and the tumor cells. Thus, SP could induce mitogenesis via the following mechanisms (Fig. 1): (1) autocrine (SP is secreted from tumor cells); (2) paracrine (SP exerts a mitogenic action in endothelial cells); (3) SP is released from nerve terminals; (4) SP reaches the whole body through the bloodstream. This is regulated by the limbic system; and (5) endocrine (SP is released from the tumor mass into the blood vessels) [59,60]. Although the above mentioned works report that SP acts as a mitogen in normal and tumor cells, other work report that the peptide exerts a weak inhibitory effect on the proliferation of human retinal pigment epithelial cells [86]. However, it has been also reported that SP exerts in vitro a stimulating effect on retinal pigment epithelial cell growth [38]. These discrepancies could be due to species differences and/or to methodological procedures [38,86]. After the activation of the NK-1 receptor by SP, an increase in DNA synthesis has been reported in tumor cells, and it seems that via the NK-1 receptor the undecapeptide activates members of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK [45]. Once activated, ERK1/2 is translocated into the nucleus, inducing proliferation and protecting the cell from apoptosis [13,60] (Fig. 1). In tumor cells, SP increases the phosphorylation and activity of Akt or protein kinase B, a serine-threonine protein kinase that becomes activated via phosphatidyl-3-kinase (PI3K); the activation of Akt suppresses apoptosis [64,85]. By contrast, NK-1 receptor antagonists inhibit the basal activity of Akt [1]. Moreover, other effects are exerted by SP after binding to the NK-1 receptor: in human astrocytoma cell lines SP activates phospholipase D and enhances forskolin-stimulated cyclic AMP-production; it induces the release of interleukins, taurine and glutamate; it mobilizes intracellular calcium, induces the formation of inositol phosphate,

stimulates glycogen breakdown and influences glutamate and K+ transport [22,26,35,49,60,87]. The release of interleukins, taurine and glutamate by tumor cells induces an inflammatory process, increasing the levels of SP and hence increasing tumor cell proliferation. In astrocytoma cells, SP stimulates glycogen breakdown and increases the intracellular Ca2+ concentration. Both effects occur in a concentration-dependent manner. These effects are completely blocked by the NK-1 receptor antagonist CP-96,345 [49] and this suggests that such effects are mediated by the NK-1 receptor. Moreover, the Warburg effect occurs because most cancer cells predominantly produce energy by means of a high rate of glycolysis followed by lactic acid fermentation [91]. Growing tumor cells typically have glycolytic rates up to 200 times higher than those of their normal tissues of origin; this occurs even if oxygen is plentiful. Thus, the release of SP from tumor cells produces glycogen breakdown and the glucose obtained would be used by tumor cells to increase their metabolism [49]. This mechanism could in part explain the Warburg effect. By contrast, NK-1 receptor antagonists block the glycogen breakdown in tumor cells [49], and in this way they can counteract the Warburg effect. 2.7. SP induces the migration of tumor cells The migration of tumor cells is a crucial requirement for the development of metastasis and the progression of cancer. At present, over 90% of cancer deaths are derived not from the primary tumor but from the development of metastases [83]. Thus, a major goal in the treatment of cancer should be to inhibit the development of these. In this sense, it is known that tumor cell migration is induced by classical neurotransmitters (dopamine, noradrenaline) and peptides (e.g., SP) and that such migration is inhibited after the administration of D2 receptor, adrenoceptor or NK-1 receptor antagonists [41,60] (Fig. 1). Moreover, it is known that after binding to the NK-1 receptor the undecapeptide induces a rapid change in cellular shape (including blebbing) and that membrane blebbing is important in cell movement, cell spreading, and cancer cell infiltration [18,50]. Moreover, it has recently been reported that SP is involved in pancreatic cancer perineural invasion and that SP induces cancer cell proliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells. SP also promotes neurite outgrowth and the migration of pancreatic cancer cell clusters to the dorsal root ganglia of newborns [44]. In sum, chemical substances regulate metastasis [41,60] and hence new strategies focused on the above data should be developed in the future for the treatment of cancer. 2.8. SP stimulates neoangiogenesis Neoangiogenesis, a hallmark of tumor development, is stimulated by SP (the peptide induces the proliferation of endothelial cells) [94] (Fig. 1). In most tumors investigated, both SP and NK1 receptors are found in intra- and peri-tumoral blood vessels, and in fact during neoangiogenesis both tissue innervation and the expression of NK-1 receptors are increased [31,60]. This means that SP, via the NK-1 receptor, influences vascular structure and function [31,57] both within and around tumors by increasing tumoral blood flow [94] and by fostering stromal development, since NK-2 and NK-3 receptor agonists do not exert a significant effect on the proliferation of endothelial cells [31,60]. 2.9. The SP/NK-1 receptor system is altered in chronic inflammation It has been suggested that chronic inflammation could be correlated with an increased risk of developing cancer (e.g., in the

