Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues

Abstract

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.

Fig. 1.

Immunohistochemical analysis. (A–D) Adrenal medullary tissue and PC-12 cell line: (A) no sst2 expression was found in normal adrenal medulla; (B) relatively weak sst2 staining was seen in benign PHEO; (C) more pronounced sst2 receptor expression in malignant PHEO and (D) PC-12 cells. (E–H) Adrenal cortical tissue and SW-13 cell line: (E) normal cortex is strongly immunostained for sst2; (F) cortical adenoma, (G) cortical carcinoma, and (H) malignant SW-13 cells show even more pronounced sst2 expression (n = 3 for all tissues and cell lines analyzed). (Scale bars, 20 μm.)

Fig. 2.

Receptors for sst2 (168 bp), SV-1 (121 bp), and LHRH (319 bp) are expressed in SW-13 cells as shown by RT-PCR (A–C). (D and E) Ultrastructural analysis demonstrates the morphology of an undifferentiated cell, with abundant characteristic elongated cristae-like mitochondria, as well as ample endoplasmic reticulum under normal culture conditions (D). (Scale bar, 3 μm.) SW-13 cells (E) are used as a model for malignant adrenocortical tumors. Cells treated with RC-160 exhibited swollen mitochondria, containing amorphous inclusions, and showing disruption of nuclear and mitochondrial membranes, indicating primarily a necrotic mode of cell death. (Scale bar, 0.1 μm.) DCV, dense core vesicles; Mit, mitochondria; Nuc, nucleus.

Fig. 3.

Effects of somatostatin analogue RC-160, GHRH antagonist MZ-4–71, and LHRH antagonist Cetrorelix on proliferation of human adrenocortical carcinoma cells in culture. This figure illustrates changes in the percent-confluence over 6 days incubation of SW-13 cell cultures under proliferative culture conditions (10% FCS), with and without the addition of the peptides MZ-4–71 and RC-160 (P < 0.05 for both) and for Cetrorelix (P < 0.01), relative to control for each day.

Fig. 4.

The somatostatin receptor subtype 2 (276 bp) is expressed in PC-12 cells as shown by RT-PCR (A). Ultrastructural analysis shows a relatively small number of dense core vesicles, typical of chromaffin cells under normal culture conditions (B). (Scale bar, 0.2 μm.) Incubation of PC-12 cells with AN-238 (C) induces characteristic apoptotic changes, including shrinking of the cytoplasm away from the cell wall, apoptotic bodies, internucleosomal DNA fragmentation, or condensation of the cytoplasm while retaining mitochondria and endomembrane structure. (Scale bar, 0.2 μm). DCV, dense-core vesicles; MIT, mitochondria; NTC, no template control; Nuc, nucleus.

Fig. 5.

Effects of RC-160, AN-162, and AN-238 on uninduced and NGF (20 ng/ml)-induced PC-12 cells. (A) Cell-count measurements. AN-238 and, to a lesser extent, AN-162 significantly reduced cell number of uninduced and NGF-induced PC-12 cells after 24 h (n = 4). RC-160 did not affect PC-12 cell number. (B) Apoptosis assays. AN-238 led to a highly significant activation of caspase 3/7 activation after 24 h (n = 4). There was no effect of RC-160 and AN-162. (C) Cytotoxicity assays. LDH release was significantly increased after incubation with AN-238 for 24 h of induced or NGF-induced cells as compared to control (n = 6) *P < 0.05, **P < 0.01, ***P < 0.001 as compared to control for all assays.