A new sol-gel synthesis of 45S5 bioactive glass using an organic acid as catalyst.

In this paper a new sol-gel approach was explored for the synthesis of the 45S5 bioactive glass. We demonstrate that citric acid can be used instead of the usual nitric acid to catalyze the sol-gel reactions. The substitution of nitric acid by citric acid allows to reduce strongly the concentration of the acid solution necessary to catalyze the hydrolysis of silicon and phosphorus alkoxides. Two sol-gel powders with chemical compositions very close to that of the 45S5 were obtained by using either a 2M nitric acid solution or either a 5mM citric acid solution. These powders were characterized and compared to the commercial Bioglass®. The surface properties of the two bioglass powders were assessed by scanning electron microscopy (SEM) and by Brunauer-Emmett-Teller method (BET). The Fourier transformed infrared spectroscopy (FTIR) and the X-ray diffraction (XRD) revealed a partial crystallization associated to the formation of crystalline phases on the two sol-gel powders. The in vitro bioactivity was then studied at the key times during the first hours of immersion into acellular Simulated Body Fluid (SBF). After 4h immersion into SBF we clearly demonstrate that the bioactivity level of the two sol-gel powders is similar and much higher than that of the commercial Bioglass®. This bioactivity improvement is associated to the increase of the porosity and the specific surface area of the powders synthesized by the sol-gel process. Moreover, the nitric acid is efficiently substituted by the citric acid to catalyze the sol-gel reactions without alteration of the bioactivity of the 45S5 bioactive glass.

In vitro antiproliferative effect and induction of apoptosis by Retama monosperma L. extract in human cervical cancer cells.

The antiproliferative effect of different extracts obtained from Retama monosperma L. was investigated on human SiHa and HeLa cervical cancer cell lines using a MTT colorimetric assay. The Retama monosperma L. dichloromethane fraction (Rm-DF) was the most active extract, exhibiting a significant cytotoxic activity on both cell lines in a dose-dependent manner, after 72 h of treatment. IC50 values obtained were 14.57 ± 4.15 µg/ml and 21.33 ± 7.88 µg/ml, for SiHa and HeLa cell lines respectively. The morphological features assessment of apoptosis in Rm-DF-treated cells showed a condensation of chromatin and apoptotic bodies, accompanied by a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species in both cell lines. The induction of apoptosis was further confirmed by Western blotting pro-caspase 3, Bcl2 and PARP; caspase 3 activity assay; and Annexin V labelling. Analysis of Rm-DF by CG/MS revealed the presence of five known quinolizidine alkaloids as well as, sparteine (10,97%), L-methyl cytisine (9.11%), 17-oxosparteine (3.49%), lupanine (0.93%) and anagyrine (39.63%). This study shows that Retama monosperma L. extract exhibits a potential anticancer activity against cervical cancer cell lines in vitro through the inhibition of proliferation and induction of apoptosis, which may involve a mitochondria-mediated signaling pathway.

The C-terminal CD47/IAP-binding domain of thrombospondin-1 prevents camptothecin- and doxorubicin-induced apoptosis in human thyroid carcinoma cells.

Camptothecin and doxorubicin belong to a family of anticancer drugs that exert cytotoxic effects by triggering apoptosis in various cell types. However there have only been few investigations showing that matricellular proteins like thrombospondin-1 (TSP-1) could be involved in the underlying mechanism of this cytotoxicity. In this report, using Hoechst reagent staining, reactive oxygen species production and caspase-3 activity measurement, we determined that both camptothecin and doxorubicin induced apoptosis in human thyroid carcinoma cells (FTC-133). On the one hand, we demonstrated that camptothecin and doxorubicin inhibited TSP-1 expression mainly occurring at the transcriptional level. On the other hand, drug-induced apoptosis determined by western blot analysis for PARP cleavage and caspase-3 activity measurement, was significantly decreased in presence of exogenous TSP-1. In order to identify the sequence responsible for this effect, we used the CD47/IAP-binding peptide 4N1 (RFYVVMWK), derived from the C-terminal domain of TSP-1, and known to play a role in apoptosis. Thus, in presence of 4N1, camptothecin and doxorubicin-induced pro-apoptotic activity was considerably inhibited. These findings suggest that induction of apoptosis by camptothecin or doxorubicin in FTC-133 cells is greatly dependent by a down-regulation of TSP-1 expression and shed new light on a possible role for TSP-1 in drug resistance.

Thrombospondin-1 C-terminal-derived peptide protects thyroid cells from ceramide-induced apoptosis through the adenylyl cyclase pathway.

Thrombospondin-1, a multi-modular matrix protein is able to interact with a variety of matrix proteins and cell-surface receptors. Thus it is multifunctional. In this work, we examined the role of thrombospondin-1 in ceramide-induced thyroid apoptosis. We focused on the VVM containing sequence localized in the C-terminal domain of the molecule. Primary cultured thyroid cells synthesize thrombospondin-1 depending on their morphological organization. As it leads thyrocytes to organize into monolayers before inducing apoptosis ceramide can modulate this organization. Here, we established that C(2)-ceramide treatment decreased thrombospondin-1 expression by interfering with the adenylyl cyclase pathway, thus leading to apoptosis. Furthermore, we demonstrated that the thrombospondin-1-derived peptide 4N1 (RFYVVMWK) abolished ceramide-induced thyroid cell death by preventing intracellular cAMP levels from dropping. Finally, we reported that 4N1-mediated inhibition of ceramide-induced apoptosis was consistently associated with a down-regulation of the caspase-3 processing. Integrin-associated protein receptor (IAP or CD47) was identified as a molecular relay mediating the observed 4N1 effects. Taken together, our results shed light for the first time on anti-apoptotic activities of the thrombospondin-1-derived peptide 4N1 and provide new information on how thrombospondin-1 may control apoptosis of non-tumoral cells.

