Midgut neuroendocrine tumor patients have a depleted gut microbiome with a discriminative signature.

RATIONALE: When compared to other types of cancer, the prevalence of midgut neuroendocrine tumors (NET) has disproportionally increased over the past decades. To date, there has been very little progress in discovering (epi)genetic drivers and treatment options for these tumors. Recent microbiome research has revealed that enteroendocrine cells communicate with the intestinal microbiome and has provided novel treatment targets for various other cancer types. Hence, our aim was to analyze the role of the gut microbiome in midgut NET patients. METHODS: Fecal samples, prospectively collected from patients and control subjects, were analyzed with next generation 16S sequencing. Patients with neuroendocrine carcinomas and recent antibiotics use were excluded. Relevant variables were extracted from questionnaires and electronic health records. Microbial composition was compared between patients and controls as well as between groups within the patient cohort. RESULTS: 87 midgut NET patients and 95 controls were included. Midgut NET patients had a less rich and diverse gut microbiome than controls (p < 0.001). Moreover, we identified 31 differentially abundant species and a gut microbial signature consisting of 17 species that was predictive of midgut NET presence with an area under the receiver operating characteristic curve of 0.863. Gut microbial composition was not directly associated with the presence of the carcinoid syndrome, tumor grade or multifocality. Nonetheless, we did observe a potential link between microbial diversity and the presence of carcinoid syndrome symptoms within the subset of patients with elevated 5-hydroxyindolacetic acid levels. CONCLUSION: Midgut NET patients have an altered gut microbiome which suggests a role in NET development and could provide novel targets for microbiome-based diagnostics and therapeutics.

MDR1 inhibition increases sensitivity to doxorubicin and etoposide in adrenocortical cancer.

Chemotherapy for adrenocortical carcinoma (ACC) has limited efficacy and is accompanied by severe toxicity. This lack of effectiveness has been associated with high tumoral levels of the multidrug resistance (MDR) pump P-glycoprotein (P-gp), encoded by the MDR1 gene. In this study, effects of P-gp inhibition on the sensitivity of ACC cells to cytotoxic drugs were evaluated. MDR1 mRNA and P-gp expression were determined in human adrenal tissues and cell lines. H295R, HAC15 and SW13 cells were treated with mitotane, doxorubicin, etoposide, cisplatin and streptozotocin, with or without the P-gp inhibitors verapamil and tariquidar. Cell growth and surviving fraction of colonies were assessed. MDR1 mRNA and P-gp protein expression were lower in ACCs than in adrenocortical adenomas (P < 0.0001; P < 0.01, respectively). MDR1 and P-gp expression were positively correlated in ACC (P < 0.0001, ρ = 0.723). Mitotane, doxorubicin, cisplatin and etoposide dose dependently inhibited cell growth in H295R, HAC15 and SW13. Tariquidar, and in H295R also verapamil, increased the response of HAC15 and H295R to doxorubicin (6.3- and 7.5-fold EC50 decrease in H295R, respectively; all P < 0.0001). Sensitivity to etoposide was increased in H295R and HAC15 by verapamil and tariquidar (all P < 0.0001). Findings were confirmed when assessing colony formation. We show that cytotoxic drugs, except streptozotocin, used for ACC treatment, inhibit ACC cell growth and colony formation at clinically achievable concentrations. P-gp inhibition increases sensitivity to doxorubicin and etoposide, suggesting that MDR1 is involved in sensitivity to these drugs and could be a potential target for cytotoxic treatment improvement in ACC.

Inhibition of Human Adrenocortical Cancer Cell Growth by Temozolomide in Vitro and the Role of the MGMT Gene.

