Dimerization of GPCRs:Novel insight into the role of FLNA and SSAs regulating SST₂ and SST₅ homo- and hetero-dimer formation

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)-based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P &lt; 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P &lt; 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulatesSST2 and SST5 intracellular trafficking induced by octreotide and pasireotide. </p

Activity of chemotherapy in mucinous ovarian cancer with a recurrence free interval of more than 6 months: results from the SOCRATES retrospective study

<p>Abstract</p> <p>Background</p> <p>Mucinous ovarian carcinoma have a poorer prognosis compared with other histological subtypes. The aim of this study was to evaluate, retrospectively, the activity of chemotherapy in patients with platinum sensitive recurrent mucinous ovarian cancer.</p> <p>Methods</p> <p>The SOCRATES study retrospectively assessed the pattern of care of a cohort of patients with recurrent platinum-sensitive ovarian cancer observed in the years 2000–2002 in 37 Italian centres. Data were collected between April and September 2005. Patients with recurrent ovarian cancer with > 6 months of platinum free interval were considered eligible.</p> <p>Results</p> <p>Twenty patients with mucinous histotype and 388 patients with other histotypes were analyzed. At baseline, mucinous tumours differed from the others for an higher number of patients with lower tumor grading (p = 0.0056) and less advanced FIGO stage (p = 0.025). At time of recurrence, a statistically significant difference was found in performance status (worse in mucinous, p = 0.024). About 20% of patients underwent secondary cytoreduction in both groups, but a lower number of patients were optimally debulked in the mucinous group (p = 0.03). Patients with mucinous cancer received more frequently single agent platinum than platinum based-combination therapy or other non-platinum schedules as second line therapy (p = 0.026), with a response rate lower than in non-mucinous group (36.4% vs 62.6%, respectively, p = 0.04). Median time to progression and overall survival were worse for mucinous ovarian cancer. Finally, mucinous cancer received a lower number of chemotherapy lines (p = 0.0023).</p> <p>Conclusion</p> <p>This analysis shows that platinum sensitive mucinous ovarian cancer has a poor response to chemotherapy. Studies dedicated to this histological subgroup are needed.</p

Feeding of preterm infants and fortification of breast milk

The administration of the adequate amount of nutrients helps to improve a correct short-term linear growth and long-term neurocognitive development. To reduce the extra-uterine growth delay in very low birth weight infants (VLBW) the best strategy of nutrition (parenteral or enteral) should be established rapidly, since the first day of life. In preterm infants, nutrition can be administered parenterally and enterally. Prematurity is the most frequent indication for parenteral nutritional support due to intestinal functional immune deficiency, deficiency of digestive enzymatic systems and reduced nutritional reserve of these infants. In terms of enteral nutrition, breast milk is the first choice. In case of preterm and VLBW infants, fortifiers are used to overcome breast milk’s protein and mineral deficiencies. When breast milk is not available, specific infant formula is the alternative

Feeding dysfunctions and failure to thrive in neonates with congenital heart diseases

Congenital heart disease (CHD) is the most common neonatal congenital malformation. The variety and severity of clinical presentation depend on the cardiac structures involved and their functional impact. The management of newborns with CHD requires a multidisciplinary approach, in which the nutritional aspect plays an important role. An adequate caloric intake during either preand post-surgical period, in fact, improves the outcome of these patients. In addition, the failure to thrive of these children in childhood has been related to long-term cognitive delay (attention deficit disorders, aggressive behaviour and poor social and emotional development). To date, there is a lack of standardized feeding protocols and caloric goals about how to feed neonates with CHD, and current practice varies widely between centres. The latest American Society for Parenteral and Enteral Nutrition guidelines reiterate the importance of proteins, and recommend early start of enteral nutrition, also in the most severe heart diseases, such as univentricular forms. Necrotizing enterocolitis (NEC), the most frequent and feared complication of early feeding of these newborns, often represents an obstacle in spreading this practice. Furthermore, as demonstrated in premature infants, breastfeeding seems to reduce the incidence of NEC. That is why breastfeeding must be encouraged, even if it can be difficult for these mothers due to delivery complications, associated with infant disease. In addition, eating difficulties may persist even after discharge, because these patients require nutritional support through nasogastric tubes or percutaneous endoscopic gastrostomies

Successful pregnancy in a patient with l-amino acid decarboxylase deficiency. therapeutic management and clinical outcome

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare inherited disorder of biogenic amine metabolism presenting with developmental delay, hypokinetic movement disorders, hypotonia, and autonomic dysfunctions

Cytoskeleton protein Filamin A is required for efficient Somatostatin receptor type 2 internalization and recycling through Rab5 and Rab4 sorting endosomes in tumor somatotroph cells

The high expression of somatostatin receptor 2 (SST2) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0±2.7% vs 4±4.3% SST2 internalization, control vs FLNA siRNA cells, respectively, P&lt;0.001) and human GH-secreting primary cultured cells (70.3±21.1% vs 24±19.2% SST2 internalization, control vs FLNA siRNA cells, respectively, P&lt;0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Co-immunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely-dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and Rab4, respectively (33.7±8.9% down to 25.9±6.9%, p&lt;0.05, and 28.4±7.4% down to 17.6±5.7%, p&lt;0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control vs FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and Rab4 sorting endosomes

β-arrestin 2 is required for dopamine receptor type 2 inhibitory effects on AKT phosphorylation and cell proliferation in pituitary tumors.

Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for PRL-secreting pitui-tary tumors but are poorly effective in non-functioning pituitary neuroendocrine tumors (NF-PitNET). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a β-arrestin2-dependent mechanism in mouse striatum. The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test β-arrestin 2 involvement. We found that DRD2 agonist BIM53097 induced a reduction of p- AKT /total-AKT ratio in MMQ (-32.8±17.6%, p&lt;0.001 vs basal) and in a subset (n=15/41,36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase of p-AKT. The ability of BIM53097 to reduce p-AKT correlated to its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097 and nearly all subgroup 2 NF-PitNETs were resistant. β-arrestin 2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs. In MMQ, β-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, β-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth. In conclusion, we demonstrated that β-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of β-arrestin 2 as a biomarker predicting NF-PitNETs responsiveness to treatment with dopamine agonists

Potential role of raltegravir-based therapy to induce rapid viral decay in highly viraemic HIV-infected neonates

We report safety and tolerability of raltegravir (RAL) as a forth HIV agent in two highly viraemic newborns. Raltegravir (6 mg/kg) was given orally twice daily. The other antiretrovirals were assumed according to standard dose for newborns. The first baby was born at week 36. An antiretroviral therapy consisting of zidovudine, lamivudine, and lopinavir/ritonavir was started 96 hour after delivery. Raltegravir was added at hour 120, being plasma HIV-1 RNA above 10\uc3\u97106 copies/ml. HIV RNA declined to 5\uc2\ub7000 copies/ml at day 30. The second baby was born at week 40. He was started on zidovudine, lamivudine, and nevirapine at day 0, while RAL was added at day 3. Plasma HIV-1 RNA declined from 6\uc2\ub76\uc3\u97106 at birth to 52 copies/ml at day 28. RAL tolerability was good in both patients, one with gamma-glutamyltransferase increase, which normalized after RAL discontinuation. Raltegravir-based four drug regimen may be effective and well tolerated in highly viraemic HIV neonates up to 4 weeks

Octreotide and pasireotide effects on medullary thyroid carcinoma (MTC) cells growth, migration and invasion

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine neoplasm of the parafollicular thyroid C cells. Although somatostatin receptors are expressed by MTCs, treatment with octreotide has shown poor efficacy, whereas recently pasireotide has demonstrated antiproliferative effects in persistent postoperative MTCs. Aim of this study was to test the effects of octreotide and pasireotide on MTC cells proliferation, cell cycle proteins expression, MAPK activation, apoptosis, calcitonin secretion, migration and invasion in TT cell line as well as in primary MTC cultured cells. Our results showed that both octreotide and pasireotide reduced TT cell proliferation (− 35.2 ± 12.1%, p &lt; 0.001, and − 25.3 ± 24.8%, p &lt; 0.05, at 10− 8 M, respectively), with concomitant inhibition of ERK phosphorylation and cyclin D1 expression. This cytostatic effect was accompanied by a proapoptotic action, with an increase of caspase3/7 activity of 1.5-fold. Moreover, both octreotide and pasireotide inhibited cell migration (− 50.9 ± 11.3%, p &lt; 0.01, and − 40.5 ± 17%, p &lt; 0.05, respectively) and invasion (− 61.3 ± 35.1%, p &lt; 0.05, and − 49.7 ± 18%, p &lt; 0.01, respectively). No effect was observed on calcitonin secretion. We then tried to extend these observations to primary cultures (n = 5). Octreotide and/or pasireotide were effective in reducing cells proliferation in 3 out of 5 tumors, and to induce cell apoptosis in 1 out of 3 MTCs. Both octreotide and pasireotide were able to reduce cell migration in all MTC tested. SST2, SST3 and SST5 were expressed in all MTC, with a tendency to increased expression of SST2 in RET mutated vs wild type MTCs. In agreement, inhibition of mutated RET in TT cells reduced SST2 expression. In conclusion, we demonstrated that octreotide and pasireotide inhibited cell proliferation and invasiveness in a subset of MTC, supporting their potential use in the control of tumor growth

Stage I juvenile granulosa cell tumors of the ovary: A multicentre analysis from the MITO-9 study

OBJECTIVE: Juvenile type granulosa cell tumor (JGCTs) are extremely rare, mainly diagnosed in young women and pre-pubertal girls at stage I disease. Literature is scanty and guidelines regarding the optimal management are still controversial. The aim of this study is to add on the experience of the MITO group (Multicenter Italian Trials in Ovarian Cancer).METHODS: Clinicopathological data from patients with stage I JGCTs were retrospectively collected. Descriptive statistics were used to characterize the patient population. Clinicopathological features and treatment variables were evaluated for association with relapse.RESULTS: Seventeen patients were identified. Surgical approach was laparoscopic and open for 7 (41%) and 10 (59%) patients, respectively. Fertility sparing surgery (FSS) was performed in 15 patients (88%): unilateral salpingo-oophorectomy (USO) in 11 patients, cystectomy with subsequent USO in 2 patients and cystectomy alone in the remaining 2. Adjuvant chemotherapy was given in 2 cases. After a median follow up time of 80 months, no recurrences were registered.CONCLUSIONS: Given the available data, minimally invasive surgery is safe in stage I JGCTs. Because of the good prognosis and of the young age of patients, FSS can be chosen in most of the cases. The role of cystectomy deserves further validation. The need of adjuvant chemotherapy in stage I disease is still unclear, even if available data does not seem to support treatment over surveillance