Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression.

OBJECTIVE: To study the role of mitogen-activated protein kinases (MAPKs) in human annulus fibrosus (AF) cells subjected to cyclic tensile stress (CTS). DESIGN: An in vitro system for CTS studies was established using AF cultures on fibronectin-coated silicone dishes. MAPK phosphorylation was studied by western analysis, while gene expression was followed by qRT-PCR. DNA synthesis was assessed by both tritiated thymidine incorporation and flow cytometry, and collagen synthesis using tritiated proline incorporation and the protease-free collagenase method. RESULTS: All three MAPKs studied, i.e., ERK, SAPK/JNK, and p38 were found to be phosphorylated immediately after CTS application within physiological range. A second wave of phosphorylation appeared at later time points. MAPK activation was elevated at higher CTS magnitudes, but independent of the frequency. CTS did not stimulate DNA synthesis neither extracellular matrix turnover, but it stimulated the proinflammatory genes, COX-2, IL-6, and IL-8. This stimulation was more intense at the highest magnitude (8%) tested and at the median frequency (1 Hz) and time interval (12 h). Blocking of ERK, SAPK/JNK, and p38 MAPK inhibited the CTS-induced stimulation of COX-2 and IL-8, while IL-6 expression was mediated only by SAPK/JNK and p38 MAPK. CONCLUSIONS: We have described for the first time the activation of MAPKs in human AF cells in response to CTS and showed that it drives an inflammatory reaction. These observations shed light on the mechanisms of intervertebral disc (IVD) cell responses to mechanical stress, contributing to the understanding of disc pathophysiology and possibly to the design of novel therapeutic interventions.

Ursolic acid triggers apoptosis and Bcl-2 downregulation in MCF-7 breast cancer cells.

In this report we determine the ability of ursolic acid (UA) to induce apoptosis and to modulate glucocorticoid receptor (GR) and Activator Protein-1 (AP-1) in MCF-7 cells. The UA-induced apoptosis (53 microM), the PARP cleavage, and the decrease in Bcl-2 protein (53 microM) support the notion that UA induces apoptosis through the intrinsic mitochondrial pathway. UA binds GR (relative binding affinity: 2.57) and translocates GR into nucleus, suggesting its potential as a GR modulator. UA had no effect on GRE- or TRE-driven gene expression. In summary, UA is a GR modulator and may be considered as a potential anticancer agent in breast cancer.

Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cells.

PURPOSE: Glucocorticoids are widely used as adjuvant therapy in hormonal refractory prostate cancer; their therapeutic role, however, remains unclear. Ursolic acid, a natural triterpene, structurally similar to dexamethasone, exhibits antitumor effects in various cell types. Our main objective was to investigate the effects of ursolic acid on cell viability, apoptosis and bcl-2 protein, in human hormone refractory and androgen-sensitive prostate cancer cells. METHODS: The ursolic acid-induced changes in cell viability, apoptosis and bcl-2 protein were examined in human hormone refractory prostate cancer PC-3 cells and androgen-sensitive LNCaP cells, by MTT assay, flow cytometry and western blot analysis, respectively. RESULTS: Ursolic acid inhibited significantly the cell viability and induced apoptosis in PC-3 cells at 55 microM and in LNCaP cells at 45 microM associated with a downregulation of bcl-2 protein. CONCLUSIONS: The antiproliferative and apoptotic effects of ursolic acid in PC-3 and LNCaP cells implicate its potential therapeutic use for the treatment of hormone refractory and androgen-sensitive prostate cancer. The downregulation of bcl-2 may be one of the molecular mechanisms via which it induces apoptosis in PC-3 and LNCaP cells.

Food-dependent androgen and cortisol secretion by a gastric inhibitory polypeptide-receptor expressive adrenocortical adenoma leading to hirsutism and subclinical Cushing's syndrome: in vivo and in vitro studies.

