Nicola Surico

p130Cas interacts with estrogen receptor alpha and modulates non-genomic estrogen signaling in breast cancer cells.

Steroid hormones bind to their receptors and trans-activate target genes. Rapid non-genomic action of steroid hormones has been proposed in addition to the one at the genomic level. Estrogen has been described to activate c-Src kinase and this activation has been shown to be responsible for estrogen-dependent mitogenicity. A major substrate of c-Src kinase activity is the cytoskeletal protein p130Cas, originally identified in v-Src-transformed cells. We show that in the human breast carcinoma T47D cells, upon estrogen treatment, p130Cas rapidly and transiently associates with the estrogen receptor α in a multi-molecular complex containing the c-Src kinase and the p85 subunit of PI 3-kinase. Association of p130Cas with the estrogen receptor α occurs within 3 minutes of estrogen treatment and is dependent on c-Src kinase activation. Transient overexpression of p130Cas in T47D cells increases estrogen-dependent Src kinase and Erk1/2 MAPKs activities and accelerates their kinetics of stimulation. A similar effect was detected on estrogen-dependent cyclin D1 expression, suggesting a role for p130Cas in regulating estrogen-dependent cell cycle progression. Double-stranded small RNA interference (siRNA) by silencing endogenous p130Cas protein, was sufficient to inhibit estrogen-dependent Erk1/2 MAPKs activity and cyclin D1 induction, demonstrating the requirement of p130Cas in such events. Therefore, our data show that the adaptor protein p130Cas associates with the estrogen receptor transducing complex, regulating estrogen-dependent activation of c-Src kinase and downstream signaling pathways.

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

Differential expression of microRNAs between eutopic and ectopic endometrium in ovarian endometriosis.

Endometriosis, defined as the presence of endometrial tissue outside the uterus, is a common gynecological disease with poorly understood pathogenesis. MicroRNAs are members of a class of small noncoding RNA molecules that have a critical role in posttranscriptional regulation of gene expression by repression of target mRNAs translation. We assessed differentially expressed microRNAs in ectopic endometrium compared with eutopic endometrium in 3 patients through microarray analysis. We identified 50 microRNAs differentially expressed and the differential expression of five microRNAs was validated by real-time RT-PCR in other 13 patients. We identified in silico their predicted targets, several of which match the genes that have been identified to be differentially expressed in ectopic versus eutopic endometrium in studies of gene expression. A functional analysis of the predicted targets indicates that several of these are involved in molecular pathways implicated in endometriosis, thus strengthening the hypothesis of the role of microRNAs in this pathology.

Arsenic Trioxide and Breast Cancer: Analysis of the Apoptotic, Differentiative and Immunomodulatory Effects

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia and has activity in vitro against several solid tumour cell lines, where induction of differentiation and apoptosis are the prime effects. To investigate the potential therapeutic application of As2O3 to breast cancer, we analysed the effects of As2O3 on the growth of four human breast cancer cell lines: MCF7, MDA-MB-231, T-47D and BT-20. Cells were cultured in 0.5, 2 and 5 μM As2O3, a range of pharmacologically achievable concentrations of As2O3. At ≥2 μM, As2O3 rapidly induced cell death by apoptosis in MCF7 and MDA-MB-231 while T-47D and BT-20 were partially resistant. At 0.5 μM, As2O3 was subapoptotic but induced features of differentiation consisting in upregulation of ICAM-1 (CD54), a marker of mammary epithelial differentiation, and cell cultures appeared morphologically more orga-nized. Furthermore, we demonstrate by standard cytotoxicity assays that As2O3 treatment can augment breast cancer cell lysis by lymphokine-activated killer cells and demonstrate an important role of the ICAM-1/LFA-1 interaction in this process. This additional activity of As2O3 could translate into improved antitumour immunosurveillance in vivo. In conclusion, As2O3 induced varying degrees of differentiation, apoptosis and lysis in these model cell lines, and may be a promising adjuvant to current treatments of breast cancer by virtue of its triple apoptotic, differentiative and immunomodulatory effects.

Multiple small intestinal stromal tumours in a patient with previously unrecognised neurofibromatosis type 1: Immunohistochemical and ultrastructural evaluation

Summary Neurofibromatosis type 1 could be associated with multiple gastrointestinal stromal tumours, although their presence is not considered among the major diagnostic criteria. We present here a case of a 50‐year‐old female complaining of abdominal pain, with about 100 small intestinal stromal tumours. This finding prompted us to suspect a neurofibromatosis which was clinically confirmed afterwards. Light microscopy examination revealed a low‐grade stromal tumour with skeinoid fibres. Mixed neural‐interstitial cells of Cajal origin or, alternatively, neural differentiation of interstitial cells of Cajal are discussed on the basis of immunophenotype (CD117 +, CD34 +) and ultrastructure. A 2‐year follow‐up did not indicate an aggressive course in the case of this neoplasm.

