IGF1R and MAPK15 Emerge as Potential Targets of Pentabromobenzylisothioureas in Lung Neuroendocrine Neoplasms.

Pentabromobenzylisothioureas are antitumor agents with diverse properties, including the inhibition of MAPK15, IGF1R and PKD1 kinases. Their dysregulation has been implicated in the pathogenesis of several cancers, including bronchopulmonary neuroendocrine neoplasms (BP-NEN). The present study assesses the antitumor potential of ZKKs, a series of pentabromobenzylisothioureas, on the growth of the lung carcinoid H727 cell line. It also evaluates the expression of MAPK15, IGF1R and PKD1 kinases in different BP-NENs. The viability of the H727 cell line was assessed by colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) and its proliferation by BrdU (5-bromo-2'-deoxyuridine) assay. Tissue kinase expression was measured using TaqMan-based RT-PCR and immunohistochemistry. ZKKs (10-4 to 10-5 M) strongly inhibited H727 cell viability and proliferation and their antineoplastic effects correlated with their concentrations (p < 0.001). IGF1R and MAPK15 were expressed at high levels in all subtypes of BP-NENs. In addition, the SCLC (small cell lung carcinoma) patients demonstrated higher mRNA levels of IGF1R (p = 0.010) and MAPK15 (p = 0.040) than the other BP-NEN groups. BP-NENs were characterized by low PKD1 expression, and lung neuroendocrine cancers demonstrated lower PKD1 mRNA levels than carcinoids (p = 0.003). ZKKs may suppress BP-NEN growth by inhibiting protein kinase activity. Our results suggest also a possible link between high IGF1R and MAPK15 expression and the aggressive phenotype of BP-NEN tumors.

High Expression of NEK2 and PIM1, but Not PIM3, Is Linked to an Aggressive Phenotype of Bronchopulmonary Neuroendocrine Neoplasms.

Dysregulations of the NEK2 and PIM1-3 kinase signaling axes have been implicated in the pathogenesis of several cancers, including those with a neuroendocrine phenotype. However, their impact on bronchopulmonary neuroendocrine neoplasms (BP-NENs) has not been investigated. The aim of this pilot study was to determine mRNA and protein levels of NEK2, PIM1, and PIM3 in a group of 49 patients with BP-NENs: 11 typical carcinoids, 5 atypical carcinoids, 11 large cell neuroendocrine carcinomas, 22 small cell lung carcinomas (SCLC). The expression was measured using TaqMan-based RT-PCR and immunohistochemistry. NEK2 and PIM1 mRNA levels were higher in the SCLC patients than in the other BP-NEN groups (p < 0.001). There was an association between NEK2 mRNA and protein expression (p = 0.023) and elevated NEK2 mRNA levels were related to reduced survival in BP-NEN patients (p = 0.015). Patients with higher PIM1 protein expression had also diminished survival comparing with those with weak or no PIM1 expression (p = 0.037). Elevated NEK2 and PIM1 expression were related to aggressive tumor phenotype and indirectly affected the overall survival of BP-NEN patients. Our pilot study supports the need for future investigation of the biological function of NEK2 and PIM1 in BP-NEN transformation to verify the clinical value of our findings.

Elevated serum concentrations of IGF-1 and IGF-1R in patients with thyroid cancers.

BACKGROUND: The rising incidence of thyroid cancer observed in the last few decades requires an improvement in diagnostic tools and management techniques for patients with thyroid nodules. AIMS: The aim of this study was to assess serum concentrations of IGF-1 and IGF-1R in patients diagnosed with thyroid cancers. METHODS: 36 patients diagnosed with papillary thyroid cancer (PTC), 11 subjects with follicular thyroid cancer (FTC), 9 patients with anaplastic thyroid cancer (ATC) and 19 subjects with multinodular nontoxic goiter (MNG) were enrolled to the study. The control group (CG) consisted of 20 healthy volunteers. Blood samples were collected one day before surgery. Serum IGF-1 and IGF-1R concentrations were measured using specific ELISA methods. RESULTS: Significantly higher concentrations of IGF-1 were found in patients with PTC as compared with controls but not that obtained from subjects diagnosed with MNG. The concentration of IGF-1R was significantly elevated in subjects with PTC and ATC as compared with healthy volunteers. Similarly, patients diagnosed with PTC or ATC presented significantly higher serum concentration of IGF-1R in comparison to the MNG group. CONCLUSIONS: Our results show that the IGF-1 - IGF-1R axis plays a significant role in the development of PTC and ATC and imply that serum concentrations of both cytokines may be considered as additional markers for the differentiation of malignancies during the preoperative diagnosis of patients with thyroid gland tumors. These results indicate that IGF-1R serum concentrations allow us to differentiate between MNG and PTC or ATC. Moreover IGF-1R serum values appear to be better predictor of PTC and ATC than IGF-1 concentrations.

