Effect of ghrelin on the apoptotic deletion rate of different types of cells cultured in vitro
ANNA S. BELLONI1, CARLO MACCHI1, PIERA REBUFFAT1, MARIA TERESA CONCONI2, LUDWIK K. MALENDOWICZ3, PIER PAOLO PARNIGOTTO2 and GASTONE G. NUSSDORFER1
Departments of 1Human Anatomy and Physiology, Section of Anatomy and 2Pharmaceutical Sciences, University of Padua, I-35121 Padua, Italy; 3Department of Histology and Embryology, Poznan School of Medicine, PL-60781 Poznan, Poland
Received March 2, 2004; Accepted April 23, 2004
Abstract. Evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, controls the growth of several human and rat cell types cultured in vitro. Hence, we have investigated, by using both TUNEL and ELISA assays, the effects of 10-8 M ghrelin on the basal apoptotic deletion rate of rat osteoblasts and thymocytes, rat and human adrenocortical cells, human umbilical vein endo- thelial cells, and human aldosteronoma cells cultured in vitro, as well as of the human adrenocortical carcinoma-derived cell lines NCI-H295 and SW-13. Both assays consistently showed that ghrelin did not affect apoptotic rate of normal rat and human cells, but significantly enhanced apoptotic deletion in aldosteronoma, NCI-H295 and SW-13 cell cultures. Due to the central role of apoptosis in the control of tumor growth, these findings, if confirmed in other tumor cell types, could suggest an antitumoral action of ghrelin.
Introduction
Many lines of evidence indicate that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) (1-4), variously affects the growth of in vitro cultured cells. Ghrelin was found to enhance the proliferative activity of rat zona glomerulosa cells (5), prostate cancer PC3 cell lines (6) and H9c2 cardiomyocytes (7), but to lower that of rat neuro- microvascular endothelial cell (NEC) (unpublished data), and several human breast (8) and thyroid carcinoma cell lines (9). Moreover, ghrelin has been reported to inhibit doxorubicin- induced apoptotic death of H9c2 cardiomyocytes and PAE endothelial cell line (10), but to raise basal apoptotic rate of NEC (unpublished data) and not to affect that of rat zona glomerulosa cells (5).
Correspondence to: Professor G.G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy
E-mail: gastone.nusdorfer@unipd.it
Key words: ghrelin, apoptosis, adrenocortical cells, endothelial cells, osteoblasts, thymocytes, aldosteronoma cells, adrenocortical carcinoma-derived cell lines
In light of these rather conflicting observations and the accumulating findings stressing the pivotal role of apoptosis in tumor biology (11-13), it seemed worthwhile to investigate by two different techniques the effect of ghrelin on basal apoptotic deletion rate of different rat and human cell types cultured in vitro.
Materials and methods
Sprague-Dawley male rats (200-220 g in body weight) were purchased from Charles-River (Como, Italy), and zona glomerulosa, osteoblasts and thymocytes were isolated as previously described (14-17). Fragments of normal human adrenal cortexes were obtained from two adult patients under- going unilateral nephrectomy/adrenalectomy for kidney cancer, and fragments of aldosteronoma from other two patients. Adrenocortical and aldosteronoma cells were isolated as detailed earlier (18,19). The study protocol was approved by the local Ethics Committee for Animals and Human Studies. Human umbilical vein endothelial cells (HUVEC) were provided by Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna (Brescia, Italy), and human adrenocortical carcinoma cell lines NCI-H295 and SW-13 cell lines by the American Type Culture Collection (Rockville, MD).
Cells were seeded (1.5x104 cells/cm2) into a 24-well plate, and cultured for 24 h at 37℃ in Dulbecco’s modified minimum essential medium (Sigma Chemical Co., St. Louis, MO) or, in the case of HUVEC, endothelial cell growth medium EBM medium (Biowhittaker, Verviers, Belgium). Growth media contained 1% amphotericin-B/gentamycin and 5% fetal calf serum (FCS; Sigma). Growth medium was replaced with a fresh one, containing or not 10-8 M human or rat ghrelin (Phoenix Pharmaceuticals, Belmon, CA), and cells were incubated for other 24 h at 37℃.
