M. Antonia Lizarbe

Bioactive sol-gel glasses with and without a hydroxycarbonate apatite layer as substrates for osteoblast cell adhesion and proliferation.

The biocompatibility of three sol-gel bioactive glasses with SiO(2) as the main constituent (75, 72.5 and 70 mol%), identical CaO content (25mol%), and without or with P(2)O(5) as third constituent (0, 2.5 and 5 mol%), have been analyzed (S75, S72.5P2.5, and S70P5 glasses). These studies were performed on both untreated glasses and on glasses coated with a hydroxycarbonate apatite (HCA) layer formed in vitro by soaking 7d in an acellular simulated body fluid. Cell attachment, spreading and proliferation were studied using neonatal rat calvaria osteoblasts. Cells attach to the three untreated glasses but show a higher efficiency on that with the higher phosphate content (S70P5). The formation of the HCA layer significantly enhances this process (1.7-fold). In all cases, attachment is followed by cell spreading on the surface of the materials, adopting the cells a flattened morphology and showing diverse anchoring cell projections. Mitotic activity has been detected on osteoblasts growing on the sol-gel glasses, being this process 2-4-fold higher when the apatite-like layer is already formed. Taking into account the results herein presented, these bioactive glasses can be considered biocompatible. In addition, their biocompatibility is greatly enhanced after induction of the formation of an HCA layer.

Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5

Butyrate induces differentiation and alters cell proliferation in intestinal–epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium‐dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose‐free inosine‐containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase‐IV (DPP‐IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS‐TC2, BCS‐TC2.2, HT‐29, and Caco‐2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco‐2 cells. Inosine‐differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco‐2 cells. AnxA2 down‐regulation is not due to proteasome activation and seems to be related to the butyrate‐induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth‐state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell‐to‐cell contacts. Butyrate induces changes in subcellular localization towards a vesicle‐associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up‐regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up‐regulation of AnxA1 could contribute to the reported anti‐inflammatory effects of butyrate in colon inflammatory diseases.

Midregion Parathyroid Hormone‐Related Protein Inhibits Growth and Invasion In Vitro and Tumorigenesis In Vivo of Human Breast Cancer Cells

Parathyroid hormone‐related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post‐translational processing, and PTHrP(38–94)‐amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38–94)‐amide in a panel of six breast cancer cell lines “in vitro” and in MDA‐MB231 cells “in vivo” specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38–94)‐amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38–94)‐amide into MDA‐MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti‐breast cancer effects.

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.

Calcium-Dependent Conformational Rearrangements and Protein Stability in Chicken Annexin A5

The conformational rearrangements that take place after calcium binding in chicken annexin A5 and a mutant lacking residues 3-10 were analyzed, in parallel with human annexin A5, by circular dichroism (CD), infrared spectroscopy (IR), and differential scanning calorimetry. Human and chicken annexins present a slightly different shape in the far-UV CD and IR spectra, but the main secondary-structure features are quite similar (70-80% alpha-helix). However, thermal stability of human annexin is significantly lower than its chicken counterpart (approximately 8 degrees C) and equivalent to the chicken N-terminally truncated form. The N-terminal extension contributes greatly to stabilize the overall annexin A5 structure. Infrared spectroscopy reveals the presence of two populations of alpha-helical structures, the canonical alpha-helices (approximately 1650 cm(-1)) and another, at a lower wavenumber (approximately 1634 cm(-1)), probably arising from helix-helix interactions or solvated alpha-helices. Saturation with calcium induces: alterations in the environment of the unique tryptophan residue of the recombinant proteins, as detected by near-UV CD spectroscopy; more compact tertiary structures that could account for the higher thermal stabilities (8 to 12 degrees C), this effect being higher for human annexin; and an increase in canonical alpha-helix percentage by a rearrangement of nonperiodical structure or 3(10) helices together with a variation in helix-helix interactions, as shown by amide I curve-fitting and 2D-IR.

Kinetics of in vivo degradation of sepiolite-collagen complexes: Effect of glutaraldehyde treatment

A kinetic study of the in vivo degradation of sepiolite-collagen complexes after subcutaneous implantation in rats was performed. A foreign-body reaction was the characteristic host tissue response against the implants. The resorption of the implanted materials was analyzed by measuring both the weight and the collagen persistences. This last was measured by using (14C)acetylated collagen, which was revealed to be not modified upon radioactive labeling, in terms of its ability to form a complex with sepiolite. The persistence of the implants is controllable by treatment of the collagen component with glutaraldehyde. Thus, for 1% glutaraldehyde-treated collagen complexes, 100% of persistence was observed after several months of implantation, this value decreasing to a few days for nontreated collagen samples. The collagen-sepiolite complex showed a low immunological response, almost null for 1% glutaraldehyde-treated collagen complexes, which was analyzed by measuring anti-collagen antibodies levels. Based on the performed studies, sepiolite-collagen complexes can be considered a resorbable material.

