Inhibitory effect of combined treatment with the aromatase inhibitor exemestane and tamoxifen on DMBA-induced mammary tumors in rats

The antitumor effect of exemestane (FCE 24304), an irreversible aromatase inhibitor, given alone or in combination with tamoxifen, was investigated in rats with 7, 12-dimethylbenzanthracene (DMBA)-induced mammary tumors. The compounds were given once daily, 6 days a week for 4 weeks. Exemestane, given at the dose of 20 mg/kg/day s.c., induced 26% complete (CR) and 18% partial (PR) tumor regressions, compared to 0% CR and 6% PR observed in controls. Tamoxifen, given at 1 mg/kg/day p.o., induced 16% CR and 13% PR. The combined treatment caused 41% CR and 16% PR, thus resulting in a higher antitumor effect than either single treatment. The apperance of new tumors was reduced by each single treatment and almost totally prevented by the combined treatment. Serum prolactin (PRL) levels, assayed 4 h after the last dose, were unchanged in the group treated with the combination, whereas tamoxifen alone caused a slight increase of serum PRL. These results indicate that estrogen deprivation through aromatase inhibition and estrogen receptor antagonism causes a better inhibition of DMBA-induced mammary tumors than either treatment modality alone.     

Antiestrogenic and antitumor properties of the new triphenylethylene derivative toremifene in the rat

The effects of toremifene, a new triphenylethylene derivative, on the uterus and DMBA-induced mammary tumors in rats were compared to tamoxifen. The ability of toremifene to compete with [3H]estradiol for cytoplasmic estrogen receptor from rat uterus was similar to tamoxifen, the IC50 being 26 and 23 microM respectively. In immature intact rats the two compounds, administered orally for three consecutive days, had similar intrinsic partial estrogenic efficacy, at 50 mg/kg, about 40% of that of estradiol benzoate (EB). However, at doses less than or equal to 10 mg/kg, the estrogenic effect of toremifene was seen at doses about 40 times higher than that of tamoxifen. The two compounds, administered together with a standard dose of EB, expressed the same maximal antiestrogenic efficacy (about 65% inhibition) at 50 mg/kg. However, the minimal effective antiestrogenic dose of toremifene was about 10 times that of tamoxifen and the ratio between antiestrogenic/estrogenic properties was favourable to toremifene. The duration of the antiestrogenic (antiuterotrophic) effect of a single oral dose (10 mg/kg) of the two compounds proved similar: at least 4 days in intact rats and 3 days in ovariectomized rats. In DMBA-induced tumor bearing rats toremifene was administered p.o., 6 times/week for 4 weeks at 0.08, 0.4, 2, 10 and 50 mg/kg. It was effective at the doses of 2, 10 and 50 mg/kg, inducing 39, 35 and 46% tumor regressions. The activity of toremifene at the minimal effective dose of 2 mg/kg was then compared with that of tamoxifen given at the same dose level. The compounds had comparable activity (47 vs 44% tumor regressions).     

Structure and functional analysis of the IGF-II/IGF2R interaction

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.     

Chemical characterization of humic substances extracted from organic-waste-amended soils

Humic substances were extracted from a soil treated, in a 4-year experiment, at different rates with a sludge from anaerobic treatment of combined civil and industrial wastes, and with agricultural manure. Elemental and chemical analyses, molecular weight (MW) distribution and infrared (IR) spectroscopy were performed on the purified humic acids (HA). Organic wastes significantly increased the HA content of the treated soils and improved CEC. The C/N, C/H and C/O ratios of HA extracted from the original wastes showed a higher degree of humification for sludge than for manure. This difference was also noticed for the C/N ratio of soil humic extracts, indicating a faster humification process for the sludges in soil. The content of oxygen-containing functional groups was lower than the ‘model’ HA reported in the literature, and even more so for HA from sludges, reflecting their anaerobic formation. MW distribution and E₄/E₆ ratios showed that the sludge material had a higher molecular complexity than manure, supporting the high degree of humification attributed to the former. HA extracted from sludge-treated soils revealed a molecular dimension increasing with the application doses of waste material. Infrared spectra showed that HA formed in soils after waste additions reflected the chemical composition of the original organic material, which was rich in aliphatic groups and peptides for sludge and in carbohydrates for manure. On the basis of this study, it is concluded that not only are organic waste additions able to build up the HA content in soils but the HA formed assume the chemical characteristics and the degree of humification of the original material.     

The culturing of human Schwann cells: a new contribution to the study of polyneuropathy

We have established 44 Schwann cell cultures from human peripheral nerves that underwent biopsy for diagnostic purposes using a new "sandwich" connective tissue and cut into 1 mm cubic explants which were placed between two glass coverslips coated with D-poly-L-lysine, in HAM-F10 medium, 15% FCS, 600 mg/dl glucose and antibiotics. In the first 3 weeks this new sandwich technique yields a fairly great and homogeneous amount of Schwann cell growth, with only a few fibroblasts and virtually no macrophages and provides a suitable substrate on which immuno cytochemical studies could be performed.     

