The NK1 receptor is involved in the antitumoural action of L-733,060 and in the mitogenic action of substance P on neuroblastoma and glioma cell lines

Abstract

We have carried out an in vitro study to investigate the ability of substance P to activate cell growth and the NK1 receptor antagonist L-733,060 to inhibit cell growth in the SKN-BE(2) neuroblastoma and GAMG glioma cell lines. A coulter counter was used to determine viable cell numbers, followed by application of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium], inner salt, colorimetric method to evaluate cell viability in this cytotoxicity assay. Nanomolar concentrations of substance P increased, and micromolar concentrations of L-733,060 inhibited the growth of both cell lines studied, with and without previous administration of substance P. In addition, we have demonstrated by immunoblot analysis that NK1 receptors are present in both cancer cell lines studied here. Thus, this study demonstrates that substance P acts as a mitogen in the SKN-BE(2) neuroblastoma and GAMG glioma cell lines, and that the antitumoural action of L-733,060 on both human cell lines occurs through the NK1 receptor. This action suggests that the NK1 receptor is a new and promising target in the treatment of human neuroblastoma and glioma.

Introduction

Substance P (SP) belongs to the tachykinin family of peptides, the biological actions of tachykinins being mediated by three receptors – neurokinin (NK)-1, NK-2 and NK-3, – the NK-1 receptor showing preferential affinity for SP. After binding to the NK1 receptor, this peptide regulates many biological functions (von Euler and Gaddum, 1931, Pernow, 1983, Hökfelt et al., 2001).

SP has been found in human neuroblastoma cells of the parotid gland (Shrestha et al., 1994); it is expressed in almost 90% of the metastatic neuroblastoma cells in bone marrow (Nowicki and Miskowiak, 2002), and it has been documented in human malignant gliomas (Palma et al., 1999, Palma et al., 2000). Furthermore, astrocytomas and gliomas contain significant levels of NK1 receptors (Palma et al., 1999, Palma et al., 2000) and it is also known that activation of the NK1 receptor induces mitogenesis in several cell types (Payan et al., 1983, Nilsson et al., 1985, Lotz et al., 1987, Ziche et al., 1990, Muñoz et al., 2004b) and that substance K, an elongated form of neurokinin A, induces mitogenesis (Nilsson et al., 1985, Sharif et al., 1996). In addition, in several glial brain tumour-derived cell lines, the presence of NK1 receptors is strictly correlated with the effect of SP and/or neurokinin A on increases in DNA synthesis and cellular proliferation (Luo et al., 1996, Palma et al., 1999, Sharif et al., 1996). Thus, for example, the activation of NK1 receptors induces mitogenesis in human astrocytoma (Luo et al., 1996) and glioma cells (Palma et al., 1999, Palma et al., 2000).

L-733,060 is a selective, potent and long-acting central non-peptide tachykinin NK1 receptor antagonist showing high affinity for the human NK1 receptor in vitro (Rupniak et al., 1996). It is known that administration of L-733,060 produces analgesia (Rupniak et al., 1996) and antidepressive effects (Kramer et al., 1998, Varty et al., 2003), and it has been used in the treatment of a broad range of anxiety and mood disorders (Rupniak et al., 2000) and inflammatory liver disease, most likely by inhibiting SP effects (Bang et al., 2003). Moreover, we have recently demonstrated that L-733,060 displays antitumoural activity against human SKN-BE(2) neuroblastoma and GAMG glioma cell lines (Muñoz et al., 2004a).

Currently, we are unaware of whether the cell death observed in the SKN-BE(2) and GAMG cell lines might be due to a general toxic effect of L-733,060 or to a specific action of this substance on the NK1 receptors in these tumour cell lines (Muñoz et al., 2004a). It is also unknown whether SP exerts a mitogenic action or not on both tumour cell lines, and whether the NK1 receptors are present in the SKN-BE(2) and GAMG cell lines. Thus, the aims of this study were: (1) to demonstrate, using a MTS colorimetric method to evaluate cell viability, that the antitumoural action of the NK1 receptor antagonist L-733,060 occurs through a specific action on the NK1 receptor; (2) to study the role of SP and the NK1 receptor in the induction of SKN-BE(2) and GAMG cell proliferation; and (3) to demonstrate the presence of NK1 receptors in both cancer cell lines.