Aberrant gastric inhibitory polypeptide (GIP) receptor expression in bilaterally hyperplastic adrenals or unilateral adrenal adenomas is a rare form of adrenal hyperfunction. So far, only few cases have been described. In all these cases, cortisol was the predominant steroid released in a food-dependent manner, leading to the development of non-ACTH-dependent Cushing's syndrome. In the present study, we describe a novel case of a GIP receptor-expressive adrenocortical adenomatous nodule, detected incidentally by computed tomography scanning in a 41-yr-old lady with hirsutism but no clinical signs of Cushing's syndrome, on physical examination. Hormonal investigations in morning fasting samples showed slightly elevated androgen levels, low-normal baseline cortisol, normal suppression of cortisol after dexamethasone administration, and ACTH levels that were not suppressed and did stimulate after CRH administration. The elevated urinary free cortisol excretion, in conjunction with an atypical cortisol diurnal rhythm, raised the possibility of an aberrant stimulation of cortisol production by the adrenal tumor. Further studies demonstrated food-dependent secretion of cortisol, which was abolished by prior octreotide administration. Notably, substantial amounts of adrenal androgens were also secreted after food consumption. Removal of the tumor resulted in undetectable cortisol and androgen levels that did not respond to food consumption. Histological examination of the excised tumor revealed an adrenocortical adenomatous nodule originating from the inner zona reticularis, consisting mainly of compact cells. A steroidogenic secretory pattern, indicating the concomitant release of adrenal androgens and cortisol, was also observed in vitro from tumor cells cultured in the presence of GIP. The in vitro secretory response to GIP was higher for the adrenal androgen DHEA, compared with cortisol. The expression of the GIP receptor in tumor cells, but not in the adjacent normal adrenal, was demonstrated by RT-PCR), using specific oligonucleotide probes for this receptor. In summary, we describe a patient with a GIP-expressive cortisol and androgen oversecreting adrenocortical nodule with the unusual presentation of hirsutism and not the typical clinical signs of Cushing's syndrome. It is of note that food intake in this patient provoked a substantial increase in both adrenal androgen and cortisol levels that, together with the histological appearance of this nodule, was compatible with a zona reticularis-derived tumor. Thus, aberrant expression of the GIP receptor does not exclusively involve cells of a zona fasciculata phenotype, as previously reported, but may also occur in other types of differentiated adrenocortical cells.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Fibroblast responses to exogenous and autocrine growth factors relevant to tissue repair. The effect of aging.

The aging process is often associated with impaired wound healing, but the cellular and molecular mechanisms implicated are not completely understood. Accordingly, we have investigated the response of human fibroblasts from donors of various ages to platelet-derived and autocrine growth factors, in terms of mitogenicity as well as extracellular matrix synthesis and degradation. Our data indicate that fibroblast responses persist during aging, suggesting the involvement of systemic factors in the regulation of the healing process. In this context, we have found that neutral endopeptidase-24.11, a metalloproteinase controlling the action of neuroendocrine peptides and also of immunocyte chemotaxis, is overexpressed during aging. Finally, the connection between these data and those from in vitro aging studies is discussed.

Synthesis and biological evaluation of novel daunorubicin-estrogen conjugates.

The synthesis of two novel daunorubicin-estrogen conjugates with a steroidal and a non-steroidal ligand was undertaken in an attempt to target the cytotoxicity of anthracycline to estrogen-receptor positive cells. These conjugates (3 and 4), in contrast to their corresponding ligands, displayed weak binding affinities of 0.079 and 0.851 for the estrogen receptor. Conjugate 3 was consistently more cytotoxic than 4, which however showed some selectivity to estrogen receptor positive cell lines.

Isolation and characterization of a novel bladder cancer cell line: inhibition by epidermal growth factor.

A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells' proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.

Characterization and in vitro estrogenicity of orthodontic adhesive particulates produced by simulated debonding.

