Lipophilization of Hydroxytyrosol-Enriched Fractions from Olea europaea L. Byproducts and Evaluation of the in Vitro Effects on a Model of Colorectal Cancer Cells.

A hydroxytyrosol (HTyr)-enriched fraction containing HTyr 6% w/w, derived from Olea europaea L. byproducts and obtained using an environmentally and economically sustainable technology, was lipophilized under green chemistry conditions. The effects of three fractions containing hydroxytyrosyl butanoate, octanoate, and oleate, named, respectively, lipophilic fractions 5, 6, and 7, and unreacted HTyr on the human colon cancer cell line HCT8-ß8 engineered to overexpress estrogen receptor ß (ERß) were evaluated and compared to those of pure HTyr. The experimental data demonstrated that HTyr and all fractions showed an antiproliferative effect, as had been observed by the evaluation of the cellular doubling time under these different conditions (mean control, 32 ± 4 h; HTyr 1, 65 ± 9 h; fraction 5, 64 ± 11 h; fraction 6, 62 ± 14 h; fraction 7, 133 ± 30 h). As evidenced, fraction 7 containing hydroxytyrosyl oleate showed the highest activity. These results were related to the link with ER-ß, which was assessed through simultaneous treatment with an inhibitor of ERß.

The effect of strontium chloride on human periodontal ligament stem cells.

The complete repair of periodontal structures remains an exciting challenge that prompts researchers to develop new treatments to restore the periodontium. Recent research has suggested strontium ion to be an attractive candidate to improve osteogenic activity. In this study, we have isolated a clonal finite cell line derived from human periodontal ligament (PDL) in order to assess whether and in which way different doses of SrCl2 (from 0.5 to 500 µg/ml) can influence both the proliferation and the mineralization process, for future application in oral diseases. PDL cells were cloned by dilution plating technique and characterized by FACS. Cell proliferation analysis and mineralization were performed by [3H]-thymidine incorporation and spectrofluorometric assay. Results have evidenced that the higher SrCl2 concentrations tested, from 25 to 500 µg/ml, have increased the proliferation activity after only 24 h of treatment. Interestingly, the same higher concentrations have decreased the mineralization, which was conversely increased by the lower ones, from 0.5 to 10 µg/ml. Our findings suggest the possible use of SrCl2 in appropriate delivery systems that release, at different time points, the specific dose, depending on the biological response that we want to induce on periodontal ligament stem cells, providing a more efficient periodontal regeneration.

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

Growing Strong and Healthy with Mister Bone: An Educational Program to Have Strong Bones Later in Life.

Optimal peak bone mass and bone health later in life are favored by a sufficient calcium intake in infancy, childhood and adolescence. The purpose of this study was to test a new educational program created to monitor and to improve calcium and vitamin D intake in children. Nutritional habits in children were evaluated through a food frequency questionnaire (FFQ) to assess the intake of calcium, vitamin D, dairy products, and total caloric energy at baseline and after seven months of exposure to a unique educational program applied between November 2013 and May 2014 in 176 schoolchildren (48% male, 52% female) attending the fourth and fifth grades of two selected primary schools in Florence, Italy. A significant increase of calcium (from 870 ± 190 to 1100 ± 200 mg/day, p < 0.05), and vitamin D (from 3.6 ± 1.53 to 4.1 ± 2 µg/day) intake in children was documented after the educational program. The amount of specific foods important for bone health consumed, such as milk and vegetables, increased significantly, both in male and female children (p < 0.05). The proposed educational program appears to be effective in modifying calcium intake in children, with a significant increase in the consumption of dairy products and vegetables, but without a significant change in the total caloric intake.

In Vitro Behavior of Human Adipose Tissue-Derived Stem Cells on Poly(ε-caprolactone) Film for Bone Tissue Engineering Applications.