CONTEXT: Treatment of patients with adrenocortical carcinomas (ACC) with mitotane and/or chemotherapy is often associated with toxicity and poor tumor response. New therapeutic options are urgently needed. OBJECTIVE: The objectives of the study were to evaluate the therapeutic possibilities of temozolomide (TMZ) in ACC cells and to assess the potential predictive role of the DNA repair gene O6-Methylguanine-DNA methyltransferase (MGMT) in adrenocortical tumors. METHODS: Three human ACC cell lines and eight primary ACC cultures were used to assess effects of TMZ in vitro. In the cell lines, 11 normal adrenals, 16 adrenocortical adenomas, and 29 ACC, MGMT promoter methylation and expression were determined. RESULTS: IC50 values of TMZ on cell growth were 39 µM, 38 µM, and 44 µM for H295R, HAC15, and SW13, respectively. TMZ induced apoptosis and provoked cytotoxic and cytostatic effects by reducing the surviving fraction of ACC colonies and the colony size. TMZ thereby induced cell cycle arrests in ACC cell lines. TMZ and mitotane both inhibited growth of ACC cells cultured as three-dimensional spheroids. TMZ inhibited cell amount in five of eight primary ACC cultures and induced apoptosis in seven of eight primary ACC cultures. In ACC cell lines and adrenal tissues, MGMT promoter methylation was low. In ACCs, methylation was inversely correlated with MGMT mRNA expression. MGMT protein expression was not correlated with MGMT methylation. CONCLUSIONS: For the first time, we show the therapeutic potential of temozolomide for ACC, offering an urgently needed potential alternative for patients not responding to mitotane alone or with etoposide, doxorubicin, and cisplatin. (Pre-)clinical studies are warranted to assess efficacy in vivo.

Methylation of IGF2 regulatory regions to diagnose adrenocortical carcinomas.

Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis. Discrimination of ACCs from adrenocortical adenomas (ACAs) is challenging on both imaging and histopathological grounds. High IGF2 expression is associated with malignancy, but shows large variability. In this study, we investigate whether specific methylation patterns of IGF2 regulatory regions could serve as a valuable biomarker in distinguishing ACCs from ACAs. Pyrosequencing was used to analyse methylation percentages in DMR0, DMR2, imprinting control region (ICR) (consisting of CTCF3 and CTCF6) and the H19 promoter. Expression of IGF2 and H19 mRNA was assessed by real-time quantitative PCR. Analyses were performed in 24 ACCs, 14 ACAs and 11 normal adrenals. Using receiver operating characteristic (ROC) analysis, we evaluated which regions showed the best predictive value for diagnosis of ACC and determined the diagnostic accuracy of these regions. In ACCs, the DMR0, CTCF3, CTCF6 and the H19 promoter were positively correlated with IGF2 mRNA expression (P<0.05). Methylation in the most discriminating regions distinguished ACCs from ACAs with a sensitivity of 96%, specificity of 100% and an area under the curve (AUC) of 0.997±0.005. Our findings were validated in an independent cohort of 9 ACCs and 13 ACAs, resulting in a sensitivity of 89% and a specificity of 92%. Thus, methylation patterns of IGF2 regulatory regions can discriminate ACCs from ACAs with high diagnostic accuracy. This proposed test may become the first objective diagnostic tool to assess malignancy in adrenal tumours and facilitate the choice of therapeutic strategies in this group of patients.

Preoperative normalization of cortisol levels in Cushing's disease after medical treatment: consequences for somatostatin and dopamine receptor subtype expression and in vitro response to somatostatin analogs and dopamine agonists.