OBJECTIVE: To investigate the structure and composition of ground orthodontic adhesive particulates produced under simulated clinical conditions and assess their estrogenic action in vitro. MATERIALS AND METHODS: A chemically cured and a light-cured adhesive were included in the study. Specimens were prepared by simulating bonding procedures, covering the bracket base surface with cellulose films to detach the full set material. The adhesives prepared under this method were grounded in glass chambers with an 8-fluted tungsten carbide on a high-speed handpiece; a new bur and different chamber was used for each adhesive sample and grindings were performed on different days to avoid contamination of the room. The adhesive particulates produced were subjected to FT-IR spectroscopy for the molecular characterization of particles; scanning electron microscopy for the morphologic condition and structure; and X-ray microanalysis for the elemental composition of the particles. Amounts of the ground adhesives were immersed in saline for 1 month at 37 degrees C. Eluents from solution of the two adhesives were added to media of an estrogen-responsive cell line derived from human breast adenocarcinoma (MCF-7), to assess the estrogenicity. Positive (estradiol and bisphenol-A) and negative (saline) controls were used; all assays were repeated four times and the results were averaged. Estrogenicity data were analyzed with one-way ANOVA and the Tukey test at the .05 level of significance. RESULTS: The study of the composition of particles revealed compounds related to monomers with no major differences noted. Significant structural alterations were observed between the materials studied, with the chemically cured adhesive having larger particles. The ground samples contained Si, Na and Al apparently deriving from fillers, whereas large Ba fillers were identified only in the chemically cured group, whereas no distinct molecular variation was noted between the set material and its corresponding particulate form. Both chemically cured and light-cured adhesives exhibited an estrogenic action through induction of the proliferation rate of MCF-7 cells (160% and 128%, respectively, compared to control). SIGNIFICANCE: Apart from the potentially hazardous action of adhesive particulate aerosol produced by grinding, composite resin particulates may act as endocrinological disruptors.

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes.

Lentinula edodes, known as "shiitake" is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC(50) values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI(comit)/SI(mit) ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.

In vitro studies on ultrasmall superparamagnetic iron oxide nanoparticles coated with gummic acid for T2 MRI contrast agent.

Ultrasmall superparamagnetic iron oxide nanoparticles coated with gummic acid have been investigated as possible constituents of aqueous ferrofluids for biomedical applications and especially for MRI contrast agent. The structural characteristics and the size of the nanoparticles have been analyzed as well as the magnetic properties. In order to evaluate any possible capabilities as a contrast agent, the relaxation time, T2, of hydrogen protons in the colloidal solutions of nanoparticles have been measured in order to gain information on the relaxation behavior compared to other MRI contrast agents. The in vitro cytotoxicity of the obtained magnetic nanoparticles of iron oxide coated with gummic acid was investigated by two separate methods (MTT and FACS analysis) and by using three different normal and transformed cell lines. Our results showed that the synthesized nanoparticles had no toxic effect on any of the cell lines used.

Evaluation of the anti-inflammatory and cytotoxic activities of naphthazarine derivatives from Onosma leptantha.

The root extracts of Onosma leptanhtha were evaluated for their anti-iflammatory and cytotoxic activities. The cyclohexane extract, which appeared as the most active in both assays, has been further subjected to bioassay-directed fractionation to afford the naphthazarine derivatives: beta,beta-dimethylacrylshikonin (1), isovalerylshikonin (2) and acetylshikonin (3). The evaluation of the anti-inflammatory activity was performed on carrageenan-induced rat paw edema test. All the tested compounds proved to be active, while compound 3 showed the best anti-inflammatory effect. In addition, the cytotoxic activity of the extracts and isolated compounds, was also assayed against L1210 murine lymphoblastic leukemia cell line, and human fibrosarcoma HT-1080 cells. Compound 1 exhibited remarkable cytotoxic activity (390 nM for L1210 cells), which is superior to that of shikonin, which was used as control.

Autocrine growth regulation in fetal and adult human fibroblasts.

Medium conditioned (CM) by human fetal fibroblasts stimulates proliferation in sparse cultures. This effect is inhibited by suramin and staurosporine, indicating the presence of autocrine growth factors in CM. On the contrary, CM inhibits DNA synthesis in confluent cultures, suggesting a regulatory role for the secreted factors. The growth regulatory profile of CM persists during in vitro ageing. However, it changes dramatically during the fetal-to-adult transition, as adult human fibroblasts are stimulated by CM, regardless of the culture density. These effects are similar to those that TGF-beta is known to have on fetal and adult human fibroblasts. Indeed TGF-beta is present in media conditioned by human fibroblasts, but CM-activity cannot be ascribed solely to this factor. Fibroblasts originating from different tissues exhibit the same autocrine regulatory features, suggesting the general character of this mechanism.

Hydrolyzable tannins, the active constituents of three Greek Cytinus taxa against several tumor cell lines.

Hydrolyzable tannins were found to be the active cytotoxic constituents of three Greek Cytinus taxa: Cytinus ruber, Cytinus hypocistis subsp. hypocistis and Cytinus hypocistis subsp. orientalis. The cytotoxic activity was evaluated against a broad spectrum of cancer cell lines. The structure of the active compounds was investigated with NMR and electrospray-MS/MS techniques.