The effect of clozapine on prolactin secretion at the level of the lactotroph

Abstract Clozapine is an antipsychotic drug which is unusual in that it has no dopamine receptor-blocking activity. Previous studies gave conflicting results whether administration of clozapine induces hyperprolactinemia. In the present study it was shown that a wide concentration range of clozapine does not interfere with dopamine-mediated inhibition of prolactin (PRL) secretion by normal cultured rat pituitary cells. This in contrast to other neuroleptics, like haloperidol and trifluoperazine. Clozapine does also not antagonize norepinephrine-mediated inhibition of PRL secretion. Clozapine exerts at micromolar concentrations a direct inhibitory action on PRL release by cultured normal rat pituitary cells. In cultured rat pituitary tumor cells, these high concentrations of clozapine directly inhibit PRL release as well as the DNA content of the cells, suggesting a direct antimitotic action. In this model clozapine was about 5–10 times less potent than trifluoperazine. Clozapine and trifluoperazine exert an additive inhibitory action both on PRL release and on the DNA content of the pituitary tumor cells. It is concluded that clozapine does not interfere at the pituitary level with dopamine-mediated inhibition of PRL release. At micromolar concentrations clozapine may act on lactotrophs as a calmodulin-inhibitor. These observations suggest that the transient PRL-releasing effects which have been observed in both animal and human studies after clozapine administration are mediated via supra-pituitary actions of the drug

Percoll density gradient centrifugation of rat pituitary tumor cells

Abstract Tumor cells prepared from PRL-secreting rat pituitary 7315b tumors of increasing weight were separated on continuous Percoll density gradients, according to differences in their density. Whether the cell subpopulations obtained by density gradient separation showed differences in protein content per cell, PRL production per cell, growth rates and responsiveness to the somatostatin analog SMS 201–995 in vitro was investigated. In addition, we studied PRL release by individual 7315b tumor cells, using the reverse hemolytic plaque assay (RHPA). The tumor cells from tumors of increasing weight were recovered within a narrow density range (1.060–1.070 g/ml) and showed a normal distribution profile. There were no differences between the subpopulations with respect to the parameters mentioned above. Moreover, no differences were found with respect to these parameters between tumor cells derived from tumors of increasing weight. In agreement with the above data we found no evidence for subtypes of adenoma cells being preferentially responsive to SMS 201–995, using the RHPA. Conclusions: (1) the transplantable PRL-secreting rat pituitary tumor 7315b consists of a functionally homogeneous cell population; (2) growth of this tumor in vivo does not lead to the induction of functionally heterogeneous cell subpopulations within this tumor; (3) the escape of this tumor from the tumor growth-inhibitory effect of SMS 201–995, which has previously been demonstrated in vivo, may not have been the result of clonal selection of somatostatin-unresponsive cells

Role of the mTOR pathway in normal and tumoral adrenal cells

The mammalian target of rapamycin (mTOR) is a kinase of the phosphoinositide 3-kinase (PI3Ks)/protein kinase B (PKB or AKT) signaling pathway, which is one of the most important intracellular mediators of the activity of growth factors receptors, including vascular endothelial growth factor (VEGF) and insulin-like growth factors (IGFs). Dysregulation of the mTOR pathway has been found in many human tumors. Therefore, the mTOR pathway is considered as a target for antineoplastic therapy in several malignancies. Presently, the role and functions of mTOR and its signaling pathway in the normal and pathological adrenal gland has not been clarified yet. However, many growth factors and growth factor receptors, which are considered to play a role in the pathogenesis of adrenal tumors, can at least in part exert their effects through the activation of PI3K/AKT/mTOR pathway. Dysregulation of AKT has been reported in adrenocortical carcinomas and adrenomedullary tumors, named pheochromocytomas. Adrenocortical carcinomas and malignant pheochromocytomas are aggressive tumors with poor prognosis and scant treatment options. Therefore, new treatment options are warranted for these malignancies. On the basis of the current knowledge, mTOR could play a role in the pathogenesis of both adrenocortical carcinomas and pheochromocytomas. Moreover, mTOR inhibitors, interfering with the activation of several mitogenic and angiogenic factors, could be considered as a novel treatment opportunity for the management of malignant adrenal tumors

Pro-inflammatory cytokines affect pancreatic carcinoma cell. Endothelial cell interactions