Interleukin-1beta (IL-1beta) induces a crosstalk between cAMP and ceramide signaling pathways in thyroid epithelial cells.

Interleukin-1 beta (IL-1beta) is an important regulator of the thyroid cell function. This cytokine has been largely described to trigger an important biological signaling cascade: the sphingomyelin/ceramide pathway. In this report, we show that IL-1beta induces the transient activation of a neutral sphingomyelinase in porcine thyroid cells. Moreover, IL-1beta and ceramides are demonstrated to inhibit the TSH-induced cAMP production via the implication of alphaGi subunit of the adenylyl cyclase system. This crosstalk between cAMP and ceramide pathways constitutes a preponderant process in the TSH-controlled differentiation state of thyrocytes. All these results argue for the involvement of ceramides and IL-1beta in the thyroid function regulation, leading to a cell dedifferentiated state.

Thrombospondin 1: a multifunctional protein implicated in the regulation of tumor growth.

Thrombospondins belong to a family of extracellular matrix (ECM) proteins widely found from embryonic to adult tissues. The modular structure of thrombospondins contains a series of peptide sequences implicated in a multiplicity of biological functions. Extracellular matrix undergoes important alterations under proteolysis that occurs in pathological processes like tumorigenesis. An elevated secretion of thrombospondin 1 (TSP1) is often observed in tumors and is sometimes considered as a predictive factor. However, the role of TSP1 in cancer progression remains controversial and must be carefully apprehended. The regulation of cell adhesion, proliferation, apoptosis by TSP1 is examined in the present review and it is clear from the literature and from our investigations that TSP1 presents both stimulatory and inhibitory effects. The exposition of cryptic sites upon conformational changes can partially explain this contradiction. More interestingly, the analysis of TSP1-directed intracellular signaling pathways activated through specific receptors or supramolecular receptors docking systems may be useful to discriminate the precise function of TSP1 in tumor progression. The central role played by TSP1 in the control of matrix-degrading enzyme activation and catabolism reveals attractive tracks of research and highlights the involvement of the lipoprotein receptor-related protein (LRP) receptor in these events. Therefore, TSP1-derived peptides constitute a source of potentially active matrikins which could provide essential tools in cancer therapy.

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells.

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl-2, Bcl-x(L)/Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We demonstrate that caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose-response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre-treatment with the caspase inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment.

Interleukin 1 beta (IL-1 beta) action in porcine thyroid cells involves the ceramide signalling pathway.

Interleukin 1 beta (IL-1beta) is often associated with thyroidal autoimmune diseases. This cytokine has been largely described to trigger an important biological signalling pathway: the sphingomyelin/ceramide pathway. In this report we show that IL-1beta induces ceramide formation and sphingomyelin degradation in porcine thyroid cells via the activation of a neutral sphingomyelinase. Among the potential targets of IL-1beta and ceramides action, we have investigated the role of an atypical protein kinase C (PKC), the PKC zeta. We show that both IL-1beta and ceramides lead to an increase of PKCzeta activity. All these results suggest an important role for ceramides and IL-1beta in regulation of thyroid function, leading to cell survival or to apoptosis.

Interferon-gamma signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii.

We assayed mitogen-activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii. In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii. The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN-gamma. However, IFN-gamma treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN-gamma altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN-gamma directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN-gamma may be involved in mediating their protective signals.

In vivo modulation of annexins I, II and V expression by thyroxine and methylthiouracil.

Regulation of annexin concentration and localization were investigated in thyroid tissues of hypothyroid [methylthiouracil (MeSur) treatment], euthyroid (control) and hyperthyroid [thyroxine (T4) treatment] rats. A low level of circulating thyroid hormones induces a decrease of total thyroid calcium-binding protein concentration when compared with the concentration in unstimulated animals. Conversely, concentrations of annexins I, II and V increase. The accumulation of these proteins in two subcellular compartments (cytosolic and particulate fractions) can be reversed by addition of thyroid hormones. The finding of a specific increase in annexins concentration in thyroid-hormone-deficient rats, with a general decrease of the total calcium-binding protein content points to a very important role of these proteins in the cells. Furthermore, hyperthyroidisnt gives opposite results. To investigate the transduction pathway of annexins I-, II- and V-induced biosynthesis by thyroid hormones in thyroid glands, we used cultured pig thyroid cells as in vitro model system. In previous work [16], we have shown that annexin concentrations and localization are under TSH control via the adenylate cyclase pathway. In the presence of MeSur (in the culture medium), the protein-binding iodine remains low, indicative of weak thyroid hormone synthesis (data not shown) and that the annexins content is unchanged. These results suggest that, in thyroid tissue, an indirect mechanism links thyroid hormones to annexin expressions via the TSH feed-back loop, and excludes autocrine regulation.