Differential effect of intestinal neuropeptides on invasion and migration of colon carcinoma cells in vitro

doi:10.1016/S0304-3835(97)00167-5

Cancer Letters

Volume 116, Issue 1, 3 June 1997, Pages 111-116

https://doi.org/10.1016/S0304-3835(97)00167-5 Get rights and content

Abstract

We investigated the effect of neuropeptides, which are vasoactive intestinal polypeptide (VIP), substance P, (SP), neuropeptide Y (NPY), neurokinin A (NKA), somatostatin (SOM), calcitonin gene-related peptide (CGRP), and leucine-enkephalin (L-ENK), on the invasion of murine Colon 26-L5 adenocarcinoma cells through a reconstituted basement membrane (Matrigel) using a Transwell cell culture chamber assay. VIP, SP, NPY, and L-ENK reduced invasive potential of tumor cells in a concentration-dependent manner, whereas SOM, CGRP, and NKA had no effect. Especially, VIP showed the most effective in inhibiting tumor invasion, and achieved 50% reduction at 10⁻⁶ M. A similar effect by VIP was also observed in cell migration to fibronectin. VIP had no effect on the growth of tumor cells at the concentrations ranging from 10⁻¹⁰ to 10⁻⁶ M. The suppressed ability of the tumor cell motility by VIP (10⁻⁶ M) was practically recovered by co-treatment with 2′,5′-dideoxyadenosine, an adenylate cyclase inhibitor. These results indicate that VIP, among the neuropeptides used, could inhibit Matrigel invasion of Colon 26-L5 carcinoma cells through partial suppression of their motility, and the reduction was associated with an intracellular cAMP-mediated pathway.

Introduction

Metastasis of cancer is composed of multiple steps via interactions between cancer cells and extracellular matrix (ECM). The process is divided into three steps [1]: (1) tumor cell attachment to the matrix components, (2) local degradation of matrix by tumor cell associated proteases, and (3) tumor cell locomotion into the region of the matrix modified proteolysis. During the sequential steps of metastatic cascade, tumor cells should be affected by many kinds of organ-derived factors, such as cytokines [2]and ECM components [3]in a microenvironment.

Neuropeptides, released from peripheral as well as central nervous system, are important for functional regulation in a microenvironment of the tissues in physiological states. In colon tissue, several neuropeptides such as vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), neurokinin A (NKA), somatostatin (SOM), and leucine-enkephalin (L-ENK) are observed [4], and the contribution of neuropeptides to the local regulation of the function in the tissues have been elucidated. Some of these neuropeptides such as VIP, NPY, NKA, and SP affect intestinal water and electrolyte secretion via epithelial cell surface receptor 5, 6. Moreover, VIP, SP, and CGRP show vasodilative effect 7, 8, 9, whereas NPY shows vasoconstrictive effect [10]. L-ENK inhibits intestinal contraction [11], while SP promotes it [12]. In the regulation of tumor invasion, it is reported that VIP increased the invasive potential of prostatic carcinoma cells [13]. However, the role of these neuropeptides on invasive ability of colon carcinoma cells remains unclear.

In this study, we investigated the effect of colon tissue-associated neuropeptides, which are VIP, SP, NPY, NKA, SOM, CGRP, and L-ENK, on invasion of reconstituted basement membrane (Matrigel) by Colon 26-LS carcinoma cells in vitro.

Section snippets

Cells and cell culture

Murine Colon 26-L5 adenocarcinoma cell line (highly liver-metastatic), derived from Colon 26, was kindly provided by Dr. Y. Sato (Hokkaido University School of Medicine, Sapporo, Japan). The cell line was maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), l-glutamine, and 2-mercaptoethanol, 10² U/ml penicillin, and 0.1 mg/ml streptomycin.

Reagents

Synthetic VIP, SP, NPY, CGRP, NKA, SOM, and L-ENK were purchased from Peptide Institute, Inc., Osaka, Japan.

Cell invasion assay

Tumor cell invasion through

Effect of neuropeptides on invasion of Matrigel by Colon 26-L5 adenocarcinoma cells

We first investigated the effect of neuropeptides on tumor cell invasion of Matrigel in Transwell cell culture chamber assay. Fig. 1 shows that VIP, SP, NPY, and suppressed the invasive ability of Colon 26-LS cells in a concentration-dependent manner. In particular, VIP showed the most potent inhibitory effect among these neuropeptides, and achieved 50% inhibition of control at 10⁻⁶ M. At this concentration VIP morphological alteration of tumor cells was not observed under a microscope (data

Discussion

Several organ-derived factors such as cytokines and ECM have been believed to regulate tumor growth and invasion 2, 3. However, the role of neuropeptides on tumor invasion is not well defined. In this study, we examined the effect of seven kinds of neuropeptides, which occurred in colon tissue, on invasive potential of colon carcinoma cells in a Transwell chamber assay. The invasion assay is subdivided into at least three possible steps: (1) cell attachment to Matrigel, (2) enzymatic

Acknowledgements

This work was supported in part by Grants-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, Sports and Culture (Nos. 06282122 and 07273106), and by a Grant from the Hokuriku Industrial Advancement Center, and by Grant-in-Aid for Special Project Research from Toyama Medical and Pharmaceutical University, Japan.