CONTEXT: Corticotroph pituitary adenomas often highly express the dopamine 2 receptor (D2R) and somatostatin receptor subtype 5 (sst5). The sst2 expression is relatively low, likely resulting from downregulating effects of high cortisol levels. This may explain why the sst2-preferring somatostatin analog octreotide, compared with the multi-receptor-targeting somatostatin analog pasireotide, is generally ineffective in Cushing's disease. OBJECTIVE: Our objective was to compare sst and D2R expression levels between adenomas from patients with elevated and normalized preoperative urinary free cortisol excretion. PATIENTS AND DESIGN: Corticotroph adenoma tissue was examined from patients from group 1 (n = 22; elevated preoperative urinary free cortisol) and group 2 (n = 11; mean duration of preoperative normocortisolism 10 weeks). Somatotroph adenoma tissue from 10 acromegalic patients was examined to compare receptor expression profiles. MAIN OUTCOME MEASURES: We evaluated receptor mRNA and protein expression levels and effects of octreotide, pasireotide, and cabergoline on ACTH secretion by cultured human corticotroph adenoma cells. RESULTS: The sst2 mRNA expression in group 2 was 10-fold higher than in group 1 (P < .01), even comparable to that in somatotroph adenomas. There were no statistically significant differences in sst5 and D2R mRNA expression or in sst2, sst5, and D2R protein expression between both groups of corticotroph adenomas. In responders, octreotide (n = 2 out of 4; -30.5% ± 10.4%) was less potent than pasireotide (n = 5 out of 6; -47.0% ± 4.2%) and cabergoline (n = 3 out of 4; -41.9% ± 3.1%) with respect to inhibition of ACTH secretion by adenomas from group 2. CONCLUSIONS: After achieving normocortisolism induced by medical therapy, cortisol-mediated sst2 downregulation on corticotroph adenomas appears to be a reversible process at the mRNA but not at the protein level. Octreotide remains less potent than pasireotide and cabergoline with respect to in vitro inhibition of ACTH secretion. Whether sustained normocortisolism induced by medical therapy induces re-expression of functional sst2 protein in corticotroph adenomas and whether this increases the ACTH-lowering potency of octreotide remains to be established.

Chromogranin A, Ki-67 index and IGF-related genes in patients with neuroendocrine tumors.

Chromogranin A (CgA) and the Ki-67 proliferation index are considered as important biochemical and pathological markers for clinical behaviour of gastroenteropancreatic neuroendocrine tumors (GEP NETs), respectively. The IGF system has been suggested as an important regulator of GEP NET proliferation and differentiation. A possible relationship between serum CgA (sCgA), Ki-67 proliferation index, and expression of IGF-related genes in patients with GEP NETs has not been demonstrated yet. This study investigates the relationship between sCgA, the Ki-67 proliferation index, and the expression of IGF-related genes in GEP NET tissues and their relation with 5-year survival. Tumor and blood samples from 22 GEP NET patients were studied. TUMORAL MRNA EXPRESSION OF IGF-RELATED GENES (IGFS: IGF1, IGF2; IGF receptors: IGF1R, IGF2R; insulin receptors: subtype A (IR-A) and B (IR-B); IGF-binding proteins (IGFBPs): IGFBP1, IGFBP2, IGFBP3, and IGFBP6) was measured using quantitative RT-PCR. Ki-67 proliferation index was determined using immunohistochemistry. sCgA was measured with ELISA. Five-year survival in patients with nonelevated sCgA (n=11) was 91 vs 46% in patients with elevated sCgA (n=11) (P=0.006). IR-A mRNA expression was significantly higher in tumors obtained from patients with elevated sCgA than in those from patients with nonelevated sCgA (6.42±2.08 vs 2.60±0.40; P=0.04). This data suggests that sCgA correlates well with 5-year survival of GEP NET patients, and that IR-A mRNA expression correlates well with tumor mass in GEP NET patients.

Fluconazole inhibits human adrenocortical steroidogenesis in vitro.

The antifungal agent ketoconazole is often used to suppress cortisol production in patients with Cushing's syndrome (CS). However, ketoconazole has serious side effects and is hepatotoxic. Here, the in vitro effects of ketoconazole and fluconazole, which might be less toxic, on human adrenocortical steroidogenesis were compared. The effects on steroidogenesis were examined in primary cultures of nine human adrenocortical tissues and two human adrenocortical carcinoma cell lines. Moreover, the effects on mRNA expression levels of steroidogenic enzymes and cell growth were assessed. Ketoconazole significantly inhibited 11-deoxycortisol (H295R cells; maximum inhibition 99%; EC(50) 0.73 µM) and cortisol production (HAC15 cells; 81%; EC(50) 0.26 µM and primary cultures (mean EC(50) 0.75 µM)). In cultures of normal adrenal cells, ketoconazole increased pregnenolone, progesterone, and deoxycorticosterone levels, while concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, DHEA, and androstenedione decreased. Fluconazole also inhibited 11-deoxycortisol production in H295R cells (47%; only at 1 mM) and cortisol production in HAC15 cells (maximum inhibition 55%; EC(50) 35 µM) and primary cultures (mean EC(50) 67.7 µM). In the cultures of normal adrenals, fluconazole suppressed corticosterone, 17-hydroxypregnenolone, and androstenedione levels, whereas concentrations of progesterone, deoxycorticosterone, and 11-deoxycortisol increased. Fluconazole (1 mM) slightly increased STAR mRNA expression in both cell lines. Neither compound affected mRNA levels of other steroidogenic enzymes or cell number. In conclusion, by inhibiting 11ß-hydroxylase and 17-hydroxylase activity, pharmacological concentrations of fluconazole dose dependently inhibit cortisol production in human adrenocortical cells in vitro. Although fluconazole seems less potent than ketoconazole, it might become an alternative for ketoconazole to control hypercortisolism in CS. Furthermore, patients receiving fluconazole because of mycosis might be at risk for developing adrenocortical insufficiency.

