Maurizio Spinello

Meta-Analysis on the Effects of Octreotide on Tumor Mass in Acromegaly

Background The long-acting somatostatin analogue octreotide is used either as an adjuvant or primary therapy to lower growth hormone (GH) levels in patients with acromegaly and may also induce pituitary tumor shrinkage. Objective We performed a meta-analysis to accurately assess the effect of octreotide on pituitary tumor shrinkage. Data Sources A computerized Medline and Embase search was undertaken to identify potentially eligible studies. Study Eligibility Criteria Eligibility criteria included treatment with octreotide, availability of numerical metrics on tumor shrinkage and clear definition of a clinically relevant reduction in tumor size. Primary endpoints included the proportion of patients with tumor shrinkage and mean percentage reduction in tumor volume. Data Extraction and Analysis The electronic search identified 2202 articles. Of these, 41 studies fulfilling the eligibility criteria were selected for data extraction and analysis. In total, 1685 patients were included, ranging from 6 to 189 patients per trial. For the analysis of the effect of octreotide on pituitary tumor shrinkage a random effect model was used to account for differences in both effect size and sampling error. Results Octreotide was shown to induce tumor shrinkage in 53.0% [95% CI: 45.0%–61.0%] of treated patients. In patients treated with the LAR formulation of octreotide, this increased to 66.0%, [95% CI: 57.0%–74.0%). In the nine studies in which tumor shrinkage was quantified, the overall weighted mean percentage reduction in tumor size was 37.4% [95% CI: 22.4%–52.4%], rising to 50.6% [95% CI: 42.7%–58.4%] with octreotide LAR. Limitations Most trials examined were open-label and had no control group. Conclusions Octreotide LAR induces clinically relevant tumor shrinkage in more than half of patients with acromegaly.

Bone turnover, bone mineral density, and fracture risk in acromegaly: a meta-analysis.

CONTEXT GH excess causes an increase in bone turnover, but the consequences in terms of skeletal fragility have long been uncertain due to the heterogeneity of studies dealing with this topic. OBJECTIVE We conducted a meta-analysis of studies examining the effects of acromegaly on bone turnover, bone mineral density (BMD), and fractures. Furthermore, we evaluated the effects of sex, gonadal status, and activity of disease on skeletal end-points in acromegaly. DATA SOURCES We conducted MEDLINE and EMBASE systematic searches up to December 31, 2013. STUDY ELIGIBILITY CRITERIA Studies conducted in patients with acromegaly and reporting at least one determinant of skeletal fragility. DATA EXTRACTION AND ANALYSIS Study design, patient characteristics, interventions, and outcomes were independently extracted by two authors. We calculated the standardized mean difference (SMD) of bone turnover and BMD differences, whereas fractures were presented as relative frequencies in acromegaly and odds ratios between patients and controls. RESULTS Forty-one studies fulfilled eligibility criteria and were therefore selected for data extraction and analysis. A total of 1935 patients were included (eight to 206 per study). Acromegaly patients had higher bone formation (SMD, 1.49; 95% confidence interval [CI], 0.97-2.01; P < .0001) and bone resorption (SMD, 1.57; 95% CI, 1.03-2.10; P < .0001) as compared to control subjects, without significant differences in lumbar spine BMD. BMD at the femoral neck tended to be higher in acromegaly patients vs control subjects (SMD, 0.67; 95% CI, 0.07-1.27; P = .03). Patients with acromegaly had high frequency of vertebral fractures (odds ratio, 8.26; 95% CI, 2.91-23.39; P < .0001), in close relationship with male gender, hypogonadism, and active acromegaly. LIMITATIONS LIMITATIONS included heterogeneous study protocols with possible variability in the assessment of skeletal end-points. CONCLUSIONS Skeletal fragility is an emerging complication of acromegaly.

Effects of high-dose octreotide LAR on glucose metabolism in patients with acromegaly inadequately controlled by conventional somatostatin analog therapy

OBJECTIVE In this study, the effect of high-dose octreotide LAR on glucose metabolism in patients with acromegaly was investigated. DESIGN A post-hoc analysis of a clinical trial enrolling 26 patients with acromegaly not controlled by standard maximal somatostatin analog (SSAs) dose and randomized to receive high-dose (60  mg/28 days) or high-frequency (30  mg/21 days) octreotide i.m. injection (octreotide LAR) for 6 months. METHODS Glucose metabolic status was defined as worsened when a progression from normoglycemia to impaired fasting glucose (IFG) or from IFG to diabetes occurred or when an increase of HbAlc by at least 0.5% was demonstrated. An improvement of glucose metabolism was defined in the presence of a regression from IFG to normoglycemia and/or when HbAlc decreased by at least 0.5%. RESULTS Glucose metabolic status remained unchanged in a majority of patients (16/26 patients, 65.3%), worsened in six patients, and improved in four patients. Pre-existing metabolic status did not predict worsening of glucose metabolism, which, conversely, was significantly related to persistent biochemical activity of the disease. In fact, patients with worsened glucose metabolism exhibited a less frequent decrease in serum GH and IGF1 levels, compared with patients with improved or unchanged glucose metabolism (2/6 vs 18/20; P=0.01). CONCLUSION An increase in octreotide LAR dose or frequency did not impact on glucose metabolism in most patients. Worsening of glucose metabolic status occurred in close relation with persistently uncontrolled acromegaly.

