Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) binds steroids in the blood but is also present in the extravascular compartments of some tissues. Mice expressing a human SHBG transgene in the liver have human SHBG in their blood. In these animals, human SHBG accumulates within the stromal matrix of the endometrium and epididymis. This is remarkable because these tissues do not express the transgene. Human SHBG administered intravenously to wild-type mice in the presence of estradiol is rapidly sequestered within the endometrial stroma, and this prompted us to search for SHBG interacting proteins. Yeast two-hybrid screens revealed that fibulin-1D and fibulin-2 interact with the amino-terminal laminin G domain of SHBG. These interactions were verified in GST-pull down assays in which human SHBG bound the carboxyl-terminal domains of fibulin-1D and fibulin-2 in a steroid-dependent manner, with estradiol being the most effective ligand, and were enhanced by reducing the N-glycosylation of human SHBG. Like human SHBG, fibulin-1 and fibulin-2 concentrate within the endometrial stroma. In addition, SHBG co-immunoprecipitates with these fibulins in a proestrus uterine extract. These matrix-associated proteins may therefore sequester plasma SHBG within uterine stroma where it can control sex-steroid access to target cells. Given the interplay between fibulins and numerous proteins within the basal lamina, interactions between SHBG and matrix proteins may exert novel biological effects.     

Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit

How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.     

Nandrolone and stanozolol induce leydig cell tumor proliferation through an estrogen‐dependent mechanism involving IGF‐I system

Several substances such as anabolic androgenic steroids (AAS), peptide hormones like insulin‐like growth factor‐I (IGF‐I), aromatase inhibitors and estrogen antagonists are offered via the Internet, and are assumed without considering the potential deleterious effects that can be caused by their administration. In this study we aimed to determine if nandrolone and stanozolol, two commonly used AAS, could have an effect on Leydig cell tumor proliferation and if their effects could be potentiated by the concomitant use of IGF‐I. Using a rat Leydig tumor cell line, R2C cells, as experimental model we found that nandrolone and stanozolol caused a dose‐dependent induction of aromatase expression and estradiol (E2) production. When used in combination with IGF‐I they were more effective than single molecules in inducing aromatase expression. AAS exhibited estrogenic activity and induced rapid estrogen receptor (ER)‐dependent pathways involving IGF1R, AKT, and ERK1/2 phosphorylation. Inhibitors for these kinases decreased AAS‐dependent aromatase expression. Up‐regulated aromatase levels and related E2 production increased cell proliferation as a consequence of increased cyclin E expression. The observation that ER antagonist ICI182,780 was also able to significantly reduce ASS‐ and AAS + IGF‐induced cell proliferation, confirmed a role for estrogens in AAS‐dependent proliferative effects. Taken together these data clearly indicate that the use of high doses of AAS, as it occurs in doping practice, enhances Leydig cell proliferation, increasing the risk of tumor development. This risk is higher when AAS are used in association with IGF‐I. To our knowledge this is the first report directly associating AAS and testicular cancer. J. Cell. Physiol. 227: 2079–2088, 2012. © 2011 Wiley Periodicals, Inc.     

Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation

Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents.     

Thermodynamic characterization of viral procapsid expansion into a functional capsid shell

The assembly of "complex" DNA viruses such as the herpesviruses and many tailed bacteriophages includes a DNA packaging step where the viral genome is inserted into a preformed procapsid shell. Packaging triggers a remarkable capsid expansion transition that results in thinning of the shell and an increase in capsid volume to accept the full-length genome. This transition is considered irreversible; however, here we demonstrate that the phage λ procapsid can be expanded with urea in vitro and that the transition is fully reversible. This provides an unprecedented opportunity to evaluate the thermodynamic features of this fascinating and essential step in virus assembly. We show that urea-triggered expansion is highly cooperative and strongly temperature dependent. Thermodynamic analysis indicates that the free energy of expansion is influenced by magnesium concentration (3-13?kcal/mol in the presence of 0.2-10?mM Mg(2+)) and that significant hydrophobic surface area is exposed in the expanded shell. Conversely, Mg(2+) drives the expanded shell back to the procapsid conformation in a highly cooperative transition that is also temperature dependent and strongly influenced by urea. We demonstrate that the gpD decoration protein adds to the urea-expanded capsid, presumably at hydrophobic patches exposed at the 3-fold axes of the expanded capsid lattice. The decorated capsid is biologically active and sponsors packaging of the viral genome in vitro. The roles of divalent metal and hydrophobic interactions in controlling packaging-triggered expansion of the procapsid shell are discussed in relation to a general mechanism for DNA-triggered procapsid expansion in the complex double-stranded DNA viruses.     