Bone tissue engineering is an emerging field, representing one of the most exciting challenges for scientists and clinicians. The possibility of combining mesenchymal stem cells and scaffolds to create engineered tissues has brought attention to a large variety of biomaterials in combination with osteoprogenitor cells able to promote and regenerate bone tissue. Human adipose tissue is officially recognized as an easily accessible source of mesenchymal stem cells (AMSCs), a significant factor for use in tissue regenerative medicine. In this study, we analyze the behavior of a clonal finite cell line derived from human adipose tissue seeded on poly(ε-caprolactone) (PCL) film, prepared by solvent casting. PCL polymer is chosen for its good biocompatibility, biodegradability, and mechanical properties. We observe that AMSCs are able to adhere to the biomaterial and remain viable for the entire experimental period. Moreover, we show that the proliferation process and osteogenic activity of AMSCs are maintained on the biofilm, demonstrating that the selected biomaterial ensures cell colonization and the development of an extracellular mineralized matrix. The results of this study highlight that AMSCs and PCL film can be used as a suitable model to support regeneration of new bone for future tissue engineering strategies.

In vitro effects of extracts of extra virgin olive oil on human colon cancer cells.

The Mediterranean diet is associated with a lower incidence of atherosclerosis, cardiovascular diseases, and some types of cancer. Recent interest has been focused on the biological activity of phenolic compounds present in extra virgin olive oils (EVOOs). Both in vivo and in vitro studies have shown that EVOO components have positive effects on metabolic parameters, such as plasma lipoproteins, oxidative damage, inflammatory markers, platelet function, and antimicrobial activity. We have investigated the possible interactions between 2 extracts of extra virgin olive oil and estrogen receptor ß (ERß) in an in vitro model of colon cancer. The qualification and quantification of the components of the 2 samples tested showed that phenolic compounds-hydroxytyrosol, secoiridoids, and lignans-are the major represented compounds. EVOO extracts were tested on a colon cancer cell line engineered to overexpress ERß (HCT8-ß8). By using custom made Oligo microarray, gene expression profiles of colon cancer cells challenged with EVOO-T extracts when compared with those of cells exposed to 17ß-estradiol (17ß-E2). This study demonstrated that the EVOO extracts tested showed an antiproliferative effect on colon cancer cells through the interaction with estrogen-dependent signals involved in tumor cell growth. Specifically, the ability of EVOO extracts to inhibit cell proliferation was superimposable to the activation of the ERß receptor, similar to what was observed after 17ß-E2 challenge.

In vitro effects of polyphenols on colorectal cancer cells.

AIM: To investigate the effects of quercetin and genistein on colon cancer cell proliferation and their estrogen receptor ß (ERß) expression. METHODS: Colon cancer cells were stably transfected with a mammalian expression vector to overexpress ERß (HCT8-ß8-expressing cells) or a control vector (HCT8-pSV2neo-expressing cells). The proliferation of these cells was examined after treatment with quercetin or genistein (5-100 µmol/L), or 10 nmol/L 17ß-estradiol (17ß-E2). Cell viability was examined by acridine orange staining following treatments for 48 or 144 h. Effects of quercetin and genistein on ERß transcriptional transactivation were examined by luciferase activity in HCT8-ß8-expressing cells transiently transfected with a pEREtkLUC reporter vector. In addition, the regulation of ERß transcription by phytoestrogens and 17ß-E2 was examined by quantitative polymerase chain reaction. RESULTS: Proliferation of HCT8-ß8-expressing cells was not reduced low doses (5 µmol/L) of quercetin and genistein, while it was reduced at 25-50 µmol/L with an effect similar to 10 nmol/L 17ß-E2. Treatment with doses of phytoestrogens ≥ 75 µmol/L completely blocked cell growth and reduced overall cell counts, however no effects at any dose were observed in HCT8-pSV2neo-expressing cells. These results were supported by viability staining that revealed acridine orange-stained lysosomes with high doses or extended treatment periods. Genistein and quercetin (50 µmol/L) significantly increased ER-responsive luciferase activity similar to 10 nmol/L 17ß-E2 (P < 0.05). Furthermore, genistein and quercetin (50 µmol/L), as well as 10 nmol/L 17ß-E2 significantly increased ERß mRNA levels in HCT8-ß8-expressing cells (P < 0.05). In addition, treatment of HCT8-pSV2neo-expressing cells with 50 µmol/L quercetin or 10 nmol/L 17ß-E2 significantly increased ERß mRNA levels compared to untreated controls (P < 0.05), though the absolute levels were much lower than in HCT8-ß8-expressing cells. CONCLUSION: The antitumorigenic effects of the phytoestrogenic compounds quercetin and genistein on colon cancers cells occur through ERß activity and expression.