AMP-activated protein kinase regulates normal rat somatotroph cell function and growth of rat pituitary adenomatous cells.

AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP and elevate AMP levels such as glucose deprivation and hypoxia. The AMPK system is primarily thought of as a regulator of metabolism and cell proliferation. Little is known about the regulation and the effects of AMPK in somatotroph cells. We present results from “in vitro” studies showing that AMPK activity has a role in regulating somatotroph function in normal rat pituitary and is a promising target for the development of new pharmacological treatments affecting cell proliferation and viability of pituitary adenomatous cells. In parallel, we show “in vivo” data obtained in the rat suggesting that AMPK is an intracellular transducer that may play a role in mediating the effects of the pharmacological treatment with dexamethasone on somatotrophs. In rat pituitary cell cultures, the AMP analog AICAR induced a rapid and clear-cut activation of AMPK. AICAR decreased GH release and total cellular GH content. An appropriate level of AMPK activation was essential for GH3 adenomatous cells. Remarkably, over-activation by AICAR induced apoptosis of GH3 whereas the AMPK inhibitor compound C was more effective at reducing cell proliferation. The role of endocrine or paracrine factors in regulating AMPK phosphorylation and activity in GH3 cells has been also studied. As to “in vivo” studies, western blot analysis revealed a significant decrease of phosphorylated AMPK alpha-subunit in pituitary homogenates of DEX-treated rats versus controls, suggesting reduced AMPK activity. In conclusion, our studies showed that AMPK has a role in regulating somatotroph function in normal rat pituitary and proliferation of pituitary adenomatous cells.

Effects of AMPK activation and combined treatment with AMPK activators and somatostatin on hormone secretion and cell growth in cultured GH-secreting pituitary tumor cells

We investigated the effects of the AMPK activator AICAR as compared to somatostatin-14 on cell viability and GH secretion in human GH-secreting pituitary adenomas in vitro and in rat GH3 cells. Overnight treatment with AICAR increased phospho-(threonine-172) AMPK levels (activated AMPK) in cultured human adenomas. As to the effects on cell viability, four adenomas out of 15 were responsive to AICAR (0.4mM) and five adenomas were responsive to SS-14 (100 nM). One adenoma was responsive to both somatostatin and AICAR. The effects of cotreatment with SS-14 and AICAR were investigated in eight adenomas. In two adenomas, the effects of AICAR+SS-14 did not exceed the effect of AICAR. In two adenomas which were not responsive to either AICAR or SS-14, the cotreatment was able to reduce cell viability versus control. Two adenomas were not responsive to any treatment. As to the effects on GH secretion, nine adenomas out of 15 were responsive to AICAR. Twelve adenomas were responsive to SS-14. Eight adenomas were responsive to both AICAR and SS-14. Cotreatment exceeded the effect of single treatments in 4 out of 10 adenomas. In GH3 cells, AICAR reduced the activity of p70S6 kinase, which plays an important role in cell growth. SS-14 did not affect significantly AMPK phosphorylation and p70S6K activity but it was able to enhance the inhibitory effect of AICAR on phospho-S6 levels. Moreover, AICAR and SS-14 reduced ERK phosphorylation with a different time course. The combined treatment reduced phospho-ERK levels at any time point.

ACROSCORE: A new and simple tool for the diagnosis of acromegaly, a rare and underdiagnosed disease

Acromegaly, a disease caused by GH/IGF‐I hypersecretion, is associated with a high mortality rate; early recognition is therefore necessary to ensure successful treatment and to avoid comorbidities. We have created a symptom/sign scoring tool (ACROSCORE) for physicians to use to identify acromegaly.

Role of AMP‐Activated Protein Kinase Activators in Antiproliferative Multi‐Drug Pituitary Tumour Therapies: Effects of Combined Treatments with Compounds Affecting the mTOR‐p70S6 Kinase Axis in Cultured Pituitary Tumour Cells

AMP‐activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)‐secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol‐3‐kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down‐regulation by specific small‐interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR‐p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF‐4708671, with the effects of the AMPK activator 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor‐induced p70S6K activity, as shown by the decrease of phospho‐p70S6K levels. However, it was far less effective than rapamycin and PF‐4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF‐4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co‐treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor‐induced extracellular signal regulated kinase 1/2 phosphorylation. In conclusion, the results of the present study suggest the increased efficacy of combined antiproliferative therapies, including rapamycin analogues and AMPK activators in GH‐secreting pituitary tumours, as a result of complementary and only partially overlapping mechanisms of action.

Interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK) and the estrogen receptor activities in regulating rat pituitary tumor cell (GH3) growth in vitro

Estrogen receptor α has a role in regulating rat somatolactotroph tumor cell growth (GH3 cells). AMP-activated protein kinase (AMPK) is a metabolic checkpoint which is able to negatively regulate intracellular signaling downstream of growth factors receptors in conditions increasing cellular AMP levels. We have recently reported on the role of AMPK activation in affecting viability and proliferation of GH3 cells. In the present study, we investigated the interplay between ER- and AMPK-pathways. Results can be regarded as relevant to the development of novel multi-targeted pharmacological therapies against pituitary tumors. We confirmed that estradiol (E2) and the ER antagonist fulvestrant exert stimulatory and inhibitory effects, respectively on GH3 cell growth in a competitive manner. The upstream kinase LKB1 is known to phosphorylate and activate AMPK. Here we showed that neither E2 nor fulvestrant caused a downregulation of LKB1 expression and phospho-AMPK levels in GH3 cells. Actually, fulvestrant strongly reduced the phosphorylation of ACC, which is a direct target of AMPK and a known index of AMPK activity. 2-deoxyglucose, a compound reducing glucose utilization, caused an increase in AMPK activity vs baseline and was able to hinder the stimulatory effect of E2 on cell viability, confirming that the exposure of GH3 cells to estrogens does not prevent them from being responsive to the inhibitory activity of compounds activating AMPK. Finally, the AMPK activator AICAR (AMP analog) did not cause further decrease in cell viability in the course of co-treatments with fulvestrant versus fulvestrant alone, in agreement with impaired phospho-AMPK activity in the presence of the anti-estrogen.

Effects of pasireotide (SOM230) on protein turnover and p70S6 kinase-S6 ribosomal protein signaling pathway in rat skeletal muscle cells

The multiligand somatostatin (SS-14) analog pasireotide (SOM230) has been approved to treat patients harboring ACTH-secreting pituitary adenomas [1]. The higher binding affinity of pasireotide for the sst5 receptor subtype compared to the first generation analogs, octreotide and lanreotide [2, 3], joint to a functional selectivity of distinct SS14 analogs at sst2 receptor [4, 5], suggest to explore its potential effects on extra-pituitary tissues. Indeed, SS-14 receptor subtypes (sst1–5) are heterogeneously expressed in different tissues [6]. There is evidence that SS-14 is transiently expressed in motoneurons during early postnatal development in the rat and the expression of sst2, sst3, and sst4 receptor subtypes in rat skeletal muscle decreases progressively during development [7]. To our knowledge, the hypothesis that stable SS-14 analogs may exert some direct effects in skeletal muscle cells has not been tested yet. These effects, if any, would certainly be of interest as to the use of pasireotide in the treatment of diseases entailing an excess of catabolism and loss of lean mass. We decided to assess the direct effects of pasireotide on protein turnover and the interaction between pasireotide and the synthetic glucocorticoid dexamethasone (DEX) in skeletal muscle cells. Then, we investigated the effects of pasireotide on the activation of the mTOR-p70S6 kinase-S6 ribosomal protein signalling pathway, an important anabolic pathway [1, 8, 9] in L6 cells compare to rat pituitary GH3 cells. Rat myoblast-like cells (L6 cell line) and rat pituitary tumor cells (GH3 cell line) were purchased from IZSLER (Brescia, Italy). Before performing a study, L6 cells were grown to confluence. Then, they were maintained in high glucose DMEM supplemented with 2% Horse serum (HS), for 5–7 days to induce their differentiation into polynucleated skeletal muscle cells. Pasireotide (SOM230, Novartis, Basel, Switzerland) was dissolved in water. DEX (Sigma-Aldrich) was dissolved in ethanol. The antibodies used in the present study were obtained from the following sources: anti-phospho-p70S6K (Thr-389) and anti-phosphoS6 Ribosomal Protein (Ser-235/236) from Cell Signalling; anti-α-tubulin from Sigma-Aldrich; anti-ribosomal protein S6 from Santa Cruz Biotech. Radiolabelled compounds were purchased by Perkin–Elmer. New protein synthesis was determined by measuring the incorporation of Trichloroacetic acid (TCA) insoluble radioactivity into cellular proteins, according to the method described by Menconi et al. [10], with some modifications. Briefly, after differentiation, L6 cells were exposed to the different drugs or to their respective vehicles for the appropriate time intervals and then serum-starved for 12 h. After starvation, they were incubated in fresh DMEM supplemented with 2% HS and with 2.0 mCi/ml of L-[3,5−H]tyrosine for the next 4 h. The treatments with drugs were renewed during serum-starvation and during labelling with H-tyrosine. The overall treatment with pasireotide lasted 24 h. Finally, the medium with H-tyrosine was removed and * Giovanni Tulipano giovanni.tulipano@unibs.it