Ghrelin inhibits proliferation and increases T-type Ca2+ channel expression in PC-3 human prostate carcinoma cells

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Abstract

Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca2+ levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca2+ channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca2+ channel expression.

Research highlights

► Ghrelin decreases prostate carcinoma PC-3 cells proliferation. ► Ghrelin favors apoptosis in PC-3 cells. ► Ghrelin increase in intracellular free Ca2+ levels in PC-3 cells. ► Grelin up-regulates expression of T-type Ca2+ channels in PC-3 cells. ► PC-3 cells express T-channels of the CaV3.1 and CaV3.2 subtype.

Introduction

Ghrelin is a 28-amino acid peptide with an n-octanoylation indispensable for its biologic activity originally isolated from the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GSH-R) [1], [2]. Ghrelin is essentially a multifunctional hormone with roles in growth hormone release, food intake and cell proliferation [2], [3]. However, in spite of its widespread and important physiologic actions, its precise regulatory mechanisms remain ambiguous.

The role of ghrelin in the control of cell proliferation and cancer has received considerable attention [3]; however, discordant results have been reported. Hence, proliferative actions of the hormone (or its synthetic analogs) have been observed in hepatoma HepG2 cells, as well as in prostate and breast cancer cell lines [4], [5], whereas antiproliferative effects have been reported in non-tumoral thyroid cell lines, as well as in lung, breast and prostate cancer cell lines [6], [7], [8], [9]. Although the reason for such discrepancy is unknown, possible explanations could include differences in the methodological approaches, in the cell types investigated and/or the expression of GHS-Rs.

Ghrelin and the GHS-R1a are both present in the prostate [4], [10]. However, modified prostatic epithelial cells (both hyperplastic or neoplastic) express higher levels of ghrelin than normal prostate, suggesting the possible existence of an autocrine/paracrine role of the hormone in the neoplastic processes [8], [10]. More specifically, in the androgen-independent human prostate carcinoma PC-3 cells, Jeffery and her colleagues reported that ghrelin at nM concentrations stimulated cell proliferation [4], [10]. In contrast, Cassoni et al. demonstrated that ghrelin had a biphasic effect on the growth of PC-3 cells [8], with a stimulating activity at low concentrations (10–100 pM) and an inhibiting effect at higher doses (1 μM). It is worth noting however that the molecular mechanism underlying the actions of ghrelin on PC-3 cells proliferation remain virtually unexplored. In the present report, we have examined the role exerted by ghrelin on prostatic cancer cells growth in vitro at concentrations quite close to those found in ghrelin-producing tissues (1–50 nM), and further investigated some of the mechanisms by which the hormone was able to inhibit cell proliferation.

Section snippets

Materials

[3H]-Thymidine was obtained from Amersham Life Science; fetal bovine serum was purchased from Invitrogen. Ghrelin (55-0-03A) and GHRP-6 (52-1-80B) were purchased from American Peptide Company Inc. Pimozide (EK-400) was from Alomone Labs and mibefradil was a gift of Dr. J.C. Gómora (Institute of Cellular Physiology, UNAM, Mexico). All other chemicals were reagent grade purchased from Sigma–Aldrich. Plasticware was from Sarstedt.

Cell culture

Human prostate PC-3 cells were a gift of Dr. A. Zentella (National

Results and discussion

As previously described for ghrelin production in prostate cancer, divergent observations have been reported on the GHS-Rs expression in prostate carcinoma cell lines and tissues. Though the presence of GHS-Rs (1a and 1b) at both mRNA and peptide level has been reported in various prostate carcinoma cell lines including PC-3 by Chopin and co-workers [4], [10], neither GHS-R1a mRNA nor protein was detected in the same cells by Cassoni et al. [8]. However, despite the absence of GHS-Rs mRNA, the

Acknowledgments

We thank Drs. J.A. Arias-Montaño (Cinvestav-IPN, Mexico) and J.E. Soria-Jasso (University of Zacatecas, Mexico) for their advise and help in performing the Ca2+i measurements experiments. This work was partially supported by a grant from Consejo Nacional de Ciencia y Tecnologia (CONACyT, Mexico) to R. Felix.

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