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Fig. 2. Involvement of the SP/NK-1 receptor system in emotional stress, chronic inflammation and cancer. In all the cases, there is an upregulation of such system. NK-1 receptor antagonists exert antiinflammatory, anxiolytic, antidepressive and antitumor actions.

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cancer progression by activating the SP/NK-1 receptor system and that emotional behavior [14,20], cancer and metastasis [41] might be related through alterations in this system. Accordingly, through this system there could be an interrelationship between the progression of cancer and cerebral mechanisms. The NK-1 receptor expressed in the limbic system is similar to the NK-1 receptor overexpressed in human tumor cells and samples. After binding to NK-1 receptors located in the neurons of the limbic system, SP produces anxiety and depression, whereas after binding to the NK-1 receptors located in human tumor cells it induces cell proliferation, angiogenesis and the migration of these cells for invasion, infiltration and metastasis [59,60]. This mechanism could partly explain the link between the emotional stress (e.g., depression) and cancer progression (Fig. 2). In sum, according to the data reported above the SP/NK-1 receptor system is profoundly involved in cancer and hence several strategies could be developed in order to search for an antitumor action, such as the use of NK-1 receptor antagonists, the use of antibodies against tachykinin receptors or against SP, and/or the use of proteins than can bind to SP. We shall focus the review below on NK-1 receptor antagonists.

gastrointestinal tract), since during the inflammatory processes an increase of mitogenesis and mutagenesis occurs [3,8,46,92]. It is known that tachykinins control the activity of inflammatory cells, that SP induces edema formation, and that both SP and NK1 receptors are up-regulated during the inflammation processes [12,36,37,89]. For example, elevated levels of SP and up-regulated NK-1 receptor expression have been reported in the rectum and colon of patients with inflammatory bowel disease [67]. Moreover, in rats, NK-1 receptor antagonists have been used as antiinflammatory agents [4]. All these data suggest that the SP/NK-1 receptor system is up-regulated in chronic inflammation and that the alteration of this system could facilitate the development of cancer (Fig. 2). Moreover, it has recently been reported that the truncated NK-1 receptor is overexpressed in colonic epithelial cells from patients with colitis-associated cancer, whereas the full-length is not affected [25]. Thus, the overexpression of NK-1 receptors could be used as a diagnostic marker to identify patients at risk of neoplasms and may serve as a useful therapeutic target in the treatment of chronic inflammation associated cancer.

NK-1 receptor antagonists are classified in peptide or non-peptide (WIN- 51,708, RP-67,580, RP-73,467, RPR-100,893, CP-96,345, L-709,210, CP-99,994, GR-203,040, GR-205,171 (Vofopitant), CP-122,721, L-733,060, L-741,671, L-742,694, L-732,138, aprepitant) antagonists [59,60]. We shall focus our review on the second type, since peptide NK-1 receptor antagonists suffer from a number of drawbacks (e.g., poor potency and ability to discriminate between tachykinin receptors, neurotoxicity. . .). After binding to the NK-1 receptor, NK-1 receptor antagonists block the pathophysiological actions mediated by NK-1 receptor agonists and could therefore be used as therapeutic agents. In fact, these antagonists exert analgesic, antidepressive, antiinflammatory, anxiolytic, antimigraine, neuroprotector, hepatoprotector, anticonvulsant, anticolestasis, antipruritus, antiviral and antiemetic effects, as well as exerting antitumor, antiangiogenic and antimigration actions [59,60,68].