CYP19 and ESR1 gene polymorphisms: response of the bone mineral density in post-menopausal women to hormonal replacement therapy.

OBJECTIVES: Sex steroids are important regulators of bone physiology and play an essential role in the maintenance of bone health throughout the life. Hormonal replacement therapy (HRT) is a treatment commonly used to relieve symptoms and some undesirable consequences of menopause such as osteoporosis. Osteoporosis, characterized by the loss of bone mass and deterioration of microarchitecture with a consequent higher risk of fragility fractures, is under genetic influence. A tetranucleotide (TTTA)n microsatellite repeat polymorphism, at intron 4 of the CYP19 (aromatase) gene, has been previously associated with higher lumbar spine bone mineral density (LS-BMD) and lower risk of spine fracture in postmenopausal women. Moreover, the ERα encoded by the ESR1 gene is another important candidate for the regulation of bone mass of menopause. Moreover prospective analysis from >18.000 subjects at the GENOMOS study indicated that XX homozygotes genotype had a reduced risk of fracture independently from BMD. In the present study, we investigated in postmenopausal Italian women, at baseline and after 1 year of HRT, whether ESR1 and CYP19 gene polymorphisms could affect BMD through different statistical models. METHODS: This study has been performed on 100 post-menopausal Italian women, from a larger group of 250. The study group was administred HRT and LS-BMD was measured at baseline and after 1 year of therapy. Genetic analysis evaluating ESR1 and CYP19 gene polymorphisms was performed. RESULTS: Generalized Linear Models (GLMs) test showed that women with normal LS-BMD at the baseline had a major statistically significant BMD increase of 0.1426 gr/cm(2) (p= 0.0001) with respect to the osteoporotic patients. In addition, subjects with genotype 1 and 2 of CYP19 gene had a lower modification in LS-BMD after 1 year of HRT (0.0837 gr/cm(2) and 0,076 g/cm(2); p=0.0470 and 0,0547 respectively) when compared to genotype 3. No influences of the aromatase genotypes were observed in the variable difference using both Anova and GLMs test. Regarding the ESR1 gene polymorphism, the LS-BMD after 1 year of HRT was influenced by the diagnosis at the baseline and height and ERα genotypes were able to influence difference with statistical significant results with both test. CONCLUSIONS: In the present study, we have demonstrated that CYP19 gene polymorphism is able to influence the effect of 1 year HRT on LS-BMD with no influence on pre-/ and post-/HRT LS-BMD differences. Although ESR1 gene polymorphism is not able to influence the LS-BMD after 1 year HRT, it influences the observed modifications during the year of therapy. These data underlie the complexity of the genetics of the bone mass and its importance in influencing the response to HRT.

PTH-C1: a rat continuous cell line expressing the parathyroid phenotype.

The lack of a continuous cell line of epithelial parathyroid cells able to produce parathyroid hormone (PTH) has hampered the studies on in vitro evaluation of the mechanisms involved in the control of parathyroid cell function and proliferation. The PT-r cell line was first established from rat parathyroid tissue in 1987, but these cells were known to express the parathyroid hormone-related peptide (Pthrp) gene, but not the Pth gene. In an attempt to subclone the PT-r cell line, a rat parathyroid cell strain was isolated and named PTH-C1. During 3 years, in culture, PTH-C1 cells maintained an epithelioid morphology, displaying a diploid chromosome number, a doubling time around 15 h during the exponential phase of growth, and parathyroid functional features. PTH-C1 cell line produces PTH and expresses the calcium sensing receptor (Casr) gene and other genes known to be involved in parathyroid function. Most importantly, the PTH-C1 cells also exhibit an in vitro secretory response to calcium. Altogether these findings indicate the uniqueness of the PTH-C1 cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells.