Dysregulation in IGF-1R, FGFR4 and ßKlotho signaling in patients with medullary thyroid cancer.

BACKGROUND: Medullary thyroid cancer (MTC) is a relatively rare thyroid neoplasm derived from neuroendocrine C cells which secrete calcitonin. αKlotho (αKL) and ßKlotho (ßKL) are transmembrane proteins which modulate different signaling systems, such as endocrine FGFs and IGF1 pathways. Dysregulation of the FGF19/FGFR4/ßKL and IGF-1/IGF-1R/αKL signaling axes has been implicated in the pathogenesis of several cancers. However, their role in the pathogenesis of MTC has not been determined. METHODS: The aim of this study was to assess αKL, ßKL, FGF19, IGF-1, FGFR4, and IGF-1R concentrations in a group of 11 patients with medullary thyroid cancer (MTC). The control group consisted of 20 healthy volunteers. Serum concentrations of these factors were measured using specific ELISA methods. RESULTS: Significantly lower concentrations of ßKL and higher concentrations of FGFR4 and IGF-1R were found in patients with MTC as compared to controls. CONCLUSIONS: Our results indicate that a disrupted signaling pathway for ßKL, FGFR4 and IGF-1R may play a role in the development of medullary thyroid cancers. However, further studies are required to confirm these findings and to use this knowledge in clinical practice.

Estimation of vitamin D status in patients with secondary and primary hypothyroidism of different etiology.

BACKGROUND: Though vitamin D deficiency is a global problem with wide spectrum of severe public health consequences, inadequate vitamin D status still remains one of the most common and untreated medical conditions. Thyroid diseases, including hypothyroidism, also represent the most frequent endocrinopathies in general population. OBJECTIVES: To determine the vitamin D status in hypothyroid patients and to ascertain the status of thyroid hormone replacement. METHODS: The 25(OH)D concentrations (ECLIA) in 71 hypothyroid patients recruited in the Outpatient Clinic of Endocrinology or Department of Clinical Endocrinology were assessed. The examined group was composed of 59 subjects diagnosed with primary hypothyroidism of different etiology and 12 patients with secondary hypothyroidism. The control group included 16 healthy individuals. RESULTS: Mean serum 25(OH)D concentration in healthy volunteers was significantly lower than in hypothyroid subjects (13.09±1.63 vs. 19.92±1.37 ng/mL). Patients with a history of thyroidectomy presented with significantly higher mean 25(OH)D concentration than controls (23.25±2.75 vs. 13.09±1.63 ng/mL). Mean serum 25(OH)D concentration in effectively treated hypothyroidism was significantly higher than in controls (21.90±1.47 vs. 13.09±1.63 ng/mL) or undertreated hypothyroidism (21.90±1.47 vs. 13.52±3.39 ng/mL). Hypothyroid patients aged under 60 years presented with significantly lower mean 25(OH)D concentration than elders (16.46±1.54 vs. 24.39±1.18 ng/mL). The major 25(OH)D deficient (≤10 ng/mL) or deficient (≤20 ng/mL) hypothyroid patients were significantly younger than those with 25(OH)D concentrations exceeding 10 ng/mL or 20 ng/mL respectively. CONCLUSIONS: These findings confirm the necessity for vitamin D status improvement in the general population and more effective healthcare of hypothyroid patients.

Alteration in the serum concentrations of FGF19, FGFR4 and ßKlotho in patients with thyroid cancer.