2.10. The SP/NK-1 receptor system and stress

3.1. NK-1 receptor antagonists inhibit tumor cell growth

It is known that depression and cancer co-occur commonly. The prevalence of depression among cancer patients increases with disease severity and with symptoms such as pain and fatigue. Moreover, chronic and severe depression may be associated with an elevated risk of developing cancer [82]. There is evidence suggesting that depression predicts cancer progression and mortality and that by providing psychosocial support to cancer patients’ depression, anxiety, and pain can be reduced [82]. Moreover, such support may increase the survival time of patients with cancer [82]. Thus, there is a bi-directional relationship between cancer and depression, offering new opportunities for therapeutic interventions [82]. All these data suggest that psychological factors are involved in cancer progression [32], but although the link is absolutely clear the underlying mechanism remains unknown. SP and NK1 receptors are present in the limbic system, including the hypothalamus and the amygdala. In addition, SP may be involved in the integration of emotional responses to stress, suggesting the possibility that the pathogenesis of depression could be due to an alteration of the SP/NK-1 receptor system [40]. In fact, in depression an increase in the production of SP has been reported [40]. The increase level of SP could accelerate cancer progression. The above data suggest that depression could induce

NK-1 receptor antagonists (L-733,060, L-732,138, the drug aprepitant. . .) exert an antitumor action [55–57,59–63,76] (Fig. 1). In particular, these antagonists exert such action against human glioma, larynx carcinoma, neuroblastoma, rhabdomyosarcoma, leukemia, astrocytoma, osteosarcoma, lymphoma, retinoblastoma, melanoma, lung, breast, and gastric, pancreas and colon carcinoma cell lines [7,33,55–57,59–63,76]. It is known that the antitumor action of L-733,060 against human cancer cell lines is more potent than that of aprepitant, and that the antitumor action of aprepitant is more potent than that of L-732,138 [59,60]. NK-1 receptor antagonists block the SP-induced mitogen stimulation of tumor cells, as well as inhibiting tumor cell growth in a dose-dependent manner [59,60]. After binding to the NK-1 receptors overexpressed in tumor cells, NK-1 receptor antagonists activate the apoptotic machinery and these cells (e.g., neuroblastoma...) die by apoptosis [55–57,59,60,76] (Fig. 1). Thus, the induction of apoptosis represents a highly suitable approach to cancer treatment, although currently the mechanisms responsible for inducing apoptosis in tumor cell are in general unknown. Despite this, it has been reported that the blockade of NK-1 receptors by NK-1 receptor antagonists inhibits the basal kinase activity of Akt. Tumor cells

