The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence.

OBJECTIVE: Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. DESIGN: This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor ß1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated ß galactosidase activity (SA-ß-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. RESULTS: Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-ß-gal high, p = 0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. CONCLUSIONS: Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-ß-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

H2O2 promotes the aging process of melanogenesis through modulation of MITF and Nrf2.

The purpose of this study is to investigate the effect of H2O2 on the aging of melanogenesis in human melanocytes. The staining of SA-ß-galactosidase, an aging marker, was remarkably increased in the cells aged with H2O2 at 62.5 µM or more compared with young cells. The intracellular H2O2 level and melanin synthesis were also reduced in both H2O2-treated cells and senescent cells compared with young cells in DCFH-DA assay. Both the senescent cells and the H2O2-treated cells showed higher expression level of Catalase than young cells in western blot and immunofluorescence staining. Furthermore, the expression levels of TRP-1, TRP-2 and p300 were reduced in both senescent cells and the H2O2-treated cells, but that of SIRT-1 was inverted compared with young cells. In addition, H2O2 reduced the expression level of MITF but increased that of Nrf2 in nucleus. Those results indicate that the expression levels of antioxidant enzymes in senescent cells and H2O2-treated cell are upregulated, but the expression levels of proteins involved in melanin synthesis are downregulated. Above findings suggest that H2O2 could play a key role in the aging process of melanogenesis through modulation of MITF and Nrf2.

Ultrahigh Enzyme Activity Assembled in Layered Double Hydroxides via Mg(2+)-Allosteric Effector.

It is well-known that some metal ions could be allosteric effectors of allosteric enzymes to activate/inhibit the catalytic activities of enzymes. In nanobiocatalytic systems constructed based on the positive metal ion-induced allosteric effect, the incorporated enzymes will be activated and thus exhibit excellent catalytic performance. Herein, we present an environmentally friendly strategy to construct a novel allosteric effect-based ß-galactosidase/Mg-Al layered double hydroxide (ß-gal/Mg-Al-LDH) nanobiocatalytic system via the delamination-reconstruction method. The intercalated ß-gal in the LDH galleries changes its conformation significantly due to the Mg(2+)-induced allosteric interactions and other weak interactions, which causes the activation of enzymatic activity. The ß-gal/Mg-Al-LDH nanobiocatalytic system shows much higher catalytic activity and affinity toward its substrate and about 30 times higher catalytic reaction velocity than the free ß-gal, which suggests that Mg(2+)-induced allosteric effect plays a vital role in the improvement of enzymatic performance.

Glucocorticoids induce senescence in primary human tenocytes by inhibition of sirtuin 1 and activation of the p53/p21 pathway: in vivo and in vitro evidence.

UNLABELLED: Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. OBJECTIVE: To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. METHODS: Features of senescence (ß-galactosidase activity at pH 6 (SA-ß-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. RESULTS: Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-ßgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. CONCLUSION: As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue.

Anti-estrogenic activity of mansonone G and mansorin A derivatives.

CONTEXT: Mansonone G and mansorin A are major bioactive constituents from Mansonia gagei Drumm (Sterculiaceae) wood, and their mild anti-estrogenic activity was reported previously by the authors. OBJECTIVE: In order to increase the potency of their anti-estrogenic effect and to clarify their binding way to estrogen receptor on a molecular level, several derivatives of both compounds will be prepared and a docking study of the original compounds and their derivatives on estrogen receptor alpha (ERα) was carried out. MATERIALS AND METHODS: The original compounds were isolated from the heartwood of M. gagei. Nine alkyl derivatives were prepared by acetylation, methylation, or adding a basic side chain to the free hydroxyl group of both compounds. The estrogenic/anti-estrogenic activities of the derivatives compared to the original compounds were carried out using ERα competitive binding screen and yeast two-hybrid assay expressing ERα and ERß using concentrations ranging from 10 to 100 µM. RESULTS: Acetyl mansonone G showed a 10-fold increase in its binding ability to ERα compared to mansonone G with an IC50 630 µM. Similarly, methyl mansonone G and acetyl mansonone G showed 50% and 35% inhibition of 17ß-estradiol-induced ß-galactosidase activity at 10 µM in the yeast expressing ERα, and 42% and 30%, respectively, at 10 µM in the yeast expressing ERß. Virtual docking of acetyl mansonone G to ERα showed that it binds, with its acetyl oxygen, in a similar way to the 17ß-OH of estradiol. DISCUSSION AND CONCLUSION: The phenolic hydroxyl group in mansonones and mansorins was not essential for binding to estrogen receptors. In addition, acetyl mansonone G could represent a promising starting material for the synthesis of anti-estrogenic agents.

