Camillo Rosano

Bisphenol A Induces Gene Expression Changes and Proliferative Effects through GPER in Breast Cancer Cells and Cancer-Associated Fibroblasts

Background: Bisphenol A (BPA) is the principal constituent of baby bottles, reusable water bottles, metal cans, and plastic food containers. BPA exerts estrogen-like activity by interacting with the classical estrogen receptors (ERα and ERβ) and through the G protein-coupled receptor (GPR30/GPER). In this regard, recent studies have shown that GPER was involved in the proliferative effects induced by BPA in both normal and tumor cells. Objectives: We studied the transduction signaling pathways through which BPA influences cell proliferation and migration in human breast cancer cells and cancer-associated fibroblasts (CAFs). Methods and results: We used as a model system SKBR3 breast cancer cells and CAFs that lack the classical ERs. Specific pharmacological inhibitors and gene-silencing procedures were used to show that BPA induces the expression of the GPER target genes c-FOS, EGR-1, and CTGF through the GPER/EGFR/ERK transduction pathway in SKBR3 breast cancer cells and CAFs. Moreover, we observed that GPER is required for growth effects and migration stimulated by BPA in both cell types. Conclusions: Results indicate that GPER is involved in the biological action elicited by BPA in breast cancer cells and CAFs. Hence, GPER-mediated signaling should be included among the transduction mechanisms through which BPA may stimulate cancer progression.

Synthesis and biological evaluation of novel pyrazole derivatives with anticancer activity.

We synthesized thirty-six novel pyrazole derivatives and studied their antiproliferative activity in human ovarian adenocarcinoma A2780 cells, human lung carcinoma A549 cells, and murine P388 leukemia cells. Four of these substances were selected because of their higher antiproliferative activity and further analyses showed that they were all able to induce apoptosis, although to a different extent. The expression of p53 and p21(waf1), which induce apoptosis and cell cycle arrest, was evaluated by western blot analysis in cells treated with compound 12d. The analysis of the cell cycle showed that all the selected compounds cause a partial G2/M block and the formation of polyploid cells. Furthermore, the four selected compounds were tested for their interaction with the microtubular cytoskeletal system by docking analysis, tubulin polymerization assay and immunofluorescence staining, demonstrating that the compound 12d, unlike the other active derivatives, was able to significantly bind dimers of α- and β-tubulin, probably causing a molecular distortion resulting in the disassembly of microtubules.

Estriol acts as a GPR30 antagonist in estrogen receptor-negative breast cancer cells.

Estrogens are structurally related steroids that regulate important physiological processes. 17beta-estradiol (E2) is reversibly oxidized to estrone (E1) and both E2 and E1 can be irreversibly converted to estriol (E3), which also originates directly from androstenedione. The action of E2 has been traditionally explained by the binding to the estrogen receptor (ER) alpha and ER beta, however the G protein-coupled receptor (GPR) 30 has been recently involved in the rapid signaling triggered by estrogens. Although the role of E2 in the development of breast cancer has been largely documented, the contribution of E3 still remains to be completely evaluated. Here, we demonstrate for the first time that E3 acts as a GPR30 antagonist since it was able to inhibit the GPR30-mediated responses such as the rapid ERK activation, the up-regulation of target genes like c-fos and connective tissue growth factor, the proliferative effects observed in ER-negative SkBr3 cells.

The X-ray three-dimensional structure of avidin.

Avidin is a basic, highly stable, homotetrameric protein, isolated from bird egg-white, binding up to four molecules of D-biotin with extremely high affinity (Kd approximately 10(-15) M). The protein has been the object of different crystallographic investigations. In all the crystal structures, the four avidin subunits display almost exact 222 symmetry. Each avidin chain (128 amino acids) is arranged in a eight-stranded antiparallel beta-barrel, whose inner region defines the D-biotin binding site. The molecular bases of D-biotin affinity can be recognised in a fairly rigid binding site, which is sterically complementary to the shape and polarity of the incoming vitamin, and is readily accessible in the apoprotein structure. Avidin displays remarkable structural and functional relationships to the acidic protein sretpavidin, isolated from Streptomyces avidinii.

Cyanide Binding to Lucina pectinata Hemoglobin I and to Sperm Whale Myoglobin: An X-Ray Crystallographic Study

The x-ray crystal structures of the cyanide derivative of Lucina pectinata monomeric hemoglobin I (L. pectinata HbI) and sperm whale (Physeter catodon) myoglobin (Mb), generally taken as reference models for monomeric hemoproteins carrying hydrogen sulfide and oxygen, respectively, have been determined at 1.9 A (R-factor = 0. 184), and 1.8 A (R-factor = 0.181) resolution, respectively, at room temperature (lambda = 1.542 A). Moreover, the x-ray crystal structure of the L. pectinata HbI:cyanide derivative has been studied at 1.4-A resolution (R-factor = 0.118) and 100 K (on a synchrotron source lambda = 0.998 A). At room temperature, the cyanide ligand is roughly parallel to the heme plane of L. pectinata HbI, being located approximately 2.5 A from the iron atom. On the other hand, the crystal structure of the L. pectinata HbI:cyanide derivative at 100 K shows that the diatomic ligand is coordinated to the iron atom in an orientation almost perpendicular to the heme (the Fe-C distance being 1.95 A), adopting a coordination geometry strictly reminescent of that observed in sperm whale Mb, at room temperature. The unusual cyanide distal site orientation observed in L. pectinata HbI, at room temperature, may reflect reduction of the heme Fe(III) atom induced by free radical species during x-ray data collection using Cu Kalpha radiation.