3. NK-1 receptor antagonists as antitumor drugs

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develop strategies to neutralize the multiple pathways leading to cell death, and it has been suggested that one of the most important of these is the expression of the NK-1 receptor [17]. This strategy renders tumor cells highly dependent on the SP stimulus, which provides a potent mitotic signal. This signal could counteract the different death signal pathways activated in tumor cells. The absence of the mitotic signal when the receptor is blocked with NK-1 receptor antagonists could tilt the balance within the cell to favoring apoptotic/death signals, and hence the cell dies [17]. The data reported suggest that NK-1 receptor antagonists could inhibit a large number of tumor cell types in which NK-1 receptors are overexpressed [56,57,59,60,63], and that NK-1 receptor antagonists could be candidates for broad-spectrum antineoplastic drugs [59,60]. In general, the administration of NK-1 receptor antagonists does not induce serious side-effects [11,54,60,72,75,84], although headaches, hiccupping, vertigo and drowsiness have been reported in humans after their administration [54,75,84]. The safety of aprepitant against human fibroblasts has been demonstrated, the IC50 for fibroblasts is three times higher than the IC50 for tumor cells [55]. Moreover, the IC50 for non-tumor cells is 90 ␮M but the IC100 for tumor cells is 60 ␮M approximately [55]. In a murine model, it has been reported that SP provides in vivo protection against tumor growth (prevents or delays tumor establishment), this action being mediated by natural killer and T cells [47]. In animal models the antitumor action of NK-1 receptor antagonists has also been reported. For example, it has been demonstrated that SP analog antagonists (synonymous of peptide NK-1 receptor antagonists) cause tumor regression in athymic nude mice [73], inhibit the tumor growth of an H-69 xenograft in nude mice [79] and attenuate tumor growth in HPAF-II xenografts in nude mice, involving both antiproliferative and antiangiogenic mechanisms [30], whereas the non-peptide NK-1 receptor antagonist MEN-11,467 (11.7 ␮M–1.4 mg/kg/day, 2 weeks) diminishes tumor volume [7,69]. However, it has been reported that aprepitant (doses ranging from 3 to 300 mg/kg/day, 3 days treatment) does not exert an antitumoral effect in vivo experiments [42]. The lack of results of aprepitant could be due to the methodology (e.g., the animals were only treated with aprepitant for 3 days; the animals were sacrificed 48 h after the administration of the last doses of aprepitant; aprepitant was administered parenterally, not orally, and its intravenously administered prodrug fosaprepitant was not used in the study. . .) and/or to the animal used. Thus, before developing clinical trials in humans more in-depth in vivo studies must be carried out. It has been suggested that the coadministration of NK-1 receptor antagonists and microtubule destabilizing agents (e.g., vinblastine) could be useful in cancer, since these compounds have a synergic effect [39,60]. This combination is synergistic for the growth inhibition of NK-1 receptor-possessing cancer cells, but not for normal cells. A better understanding of the mechanisms underlying this interaction is needed in order to assess the clinical relevance of this novel synergistic combination. Moreover, it has been reported that the use of chemotherapy and/or radiation therapy and NK1 receptor antagonists affords a synergistic antitumor action and decreases the side effects of chemotherapy and radiation therapy [2,39,60]. Carcinogenicity studies have been carried for aprepitant and fosaprepitant in mice and in rats [19]. In male mice, aprepitant induced an increase in hepatocellular adenomas and/or carcinomas (1000–2000 mg/kg/day) and skin fibrosarcomas (125–500 mg/kg/day). In male rats, treatment with aprepitant (1000 mg/kg twice daily) caused an increase in thyroid parafollicular cell carcinoma and in thyroid follicular cell adenomas and carcinomas (5–1000 mg/kg twice daily), whereas in female rats hepatocellular adenomas (5–1000 mg/kg twice daily) and hepatocellular carcinomas and thyroid follicular adenomas

(125–1000 mg/kg twice daily) were reported. It seems that the carcinogenetic effects of the drug aprepitant could be involved with the hepatic CYP metabolism: liver and thyroid tumors of these types are a species-specific consequence of hepatic CYP enzyme induction in rodents, and are consistent with the changes observed in rodents with other structurally and pharmacologically dissimilar compounds that have been shown to induce hepatic CYP enzymes. Regarding skin fibrosarcomas, very high doses during a long period of time are required; this paradoxical effect could be involved in the non-specific toxic property of the chemical compound at very high doses. However, the carcinogenetic safety of aprepitant compared with cytostatic drugs is the antithesis, because cytostatics are not specific drugs that act in all types of cell and moreover they induce genetic alteration in both normal and tumor cells. By contrast, NK-1 receptor antagonists could act selectively against tumor cells. Moreover, extrapolating the aprepitant concentrations used as an antitumor agent in vitro studies, the doses of aprepitant for the possible treatment of cancer would be very low in comparison with carcinogenetic doses. 3.2. NK-1 receptor antagonists inhibit the migratory activity of tumor cells NK-1 receptor antagonists block changes in cellular shape (including blebbing) mediated by SP [41,50]. This is very interesting, since membrane blebbing is important in cell spreading and cancer cell invasion [18,60]. In fact, it is known that L-733,060 inhibits SP-mediated increased migratory activity of tumor cells and that SP induces the migration and invasion of gastric adenocarcinoma cells [21,60] (Fig. 1). Moreover, SP induces cancer cell proliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells, whereas NK-1 receptor antagonists inhibit these effects. SP also promotes neurite outgrowth and the migration of pancreatic cancer cell clusters to the dorsal root ganglia of newborns, which is blocked by NK-1 receptor antagonists [44]. In sum, these data open up new perspectives for a specific inhibition of tumor cell invasion and metastasis, since the migration of tumor cells is a prerequisite for invasion and metastasis and is dependent on chemical substances (e.g., SP). 3.3. NK-1 receptor antagonists exert antiangiogenic properties NK-1 receptors are highly expressed in blood vessels located in both the tumor mass and peritumoral tissues [23]. NK-1 receptor antagonists decrease tumor-associated angiogenesis and block the mitogenesis of endothelial cells mediated by SP [30,59,60] (Fig. 1). By contrast, angiogenesis is enhanced after the administration of SP/NK-1 receptor agonists, since these increase the mitogenesis of the endothelial cells, stimulating vessel growth [94]. These data indicate that the SP/NK-1 receptor system controls angiogenesis. 3.4. NK-1 receptor antagonists exert antiinflammatory, antidepressant and anxiolytic effects SP and NK-1 receptors are overexpressed in chronically inflammation and in emotional stress (depression and anxiety) [4,40,59,60,77]. By contrast, after binding specifically to NK-1 receptors NK-1 receptor antagonists could improve these diseases in a concentration-dependent manner by blocking the pathophysiological actions mediated by SP (Fig. 2). In fact, the use of NK-1 receptor antagonists could not only improve cancer treatment but could also prevent the appearance/development of the above-mentioned diseases. It has been demonstrated that the NK-1 receptor aprepitant (300 mg/day) exerts a similar antidepressant effect than paroxetine and that it was well tolerated and no