INTRODUCTION: ßKlotho (ßKL) is known to act as co-receptor for fibroblast growth factor receptor 4 (FGFR4) which is the main cognate receptor for fibroblast growth factor 19 (FGF19). Dysregulation of this FGF19/FGFR4/ßKL signaling axis has been implicated in the pathogenesis of several cancers. However, its role in the pathogenesis of thyroid cancer has not been determined. MATERIALS AND METHODS: The aim of this study was to assess FGF19, FGFR4 and ßKL concentrations in a group of 36 patients with papillary thyroid cancer (PTC), 11 patients with follicular thyroid cancer (FTC), 9 patients with anaplastic thyroid cancer (ATC) and a group of 19 subjects with multinodular nontoxic goiter (MNG). The control group consisted of 20 healthy volunteers. Serum FGF19, FGFR4 and ßKL concentrations were measured using specific ELISA methods. RESULTS: Significantly lower concentrations of ßKL and higher concentrations of FGF19 were found in patients with PTC, FTC and ATC as compared with MNG group and controls. An elevation of FGFR4 serum concentration was observed in all thyroid cancer groups in comparison to MNG group and controls; however, in FTC group it was statistically insignificant. A positive correlation was found between ßKL and FGFR4 concentrations in PTC patients. The levels of ßKL, FGF19 and FGFR4 did not differ significantly between MNG group and healthy controls. CONCLUSIONS: Our results indicate that a disrupted FGF19/FGFR4/ßKL signaling pathway may play a role in the development of thyroid cancers. However, further studies are needed to elucidate the molecular mechanism of the neoplastic transition of thyroid epithelial cells.

Elevated Concentrations of SERPINE2/Protease Nexin-1 and Secretory Leukocyte Protease Inhibitor in the Serum of Patients with Papillary Thyroid Cancer.

Introduction. SERPINE2 and secretory leukocyte protease inhibitor (SLPI) are proteins with anticoagulant properties which could promote solid tumor growth. However, their role in the pathogenesis of thyroid cancer has not been determined. Materials and Methods. The aim of this study was to assess serum SERPINE2 and SLPI concentrations in a group of 36 patients with papillary thyroid cancer (PTC) and a group of 19 subjects with multinodular nontoxic goiter (MNG). The control group (CG) consisted of 20 healthy volunteers. Blood samples were collected one day before surgery. Serum SERPINE2 and SLPI concentrations were measured using specific ELISA methods. Results. Significantly higher concentrations of SERPINE2 and SLPI were found in patients with PTC as compared with MNG and controls. Positive correlation was found between SERPINE2 and SLPI concentrations in PTC patients. The levels of SERPINE2 and SLPI did not differ significantly between MNG and healthy controls. Conclusions. Our results indicate that SERPINE2 and SLPI play a significant role in the development of papillary thyroid cancer and imply that the evaluation of serum concentrations of both anticoagulant molecules may be considered as additional marker for the differentiation of malignancies during the preoperative diagnosis of patients with thyroid gland tumors.

Somatostatin Analogs and Tumor Localization Do Not Influence Vitamin D Concentration in Patients with Neuroendocrine Tumors.

Patients with neuroendocrine tumors (NETs), malignancies of rare but still rising incidence, may be a group at higher risk of vitamin D insufficiency. The gastrointestinal tumor prevalence and somatostatin analog (SSA) therapy may cause vitamin D malabsorption. The aim of this study was to evaluate the serum level of vitamin D in NET patients. A total of 36 NET patients were enrolled into the experimental group and 16 individuals were enrolled into the control group. All patients were further classified into subgroups according to primary tumor localization (gastropancreatic, lung, and other NETs) or therapy (with or without SSA treatment). The concentrations of total 25(OH)D were assayed with Electrochemiluminescence immunoassay (ECLIA). Serum concentration of 25(OH)D in NET patients did not differ significantly from that of the control group. However, the average level of 25(OH)D in both groups met the criteria of vitamin D deficiency. Importantly, SSA therapy did not aggravate vitamin D deficiency. Moreover, the concentration of 25(OH)D in the studied group was not significantly influenced by primary tumor localization, patient age, or season. Vitamin D deficiency is a widespread disorder affecting both NET patients and individuals without other health problems, and SSA and gastrointestinal tumor localization do not exacerbate this condition.

Decreased serum level of IL-7 in patients with active Graves' disease.