Scanning electron microscopy of stromal cells of human placental villi throughout pregnancy

Summary Morphological changes in fixed stromal cells and Hofbauer cells were studied throughout pregnancy in different types of placental chorionic villi by scanning electron microscopy. In the mesenchymal villus the fixed stromal cells were characterized by thin cytoplasmic processes. Hofbauer cells exhibited blebs on their surface. Large sail-like processes with a crescent profile which surrounded well developed stromal channels and a small cell body typified the small reticulum cells of the immature intermediate villus. The Hofbauer cells here displayed blebs, microplicae and large lamellipodia. Short cytoplasmic expansions and a large cell body characterized the fibroblasts present inside the stem villus. Hofbauer cells were rare, having blebs or a few short lamellipodia. The mature intermediate villus contained small and large reticulum cells. The latter had a much larger cell body than the small ones and displayed a few short cytoplasmic processes partly delimiting narrow incomplete stromal channels. Occasional Hofbauer cells with small microplicae and/or blebs were present. The small reticulum cells and fibroblasts present in the terminal villus showed similar morphological features as above. However, the former exhibited less developed cytoplasmic extensions and therefore no stromal channels were observed. In the terminal villus, the morphology of the rare Hofbauer cells was similar to that found in the mature intermediate villus.     

A three-dimensional study of the normal human placental villous core

Summary In order to study the three-dimensional ultrastructure of the Hofbauer cells, human placentae from the 6th to the 21 st week of gestation and also from the end of pregnancy were cryofractured and observed by scanning electron microscopy. Hofbauer cells were found in the villous core at all the gestation stages examined. Their surface morphology was characterized by lamellipodia, funnel-like structures, blebs and microplicae. This pleomorphic aspect was probably related to functional or environmental conditions. In addition, thin cytoplasmic processes connected the Hofbauer cells with each other and with the components of the villous stroma. Fractured Hofbauer cells revealed large vacuoles in the cytoplasm; the vacuoles were smaller in size both at the beginning and at the end of pregnancy. This study further attests to the macrophagic nature of these cells.     

Novel aromatase and 5α-reductase inhibitors

Inhibitors of aromatase and 5α-reductase may be of use for the therapy of postmenopausal breast cancer and benign prostatic hyperplasia, respectively. FCE 27993 is a novel steroidal irreversible aromatase inhibitor structurally related to exemestane (FCE 24304). The compound was found to be a very potent competitive inhibitor of human placental aromatase, with a K_i of 7.2 nM (4.3 nM for exemestane). In preincubation studies with placental aromatase FCE 27993, like exemestane, was found to cause time-dependent inhibition with a higher rate of inactivation ( ) and a similar K_i(inact) (56 vs 66 nM). The compound was found to have a very low binding affinity to the androgen receptor (RBA 0.09% of dihydrotestosterone) and, in contrast to exemestane, no androgenic activity up to 100 mg/kg/day s.c. in immature castrated rats. Among a series of novel 4-azasteroids with fluoro-substituted-17β-amidic side chains, three compounds, namely FCE 28260, FCE 28175 and FCE 27837, were identified as potent in vitro and in vivo inhibitors of prostatic 5α-reductase. Their IC₅₀ values were found to be 16, 38 and 51 nM for the inhibition of the human enzyme, and 15, 20 and 60 nM for the inhibition of the rat enzyme, respectively. When given orally for 7 days in castrated and testosterone (Silastic implants) supplemented rats, the new compounds were very effective in reducing prostate growth. At a dose of 0.3 mg/kg/day inhibitions of 42, 36 and 41% were caused by FCE 28260, FCE 28175 and FCE 27837, respectively.     

Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells

Amyloid beta-protein (Abeta) aggregation produces an oxidative stress in neuronal cells that, in turn, may induce an amyloidogenic shift of neuronal metabolism. To investigate this hypothesis, we analyzed intra- and extracellular Abeta content in NT2 differentiated cells incubated with 4-hydroxy-2,3-nonenal (HNE), a major product of lipid peroxidation. In parallel, we evaluated protein kinase C (PKC) isoenzymes activity, a signaling system suspected to modulate amyloid precursor protein (APP) processing. Low HNE concentrations (0.1-1 microM) induced a 2-6 fold increase of intracellular Abeta production that was concomitant with selective activation of betaI and betaII PKC isoforms, without affecting either cell viability or APP full-length expression. Selective activation of the same PKC isoforms was observed following NT2 differentiation. Our findings suggest that PKC beta isoenzymes are part of cellular mechanisms that regulate production of the intracellular Abeta pool. Moreover, they indicate that lipid peroxidation fosters intracellular Abeta accumulation, creating a vicious neurodegenerative loop.