Mifepristone effects on tumor somatostatin receptor expression in two patients with Cushing's syndrome due to ectopic adrenocorticotropin secretion.

CONTEXT: Two patients presented with Cushing's syndrome due to ectopic ACTH secretion. Initial localization studies included computed tomography, magnetic resonance imaging, and octreoscans ((111)In-pentreotide scintigraphy), which were negative in both patients. They were treated with the glucocorticoid receptor antagonist mifepristone, with improvement in their clinical symptoms. Follow-up octreoscans after, respectively, 6 and 12 months showed the unequivocal presence of a bronchial carcinoid in both patients. OBJECTIVE: The objective of the study was to correlate in vivo and in vitro findings in patients with ectopic ACTH-producing syndrome. METHODS: We determined the expression of somatostatin and dopamine receptors by immunohistochemistry (patients 1 and 2), quantitative PCR, and in vitro culturing of tumor cells (patient 1 only). IN VITRO RESULTS: Both tumors were strongly positive for somatostatin receptor type 2 (sst(2)) on immunohistochemistry, whereas one of the tumors (patient 1) was also dopamine receptor subtype 2 (D(2)) positive on both immunohistochemistry and quantitative PCR. Octreotide (a sst(2) preferring analog) and cabergoline (D(2) agonist) both decreased the ACTH levels in the cultured tumor cells of patient 1. CONCLUSION: We describe two patients with ACTH-producing bronchial carcinoids, in whom a direct down-regulatory effect of glucocorticoid levels on tumoral sst(2) receptor expression is suggested by a remarkable change in octreoscan status after successful mifepristone therapy. Further studies will have to demonstrate whether glucocorticoid lowering or antagonizing therapy may be used to improve the diagnostic accuracy of somatostatin receptor scintigraphy in patients with ectopic ACTH production of unknown primary origin.

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.

Aberrant expression of multiple hormone receptors in ACTH-independent macronodular adrenal hyperplasia causing Cushing's syndrome.

OBJECTIVE: Aberrant adrenal expression of various hormone receptors has been identified in ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing cortisol hypersecretion regulated by hormones other than ACTH. We aimed to determine aberrant expression of multiple hormone receptors in vivo and in vitro in adrenal tissue of a patient with AIMAH. DESIGN: The design of the study includes clinical case description, and biochemical and immunohistochemical analysis to demonstrate aberrant expression of multiple hormone receptors in AIMAH. METHODS: The subject of the study is a male diagnosed with Cushing's syndrome because of AIMAH. Directly after laparoscopic removal of the adrenals, adrenal tissue was incubated with and without test substances (ACTH, forskolin, arginine vasopressin (AVP), desmopressin, epinephrine, norepinephrine, purified human chorionic gonadotropin (hCG), metoclopramide and the combinations of AVP with ACTH, epinephrine and metoclopramide). LH/hCG-receptor (hCG-R) immunohistochemistry and RT-PCR analyses were performed to demonstrate aberrant expression of LH/hCG-R and V(1-3)-AVPR. RESULTS: AIMAH was characterized by in vivo cortisol responsiveness to AVP and in vitro cortisol responses to AVP, hCG, epinephrine, and norepinephrine suggesting aberrant adrenal expression of the receptors for AVP (the V(1-3)-AVPRs), catecholamines (the beta-AR), and LH (the LH/hCG-R). Incubation with combinations of AVP and ACTH and of AVP with epinephrine induced a stronger cortisol response compared with incubation with the individual agents. Moreover, we demonstrated adrenal V(1-3)-AVPR and LH/hCG-R expression. CONCLUSIONS: AIMAH tissue may simultaneously express multiple aberrant hormone receptors, and individual ligands may potentiate each other regarding cell proliferation and cortisol production.

