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Ghrelin immunoreactivity and carcinoma cell proliferation inhibition effects in fetal thyroid and follicular tumors
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Ghrelin-related products |
| Ghrelin, a growth hormone-releasing hormone produced by gastroenteropancreatic endocrine cells, hypothalamus, and pituitary, was recently identified in medullary thyroid carcinomas and derived cell lines. However, no data exist on its expression in either normal or neoplastic thyroid follicular cells. We analyzed ghrelin expression by immunohistochemistry, in situ hybridization, and reverse transcriptase-polymerase chain reaction in 15 fetal, 4 infant, and 10 adult thyroids, and in 54 tumors of follicular origin. We also analyzed the effects of ghrelin on cell proliferation in N-PAP and ARO thyroid carcinoma cell lines. Ghrelin-binding sites were investigated using reverse transcriptase-polymerase chain reaction to detect its growth hormone secretagogue receptor (GHS-R) mRNA and an in situ-binding localization procedure. Strong ghrelin immunoreactivity was found in fetal but not in infant or adult thyroids. Ghrelin protein and mRNA were present, in variable amounts, in benign and malignant tumors. Normal thyroids, thyroid tumors, and cell lines showed ghrelin binding sites by binding localization, in the absence of the specific GHS receptor mRNA (with the exception of one normal thyroid). Moreover, ghrelin induced dose-dependent inhibition of growth in cell lines. In conclusion, ghrelin is expressed in fetal but not in adult thyroid, and is re-expressed in tumors; the presence of ghrelin receptors other than GHS-R in normal and neoplastic adult thyroid is suggested; ghrelin inhibits cell proliferation of thyroid carcinoma cell lines in vitro. |
| Volante M., et al. American Journal of
Pathology. 2003;162:645-654 |
| Immunohistochemistry (IHC) |
Sections serial to those used for conventional histology were collected onto poly-L-lysine-coated slides and stained for ghrelin using a standard manual immunoperoxidase procedure with streptavidin-peroxidase (LSAB2 kit; DAKO, Glostrup, Denmark) and diaminobenzidine as the final reaction product. Ghrelin antibody was an anti-human ghrelin polyclonal serum (amino acids 24 to 51, code H-031-30; Phoenix Pharmaceuticals, Inc.), diluted 1/15,000, incubated or 1 hour at room temperature, with no previous antigen retrieval and revealed applying catalyzed reporter deposition (CARD) amplification with slight modifications. Negative control reactions forghrelin included omission of the primary antibody and preabsorption with a 100-fold excess of the antigen, as described elsewhere.The positive control for ghrelin were the endocrine cells of the oxyntic mucosa of the stomach. The follicular origin of the tumors was evaluated by IHC with polyclonal anti-calcitonin (diluted 1/80, DAKO) and anti-thyroglobulin (diluted 1/3000; Biogenex, San Ramon CA) antibodies. Selected cases were also stained with a double-immunohistochemical procedure using the same antibodies that react with ghrelin and thyroglobulin. This procedure was based on an immunoalkaline phosphatase (LSAB plus kit, DAKO)/fast red (BioGenex) reaction for ghrelin, followed by immunoperoxidase staining of thyroglobulin, using an anti-rabbit secondary antibody and the EnVision kit (DAKO). Ghrelin protein expression was also evaluated in the two cell lines applying the same ghrelin antibody at a working dilution of 1/400, and visualized in immunofluorescence with an anti-rabbit fluorescein isothiocyanate-conjugated antibody (diluted 1/100; Sigma Aldrich, Steinheim, Germany). |
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| Ghrelin expression in the human fetal thyroid. Thyroids from the early fetal period (a–d, gestational age 10 weeks; e–g, gestational age 12 weeks) show co-expression of thyroglobulin (b and f) and ghrelin (c and g) in the same cell population, as detected by IHC. Staining is abolished by preabsorption with full-length ghrelin peptide (d). Ghrelin expression is weaker in late fetal thyroid (h–l, gestational age 28 weeks), as shown by both IHC (i) and in situ hybridization (l). a, e, h: H&E; b–d, f, g, i: immunoperoxidase; l: in situ hybridization. Original magnifications, x200. |
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| Ghrelin expression in thyroid tumors. Immunoreactive
ghrelin is absent in peritumoral thyroid tissue (a), focally
present in a follicular adenoma (b) and in an anaplastic carcinoma
(h), and strongly expressed in classic (c) and follicular
variant (e) papillary, and in poorly differentiated (g)
carcinomas. Immunohistochemical data are paralleled by a specific
in situ hybridization signal for ghrelin mRNA (d, same
area as c). Lymph node metastases retain the ghrelin expression
capacity of the primary sites (f, same case as e). a–h:
Immunoperoxidase. Original magnifications: x200
(a, c–e, g); x400
(b, f, h). |
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