BACKGROUND: Graves' disease (GD) is a common autoimmune disease which is one of the major causes of hyperthyroidism. Interleukin 7 (IL-7) has been recently reported to play an important role in various autoimmune diseases, but its role in the pathogenesis of GD has not been assessed. The aim of this study was to evaluate the levels of IL-7 and the soluble form of its receptor (sIL-7R) in the serum of GD patients, and to identify their association with disease activity. METHODS: A total of 37 GD patients were enrolled into the experimental group and 16 individuals into the control group. All patients were further classified into three subgroups: a GD-active group (hyperthyroidism and TRAb (thyroid stimulating hormone receptor antibody) >7.5 U/L) (N=15), a GD-inactive group (euthyreosis and TRAb<1 U/L) (N=8), and other GD patients (euthyreosis and TRAb>1 U/L) (N=14). Concentrations of IL-7 and sIL-7R were assayed with ELISA. Additionally, the relationship between IL-7 and sIL-7R serum concentrations with disease activity (free triiodothyronine [FT3], free thyroxine [FT4], thyroid stimulating hormone [TSH] and TRAb) was also analyzed. RESULTS: The serum concentrations of IL-7 in GD-active patients were significantly lower than those of the control group as well as the GD-inactive and GD-other groups. The serum level of IL-7 in GD patients negatively correlated with FT4 and TRAb concentrations. Moreover, no significant difference was observed in the serum level of sIL-7R in GD patients compared to the control group. CONCLUSIONS: These observations suggest that IL-7 may play a role in the pathogenesis of GD and may be associated with its clinical activity. To this end, the serum level of IL-7 could be an additional diagnostic biomarker predictive of the disease and could be particularly valuable for TRAb-negative GD patients.

Concentration of angiopoietins 1 and 2 and their receptor Tie-2 in peripheral blood in patients with chronic obstructive pulmonary disease.

INTRODUCTION: Both angiopoietins (angiopoietin 1 - Ang-1, angiopoietin 2 - Ang-2) and angiopoietin receptors (Tie) are involved in angiogenesis and vascular remodeling. AIM: To assess concentrations of Ang-1, Ang-2 and Tie-2 in blood of patients with chronic obstructive pulmonary disease (COPD) and evaluate if their concentrations depend on the severity of the disease. MATERIAL AND METHODS: Thirty patients with COPD (stage II-IV) and 8 healthy smokers as well as 8 healthy non-smokers were included in the study. Detailed history was taken, physical examination and spirometry tests were done and blood samples were taken for evaluation of serum concentrations of Ang-1, Ang-2 and Tie. RESULTS: Among COPD patients, 8 patients suffered from moderate disease, 8 patients had severe, while 14 patients had very severe disease. The concentrations of Ang-1 and Ang-2 were not significantly greater in patients with COPD than in healthy controls. The highest concentrations of Ang-1 and Ang-2 were observed in patients with moderate COPD, and levels of Ang-2 correlated with Tie-2 in this group of patients. The levels of Ang-1 were the lowest in healthy non-smokers and in patients with severe COPD, where they inversely correlated with Tie-2. The concentrations of Ang-2 were not significantly higher in patients with moderate COPD when compared with those with severe and very severe disease and healthy smokers, and were significantly higher than in healthy non-smokers. CONCLUSIONS: It is possible that Ang-1, Ang-2 and Tie-2 play an important role especially in the early stage of COPD but not in the late phase when vascular complications of the disease occur.

Interferon alpha and rapamycin inhibit the growth of carcinoid and medullary thyroid cancer in vitro.

Neuroendocrine tumors (NETs) are highly vascularized neoplasms characterized by rising incidence. Moreover, the neuroendocrine cells were shown to express vascular endothelial growth factor (VEGF) and VEGF receptors. Therefore, angiomodulators could be potentially a new group of drugs enhancing still unsatisfactory effectiveness of NET therapy. The aim of this study was to assess the direct influence of angiomodulators: VEGF and five endogenous and exogenous antiangiogenic compounds (endostatin, interferon alpha [IFNα], rapamycin, JV1-36, semaxinib [SU5416]) on the growth of two NET cell lines: lung carcinoid H727 cell line and medullary thyroid cancer TT cell line in vitro. IFNα and rapamycin induced the inhibitory effect on H727 and TT cell viability and proliferation, increasing apoptosis and arresting the cell cycle. Also semaxinib (10(-5)M) inhibited proliferation of both cell lines. VEGF and endostatin did not influence the growth of H727 and TT cells. The inhibitory effect of IFNα, rapamycin and semaxinib on carcinoid and medullary thyroid cancer growth was revealed in our in vitro study, although some other antiangiogenic agents did not directly influence H727 and TT cell growth. Thus, IFNα and mTOR inhibitors as multidirectionally acting drugs with antiangiogenic effect could be potentially efficient in treatment of neuroendocrine tumors and are worth further studies.