Effects of somatostatin analogs on a growth hormone-releasing hormone secreting bronchial carcinoid, in vivo and in vitro studies.

CONTEXT: A 56-yr-old woman presented with acromegaly, a pulmonary mass, and elevated levels of GHRH, GH, and IGF-I. Histological examination revealed a bronchial carcinoid with positive staining for GHRH. Somatostatin analogs (SAs) can play an important role in the treatment of neuroendocrine tumors, dependent on the somatostatin receptor subtype (sst) expression pattern. The sst pattern in bronchial carcinoids and effects of SAs have not been extensively investigated, particularly not for the recently developed universal SA SOM230 (Pasireotide) that has high affinity for sst1, 2, 3, and 5. OBJECTIVE: Our objective was to investigate the in vivo response of a GHRH-producing bronchial carcinoid to octreotide (OCT), its sst-expression profile, and in vitro responses to different SAs, including SOM230. METHODS: In vivo, 50 microg OCT was administered, and plasma GH and GHRH responses were determined. In vitro, the expression of ssts was analyzed by quantitative PCR. Furthermore, the effects of SOM230 and OCT on GHRH secretion were evaluated in primary cell cultures of the carcinoid tissue. RESULTS: In vivo, OCT administration fully suppressed GH and GHRH levels. In vitro, sst1 mRNA was most abundant, followed by sst2 and sst5. Both SOM230 and OCT inhibited GHRH production dose dependently (SOM230 100 nm vs. control, P = 0.01; OCT 110 nm vs. control, P = 0.05). CONCLUSIONS: In this case of a GHRH-producing bronchial carcinoid, we demonstrated that SOM230 was a potent inhibitor of GHRH production in vitro and was at least equally potent compared with OCT. Therefore, SOM230 may be a potential therapeutic agent to control GHRH secretion in ectopic acromegaly.

Differential regulation of human dopamine D2 and somatostatin receptor subtype expression by glucocorticoids in vitro.

Dopamine agonists (DA) and somatostatin (SS) analogues have been proposed in the treatment of ACTH-producing neuro-endocrine tumours that cause Cushing's syndrome. Inversely, glucocorticoids (GCs) can differentially influence DA receptor D(2) or SS receptor subtype (sst) expression in rodent models. If this also occurs in human neuro-endocrine cells, then cortisol-lowering therapy could directly affect the expression of these target receptors. In this study, we investigated the effects of the GC dexamethasone (DEX) on D(2) and sst expression in three human neuro-endocrine cell lines: BON (carcinoid) and TT (medullary thyroid carcinoma) versus DMS (small cell lung cancer), which is severely GC resistant. In BON and TT, sst(2) mRNA was strongly down-regulated in a dose-dependent manner (IC(50) 0.84 nM and 0.16 nM), whereas sst(5) and especially D(2) were much more resistant to DEX treatment. Sst(2) down-regulation was abrogated by a GC receptor antagonist and reversible in time upon GC withdrawal. At the protein level, DEX also induced a decrease in the total number of SS (-52%) and sst(2)-specific (-42%) binding sites. Pretreatment with DEX abrogated calcitonin inhibition by sst(2)-preferring analogue octreotide in TT. In DMS, DEX did not cause significant changes in the expression of these receptor subtypes. In conclusion, we show that GCs selectively down-regulate sst(2), but not D(2) and only to a minor degree sst(5) in human neuro-endocrine BON and TT cells. This mechanism may also be responsible for the low expression of sst(2) in corticotroph adenomas and underwrite the current interest in sst(5) and D(2) as possible therapeutic targets for a medical treatment of Cushing's disease.

Low circulating insulin-like growth factor I bioactivity in elderly men is associated with increased mortality.