Synthesis, characterization, crystal structures and in vitro antistaphylococcal activity of organotin(IV) derivatives with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine

New organotin(IV) complexes of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp) with 1:1 and/or 1:2 stoichiometry were synthesized and investigated by X-ray diffraction, FT-IR and ¹¹⁹Sn Mössbauer in the solid state and by ¹H and ¹³C NMR spectroscopy, in solution. Moreover, the crystal and molecular structures of Et₂SnCl₂(dbtp)₂ and Ph₂SnCl₂(EtOH)₂(dptp)₂ are reported. The complexes contain hexacoordinated tin atoms: in Et₂SnCl₂(dbtp)₂ two 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine molecules coordinate classically the tin atom through N(3) atom and the coordination around the tin atom shows a skew trapezoidal structure with axial ethyl groups. In Ph₂SnCl₂(EtOH)₂(dptp)₂ two ethanol molecules coordinate tin through the oxygen atom and the 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the OH group of the ethanol moieties; Ph₂SnCl₂(EtOH)₂(dptp)₂ has an all-trans structure and the C-Sn-C fragment is linear. On the basis of Mössbauer data, the 1:2 diorganotin(IV) complexes are advanced to have the same structure of Et₂SnCl₂(dbtp)₂, while Me₂SnCl₂(dptp)₂ to have a regular all-trans octahedral structure. A distorted cis-R₂ trigonal bipyramidal structure is assigned to 1:1 diorganotin(IV) complexes. The in vitro antibacterial activities of the synthesized complexes have been tested against a group of reference pathogen micro-organisms and some of them resulted active with MIC values of 5 μg/mL, most of all against staphylococcal strains, which shows their inhibitory effect.     

CB(1) antagonism restores hepatic insulin sensitivity without normalization of adiposity in diet-induced obese dogs

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.     

Sensitivity of double centrifugation sugar fecal flotation for detecting intestinal helminths in coyotes (Canis latrans)

Fecal analysis is commonly used to estimate prevalence and intensity of intestinal helminths in wild carnivores, but few studies have assessed the reliability of fecal flotation compared to analysis of intestinal tracts. We investigated sensitivity of the double centrifugation sugar fecal flotation and kappa agreement between fecal flotation and postmortem examination of intestines for helminths of coyotes (Canis latrans). We analyzed 57 coyote carcasses that were collected between October 2010 and March 2011 in the metropolitan area of Calgary and Edmonton, Alberta, Canada. Before analyses, intestines and feces were frozen at -80 C for 72 hr to inactivate Echinococcus eggs, protecting operators from potential exposure. Five species of helminths were found by postmortem examination, including Toxascaris leonina, Uncinaria stenocephala, Ancylostoma caninum, Taenia sp., and Echinococcus multilocularis. Sensitivity of fecal flotation was high (0.84) for detection of T. leonina but low for Taenia sp. (0.27), E. multilocularis (0.46), and U. stenocephala (0.00). Good kappa agreement between techniques was observed only for T. leonina (0.64), for which we detected also a significant correlation between adult female parasite intensity and fecal egg counts (R(s)=0.53, P=0.01). Differences in sensitivity may be related to parasite characteristics that affect recovery of eggs on flotation. Fecal parasitologic analyses are highly applicable to study the disease ecology of urban carnivores, and they often provide important information on environmental contamination and potential of zoonotic risks. However, fecal-based parasitologic surveys should first assess the sensitivity of the techniques to understand their biases and limitations.     

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Background

Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings

LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion

LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.     

Q-Elastography in the Presurgical Diagnosis of Thyroid Nodules with Indeterminate Cytology

Quantitative ultrasound (US) elastography (Q-USE), able to evaluate tissue stiffness has been indicated as a new diagnostic tool to differentiate benign from malignant thyroid lesions. Aim of this prospective study, conducted at the Department of Surgical Sciences, of the “Sapienza” University of Rome, was to evaluate the diagnostic accuracy of Q-USE, compared with US parameters, in thyroid nodules with indeterminate cytology (Thy3).The case study included 140 nodules from 140 consecutive patients. Patient’s thyroid nodules were evaluated by Q-USE, measuring the strain ratio (SR) of stiffness between nodular and surrounding normal thyroid tissue, and conventional US parameters prior fine-needle aspiration cytology. Those with Thy3 diagnosis were included in the study. Forty of the nodules analyzed harbored a malignant lesion. Q-USE demonstrated that malignant nodules have a significant higher stiffness with respect to benign one and an optimun SR cut-off value of 2.05 was individuated following ROC analysis. Univariate analysis showed that hypoechogenicity, irregular margins and SR >2.05 associated with malignancy, with an accuracy of 67.2%, 81,0% and 89.8%, respectively. Data were unaffected by nodule size or thyroiditis. These findings were confirmed in multivariate analysis demonstrating a significant association of the SR and the irregular margins with thyroid nodule’s malignancy. In conclusion, we demonstrated the diagnostic utility of Q-USE in the differential diagnosis of thyroid nodules with indeterminate cytology that, if confirmed, could be of major clinical utility in patients’ presurgical selection.