Interferon alpha and rapamycin inhibit the growth of pheochromocytoma PC12 line in vitro.

INTRODUCTION: Pheochromocytomas are benign or malignant neuroendocrine tumours. The unsatisfactory efficacy of the traditional therapeutic methods for patients with metastatic disease results in a continuing search for more effective and targeted agents. Due to the increased vascularisation of these tumours, inhibitors of angiogenesis could be potentially a new group of drugs in pheochromocytoma/paraganglioma therapy. MATERIAL AND METHODS: The aim of this study was to evaluate the influence of angiomodulators: VEGF (vascular endothelial growth factor) and five endogenous and exogenous antiangiogenic compounds (endostatin; IFN-alpha [interferon alpha]; rapamycin - mTOR [mammalian target of rapamycin] inhibitor; JV1-36 and SU5416 (semaxinib]) on the growth of rat pheochromocytoma PC12 cell line. RESULTS: IFN-alpha (10(5) U/mL) strongly inhibited PC12 growth in a 72 h culture, increasing apoptosis and arresting the cell cycle. Rapamycin in a wide range of concentrations (10(-5) to 10(-8) M) induced a slight inhibitory effect on PC12 viability and decreased cell proliferation at the concentration of 10(-5) M. VEGF, endostatin and JV1-36 did not influence the growth of PC12. CONCLUSIONS: The study has shown for the first time that IFN-a inhibited the growth of pheochromocytoma PC12 line and confirmed the inhibitory action of rapamycin on these cells. The results suggest that IFN-alpha and mTOR inhibitors could be potentially effective in the therapy of malignant pheochromocytoma, and encourage further study in this field.

Immunohistochemical detection of follicle stimulating hormone receptor (FSHR) in neuroendocrine tumours.

INTRODUCTION: Follicle stimulating hormone receptors (FSHR) are well known to be expressed in gonads and in gonadal tumours. Recently,their incidence has also been revealed in endocrine non-gonadal tumours such as adrenal and pituitary tumours. Moreover, FSHR immunostaininghas also been reported in endothelium of intra- and peritumoral blood vessels of a large series of cancers. The presentpaper reports on the incidence of FSHR in both tumoral cells and some intratumoral blood vessels of neuroendocrine tumours (NETs). MATERIAL AND METHODS: Sixteen NETs samples were taken from 14 patients. The tumour samples were immunostained using the antibodyraised against 1-190 amino acid sequence from the human FSH-R and anti-Ki67 antibody. RESULTS: In all the samples examined, the majority of tumoral cells were immunostained with anti-FSHR antibody. Positive immunostainingconcerned also the intratumoral blood vessels endothelia in a half of the examined samples. Immunopositive blood vessels were foundmore often in tumours with higher Ki-67 index. CONCLUSION: FSHR expressed in NETs, if they are functional, may mediate the signals which can enhance further tumour growth.

Long-term impact of vertical banded gastroplasty (VBG) on plasma concentration of leptin, soluble leptin receptor, ghrelin, omentin-1, obestatin, and retinol binding protein 4 (RBP4) in patients with severe obesity.

Restrictive type bariatric surgery is an effective therapeutic approach that decreases overall mortality in patients with severe obesity. Several new cytokines, including adipocytokines that control energy metabolism, have been discovered recently, but their role in obesity is not fully recognized. The aim of the study was to evaluate the influence of vertical banded gastroplasty (VBG), one of restrictive type bariatric surgery, on peripheral blood concentrations of some adipocytokines and hormones involved in the control of food intake and energy turnover. The studied group comprised 12 females and 2 males aged from 31 to 59years (46.6±7.4) with simple obesity (BMI: 44.9±7.2) and metabolic syndrome. The patients were examined both before and 3, 6, 12, 24months after bariatric surgery (eight patients were also checked after 36 and six patients after 48months). Measurements of peripheral blood concentration of glucose, insulin, leptin, soluble leptin receptor, obestatin, ghrelin, omentin-1, and retinol binding protein 4 (RBP4) by ELISA method have been performed. After the surgery body weight, BMI and waist circumference significantly decreased. Positive changes considering the components of metabolic syndrome have been noted. Namely glucose, insulin and triglycerides' levels decreased, accompanied by the significantly lower HOMA index. Conversely, HDL cholesterol concentrations increased. Furthermore, peripheral blood concentration of leptin decreased, but the blood levels of soluble leptin receptor and ghrelin gradually increased. The positive correlations between leptin and body weight and BMI were noted as well as between the RBP4 and total cholesterol and LDL cholesterol levels. We did not observe significant differences in levels of obestatin, omentin-1 and RBP4 after surgery. In conclusion, VBG is an effective type of bariatric surgery. Fast decrease of body weight in morbidly obese patients treated by restrictive bariatric surgery leads to significant changes in peripheral blood levels of some adipokines and hormones controlling energy turnover and appetite (leptin and soluble leptin receptor) as well as ghrelin but not omentin-1, obestatin or retinol binding protein (RBP-4).