Crystal Structure and Anion Binding in the Prokaryotic Hydrogenase Maturation Factor HypF Acylphosphatase-like Domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

IntroductionThe multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells.MethodsMolecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells.ResultsMIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments.ConclusionsOur findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist.

IDUA mutational profiling of a cohort of 102 European patients with mucopolysaccharidosis type I: identification and characterization of 35 novel α-L-iduronidase (IDUA) alleles

Mutational analysis of the IDUA gene was performed in a cohort of 102 European patients with mucopolysaccharidosis type I. A total of 54 distinct mutant IDUA alleles were identified, 34 of which were novel including 12 missense mutations, 2 nonsense mutations, 12 splicing mutations, 5 micro‐deletions, 1 micro‐duplication 1 translational initiation site mutation, and 1 ‘no‐stop’ change (p.X654RextX62). Evidence for the pathological significance of all novel mutations identified was sought by means of a range of methodological approaches, including the assessment of evolutionary conservation, RT‐PCR/in vitro splicing analysis, MutPred analysis and visual inspection of the 3D‐model of the IDUA protein. Taken together, these data not only demonstrate the remarkable mutational heterogeneity characterizing type 1 mucopolysaccharidosis but also illustrate our increasing ability to make deductions pertaining to the genotype‐phenotype relationship in disorders manifesting a high degree of allelic heterogeneity.

Oleuropein and hydroxytyrosol activate GPER/ GPR30‐dependent pathways leading to apoptosis of ER‐negative SKBR3 breast cancer cells

SCOPE We have previously demonstrated that oleuropein (OL) and hydroxytyrosol (HT) reduce 17β-estradiol-mediated proliferation in MCF-7 breast cancer (BC) cells without affecting the classical genomic action of estrogen receptor (ER), but activating instead the ERK1/2 pathway. Here, we hypothesized that this inhibition could be mediated by a G-protein-coupled receptor named GPER/GPR30. Using the ER-negative and GPER-positive SKBR3 BC cells as experimental model, we investigated the effects of OL and HT on GPER-mediated activation of downstream pathways. METHODS AND RESULTS Docking simulations and ligand-binding studies evidenced that OL and HT are able to bind GPER. MTT cell proliferation assays revealed that both phenols reduced SKBR3 cell growth; this effect was abolished silencing GPER. Focusing on OL and HT GPER-mediated pathways, using Western blot analysis we showed a sustained ERK1/2 activation triggering an intrinsic apoptotic pathway. CONCLUSION Showing that OL and HT work as GPER inverse agonists in ER-negative and GPER-positive SKBR3 BC cells, we provide novel insights into the potential of these two molecules as tools in the therapy of this subtype of BC.

Synthesis, antiproliferative and apoptotic activities of N-(6(4)-indazolyl)-benzenesulfonamide derivatives as potential anticancer agents.

Recently, it has been reported that compounds bearing a sulfonamide moiety possess many types of biological activities, including anticancer activity. The present work reports the synthesis and antiproliferative evaluation of some N-(6(4)-indazolyl)benzenesulfonamides and 7-ethoxy-N-(6(4)-indazolyl)benzenesulfonamides. All compounds were evaluated for their in vitro antiproliferative activity against three tumor cell lines: A2780 (human ovarian carcinoma) A549 (human lung adenocarcinoma) and P388 (murine leukemia). The results indicated that sulfonamides 2c, 3c, 6d, 8, 13, 3b and 16 were endowed with a pharmacologically interesting antiproliferative activity with compounds 2c and 3c showing the lower IC(50) (from 0.50 ± 0.09 to 1.83 ± 0.52 μM and from 0.58 ± 0.17 to 5.83 ± 1.83 μM, respectively). Moreover, these indazoles were able to trigger apoptosis through the upregulation of the typical apoptosis markers p53 and bax. As regard to the hypothetic targets of these compounds, a preliminary docking analysis showed that all compounds seemed to interact with β-tubulin, in particular compound 3b that showed the lower Ki. The cytofluorimetric analysis of the cell cycle phases indicates that all compounds, when administered at their IC(75), caused a block in the G2/M phase of the cell cycle with the generation of subpopulations of cells with a number of chromosome >4n. When the IC(50)s were applied we observed a prevalent block in the G0/G1 phase except for compounds 16 and 8 where a partial G2/M block was present with a concomitant decrease of cells in the G0/G1 and S phases of the cell cycle. Altogether these results suggest a possible, but not exclusive, interaction with microtubules.