References (18)

E. Ekblad et al.
Projections of peptide-containing neurons in rat colon
Neuroscience
(1988)
G.J. Krejs et al.
Effect of VIP on infusion on water and ion transport in the human jejunum
Gastroenterology
(1980)
H. Itoh et al.
Relaxation of isolated bovine coronary arteries by vasoactive intestinal peptide
Eur. J. Pharmacol.
(1990)
P. Macho et al.
Neuropeptide Y (NPY): a coronary vasoconstrictor and potentiator of catecholamine-induced coronary constriction
Eur. J. Pharmacol.
(1989)
N.M. Hoosein et al.
Differential effects of peptide hormones bombesin, vasoactive intestinal polypeptide and somatostatin analog RC160 on the invasive capacity of human prostatic carcinoma cells
J. Urol
(1993)
L.A. Liotta
Tumor invasion and metastasis: role of the extracellular matrix: Rhoads memorial award lecture
Cancer Res.
(1986)
R.D. Leek et al.
Cytokine networks in solid human tumors: regulation of angiogenesis
J. Leukoc. Biol.
(1994)
M.L. Stracke et al.
The role of the extracellular matrix in tumor cell metastasis
In Vivo
(1994)
H.M. Cox et al.
Pharmacological characterization of neurokinin receptors mediating anion secretion in rat descending colon mucosa
Naunyn-Schmiedeberg's Arch. Pharmacol.
(1993)

There are more references available in the full text version of this article.

Cited by (34)