Modulation of ghrelin axis influences the growth of colonic and prostatic cancer cells in vitro.

BACKGROUND: The risk of different cancers seems to be associated with obesity. Moreover, low ghrelin levels observed in obese people may be implicated in cancer development and progression. The aim of this study was to examine the direct effects of both forms of ghrelin (acylated and unacylated) and ghrelin receptor type 1a antagonist (D-Lys-GHRP-6) on the growth of murine colon cancer MC38 and human prostate cancer DU145 cell lines in vitro. METHODS: The cells were cultured for 72 h in the presence of rat or human acylated ghrelin (rG, hG), human unacylated ghrelin (hUAG), D-Lys-GHRP-6 (GHS-RA) applied either alone or jointly. The cell line growth was assessed by the colorimetric Mosmann method. RESULTS: hUAG (10(-6), 10(-7) and 10(-10) M) inhibited MC38 cancer cell growth and, at some concentrations (10(-8), 10(-9), 10(-10) M), enhanced the antineoplastic effect of GHS-RA(10(-4) M). In turn, GHS-RA evoked a biphasic effect on MC38 cancer growth: inhibitory at 10(-4) M and stimulatory at 10(-5) and 10(-6) M. Moreover, GHS-RA at the highest examined concentration (10(-4) M) enhanced the cytostatic effect of FU. Human acylated and unacylated ghrelin and GHS-RA inhibited DU145 cancer growth with moderate and different potencies. A dose-response effect was observed for the inhibitory action of hG together with the synergistic effect of hUAG and GHS-RA. CONCLUSION: The obtained results indicate an involvement of the ghrelin axis in the growth regulation of colon and prostate cancers and may suggest new therapeutic options for these neoplasms.

Immunohistochemical detection of FSH receptors in pituitary adenomas and adrenal tumors.

OBJECTIVES: Follicle stimulating hormone (FSH) receptors (FSHR) are physiologically expressed in the ovary and testis. It is well known that FSHR are also expressed in gonadal cancers, but the data on their incidence in extra-gonadal tumors are scarce. Recently, the expression of FSHR in the vascular endothelium within different human cancers was found, but nothing is known on FSHR appearance in non-gonadal endocrine tumors. The present paper reports on the immunohistochemical detection of FSHR in human pituitary adenomas and adrenal tumors. MATERIALS AND METHODS: The study included samples of 28 pituitary adenomas and 36 adrenal tumors. Moreover, 2 samples of non-tumoral adrenal glands were also studied. FSH receptor immunostaining was performed on paraffin sections using the rabbit anti-human FSH-R polyclonal antibody raised against 1-190 amino acid sequence from the human FSH-R (sc-13935). The pituitary adenomas were immunostained to reveal the pituitary hormones and the proliferation marker Ki-67. RESULTS: In the pituitary adenomas, positive immunostaining with anti-FSHR antibody occurred in the adenoma cells cytoplasm and endothelia of the intra- and peritumoral blood vessels. The cytoplasmic immunostaining was found in the majority of investigated tumors but the intensity of staining was weak to moderate. There is some tendency towards the higher cytoplasmic FSHR score in tumors with higher Ki-67 index (atypical adenomas). In contrast to the cytoplasm, the FSHR immunostaining in blood vessels is strong and concerns all the investigated samples. Strong FSHR immunostaining is present in the endothelium of intra- and/or peritumoral blood vessels in the majority of pheochromocytomas, approximatively one half of the adrenocortical adenomas and both cases of the adrenal cancers. The immunostaining is detectable also in the tumoral cell cytoplasm in all but one examined pheochromocytomas. All the investigated adrenocortical adenomas presented strong immunostaining of cell membranes. No immunostained cell membranes were found. in adrenal cancers. The positive immunostaining was found in glandular cells, but not in blood vessels, of non-tumoral adrenal cortex and medulla. CONCLUSIONS: Immunostaining of FSHR often occurs in the endothelium of intra- and/or peritumoral blood vessels of pituitary adenomas and benign and malignant adrenal tumors. The immunostaining may be also present in tumoral cells. A role of FSHR expression in these tumors (stimulation of angiogenesis? stimulation of cell growth?) needs further studies to be clarified.

