Activation of bombesin receptor subtype-3 stimulates adhesion of lung cancer cells
Introduction
Bombesin (Bn) receptor subtype-3 (BRS-3) is a 399 amino acid protein that has 51 and 47% amino acid sequence homologies to gastrin-releasing peptide (GRP) and neuromedin B (NMB) receptors, respectively [1], [2]. BRS-3 is expressed in the uterus [1] and testis [2], suggesting a possible role of BRS-3 in reproduction. BRS-3 has also been found in lung carcinoma cells such as small cell lung cancer (SCLC) cells [2]. It has been reported that mice lacking BRS-3 develop metabolic defects, obesity and hypertension, suggesting that BRS-3 may be required for the metabolic processes responsible for energy balance and adiposity, and the maintenance of blood pressure [3].
The endogenous ligand for BRS-3 has not yet been identified, and BRS-3 does not interact with high affinity to known natural agonists of the Bn receptor family [4]. However, BRS-3 does bind to a synthetic peptide [d-Phe6, β-Ala11, Phe13, Nle14]Bn(6–14) in the cells such as BRS-3-transfected Balb 3T3 cells [4], [5]. NCI-N417, a SCLC cell line, expresses native BRS-3 but not GRP or NMB receptors [5]. NCI-N417 cell is therefore thought to be a suitable cell model for studying the activation of BRS-3 by [d-Phe6, β-Ala11, Phe13, Nle14]Bn(6–14), as there should be no interference from GRP and NMB receptor interactions.
NCI-N417 cells are usually non-adherent in culture and grow as tight aggregates in suspension. However, the cells attach to the surface of culture dishes coated with laminin, which is followed by outgrowth of long neurite-like processes [6], [7], [8]. Laminin, the most abundant glycoprotein in basement membranes, capable of stimulating tumor cell adhesion and cell motility may play an important role in the formation of metastasis [9]. To characterize BRS-3-mediated biological responses, we examined the effect of BRS-3 activation by [d-Phe6, β-Ala11, Phe13, Nle14]Bn(6–14) on the adhesion of NCI-N417 cells to laminin. Further studies were carried out to elucidate the mechanism of the receptor-mediated action.
Section snippets
Materials and methods
The anti-β1 integrin monoclonal antibodies, CD29-4B7R and CD29-MAR4, were purchased from Westinghouse and BD Biosciences, CA, respectively. The anti-vinculin antiserum and FITC-conjugated goat anti-mouse IgG were purchased from Sigma–Aldrich Co. GRP and NMB were obtained from Peptide Institute, Osaka, Japan. d-Nal-Cys-Tyr-d-Trp-Lys-Val-Cys-Nal-NH2 was purchased from Bachem AG, Switzerland. Fluo-3 AM was purchased from Wako Chemicals, Japan. Laminin and other chemicals were obtained from
Stimulation of the adhesion of NCI-N417 cells to laminin by BRS-3 activation
We first tested the ability of NCI-N417 cells to attach to laminin-coated wells without BRS-3 activation, and found adhesion of a fraction of the cells to laminin. We then examined whether the activation of BRS-3 by [d-Phe6, β-Ala11, Phe13, Nle14]Bn(6–14) stimulated the adhesion of NCI-N417 cells to laminin. We found that the cell adhesion was significantly increased in the presence of BRS-3 agonist from 1 nM to 10 μM (Fig. 1). The cell adhesion increased 2.5-fold in the presence of 100 nM [d-Phe6
Discussion
In this study, we report for the first time that activation of BRS-3 stimulates the adhesion of NCI-N417 cells to laminin. The adhesion of the cells increased 2.5-fold in the presence of 100 nM of the BRS-3 agonist [d-Phe6, β-Ala11, Phe13, Nle14]Bn(6–14). NMB also stimulated the adhesion of NCI-N417 cells only at very high concentrations (over 10 μM), presumably due to the interaction with BRS-3. A specific BRS-3 antagonist, d-Nal-Cys-Tyr-d-Trp-Lys-Val-Cys-Nal-NH2, inhibited the adhesion
Conclusion
The activation of bombesin receptor subtype-3 stimulates laminin-induced adhesion and haptotaxis of lung cancer cells. The increased adhesion of the cells to laminin by BRS-3 is accompanied by an increase in vinculin-like immunoreactivity, β1 integrin and mobilization of intracellular calcium from cellular calcium stores may be involved in the process of the adhesion of NCI-N417 cells to laminin by BRS-3 activation. Therefore, we suggest that activation of BRS-3 might affect the invasion and
Acknowledgments
We would like to thank Dr. Kiyoshige Takayama for useful discussions. We appreciate the excellent technical assistance of Ms. Shinobu Konno, Ms. Ryoko Tsuda, Ms. Tomoko Horie and Mr. Torao Narita. The work was supported by the Ministry of Education, Science and Culture of Japan.
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