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

Osteodifferentiation of human preadipocytes induced by strontium released from hydrogels.

In recent years, there has been an increasing interest in interactive application principles of biology and engineering for the development of valid biological systems for tissue regeneration, such as for the treatment of bone fractures or skeletal defects. The application of stem cells together with biomaterials releasing bioactive factors promotes the formation of bone tissue by inducing proliferation and/or cell differentiation. In this study, we used a clonal cell line from human adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes), named PA2-E12, to evaluate the effects of strontium (Sr(2+)) released in the culture medium from an amidated carboxymethylcellulose (CMCA) hydrogel enriched with different Sr(2+) concentrations on osteodifferentiation. The osteoinductive effect was evaluated through both the expression of alkaline phophatase (ALP) activity and the hydroxyapatite (HA) production during 42 days of induction. Present data have shown that Sr(2+) released from CMCA promotes the osteodifferentiation induced by an osteogenic medium as shown by the increase of ALP activity at 7 and 14 days and of HA production at 14 days. In conclusion, the use of biomaterials able to release in situ osteoinductive agents, like Sr(2+), could represent a new strategy for future applications in bone tissue engineering.

Subcutaneous adipocytes may become osteoblasts.

Commonly, mesenchymal stem cells derived from bone marrow (BMSCs) are mainly utilized in regenerative medicine field. BMSCs are able to differentiate into several lineages, showing immunosuppressive properties, and they are genetically stable in long-term cultures. In the last years, another mesenchymal stem cells population, obtained from adipose tissue, defined adipose-derived stem/stromal cells (ASCs), it is under assessment of scientific research, as alternative to BMSCs. In fact, ASCs show similar capacity to BMSCs, but unlike BMSCs can be harvested more easily with an higher yield and with less invasive manipulation. In this review the abilities of ASCs to differentiate in osteoblasts cells are shown.

Connections between the outcomes of osteoporotic hip fractures and depression, delirium or dementia in elderly patients: rationale and preliminary data from the CODE study.

BACKGROUND: osteoporosis, depression and other neuro-psychiatric disorders are very common after 50 years of age. Although these conditions recognize several and specific etiologic factors, they however appear to share physiologic, environmental processes and risk factors which may explain their possible association. METHODS: we have built up a specific research project (the CODE study, Connections between the outcomes of osteoporotic hip fractures and depression, delirium or dementia in elderly patients), and carried out a preliminary survey on 55 hip fractured elderly patients (42 women, mean age 85 years old and 13 men, mean age 82 years old), hospitalized at SS. Annunziata hospital in Florence from July to September 2010. RESULTS: there was a significant difference (p=0.010) in the functional recovery after surgery (as measured by Cumulated Ambulation Score, CAS) between depressed and non-depressed subjects (n=38), with a worse recovery and a lower CAS score in depressed patients (n=17). We also observed a higher prevalence of depression in the osteoporotic-fragile elderly people (69,1% of total sample). CONCLUSION: our preliminary survey has validated the suitability of the CODE study protocol in assessing connections between outcomes of osteoporotic hip fractures and depression in elderly patients, fostering the extension of the study (and suggesting also the inclusion of delirium and dementia) within a multicentric prospective study aimed to provide specific information and guidelines for osteoporotic fractured patients with concomitant depression or other neuro-psychiatric disorders.

A novel germline inactivating mutation in the CASR gene in an Italian kindred affected by familial hypocalciuric hypercalcemia.