Synthesis and characterization of a cell-permeable bimodal contrast agent targeting ß-galactosidase.

Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular ß-galactosidase (ß-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell-penetrating peptide D-Tat(49-57) and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl(3). Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8±0.6 mM(-1)s(-1), 123 MHz, ∼21°C) relative to the commercial Gd-DOTA (4.0±0.12 mM(-1)s(-1), 123MHz, ∼21 °C). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the ß-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in ß-gal expressing C6/LacZ and enzyme deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed non-specific background signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities.

Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polyethylenimine polyplex.

The potential of gene therapy to treat cancer is currently limited by the low expression of therapeutic genes in the tumors. Because amino acids are known to have excellent properties in cell penetration and gene expression regulation, we investigated if the conjugation of arginine (Arg), lysine (Lys) and leucine (Leu) onto the surface of the gene delivery system polyethylenimine (PEI) could lead to an improved gene expression in tumors. The intravenous administration of Arg-, Lys- and Leu-bearing PEI polyplexes led to a significant increase of gene expression in the tumor, with a ß-galactosidase expression amount at least threefold higher than that obtained after treatment with unmodified PEI polyplex. The three amino acid-bearing PEI polyplexes led to similar levels of gene expression in the tumor. The treatments were well tolerated by the mice. Arg-, Lys- and Leu-bearing PEI polyplexes are therefore highly promising gene delivery systems for cancer therapy.

Quillaja bark saponin effects on Kluyveromyces lactis ß-galactosidase activity and structure.

Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.

Kynurenine inhibits autophagy and promotes senescence in aged bone marrow mesenchymal stem cells through the aryl hydrocarbon receptor pathway.

Osteoporosis is an age-related deterioration in bone health that is, at least in part, a stem cell disease. The different mechanisms and signaling pathways that change with age and contribute to the development of osteoporosis are being identified. One key upstream mechanism that appears to target a number of osteogenic pathways with age is kynurenine, a tryptophan metabolite and an endogenous Aryl hydrocarbon receptor (AhR) agonist. The AhR signaling pathway has been reported to promote aging phenotypes across species and in different tissues. We previously found that kynurenine accumulates with age in the plasma and various tissues including bone and induces bone loss and osteoporosis in mice. Bone marrow mesenchymal stem cells (BMSCs) are responsible for osteogenesis, adipogenesis, and overall bone regeneration. In the present study, we investigated the effect of kynurenine on BMSCs, with a focus on autophagy and senescence as two cellular processes that control BMSCs proliferation and differentiation capacity. We found that physiological levels of kynurenine (10 and 100 µM) disrupted autophagic flux as evidenced by the reduction of LC3B-II, and autophagolysosomal production, as well as a significant increase of p62 protein level. Additionally, kynurenine also induced a senescent phenotype in BMSCs as shown by the increased expression of several senescence markers including senescence associated ß-galactosidase in BMSCs. Additionally, western blotting reveals that levels of p21, another marker of senescence, also increased in kynurenine-treated BMSCs, while senescent-associated aggregation of nuclear H3K9me3 also showed a significant increase in response to kynurenine treatment. To validate that these effects are in fact due to AhR signaling pathway, we utilized two known AhR antagonists: CH-223191, and 3',4'-dimethoxyflavone to try to block AhR signaling and rescue kynurenine /AhR mediated effects. Indeed, AhR inhibition restored kynurenine-suppressed autophagy levels as shown by levels of LC3B-II, p62 and autophagolysosomal formation demonstrating a rescuing of autophagic flux. Furthermore, inhibition of AhR signaling prevented the kynurenine-induced increase in senescence associated ß-galactosidase and p21 levels, as well as blocking aggregation of nuclear H3K9me3. Taken together, our results suggest that kynurenine inhibits autophagy and induces senescence in BMSCs via AhR signaling, and that this may be a novel target to prevent or reduce age-associated bone loss and osteoporosis.