Acquisition of Resistance to Butyrate Enhances Survival after Stress and Induces Malignancy of Human Colon Carcinoma Cells

Acquired resistance to apoptosis by tumor cells remains a major obstacle for cancer treatment, and hence the analysis of resistance to apoptosis constitutes a major goal in the development of antitumoral drugs. We have established a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) from nontumorigenic BCS-TC2 cells to analyze whether the acquisition of such phenotype confers resistance to apoptosis and stress. Although BCS-TC2.BR2 cells exhibited a more differentiated phenotype than the parental BCS-TC2 cells, higher butyrate concentrations remained capable of additionally enhancing their differentiation without inducing apoptosis. Survival rates of BCS-TC2.BR2 cells after glucose deprivation and heat shock were higher than those of parental cells, revealing a stress-resistant phenotype. These findings were accompanied by key differences between parental and butyrate-resistant cells in gene expression profiles and the acquisition of in vivo tumorigenicity. In conclusion, cells gaining resistance to an endogenous physiological modulator of growth, differentiation, and apoptosis concurrently acquired resistance to other agents that influence cell survival.

Changes in the expression of annexin A5 gene during in vitro chondrocyte differentiation: Influence of cell attachment

Several lines of evidence indicate that annexin A5, a membrane‐associated protein with calcium‐channel activity, plays a key role in cartilage calcification during endochondral ossification. As a major constituent of cartilage matrix vesicles, which are released from microvilli of hypertrophic chondrocytes, it is involved in calcium uptake necessary for the initial stages of cartilage calcification. Little is known, however, concerning transcriptional regulation of the annexin A5 gene during chondrocyte differentiation. Here, we report on changes in annexin A5 expression by measuring mRNA and protein levels during in vitro differentiation of chick sternal chondrocytes to the hypertrophic phenotype. Terminal differentiation of mature sternal chondrocytes was achieved in the presence of sodium ascorbate in high‐density cultures growing either in monolayer or over agarose as cell aggregates. Differentiation of chondrocytes to hypertrophic cells was followed by morphological analysis and by the onset of type X collagen expression. High expression levels of annexin A5 mRNA were detected in chondrocytes freshly isolated from the sterna by enzymatic digestion and subsequently in cells growing in monolayer, but annexin A5 gene transcription was rapidly downregulated when cells were grown in suspension as aggregates over agarose. However, protein levels did not decrease probably due to its low turnover rate. In suspension culture, annexin A5 mRNA reappeared after 3 weeks concomitantly with segregation of the aggregates into single cells and onset of chondrocyte hypertrophy. The downregulation of annexin A5 expression in cells growing as matrix‐rich aggregates was reverted when extracellular matrix components were removed and cells were reseeded onto tissue culture plastic, suggesting that cell spreading, formation of focal contacts and stress fibers stimulated annexin A5 expression in proliferating as well as in hypertrophic chondrocytes. J. Cell. Biochem. 84: 132–142, 2002.

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/ Smad complexes in human colon adenocarcinoma cells

MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.

Effect of Bile Acids on Butyrate-Sensitive and -Resistant Human Colon Adenocarcinoma Cells

Abstract: A controlled balance among cell proliferation, differentiation, and apoptosis is required for the maintenance of gastrointestinal mucosa; these processes are influenced by luminal components, such as butyrate and bile acids. Using butyrate-sensitive (BCS-TC2) and butyrate-resistant (BCS-TC2.BR2) human colon carcinoma cells, we wanted to establish whether colon carcinoma cells that acquire resistance to butyrate-induced apoptosis are also resistant to the cytotoxic effect of certain bile acids, contributing, in this way, to the progression of colon carcinogenesis. The effect of bile acids on BCS-TC2 cell viability is dose and time dependent and highly stereospecific. Quantification of the relative percentage of apoptotic cells and caspase-3 activity reveals that deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) induce apoptosis in BCS-TC2 cells. BCS-TC2.BR2 cells are consistently less sensitive to their cytotoxic effects, requiring concentrations to induce 50% inhibition (IC50) in cell viability of 740 μM and >1 mM for CDCA and DCA, respectively, compared with IC50 values of 310 and 540 μM for BCS-TC2 cells. DCA-treated BCS-TC2.BR2 cells show few apoptotic signs and no caspase-3 activation. On the other hand, CDCA-treated BCS-TC2.BR2 cells show caspase-3 activation and apoptotic features, although to a lower extent than BCS-TC2 cells. Our results, in an in vitro model system, point out that acquisition of butyrate resistance is accompanied by a partial resistance to the cytotoxic effects of bile acids, which may enhance the survival of tumorigenic cells.