Neuropeptide Y (NPY) in tumor growth and progression: Lessons learned from pediatric oncology
2016, Neuropeptides
Citation Excerpt :
Nevertheless, it is important to remember that despite many similarities between actions of the peptide in different tumor types, its effects are still tumor specific. For example, in colorectal cancer and cholangiocarcinoma, NPY inhibits cancer cell proliferation and invasiveness, while in prostate cancer cells, its effects vary between cell lines (Table 1) (DeMorrow et al., 2011; Ogasawara et al., 1997; Ruscica et al., 2007). Yet, studies confirming these findings in an orthotopic tumor microenvironment are still needed, as a full understanding of NPY actions in particular tumor types and their environment is crucial for designing rational therapies targeting this pathway.
Neuropeptide Y (NPY) is a sympathetic neurotransmitter with pleiotropic actions, many of which are highly relevant to tumor biology. Consequently, the peptide has been implicated as a factor regulating the growth of a variety of tumors. Among them, two pediatric malignancies with high endogenous NPY synthesis and release – neuroblastoma and Ewing sarcoma – became excellent models to investigate the role of NPY in tumor growth and progression. The stimulatory effect on tumor cell proliferation, survival, and migration, as well as angiogenesis in these tumors, is mediated by two NPY receptors, Y2R and Y5R, which are expressed in either a constitutive or inducible manner. Of particular importance are interactions of the NPY system with the tumor microenvironment, as hypoxic conditions commonly occurring in solid tumors strongly activate the NPY/Y2R/Y5R axis. This activation is triggered by hypoxia-induced up-regulation of Y2R/Y5R expression and stimulation of dipeptidyl peptidase IV (DPPIV), which converts NPY to a selective Y2R/Y5R agonist, NPY_3–36. While previous studies focused mainly on the effects of NPY on tumor growth and vascularization, they also provided insight into the potential role of the peptide in tumor progression into a metastatic and chemoresistant phenotype. This review summarizes our current knowledge of the role of NPY in neuroblastoma and Ewing sarcoma and its interactions with the tumor microenvironment in the context of findings in other malignancies, as well as discusses future directions and potential clinical implications of these discoveries.
The vasoactive intestinal peptide-receptor system is involved in human glioblastoma cell migration
2010, Neuropeptides
Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor in adults. This cancer has an infiltrative nature and the median survival of patients is about one year. Vasoactive intestinal peptide (VIP) belongs to a structurally related family of polypeptides and is a major regulatory factor in the central and peripheral nervous systems. VIP regulates proliferation of astrocytes and of numerous cancer cell lines and modulates migration in prostatic and colonic cancer cell lines. Little is known about the involvement of VIP and its receptors (VIP-receptor system) in proliferation or migration of GBM cells. The effects of VIP, PACAP and of synthetic VIP antagonists were tested in two human GBM cell lines, M059K and M059J, established from two different parts of a single tumor. In these cells, the data revealed that the VIP-receptor system did not affect proliferation but controlled cell migration. Indeed, in M059K cells which express components of the VIP receptor system, the VIP receptor antagonists and a PACAP antibody enhanced migration. The VIP receptor antagonists increased generation of typical migration-associated processes: filopodia and lamellipodia, and activation of Rac1 and Cdc42 GTPases. Reciprocally, in M059J cells which poorly express the VIP-receptor system, treatments with the agonists VIP and PACAP resulted in decreased cell migration. Furthermore, the peptides appeared to act through a subclass of binding sites displaying an uncommon very high affinity for these ligands. Taken together, these observations suggest that components of the VIP-receptor system negatively regulate cell migration, thus showing potential anti-oncogenic properties.
Synthesis and conformational studies of 3,4-di-O-acylated furanoid sugar amino acid-containing analogs of the receptor binding inhibitor of vasoactive intestinal peptide
2007, Tetrahedron Letters
Epigenetic Silencing of Genes in Human Colon Cancer
2006, Gastroenterology
Conformational studies of 3,4-dideoxy furanoid sugar amino acid containing analogs of the receptor binding inhibitor of vasoactive intestinal peptide
2004, Tetrahedron
Conformational analysis of vasoactive intestinal peptide (VIP) receptor binding inhibitor Leu¹-Met²-Tyr³-Pro⁴-Thr⁵-Tyr⁶-Leu⁷-Lys⁸ 1 by various NMR techniques and constrained molecular dynamics (MD) simulation studies revealed that the molecule had a turn structure involving its Tyr³-Pro⁴-Thr⁵-Tyr⁶ moiety with intramolecular hydrogen bond between Tyr⁶NH→Tyr³CO. In order to mimic the structure of 1, peptidomimetic analogs 2–4 were synthesized using conformationally constrained scaffolds of 3,4-dideoxy furanoid sugar amino acids (2S,5R)-ddSaa1 5 and its enantiomer (2R,5S)-ddSaa2 6. All these analogs displayed well defined three-dimensional structures akin to that found in 1. Peptides 2 and 3, which differed only in the sugar amino acid stereochemistry, show propensity of structures with identical intramolecular hydrogen bonds between ThrNH→MetCO. A similar structure with a hydrogen bond between TyrNH→MetCO was observed in 4.
Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer
2004, International Journal of Biochemistry and Cell Biology
Post-translational modification of proteins is an important regulatory event. Numerous biologically active peptides that play an essential role in cancerogenesis contain an evolutionary conserved proline residue as a proteolytic-processing regulatory element. Proline-specific proteases could therefore be viewed as important “check-points”. Limited proteolysis of such peptides may lead to quantitative but, importantly, due to the change of receptor preference, also qualitative changes of their signaling potential.
Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with CD26) was for many years believed to be a unique cell membrane protease cleaving X-Pro dipeptides from the N-terminal end of peptides and proteins. Subsequently, a number of other molecules were discovered, exhibiting various degree of structural homology and DPP-IV-like enzyme activity, capable of cleaving similar set of substrates. These comprise for example, seprase, fibroblast activation protein α, DPP6, DPP8, DPP9, attractin, N-acetylated-α-linked-acidic dipeptidases I, II and L, quiescent cell proline dipeptidase, thymus-specific serine protease and DPP IV-β. It is tempting to speculate their potential participation on DPP-IV biological function(s). Disrupted expression and enzymatic activity of “DPP-IV activity and/or structure homologues” (DASH) might corrupt the message carried by their substrates, promoting abnormal cell behavior. Consequently, modulation of particular enzyme activity using e.g. DASH inhibitors, specific antibodies or DASH expression modification may be an attractive therapeutic concept in cancer treatment. This review summarizes recent information on the interactions between DASH members and their substrates with respect to their possible role in cancer biology.

View all citing articles on Scopus

View full text

Differential effect of intestinal neuropeptides on invasion and migration of colon carcinoma cells in vitro

Abstract

Introduction

Section snippets

Cells and cell culture

Reagents

Cell invasion assay

Effect of neuropeptides on invasion of Matrigel by Colon 26-L5 adenocarcinoma cells

Discussion

Acknowledgements

Neuroscience

Gastroenterology

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Urol

Tumor invasion and metastasis: role of the extracellular matrix: Rhoads memorial award lecture

Cancer Res.

Cytokine networks in solid human tumors: regulation of angiogenesis

J. Leukoc. Biol.

The role of the extracellular matrix in tumor cell metastasis

In Vivo

Pharmacological characterization of neurokinin receptors mediating anion secretion in rat descending colon mucosa

Naunyn-Schmiedeberg's Arch. Pharmacol.