OBJECTIVES: The potential role of surgery-induced pro-inflammatory\n cytokines on the development of tumor recurrence in pancreatic cancer was\n investigated. MAIN OUTCOME MEASURES: The adhesion of 3 human pancreatic\n carcinoma cell lines, PanC1, MiaPaCa and BxPC3 to monolayers of\n microvascular endothelial cells after pre-incubation with 0.1 or 10 ng/mL\n IL-1beta, TNF-alpha or IL-6 was assessed in a reproducible human in vitro\n assay. Untreated monolayers served as controls. RESULTS: Pre-incubation of\n microvascular endothelial cells with IL-1beta or TNF-alpha, but not IL-6,\n increased adhesion of all three tumor cell lines as compared to adhesion\n in the control group. Maximally stimulated adhesion for PanC1 reached\n 159%, for MiaPaCa 204% and for BxPC3 155% (all vs. the control, P<0.001).\n Pre-incubation of microvascular endothelial cells with IL-1beta or\n TNF-alpha resulted in a significant up-regulation of E-selectin, ICAM-1\n and VCAM-1 expression. The addition of anti-E-selectin, anti-ICAM-1 or\n anti-VCAM-1 monoclonal antibodies did not decrease adhesion to\n microvascular endothelial cells pre-incubated with IL-1beta. Therefore,\n enhanced tumor cell binding seems to be independent of these adhesion\n molecules. CONCLUSIONS: Pro-inflammatory cytokines derived from surgical\n trauma may enhance tumor cell adhesion to microvascular endothelial cells\n and thus bring about more successful tumor cell implantation resulting in\n an increased risk of metastasis formation

The in vitro and in vivo effects of human growth hormone administration on tumor growth of rats bearing a transplantable rat pituitary tumor (7315b)

Abstract The direct effects of human GH and IGF-I on PRL secretion and cell proliferation were studied on PRL secreting rat pituitary tumor 7315b cells in vitro, as well as the effects in vivo of human GH administration on body weight, IGF-I levels and tumor size in rats bearing this transplantable tumor. In the in vitro studies IGF-I levels above 5 nM stimulated PRL release in a dose-dependent manner while GH, in concentrations of 0.23–45 nM, did not affect PRL release. Cell proliferation was stimulated by IGF-I in a dose-dependent manner from 0.5 nM onwards, while GH did not have an effect. The in vivo studies showed that 1 mg GH/rat/day prevented tumor-induced cachexia and normalized the suppressed IGF-I levels without stimulating tumor growth. It is concluded that tumor-induced cachexia can be prevented by exogenous GH administration without an increase in tumor mass, even if a tumor model is used whose cultured tumor cells respond to exposure to IGF-I with a mitotic response

Role of tumor-derived fibroblasts in the growth of primary cultures of human breast-cancer cells: Effects of epidermal growth factor and the somatostatin analogue octreotide

In the present study we have investigated the role of human breast-cancer-derived fibroblasts in the proliferation of primary cultures of epithelial cells derived from the same tumor. For this purpose, a co-culture system, using Transwell tissue-culture inserts with microporous membranes was employed. Fibroblasts and epithelial cells were enriched according to differences in their density on Percoll density gradients. The co-culture system was first established using MCF-7 breast cancer cells and a human fibroblast line (HF cells). Insulin, 17β-estradiol, EGF and HF cells all significantly stimulated the growth of MCF-7 breast cancer cells. The stimulatory effects of insulin, E2 and EGF were additive to the stimulatory effect of HF cells. These data suggest that (unique) factor(s), other than the above-mentioned growth-promoting compounds, are responsible for the growth-promoting effects of fibroblasts. In half of the human breast cancers investigated, tumor-derived fibroblasts stimulated tumor-derived epithelial cell proliferation. EGF significantly stimulated epithelial cell proliferation in 4 out of 6 cultures. The stimulatory effects of fibroblasts and EGF were additive or synergistic, and were observed in the additional presence of FCS, again suggesting production of unique factor(s) by the fibroblasts, in one culture the fibroblasts significantly inhibited epithelial tumor-cell proliferation. Conversely, the epithelial cells significantly stimulated proliferation of fibroblasts in 3 out of 3 cultures. The somatostatin analogue octreotide significantly inhibited epithelial cell proliferation by 46% in one tumor-cell culture in the absence, but not in the presence, of fibroblasts. In one culture, octreotide significantly inhibited the proliferation of fibroblasts co-cultured with epithelial cells

Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide

Recently, we developed a technique that allows the in vivo visualization in man of somatostatin receptor-positive neuroendocrine tumors after i.v. injection of [125I-Tyr3]octreotide or [111In-DTPA-D-Phe1]octreotide. Radiotherapy of such tumors using somatostatin analogs coupled to alpha- or beta-emitting radionuclides has been proposed as an application for radiolabeled somatostatin analogs. To develop this concept further, it is of importance to know whether the above-mentioned radiolabeled somatostatin analogs are internalized by the tumor cells, and whether it might be possible to manipulate the degree of internalization. In the present study we investigated the internalization of a stable somatostatin analog, [125I-Tyr3]octreotide, by mouse AtT20/D16V pituitary tumor cells and primary cultures of human GH-secreting pituitary tumor cells. Treatment of the cells with low pH was used to distinguish between membrane-bound (acid-releasable) and internalize (acid-resistant) radioligand. [125I-Tyr3]octreotide showed a time-dependent increasing accumulation in AtT20 cells; after 4 h of incubation, values up to 6-8% of the dose of radioligand added were obtained. Binding and internalization of [125I-Tyr3]octreotide were temperature dependent and inhibited by pertussis toxin. Inhibitors of lysosomal degradation did not increase the amount of internalized radioligand. After 4 h of incubation, 88% of the radioactivity present in the cells was still peptide bound, suggesting a low intracellular breakdown of this radioligand. Six of seven human GH-secreting adenoma cell cultures also internalized [125I-Tyr3]octreotide (variation between 0.24-4.98% of the dose radioligand added). Displacement of binding and internalization of [125I-Tyr3]octreotide by unlabeled octreotide showed a bell-shaped curve in AtT20 cells. At low concentrations (0.1 and 1 nM), binding and internalization were increased, whereas at higher concentrations, saturation occurred. In contrast to this, binding of [125I-Tyr3]octreotide to a broken cell preparation of AtT20 cells was displaced in a dose-dependent manner by unlabeled octreotide, with an IC50 of 0.1 nM. Similar observations were made in the human GH-secreting adenoma cell cultures. In conclusion, a high amount of [125I-Tyr3]octreotide is internalized in a specific-, time-, temperature-, and pertussis toxin-sensitive GTP-binding protein-dependent manner by mouse AtT20 and human GH-secreting pituitary tumor cells. In the presence of a low concentration of unlabeled octreotide, a rapid increase in the amount of [125I-Tyr3]octreotide internalized by AtT20 cells and by the majority of the human GH-secreting adenoma cell cultures was found.(ABSTRACT TRUNCATED AT 400 WORDS

Cortistatin rather than somatostatin as a potential endogenous ligand for somatostatin receptors in the human immune system

Cells of the human immune system have been shown to express somatostatin receptors (sst). The expression of sst suggests a functional role of the peptide somatostatin (SS). However, SS expression has not been demonstrated yet in different human immune tissues. Therefore, we investigated by RT-PCR the expression of both SS and cortistatin (CST), a SS-like peptide, in various human lymphoid tissues and immune cells. We detected SS mRNA expression in the human thymus only, while not in thymocytes. CST mRNA was clearly expressed in the immune cells, lymphoid tissues, and bone marrow. Using quantitative RT-PCR, significant differences in expression levels between tissues were demonstrated. Expression of CST mRNA was up-regulated during differentiation of monocytes into macrophages and dendritic cells and could be up-regulated by lipopolysaccharide stimulation. Two differently sized cDNA fragments of CST were detected in the majority of cells and tissues. However, although both fragments were detected in nearly all T-cell lines (7 of 8), most of the B-cell lines expressed the short fragment only (8 of 10). Usin

Immunohistochemical detection of somatostatin receptor subtypes sst1 and sst2A in human somatostatin receptor positive tumors

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors

Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas

Interferon-alpha (IFN alpha) may exert direct inhibitory effects on cell proliferation and on the production of different peptide hormones. We investigated the effect of IFN alpha on hormone production by 15 GH-secreting pituitary adenomas, 4 clinically nonfunctioning or gonadotroph pituitary adenomas, and 4 prolactinomas in vitro. In the GH-secreting pituitary adenoma cultures, a short term (72-h) incubation with IFN alpha (50-100 U/mL) significantly inhibited GH secretion in 3 of 7 cases and PRL secretion in 6 of 7 cultures. During prolonged incubation (14 days) with IFN alpha, GH and/or PRL secretion was significantly inhibited in 7 of 8 cultures (GH, 17-78% inhibition; PRL, 39-88% inhibition). In the clinically nonfunctioning or gonadotroph cultures