Apoptosis was detected by the following two methods: i) cultures were fixed in 4% paraformaldehyde for 60 min at room temperature, and apoptosis was detected by TUNEL assay, using the In Situ Cell Death Detection Kit of Roche Molecular Biochemicals (Mannheim, Germany). Cultures were incubated with 4’,6-diamino-2-phenylindole dilactate (DAPI) (Sigma Chemical Co.) for 15 min at 37℃, and the number of TUNEL-positive cells was estimated by double-fluorescence microscopy in a Leica DMIR2 HC FLUO inverted microscope
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(Leica Microsystems, Milan, Italy), as previously detailed (20); ii) cells were detached, pelleted, resuspended in lysis buffer, and then centrifuged. Pelletted nuclei were discarded, and aliquots of supernatants were transferred to streptavidin- coated wells of a microtiter plate, and apoptosis was detected by the Cell Death Detection ELISAPLUS Kit of Roche Molecular Biochemicals, following the procedure described earlier (20). Results were expressed as percent change from control value.
Data were expressed as means ± SEM of three separate experiments, and their statistical comparison was per- formed by ANOVA, followed by the Student’s t-test for paired data.
Results
TUNEL assay showed that ghrelin (10-8 M) did not affect the apoptotic deletion rate in the HUVEC, human normal adrenocortical cells, in rat osteoblasts, thymocytes or zona glomerulosa cells cultured in vitro. In contrast, ghrelin significantly enhanced apoptosis in both aldosteronoma cell and NCI-H295 or SW-13 cell cultures (Figs. 1 and 2, upper panels). ELISA assay confirmed these findings (Fig. 2, lower panel).
apoptotic rate [% change from control]
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TUNEL assay
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rat osteoblasts
rat zona glomerulosa
human adrenocortical
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rat thymocytes
HUVEC
aldosteronoma
cells
NCI-H295
cell line
SW-13 cell line
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-25-
apoptotic rate [% change from control]
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ELISA assay
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rat osteoblasts
rat zona glomerulosa
human adrenocortical
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cells
HUVEC
cells
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rat thymocytes
aldosteronoma
cells
NCI-H295
cell line
SW-13 cell line
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V
-25-
Discussion
Our present findings indicate that ghrelin does not alter the basal apoptotic rate of either normal human or rat cells cultured in vitro, but markedly enhances apoptosis in cultured human adrenocortical tumor cells. This last observation appears to conflict with the reported anti-apoptotic activity exerted by ghrelin on cardiomyocytes and endothelial cell lines (10). However, in this case ghrelin counteracted the doxorubicin-induced apoptosis, whose mitochondrial- dependent mechanisms are conceivably different from those underlying basal apoptotic death of cells cultured in the presence of low concentrations of FCS (21-23).
Ghrelin is known to act via the GHS-R, two subtypes of which have been identified, the fully functional GHS-Rla and the biologically inactive GHS-R1b (2,3). The expression of GHS-Rla as mRNA and protein has been demonstrated in the human and rat adrenal cortex (5,24-28), but not yet in adrenocortical tumor cells. Moreover, indirect evidence indicates the existence of another type of ghrelin receptor, which could be involved in the anti-apoptotic effect of the peptide on cardiomyocyte and endothelial-cell lines. In fact, acylation of ghrelin is needed for GHS-Rla activation (29), but des-acyl ghrelin was found to counteract, like ghrelin,
doxorubicin-induced apoptosis in cardiomyocyte and endo- thelial-cell lines (10). Hence, it is not unreasonable to hypothesize that the differential expression of the GHS-R subtypes may be involved in the different effects of ghrelin in the apoptotic deletion rate of various cell types.
Our study also suggests that the proapoptotic effect of ghrelin occurs only in tumor cells, i.e., aldosteronoma cells and NCI-H295 and SW-13 cell lines derived from adrenocortical carcinomas. If confirmed in other tumor cell types, this finding could acquire a great relevance. Apoptotic deletion is considered a pivotal mechanism that negatively regulates tumor growth (30), and our investigation could have provided evidence of the existence of an endogenous ligand mediating this antitumoral action.
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