Roberto Zonefrati

Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies

Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125l-TSH binding and are reactive with the glycoprotein component of the bovineTSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125l-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0–4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125l-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125l-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes.

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 (Sr2+) released in the culture medium from an amidated carboxymethylcellulose (CMCA) hydrogel enriched with different Sr2+ 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 Sr2+ 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 Sr2+, could represent a new strategy for future applications in bone tissue engineering.

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 β (ERβ) expression. METHODS Colon cancer cells were stably transfected with a mammalian expression vector to overexpress ERβ (HCT8-β8-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 17β-estradiol (17β-E2). Cell viability was examined by acridine orange staining following treatments for 48 or 144 h. Effects of quercetin and genistein on ERβ transcriptional transactivation were examined by luciferase activity in HCT8-β8-expressing cells transiently transfected with a pEREtkLUC reporter vector. In addition, the regulation of ERβ transcription by phytoestrogens and 17β-E2 was examined by quantitative polymerase chain reaction. RESULTS Proliferation of HCT8-β8-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 17β-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 17β-E2 (P < 0.05). Furthermore, genistein and quercetin (50 μmol/L), as well as 10 nmol/L 17β-E2 significantly increased ERβ mRNA levels in HCT8-β8-expressing cells (P < 0.05). In addition, treatment of HCT8-pSV2neo-expressing cells with 50 µmol/L quercetin or 10 nmol/L 17β-E2 significantly increased ERβ mRNA levels compared to untreated controls (P < 0.05), though the absolute levels were much lower than in HCT8-β8-expressing cells. CONCLUSION The antitumorigenic effects of the phytoestrogenic compounds quercetin and genistein on colon cancers cells occur through ERβ activity and expression.

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.

Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue

Since Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI2 on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI2 caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE2. Two optically active isomers of 5,6-dihydro-PGI2, i.e. stable synthetic analogs of PGI2, had qualitatively similar effects to PGI2. The relative potency ratio between the alpha- and beta- isomer as well as their potency compared to PGI2 were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI2 and its stable analogs had a greater effect than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI2 concentrations. TSH had no detectable effect on thyroidal PGI2 synthesis and release. In cultured thyrocytes the effects of PGI2 and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE2. These findings suggest the existence of a specific PGI2-responsive adenylate cyclase system in human thyroid cells other than thyrocytes, of possible physiologic significance.

In Vitro Effects of Strontium on Proliferation and Osteoinduction of Human Preadipocytes

Development of tools to be used for in vivo bone tissue regeneration focuses on cellular models and differentiation processes. In searching for all the optimal sources, adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes) are able to differentiate into osteoblasts with analogous characteristics to bone marrow mesenchymal stem cells, producing alkaline phosphatase (ALP), collagen, osteocalcin, and calcified nodules, mainly composed of hydroxyapatite (HA). The possibility to influence bone differentiation of stem cells encompasses local and systemic methods, including the use of drugs administered systemically. Among the latter, strontium ranelate (SR) represents an interesting compound, acting as an uncoupling factor that stimulates bone formation and inhibits bone resorption. The aim of our study was to evaluate the in vitro effects of a wide range of strontium (Sr2+) concentrations on proliferation, ALP activity, and mineralization of a novel finite clonal hADSCs cell line, named PA20-h5. Sr2+ promoted PA20-h5 cell proliferation while inducing the increase of ALP activity and gene expression as well as HA production during in vitro osteoinduction. These findings indicate a role for Sr2+ in supporting bone regeneration during the process of skeletal repair in general, and, more specifically, when cell therapies are applied.

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-β8 engineered to overexpress estrogen receptor β (ERβ) 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-β, which was assessed through simultaneous treatment with an inhibitor of ERβ.

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 17β-oestradiol (17βE2), 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 β-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERα, ERβ and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17βE2 and Tam respectively were proved in SPNC. Conclusions: 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.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

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.