Extracellular ATP Regulates CD73 and ABCC6 Expression in HepG2 Cells.

The ATP-binding cassette sub-family C member 6 transporter (ABCC6) is an ATP dependent transporter mainly found in the basolateral plasma membrane of hepatic and kidney cells. Mutations in ABCC6 gene were associated to the Pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by a progressive ectopic calcification of elastic fibers in dermal, ocular, and vascular tissues. It is reported that the over-expression of ABCC6 in HEK293 cells results in the cellular efflux of ATP and other nucleoside triphosphates, which in turn are rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since PPi is an inhibitor of mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the ectopic mineralization, a typical feature of PXE. In the extracellular environment, ATP is converted, not only into pyrophosphate, but also into AMP by an ectonucleosidase, which in turn is transformed into adenosine and phosphate. ABCC6 protein is thus involved in the production of extracellular adenosine and therefore it could have a role in the activation of the purinergic system. In the liver, purinergic signaling has been shown to regulate key basic cellular functions. Our previous studies showed that in ABCC6 knockdown HepG2 cells the expression of some genes, related with the calcification processes, is dysregulated. In this study, experiments have been carried out in order to verify if ABCC6, besides supplying the pyrophosphate required to prevent the mineralization of soft tissues, also plays a role in the activation of the purinergic system. For this purpose, the transport activity of ABCC6 was blocked with Probenecid and the expression of ABCC6 and NT5E was analyzed with real time PCR and western blotting. The results of this study showed that both proteins are downregulated in the presence of Probenecid and upregulated in the presence of adenosine or ATP.

Cytotoxic Activity of Origanum Vulgare L. on Hepatocellular Carcinoma cell Line HepG2 and Evaluation of its Biological Activity.

The potential of plant essential oils (EOs) in anticancer treatment has recently received many research efforts to overcome the development of multidrug resistance and their negative side effects. The aims of the current research are to study (i) the cytotoxic effect of the crude EO extracted from Origanum vulgare subsp hirtum and its main constituents (carvacrol, thymol, citral and limonene) on hepatocarcinoma HepG2 and healthy human renal cells HEK293; (ii) the antibacterial and phytotoxic activities of the above EO and its main constituents. Results showed that cell viability percentage of treated HepG2 by EO and its main constituents was significantly decreased when compared to untreated cells. The calculated inhibition concentration (IC50) values for HepG2 were lower than healthy renal cells, indicating the sort of selectivity of the studied substances. Citral is not potentially recommended as an anticancer therapeutic agent, since there are no significant differences between IC50 values against both tested cell lines. Results showed also that oregano EO and its main constituents have a significant antibacterial activity and a moderate phytotoxic effect. The current research verified that oregano EO and its main constituents could be potentially utilized as anticancer therapeutic agents.

Molecular and supramolecular structural studies on human tropoelastin sequences.

One of the unusual properties of elastin is its ability to coacervate, which has been proposed to play an important role in the alignment of monomeric elastin for cross-linking into the polymeric elastin matrix. The temperature at which this transition takes place depends on several factors including protein concentration, ionic strength, and pH. Previously, polypeptide sequences encoded by different exons of the human tropoelastin gene have been analyzed for their ability to coacervate and to self-assemble. Few of them were indeed able to coacervate and only one, that encoded by exon 30 (EX30), gave amyloid fibers. In this article, we report on two chemically synthesized peptides-a decapeptide and an octadecapeptide-whose sequences are contained in the longer EX30 peptide and on a polypeptide (EX1-7) of 125 amino-acid residues corresponding to the sequence coded by the exons 1-7 and on a polypeptide (EX2-7) of 99 amino-acid residues encoded by exons 2-7 of human tropoelastin obtained by recombinant DNA techniques. Molecular and supramolecular structural characterization of these peptides showed that a minimum sequence of approximately 20 amino acids is needed to form amyloid fibers in the exon 30-derived peptides. The N-terminal region of mature tropoelastin (EX2-7) gives rise to a coacervate and forms elastinlike fibers, whereas the polypeptide sequence containing the signal peptide (EX1-7) forms mainly amyloid fibers. Circular dichroism spectra show that beta-structure is ubiquitous in all the sequences studied, suggesting that the presence of a beta-structure is a necessary, although not sufficient, requirement for the appearance of amyloid fibers.