6-Bromoindirubin-3'-oxime (6BIO) prevents myocardium from aging by inducing autophagy.

6-Bromoindirubin-3'-oxime (6BIO) is a novel small molecule that exerts positive effects on several age-related alterations. However, the anti-aging effects of 6BIO on the aging heart remain unknown. Herein, we aim to investigate the effects of 6BIO on the myocardium and its underlying mechanism in vivo and vitro. Following 6BIO treatment, an increased p53 contents, a reduced p16 and ß-gal levels, and attenuation of cardiac fibrosis were observed, suggesting 6BIO retarded aging of cardiomyocytes. As observed, 6BIO reduced p62 contents, elevated the levels of Beclin-1 and the ratio of LC3II/I, indicating the induction of autophagy, while the reduction of the accumulation of ROS indicated 6BIO alleviated oxidative stress. In addition, 6BIO treatment inhibited both GSK3ß signaling and mTOR signaling. 6BIO might be a promising agent for preventing myocardium from aging.

Expression of c-fos and AP-1 activity in senescent human fibroblasts is not sufficient for DNA synthesis.

Human fibroblasts have a limited replicative life span when maintained in culture after which they become unresponsive to treatment with mitogens, a phenomenon most commonly called senescence. Experiments indicating that serum does not induce expression of the c-fos proto-oncogene in senescent fibroblasts raised the issue of a potential central role for c-fos in the phenotype of sustained growth arrest. This was directly tested by microinjection of oncogenic c-Ha-ras protein into senescent fibroblasts. While ras injection was found to induce marked nuclear c-fos expression and functional AP-1 transcription activity, this did not lead to DNA synthesis. These results suggest that the senescence phenotype cannot be solely attributed to the absence of c-fos expression and that the proliferative block in these cells is either independent of AP-1 transcriptional activity, downstream of it, or involves multiple molecular mechanisms.

Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites.

OBJECTIVES - Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by beta-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without beta-estradiol in fibrocartilaginous cells. MATERIAL AND METHODS - Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the beta-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml beta-estradiol or both hormones, and lysates assayed for CAT and beta-galactosidase activity. RESULTS - Cells transfected with the -1200/-42 or -139/-42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of beta-estradiol, respectively. Relaxin failed to induce CAT in the absence of the -137/-69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or beta-estradiol plus relaxin. CONCLUSION - These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.

Methods and reagents. Reducing background colonies with positive selection vectors.

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses the pros and cons of eliminating unwanted background colonies by using the positive selection vector pZErO. For details on how to partake in the newsgroup, see the accompanying box.

Inhibition of Ca2+-signal-dependent growth regulation by radicicol in budding yeast.

Compelled activation of Ca(2+) signaling by exposure of zds1Delta strain Saccharomyces cerevisiae cells to external CaCl(2) leads to characteristic physiological consequences such as growth inhibition in the G(2) phase and polarized bud growth. Screening of microbial metabolites for activity alleviating the deleterious physiological effects of external CaCl(2) identified the Hsp90 inhibitor radicicol as an inhibitor of Ca(2+)-signal-dependent cell-cycle regulation in yeast. Radicicol alleviated analogous physiological effects due to the expression of a constitutively active form of calcineurin or overexpression of Swe1, the negative regulatory kinase of the Cdc28-Clb complex. Western blot analysis indicated that radicicol inhibited Ca(2+)-induced accumulation of Swe1 and Cln2.

Therapeutic efficacy of Kangen-karyu against H2O2-induced premature senescence.