OBJECTIVE: Familial hypocalciuric hypercalcemia (FHH) syndrome is a rare benign condition, inherited as an autosomal dominant trait, in which inactivating mutations of the calcium-sensing receptor (CASR) gene affects the body's ability to regulate calcium homeostasis. Its outcome is featured by increased levels of serum calcium, moderate hypophosphatemia, and inadequately normal or elevated circulating parathyroid hormone levels. Affected patients are mostly asymptomatic and do not benefit from surgical resection of their mildly enlarged parathyroids. DESIGN: We evaluated for hypercalcemia an Italian family that was identified via a young adult male proband referred to our center for parathyroidectomy. METHODS: The patients and the family members were evaluated both biochemically and genetically as suspected FHH subjects. An in vitro functional study was performed by site-directed mutagenesis, and CASR activity was monitored by measuring intracellular calcium ([Ca(2)(+)](i)). RESULTS: The patient had a novel germline heterozygous CASR mutation (c.361_364GATT; p.D121del/fsX122). The mutation caused a premature stop codon at codon 122, exiting a truncated protein. The biochemical phenotype of all family members carrying the heterozygous deletion was concordant with classic FHH syndrome. CONCLUSIONS: Our findings confirm the role of CASR gene mutational analysis to offer a valuable addition for the recognition of FHH in hypercalcemic patients not yet characterized for a positive familial history of hypercalcemia, the only condition that identifies CASR gene mutations in hypercalcemia.

LRP5 gene polymorphism and cortical bone.

BACKGROUND AND AIMS: There is evidence that distinct genetic polymorphisms of LRP5 are associated with low Bone Mineral Density (BMD) and the risk of fracture. However, relationships between LRP5 polymorphisms and micro- and macro architectural bone characteristics assessed by pQCT have not been studied. The aim of the present study was to investigate the association of Ala1330Val and Val667Met polymorphisms in LRP5 gene with volumetric BMD (vBMD) and macro-architectural bone parameters in a population-based sample of men and women. METHODS: We studied 959 participants of the InCHIANTI study (451 men and 508 women, age range: 21-94 yrs). Trabecular vBMD (vBMDt, mg/cm3), cortical vBMD (vBMDc, mg/cm3), cortical bone area (CBA, mm2) and cortical thickness (Ct.Th, mm) at the level of the tibia were assessed by peripheral quantitative computed tomography (pQCT). Ala1330Val and Val667Met genotypes were determined on genomic DNA by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: In age-adjusted analyses both LRP 1330-valine and LRP 667-metionin variants were associated with lower vBMDt in men (p<0.05), and lower vBMDt (p<0.05), Ct.Th (p<0.05) and CBA (p<0.05) in women. After adjusting for multiple confounders, only the association of LRP5 1330-valine and 667-metionin with CBA remained statistically significant (p=0.04 and p=0.01, respectively) in women. CONCLUSION: These findings suggest that both Ala1330Val and Val667Met LRP5 polymorphisms may affect the determination of geometric bone parameters in women.

Identification of GDNF gene sequence variations in patients with medullary sponge kidney disease.

BACKGROUND AND OBJECTIVES: Medullary sponge kidney (MSK) is a rare nephropathy characterized by cystic anomalies of precalyceal ducts, nephrocalcinosis, renal stones, and tubule dysfunctions. Its association with various malformations and cases of familial aggregation supports the conviction that genetic factors are involved, but no genetic studies have been conducted to date. It is hypothesized that MSK is due to a disruption at the "ureteric bud/metanephric blastema" interface caused by critical developmental genes functioning abnormally. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Fifty-five apparently sporadic MSK patients were analyzed by direct DNA sequencing of all exons and exon-intron boundaries of glial cell-derived neurotrophic factor (GDNF) gene and rearranged during transfection (RET) gene, which have a leading role in renal development. RESULTS: Two novel variants were found in heterozygosity in the MSK case population: GDNF{ENST00000344622}:c.-45G>C and c.-27+18G>A in a putative binding domain for paired-box 2 transcription factor. As a whole, eight patients showed these variations: four patients carried the c.[-45G>C; -27+18G>A] complex allele, and the others had the c.-27+18G>A alone. A case-control study revealed that these two alleles were significantly associated with MSK. Five of the eight cases were found to be familial, and the allele variants cosegregated with the disease in a seemingly dominant pattern of inheritance. Patients revealed no mutations in the RET gene. CONCLUSIONS: This is the first report identifying GDNF gene sequence variations in patients with MSK and suggesting a role for this gene in the pathogenesis of some cases of the disease.