CONTEXT: Low IGF-I signaling activity prolongs lifespan in certain animal models, but the precise role of IGF-I in human survival remains controversial. The IGF-I kinase receptor activation assay is a novel method for measuring IGF-I bioactivity in human serum. We speculated that determination of circulating IGF-I bioactivity is more informative than levels of immunoreactive IGF-I. OBJECTIVE: Our objective was to study IGF-I bioactivity in relation to human survival. DESIGN, SETTING, AND STUDY PARTICIPANTS: We conducted a prospective observational study at a clinical research center at a university hospital of 376 healthy elderly men (aged 73-94 yr). MAIN OUTCOME MEASURES: IGF-I bioactivity was determined by the IGF-I kinase receptor activation assay. Total and free IGF-I were determined by IGF-I immunoassays. Mortality was registered during follow-up (mean 82 months). RESULTS: During the follow-up period of 8.6 yr, 170 men (45%) died. Survival of subjects in the highest quartile of IGF-I bioactivity was significantly better than in the lowest quartile, both in the total study group [hazard ratio (HR) = 1.8; 95% confidence interval (95% CI) = 1.2-2.8; P = 0.01] as well as in subgroups having a medical history of cardiovascular disease (HR = 2.4; 95% CI = 1.3-4.3; P = 0.003) or a high inflammatory risk profile (HR = 2.3; 95% CI = 1.2-4.5; P = 0.01). Significant relationships were not observed for total or free IGF-I. CONCLUSION: Our study suggests that a relatively high circulating IGF-I bioactivity in elderly men is associated with extended survival and with reduced cardiovascular risk.

Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway.

Ghrelin is expressed in normal human adrenocortical cells and induces their proliferation through growth hormone secretagogue receptor 1a (GHS-R1a). Consequently, it was of interest to us to determine whether acylated ghrelin and its predominant serum isoform, unacylated ghrelin, also act as factors for adrenocortical carcinoma cell growth. To examine a potential ghrelin-regulated system in adrenocortical tumors, we measured proliferative effects of acylated and unacylated ghrelin in the adrenocortical carcinoma cell lines SW-13 and NCI-H295R. We also examined the expression of ghrelin, GHS-R1a, and corticotrophin-releasing factor receptor 2 (CRF-R2). Acylated and unacylated ghrelin in the nanomolar range dose-dependently induced adrenocortical cell growth up to 200% of untreated controls, as measured by thymidine uptake and WST1 assay. The proliferative effects of acylated and unacylated ghrelin in SW-13 cells was blocked by [D-Lys(3)]growth hormone-releasing peptide 6 (GHRP6), but a CRF-R2 antagonist had no effect on unacylated ghrelin growth stimulation. Cell cycle analysis suggests that acylated and unacylated ghrelin suppress the sub-G(0)/apoptotic fraction by up to 50%. Measurement of DNA fragmentation and caspase-3 and -7 activity in SW-13 cells confirmed that acylated and unacylated ghrelin suppress apoptotic rate. SW-13 cells express preproghrelin mRNA and secrete ghrelin, and [D-Lys(3)]GHRP6 suppresses their basal proliferation rate, strongly suggesting that ghrelin could act as an auto/paracrine growth factor. Acylated and unacylated ghrelin are potential auto/paracrine factors acting through an antiapoptotic pathway to stimulate adrenocortical tumor cell growth. Unacylated ghrelin-stimulated growth is suppressed by an antagonist of GHS-R1a, suggesting either that unacylated ghrelin is acylated before its action or that ghrelin, unacylated ghrelin, and [D-Lys(3)]GHRP-6 bind to a novel receptor in these cells.

Inflammatory cytokines stimulate the adhesion of colon carcinoma cells to mesothelial monolayers.

Surgical handling of the peritoneum causes an inflammatory reaction, during which a potentially lethal cocktail of active mediators is produced, including cytokines and growth factors. The aim of this study was to investigate the effects of inflammatory cytokines on the interaction between tumor and mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. Preincubation of the mesothelial monolayer with IL-1beta or TNF-alpha resulted in enhanced tumor cell adhesion of Caco2 and HT29 colon carcinoma cells. The amount of stimulation for the Caco2 cells was between 20% and 40% and for HT29 cells between 30% and 70%. Blocking experiments with anti-IL-1beta and anti-TNF-alpha resulted in significant inhibition of the cytokine-stimulated tumor cell adhesion. The presented results prove that IL-1beta and TNF-alpha are significant stimulating factors in tumor cell adhesion in vitro and may therefore account for tumor recurrence to the peritoneum in vivo.

