Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Expression and function of endocannabinoid receptors in the human adrenal cortex.

Endogenous cannabinoids are important signaling molecules in neuroendocrine control of homeostatic and reproductive functions including stress response and energy metabolism. The hypothalamic paraventricular and supraoptic nuclei have been shown to release endocannabinoids, which act as retrograde messengers to modulate the synaptic release of glutamate during stress response. This study endeavors to elucidate possible interaction of the endocannabinoid system with the regulation of adrenocortical function at the adrenal level. Human adrenocortical NCI-H295R cells and normal human adrenal glands were used to study the possible effects of anandamide and cannabinoid receptor 1 (CB1) antagonist SR141716A on aldosterone and cortisol secretion. Our data indicate the expression of CB1 in human adrenal cortex and adrenocortical NCI-H295R cells; CB2 was not expressed. Furthermore, anandamide inhibited basal release and stimulated release of adrenocortical steroids (corticosterone and aldosterone); this effect was reversed by CB1 antagonist (SR141716A). Therefore, the endocannabinoid system at the level of the adrenal, can directly influence adrenocortical steroidogenesis.

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

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.

Toll-like receptors in endocrine disease and diabetes.

The body's ability to keep a steady homeostatic state is crucial to health and life. This involves providing an adequate response to a variety of challenges both physical and mental, such as microbial invasion and emotional distress. Interplay between the neuroendocrine and immune systems is essential in either case. Studies have demonstrated that toll-like receptors, or TLRs, play a regulatory role in both systems, and have been proposed as a possible link between the immune, hormonal and metabolic systems. As part of the innate immune system, these receptors control the identification by the body of microbial invaders and its immediate reaction in immune and inflammatory response. What are referred to as pattern recognition receptors are mostly expressed by cells involved in hematopoietic linkage, but an increasing number of studies have demonstrated their expression in other cell types such as neurons and endocrine cells on the hypothalamic-pituitary-adrenal (HPA) axis, thyrocytes, adipocytes and islets of Langerhans. Together with endocrine and metabolic dysregulation, immune system overreaction is often associated with infection and autoimmunity, clearly indicating TLR involvement at organ level which affects organ function. Several diseases such as autoimmune thyroid and pancreatic diseases, septic dysregulation of the HPA axis, diabetes and the metabolic syndrome have been linked to TLR activation and polymorphism. To gain insight into stress response and adaptation, we need to know more about TLRs and the specific physiological role they play in the endocrine and metabolic system and its processes.

Pam3CSK4 and LTA-TLRs ligands associated with microdomains induce IL8 production in human adrenocortical cancer cells.

Bacterially derived ligands, Pam3CSK4 and LPS, can directly impact adrenal glands steroidogenesis through microdomain-related TLR1/2 and 4, respectively, and indirectly via immune cell-derived cytokines. The bilateral immunoadrenal relationship plays an important role in the proper functioning of both systems. CXC chemokine-dependent immune cell infiltration into adrenocortical carcinomas (ACC), which correlates with poor prognosis, is a common phenomenon. Recently, IL8 was identified in ACC and NCI-H295R cells, and was found to contribute to ACC tumour growth. The aim of this study was to clarify the role of different TLR ligands in IL8 production in NCI-H295R cells. This is the first study to demonstrate the expression of several TLRs including TLR1, 3, 6, 7 and 9 in human adrenocortical cells by using the RT-PCR approach. Only stimulation with TLR1/6 together with TLR2 ligands resulted in IL8 peptide and mRNA induction in a dose and time-dependent manner. Our data suggest that gram-positive bacteria-related TLR1/2/6 ligands might contribute to adrenal gland tumorigenesis via IL8 production.

Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II-sensitization in human adrenocortical cells.

OBJECTIVES: Hypertension is a major complication of overweight with frequently elevated aldosterone levels in obese patients. Our previous work suggests a direct stimulation of adrenal aldosterone secretion by adipocytes. Owing to aldosterone's important role in maintaining blood pressure homeostasis, its regulation in obesity is of major importance. One objective was to determine the signaling mechanisms involved in adipocyte-induced aldosterone secretion. In addition to a direct stimulation, a sensitization toward angiotensin II (AngII) might be involved. The second objective was to determine a possible adipokines-induced sensitization of human adrenocortical cells to AngII. DESIGN: Human subcutaneous adipocytes and adrenocortical cells, and the adrenocortical cell line NCI-H295R were used. Adrenocortical cells were screened for signal transduction protein expression and phosphorylation. Subsequently, steroidogenic acute regulatory protein (StAR), cAMP response element-binding protein (CREB), cAMP and phosphorylated extracellular regulated kinase were analyzed by Western blot, enzyme-linked immunosorbent assay, quantitative PCR, reporter gene assay and confocal microscopy to investigate their role in adipocyte-mediated aldosterone secretion. RESULTS: AngII-mediated aldosterone secretion was largely increased by preincubating H295R cells with adipocyte secretory products. StAR mRNA and StAR protein were upregulated in a time-dependent way. This steroidogenic effect was independent of the cAMP-protein kinase A (PKA) pathway as cellular cAMP was unaltered and inhibition of PKA by H89 failed to reduce aldosterone secretion. However, CREB reporter gene activity was moderately elevated. Upregulation of StAR was accompanied by ERK1/2 MAP kinase activation and nuclear translocation of the kinases. Inhibition of MAP kinase by UO126 abolished adipokine-stimulated aldosterone secretion from primary human adrenocortical and H295R cells, and inhibited StAR gene activity. Adipokines stimulated steroidogenesis also in primary human adrenocortical cells, supporting a role in human physiology and/or pathology. CONCLUSIONS: Adipokines induce aldosterone secretion from human adrenocortical cells and sensitization of the cells to stimulation by AngII, possibly mediated via ERK1/2-dependent upregulation of StAR activity. This stimulation of aldosterone secretion could be one link between overweight and inappropriately elevated aldosterone levels.

Corticotropin releasing hormone and its function in the skin.

The skin, as the largest organ of the body, is strategically located as a barrier between the external and internal environments, being permanently exposed to noxious stressors such as bursts of radiation (solar, thermal), mechanical energy, or chemical and biological insults. Because of its functional domains and structural diversity, the skin must have a constitutive mechanism for dealing with the stressors. Activities of the skin are mostly regulated by local cutaneous factors and stressed skin can generate signals to produce rapid (neural) or slow (humoral) responses to local or systemic levels. Thus, the skin neuroendocrine system is comprised of locally produced neuroendocrine mediators that interact with corresponding specific receptors through para- or autocrine mechanisms. Furthermore, it is known for several years that the corticotropin-releasing hormone (CRH)/ pro-opiomelanocorticotropin (POMC) skin system fulfils analogous functions to the hypothalamic-pituitary-adrenal (HPA) stress axis. Additionally, skin cells produce hormones, neurotansmitters and neuropeptides, having the corresponding receptors and the skin itself is able to fulfill a multidirectional communication between endocrine, immune and central nervous systems as well as other internal organs. In summary, the skin expresses an equivalent of the prominent hypothalamic-pituitary-adrenal stress axis that may act as a cutaneous defense system, operating as a coordinator and executor of local responses to stress, in addition to its normal function: the preservation of body homeostasis.