The anti-aging potential of Kangen-karyu extract was investigated using the mechanisms of the cellular aging model of stress-induced premature senescence (SIPS) in TIG-1 human fibroblasts. SIPS was induced by a sublethal dose of H2O2 and chronic oxidative stress with repeat treatment of low-dose H2O2. Reactive oxygen species generation, lipid peroxidation, and senescence-associated beta-galactosidase activity were elevated in TIG-1 cells under SIPS induced by H2O2. However, Kangen-karyu extract led to significant declines in these parameters, suggesting its role in ameliorating oxidative stress-related aging. It was also observed that SIPS due to H2O2 treatment led to the loss of cell viability, whereas Kangen-karyu extract improved cell viability by attenuating H2O2-induced oxidative damage. TIG-1 cells under the condition of SIPS caused by sublethal and chronic low doses of H2O2 showed nuclear factor-kappaB (NF-kappaB) translocation to the nucleus from the cytosol, while Kangen-karyu extract inhibited NF-kappaB nuclear translocation, implying that Kangen-karyu extract could exert an anti-aging effect through NF-kappaB modulation. In addition, treatment with Kangen-karyu extract under H2O2-induced chronic oxidative stress normalized the cell cycle by reducing the number of cells in the G0/G1 phase and elevating the proportion of those in the S phase, indicating the role of Kangen-karyu extract in cell cycle regulation. On the other hand, Kangen-karyu extract did not exert such an effect on cell cycle regulation under acute oxidative stress induced by sublethal H2O2. Furthermore, treatment with Kangen-karyu extract prolonged the lifespan of TIG-1 cells under SIPS. The present study suggests that Kangen-karyu might play a therapeutic role against the aging process caused by oxidative stress.

Estradiol-antagonistic activity of phenolic compounds from leguminous plants.

Natural flavonoids are currently receiving much attention because of their estrogenic and antiestrogenic properties. Six isoflavones (isoprunetin, isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside, genistein, genistein 7-O-beta-D-glucopyranoside, daidzein), four flavones (luteolin, luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C), isolated from Genista morisii and G. ephedroides (two Leguminosae plants of the Mediterranean area) together with two structurally related pterocarpans, bitucarpin A and erybraedyn C, isolated from Bituminaria bituminosa (Leguminosae), were tested for the antagonist activity by a yeast based estrogen receptor assay (Saccharomyces cerevisiae RMY326 ER-ERE). Most compounds inhibited the estradiol-induced transcriptional activity in a concentration dependent manner. In particular, for the flavone luteolin 77% inhibition of the induced beta-galactosidase activity was observed. Interestingly, licoflavone C exhibited a dose-dependent antagonistic activity at concentrations up to 10(-4) M, but stimulated beta-galactosidase expression at higher concentrations resulting in a U-shaped-like dose-response curve.

Apoptosis changes and SA-beta galactosidase expression in stress-induced premature senescence (SIPS) model of human skin fibroblasts.

Stress-induced premature senescence (SIPS) model is in vitro model of cellular aging. In this study, apoptosis was evaluated in SIPS model and in replicative senescent fibroblasts. We also compared the activity of senescence-associated beta-galactosidase (SA-beta gal) as a biomarker of cellular aging. Our results suggested that SIPS model and senescent fibroblasts might share similar mechanism of aging and apoptosis pathway.

Enzyme and Cancer Cell Selectivity of Nanoparticles: Inhibition of 3D Metastatic Phenotype and Experimental Melanoma by Zinc Oxide.

Biomedical applications for metal and metal oxide nanoparticles are rapidly increasing. Here their functional impact on two well-characterized model enzymes, Luciferase (Luc) or ß-galactosidase (ß-Gal) was quantitatively compared. Nickel oxide nanoparticle (NiO-NP) activated ß-Gal (>400% control) and boron carbide nanoparticle (B4C-NP) inhibited Luc(<10% control), whereas zinc oxide (ZnO-NP) and cobalt oxide (Co3O4-NP) activated ß-Gal to a lesser extent and magnesium oxide (MgO) moderately inhibited both enzymes. Melanoma specific killing was in the order; ZnO > B4C ≥ Cu > MgO > Co3O4 > Fe2O3 > NiO, ZnO-NP inhibiting B16F10 and A375 cells as well as ERK enzyme (>90%) and several other cancer-associated kinases (AKT, CREB, p70S6K). ZnO-NP or nanobelt (NB) serve as photoluminescence (PL) cell labels and inhibit 3-D multi-cellular tumor spheroid (MCTS) growth and were tested in a mouse melanoma model. These results demonstrate nanoparticle and enzyme specific biochemical activity and suggest their utility as new tools to explore the important model metastatic foci 3-D environment and their chemotherapeutic potential.

Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity.

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a gamma-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a beta-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in gamma-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.