Angiotensins inhibit cell growth in GH3 lactosomatotroph pituitary tumor cell culture: a possible involvement of the p44/42 and p38 MAPK pathways.

The local renin-angiotensin system is present in the pituitary. We investigated the effects of angiotensins on GH3 lactosomatotroph cells proliferation in vitro and the involvement of p44/42 and p38 MAPK inhibitors in the growth-regulatory effects of angiotensins. Materials and Methods. Cell viability using the Mosmann method and proliferation by the measurement of BrdU incorporation during DNA synthesis were estimated. Results. Ang II and ang IV decreased the viability and proliferation of GH3 cells. Inhibitor of p44/42 MAPK attenuated the effects of ang II on cell viability and proliferation but did not affect the ang 5-8-dependent actions. Inhibitor of p38 MAPK prevented the decrease in the number of GH3 cells in ang-II- and ang-IV-treated groups. Conclusions. The growth-inhibitory effect of ang II is possibly mediated by the p44/42 MAPK. The p38 MAPK appears to mediate the inhibitory effects of both ang II and ang 5-8 upon cell survival.

The involvement of angiotensin type 1 and type 2 receptors in estrogen-induced cell proliferation and vascular endothelial growth factor expression in the rat anterior pituitary.

The aim of our study was to examine the involvement of renin-angiotensin system (RAS) in estrogen-induced lactotropes proliferation and vascular endothelial growth factor (VEGF) expression in rat pituitary. The study was performed on Fisher 344 rats underwent 8-day treatment with diethylstilboestrol (DES). The proliferation index (PCNA) and VEGF expression in pituitary sections were estimated using immunohistochemical methods. Treatment with DES increased the number of PCNA-positive cells, VEGF-positive cells, and VEGF-positive blood vessels in pituitary. Stimulatory effect of estrogen on cell proliferation and VEGF expression in blood vessels was attenuated by losartan, PD123319, and captopril. VEGF immunoreactivity in pituitary cells of DES-treated rats was decreased by AT1 antagonist and not changed by AT2 blocker and ACE inhibitor. Our findings suggest the involvement of RAS in DES-induced cell proliferation and VEGF expression in pituitary. Both the AT1 and AT2 receptors appear to mediate the estrogen-dependent mitogenic and proangiogenic effects in rat pituitary.

Effects of nitric oxide synthase inhibition on diethylstilbestrol-induced hyperprolactinaemia and pituitary tumourigenesis in rats.

INTRODUCTION: The overexpression of nitric oxide synthase (NOS) has been found in tumours, including pituitary adenomas. It has also been found that NOS is overexpressed in human spontaneous pituitary adenomas. The question arises whether NOS and its product, nitric oxide (NO), are involved in pituitary tumourigenesis. To investigate this question, in the present paper we examine the effects of NOS inhibition on the development of diethylstilbestrol (DES)-induced prolactin-secreting pituitary tumours in rats. MATERIAL AND METHODS: Thirty male Fisher 344 rats, four weeks old, were submitted to subcutaneous implantation of a silastic capsule containing DES (10 mg/capsule) or of an empty capsule. Six weeks after implantation, some of the DES-treated animals received a NOS inhibitor, N-nitro-l-arginine methyl ester (NAME), 1 mg/mL, in their drinking water, for the subsequent 14 days. Eight weeks after the implantation, all the animals were sacrificed, their pituitaries were weighed, and samples of heart blood were collected for prolactin (PRL) and vascular endothelial growth factor (VEGF) measurements. RESULTS: It was found that DES implantation significantly increased pituitary mass, as well as PRL and VEGF concentrations in blood serum. On the other hand, the administration of NAME did not affect significantly either VEGF concentration or pituitary mass. On the other hand, it did induce a further increase in PRL levels. CONCLUSIONS: These findings indicate that NO is involved in oestrogen-induced hyperprolactinaemia, but does not play a crucial role in oestrogen-induced pituitary tumourigenesis.