In vitro secretion of FSH by cultured clinically nonfunctioning and gonadotroph pituitary adenomas is directly correlated with locally produced levels of activin A.

OBJECTIVE: Expression of mRNAs encoding activin and its antagonists inhibin and follistatin has been described in human pituitary adenomas, including clinically nonfunctioning adenomas (NFAs) and gonadotroph adenomas (Gn-omas). Since many of the NFAs and Gn-omas secrete FSH in vitro, we hypothesized that locally produced activin may stimulate secretion of FSH in these pituitary adenomas. PATIENTS AND METHODS: Pituitary adenoma tissue was obtained from 38 patients diagnosed preoperatively as having NFAs (n = 17), Gn-omas (n = 5), prolactinomas (n = 6) or growth hormone (GH)-producing adenomas (n = 10). Actual protein levels of activin, inhibin, follistatin, FSH and LH were measured in media of these 38 cultured pituitary adenomas. In addition, we investigated correlations between concentrations of these growth factors and hormones in NFAs and Gn-omas. RESULTS: Gn-omas were found to secrete significantly more activin A in their culture medium than PRL- and GH-producing adenomas (P < 0.05). Inhibin A and inhibin B protein levels in culture media were very low. A positive correlation between levels of activin A and FSH (r = 0.56, P < 0.005) was found, while no correlation between activin A and LH could be detected. Furthermore, levels of follistatin were positively correlated with activin A levels (r = 0.73, P < 0.0005). Comparison of the activin A:follistatin ratio with the measured FSH protein levels showed an even stronger relationship (r = 0.79, P < 0.0005). CONCLUSIONS: It is concluded that levels of activin A, follistatin and FSH in media of cultured nonfunctioning adenomas and gonadotroph adenomas are positively correlated. This suggests that these adenomas secrete FSH in response to the relatively high locally produced levels of activin A.

Effect of inflammatory cytokines and growth factors on tumour cell adhesion to the peritoneum.

In this experimental study, the effect of inflammatory cytokines and growth factors on tumour cell adhesion to the peritoneum was investigated. A reproducible in vitro assay was developed to study the adhesion of CC531 colon carcinoma cells to an autologous monolayer of rat mesothelial cells. Tumour cell adhesion to mesothelium pre-incubated with interleukin-1beta (IL-1beta) and epidermal growth factor (EGF) resulted in at least 60% more tumour cell adhesion at maximal stimulation (p

Somatostatin receptor subtypes in human thymoma and inhibition of cell proliferation by octreotide in vitro.

Somatostatin (SS) and SS receptor (SSR) subtypes, code-named sst1-5, are heterogeneously expressed in the normal human thymus. This suggests their involvement in controlling the immune and/or neuroendocrine functions in this organ. Moreover, recently a high in vivo uptake of [111In-DTPA-D-Phe1]octreotide has been reported in patients bearing thymoma. The present study characterizes in vivo and in vitro, functional SS-binding sites in a human thymoma. A high uptake of [111In-DTPA-D-Phe1]octreotide was observed in the chest of a patient with myasthenia gravis due to a cortical thymoma. Specific binding of [125I-Tyr11] SS-14 was found on a membrane preparation of the surgically removed thymoma. Scatchard analysis showed high affinity binding sites (Kd, 47.5 +/- 2.5 pmol/L) with low maximum binding capacity (23.5 +/- 2.5 fmol/mg membrane protein). RT-PCR analysis showed the presence of sst1, sst2A, and a predominant sst3 messenger RNA (mRNA) expression in the tumor tissue. Primary cultured tumor cells expressed sst3 mRNA only. In contrast to the normal thymus, SS mRNA was not expressed. By immunohistochemistry, the tumor cells highly expressed sst3 receptors, weakly expressed sst1 receptors, and showed no immunostaining for sst2A receptors. sst2A immunoreactivity was found in the stromal compartment of the tumor, particularly on the endothelium of small intratumoral blood vessels. In primary cultured tumor cells, both SS and octreotide (10 nmol/L) significantly inhibited [3H]thymidine incorporation by 40.6% and 43.2%, respectively. The following conclusions were reached. 1) As this tumor displayed a high immunoreactivity for sst3 and the cultured tumor cells expressed the sst3 mRNA only, this SSR may be the subtype involved in the inhibition of epithelial tumor cell proliferation by octreotide in vitro. 2) A loss of endogenous SS production in this thymoma might be implicated in the uncontrolled cell growth. 3) In this case, the sst3 may play a role in determining the uptake of [111In-DTPA-D-Phe1]octreotide by in vivo SS receptor scintigraphy.