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statistically significant difference in the frequency of adverse events was observed as compared to placebo [54].

4. Conclusions The SP/NK-1 receptor system plays an important role in the development of cancer, metastasis and neoangiogenesis (Fig. 1). It seems that SP acts as a universal mitogen for tumor cells overexpressing NK-1 receptors and that NK-1 receptor antagonists also induce a universal effect on tumor cells, namely, apoptosis. Research into the involvement of the SP/NK-1 receptor system in cancer progression must be developed in depth in forthcoming years since it is necessary to explore new and effective therapeutic interventions in cancer research. It is important to seek strategies targeting tumor-specific molecular derangements. It is of huge importance to identify novel molecular targets for blocking tumor growth. This is the case of the NK-1 receptor, which is overexpressed in tumor cells. NK-1 receptor inhibitors induce the death of tumor cells by apoptosis (pharmacological (NK-1 receptor antagonists) and genetic (small interfering RNA gene-silencing) treatments induce the death of tumor cells). Accordingly, the NK-1 receptor is a promising target in cancer treatment and NK-1 receptor antagonists could be considered as antitumor drugs for the treatment of tumors. This conclusion is based in the following data (Fig. 1): (1) After binding to the NK-1 receptor, SP induces tumor cell proliferation, the migration of tumor cells (invasion and metastasis) and angiogenesis; and (2) NK-1 receptor antagonists inhibit tumor cell proliferation (tumor cells die by apoptosis), block the migratory activity of tumor cells and exert antiangiogenic properties. Thus, it is urgently necessary to test the antitumor action, the antimetastatic activity and the antiangiogenic action of NK1 receptor antagonists in human clinical trials. In this sense, the antitumor action of NK-1 receptor antagonists already available in clinical practice for the treatment of emesis (e.g., aprepitant) should be tested in clinical trials. It has previously been reported that the administration of aprepitant is well tolerated and is associated with minimal side effects. It seems that by increasing the number of days on which aprepitant is currently administered and using higher doses of aprepitant than those used in chemotherapyinduced nausea and vomiting this NK-1 receptor antagonist could be effective in cancer. These issues should be investigate in depth. By increasing the dose of aprepitant, higher and undescribed side effects may occur, although it has been reported that in patients with depression a dose of 300 mg/day of aprepitant was well tolerated and no significant difference in the frequency of adverse events was observed as compared with placebo. It is also known that, in vitro, aprepitant exerts an antitumor action (tumor cells die by apoptosis) against a broad number of different types of human tumor cells. Moreover, in the future, the combination of cytostatics and NK-1 receptor antagonists should be also studied. In sum, all the data point to the notion that the NK-1 receptor could be a therapeutic target in cancer.

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

Acknowledgments The authors thank N. Skinner (University of Salamanca, Spain) for stylistic revision of the English text. The technical assistance of ˜ (Virgen del Rocío University Hospital, Sevilla, Dr. Miguel E. Munoz

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