Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 µM) and DC-TA-46 (0.5 µM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc.

Chitosan-coated PLGA nanoparticles: a sustained drug release strategy for cell cultures.

A recently patented one-step methodology was used for the formulation of chitosan (CS) coated polylactic-co-glycolic acid (PLGA) nanoparticles containing dexamethasone (DXM) as a model drug. SEM investigations showed that nanoparticles (NPs) were spherical in shape with smooth surface. CS coating switched NPs ζ-potential from negative to positive, without modifying particle size distribution. Moreover, CS coating allowed a significant modulation of in vitro drug release, providing a sustained drug delivery in cultured cells. The uptake of fluorescent CS-coated PLGA NPs by hepatocytes (C3A) and fibroblasts (3T6) as well as the fate of internalized NPs were investigated by confocal microscopy. 3T6 and C3A cells were treated with DXM-loaded NPs and experiments were addressed to analyze the specific cell response to DXM, in order to evaluate its functional efficiency in comparison with conventional addition to culture medium. CS-coating of DXM loaded PLGA NPs allowed their uptake by cultured cells without inducing cytotoxicity.

Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation.

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands.

Proteolytic activity of bovine lactoferrin.

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.