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, incubation with IFN alpha resulted in inhibition of the secretion of gonadotropins and/or alpha-subunit in all cases (27-62%), whereas in the prolactinoma cultures PRL secretion was inhibited by IFN alpha in all cases (37-76%). The effect of IFN alpha was additive to the inhibitory effects of the dopamine agonist bromocriptine (10 nmol/L) or the somatostatin analog octreotide (10 nmol/L). The inhibition of hormone secretion by IFN alpha was accompanied by inhibition of the intracellular hormone concentrations. The effect of IFN alpha was dose dependent, with an IC50 for inhibition of hormone secretion of 2.3 +/- 0.3 U/mL (n = 5), which is relatively low compared with the concentrations that are reached in patients treated with IFN alpha for various malignancies. In conclusion, the potent antihormonal effect of IFN alpha on cultured pituitary adenomas suggests that this drug might be of benefit in the treatment of selected patients with secreting pituitary adenomas. As treatment with IFN alpha is associated with considerable adverse reactions, studies with this drug should only be considered in inoperable, invasive aggressive, and dopamine agonist- and/or somatostatin analog-resistant functioning pituitary macroadenomas.

Somatostatin receptor subtype expression in cells of the rat immune system during adjuvant arthritis.

Somatostatin is a neuropeptide that is widely distributed throughout the body. It acts as a neurohormone and a neurotransmitter and may also have an immunomodulatory role. The genes for five subtypes of somatostatin receptors (sst) have been cloned, suggesting that the diverse effects of the peptide might be mediated by different receptors. We are interested in studying the role of sst ininflammation, using an animal model. Because of the up-regulation of sst expression in inflamed joints in human rheumatoid arthritis, we chose rat adjuvant arthritis as an experimental model. In order to determine which of the sst subtypes might be important in immune modulation, subtype expression in leukocytes isolated from different lymphoid tissues of the rat was studied. Also, the expression levels of the most abundantly expressed sst mRNAs in leukocytes from spleen and blood were compared in rats with adjuvantarthritis and controls, using a semi-quantitative approach. Furthermore, the effect of systemic administration of a long-acting somatostatin analogue, octreotide, which binds selectively to sst subtypes 2 and 5 (sst2 and sst5), on the incidence and the severity of rat adjuvant arthritis, was studied. The main sst expressed in cells of the rat immune system, both resting and activated, were found to be sst3 and sst4. This contrasts with the human and murine situations, in which sst2 appears to be the main subtype expressed in the immune system. No quantitative differences in sst subtype mRNA levels in leukocytes from spleen and blood were found between rats with adjuvant arthritis and controls. Finally, no effect of systemic administration of octreotide on either the incidence or severity of adjuvant arthritis in Lewis rats was found. As octreotide binds selectively to sst2 and sst5, the absence of an immunomodulatory effect of this analogue in rat adjuvant arthritis corroborates our finding that these sst subtypes are not expressed in cells of the rat immune system. In conclusion, cells of the rat immune system appear to express a spectrum of sst (sst3 and sst4) different from that found in human granulomatous and autoimmune disease (mainly sst2). Therefore, the rat adjuvant arthritis model appears to be suitable only for studying the immunomodulatory effects of somatostatin analogues which have a high affinity for sst3 and sst4, but not for studying the immunomodulatory effects of octreotide, which has a high affinity only for sst2 and sst5.