Intracoronary Genistein Acutely Increases Coronary Blood Flow in Anesthetized Pigs through β-Adrenergic Mediated Nitric Oxide Release and Estrogenic Receptors

Various studies have suggested that the phytoestrogen genistein has beneficial cardioprotective and vascular effects. However, there has been scarce information regarding the primary effect of genistein on coronary blood flow and its mechanisms including estrogen receptors, autonomic nervous system, and nitric oxide (NO). The present study was planned to determine the primary effect of genistein on coronary blood flow and the mechanisms involved. In anesthetized pigs, changes in left anterior descending coronary artery caused by intracoronary infusion of genistein at constant heart rate and arterial pressure were assessed using ultrasound flowmeters. In 25 pigs, genistein infused at 0.075 mg/min increased coronary blood flow by about 16.3%. This response was graded in a further five pigs by increasing the infused dose of the genistein between 0.007 and 0.147 mg/min. In the 25 pigs, blockade of cholinergic receptors (iv atropine; five pigs) and alpha-adrenergic receptors (iv phentolamine; five pigs) did not abolish the coronary response to genistein, whose effects were prevented by blockade of beta(2)-adrenergic receptors (iv butoxamine; five pigs), nitric oxide synthase (intracoronary N(omega)-nitro-L-arginine methyl ester; five pigs) and estrogenic receptors (ERs; ERalpha/ERbeta; intracoronary fulvestrant; five pigs). In porcine aortic endothelial cells, genistein induced the phosphorylation of endothelial nitric oxide synthase and NO production through ERK 1/2, Akt, and p38 MAPK pathways, which was prevented by the concomitant treatment by butoxamine and fulvestrant. In conclusion, genistein primarily caused coronary vasodilation the mechanism of which involved ERalpha/ERbeta and the release of NO through vasodilatory beta(2)-adrenoreceptor effects.

Synthesis of Modified Ingenol Esters

Synthetic protocols for the manipulation of the polyhydroxylated southern region of ingenol (1a) were developed, and a series of isosteres of the anticancer compound ingenol 3,20-dibenzoate (1b) was prepared. The biological evaluation of these compounds showed that cytotoxicity was relatively tolerant to changes at C-20, while PKC activation was markedly affected by these modifications. These data suggest that chemical manipulation can effectively dissect cytotoxicity and tumour-promoting activity (or potential) of ingenoids, affording more optimal candidates for development, like 20-deoxy-20-fluoroingenol 3,20-dibenzoate (5b). In mild acidic medium, an unexpected vinylogous retro-pinacol rearrangement of ingenol to a tigliane derivative was observed.

Interobserver variability of colposcopic interpretations and consistency with final histologic results.

Objective. To evaluate the interobserver reliability of colposcopy and the prediction of final histologic results. Materials and Methods. Visibility of the squamocolumnar junction, presence and grading of atypical transformation zone (ATZ), and presence and grading of cervical intraepithelial neoplasia (CIN) were assessed by nine expert colposcopists in 100 cervigrams. Pairwise κ, group κ, and class-specific κ values were computed. Frequency and degree of interobserver disagreement on CIN and ATZ grading and correlation with histologic results were studied. Results. The median pairwise κ and the group κ values were excellent for ATZ detection, acceptable for the squamocolumnar junction, and poor for the other variables; the presence of ATZ had a sensitivity for CIN of 90.2% and a specificity of 48.6%; the colposcopic impression of high-grade CIN had a sensitivity of 54.4% and a specificity of 88%. Conclusions. Some colposcopic predictions are reproducible between observers, predictive of the histologic results, and can be integrated in the algorithm for the management of patients with abnormal Pap smear results.

Diacylglycerol kinase α mediates 17-β-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30

Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-β-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy.

Human Chorionic Gonadotropin Protects Vascular Endothelial Cells from Oxidative Stress by Apoptosis Inhibition, Cell Survival Signalling Activation and Mitochondrial Function Protection.

Background/Aim: Previous reports have made it hypothetically possible that human chorionic gonadotropin (hCG) could protect against the onset of pregnancy-related pathological conditions by acting as an antioxidant. In the present study we planned to examine the effects of hCG against oxidative stress in human umbilical vein endothelial cells (HUVEC). Methods: HUVEC were subjected to peroxidation by hydrogen peroxide. The modulation of nitric oxide (NO) release by hCG and its effects on cell viability, glutathione (GSH) levels, mitochondrial membrane potential and mitochondrial transition pore opening (MPTP) were examined by specific dyes. Endothelial and inducible NO synthase (eNOS and iNOS), Akt and extracellular -signal-regulated kinases 1/2 (ERK1/2) activation and markers of apoptosis were analyzed by Western Blot. Results: In HUVEC, hCG reduced NO release by modulating eNOS and iNOS. Moreover, hCG protected HUVEC against oxidative stress by preventing GSH reduction and apoptosis, by maintaining Akt and ERK1/2 activation and by keeping mitochondrial function. Conclusion: The present results have for the first time shown protective effects exerted by hCG on vascular endothelial function, which would be achieved by modulation of NO release, antioxidant and antiapoptotic actions and activation of cell survival signalling. These findings could have clinical implications in the management of pregnancy-related disorders.