Thyroid cancer: current molecular perspectives.

The thyroid cancer is a rare oncological entity, representing no more than 1% of all human malignant neoplasms. Recently, it has been demonstrated a sharp increase in incidence of differentiated thyroid carcinoma, equally occurring in both sexes. So far, multiple genetic alterations have been identified in differentiated thyroid carcinoma, leading to investigate the clinical utility of genetic studies. In particular, molecular genetic approaches searching for gene mutations in the material collected by fine needle ago-biopsy may have a particular utility in small nodules and in those specimens with an indeterminate cytology. The expansion of knowledge about genetic mutations occurring in different thyroid tumors has characterized recent years, allowing the identification of a correlation between specific mutations and phenotypic characteristics of thyroid cancers, essential for their prognosis. This review will briefly report on the histological features and the new entity represented by thyroid microcarcinoma and will focus on both environmental and genetic aspects associated with the occurrence of thyroid cancer.

In vitro effects of oestrogens, antioestrogens and SERMs on pancreatic solid pseudopapillary neoplasm-derived primary cell culture.

BACKGROUND: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development. METHODS: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17beta-oestradiol (17betaE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined. RESULTS: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and beta-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERalpha, ERbeta and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17betaE2 and Tam respectively were proved in SPNC. CONCLUSION: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.

HPLC/DAD/MS and antioxidant activity of isoflavone-based food supplements.

Isoflavones are polyphenolic compounds found mainly in legumes the benefits of which have been widely studied and attributed in particular to their phytoestrogenic activity. The aim of this study was to evaluate the quali-quantitative composition of food supplements based on soy isoflavones (Glycine max L.) and red clover (Trifolium pratense). Six commercial food supplements (five soy-based and one red clover-based) were analyzed by HPLC/DAD/MS. Genistein, daidzein, glycitein, biochanin A and formononetin derivatives (glycosides and acylglycosides) were identified in the analyzed samples. Also the antiradical activities (towards the DPPH* radical) and Fe2+ chelating abilities were compared.

Estrogen receptor expression in cutaneous melanoma: a real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study.

OBJECTIVE: To evaluate estrogen receptor (ER) expression in human melanoma tissues and in the adjacent healthy skin with the aim of explaining whether the ERalpha:ERbeta expression ratio has a role in neoplastic progression. DESIGN: Prospective study. SETTING: Department of Dermatology, University of Florence, Florence, Italy. Patients Fourteen patients, 12 with cutaneous melanoma (6 women and 6 men) and 2 with melanocytic nevi (1 woman and 1 man). MAIN OUTCOME MEASURES: Using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, we analyzed ERalpha and ERbeta messenger RNA (mRNA) and ERbeta protein expression in cutaneous melanoma and in the healthy skin surrounding the lesions. RESULTS: All melanocytic lesions expressed detectable levels of ERalpha and ERbeta mRNA as well as ERbeta protein. Dividing melanoma cases into 2 groups according to Breslow thickness, we found lower ERalpha and ERbeta mRNA levels and lower ERbeta protein levels in thicker, more invasive tumors. CONCLUSIONS: These observations suggest a role for ERs in the metastatic process of melanoma cells, pointing at the possibility of using ERbeta expression as a prognostic indicator of melanoma. The possibility of distinguishing proliferative melanomas, which are associated with dismal prognosis, from the so-called dormant melanomas opens up novel avenues in tailoring individual treatments, as already happens for other tumors.