Expression levels of the metalloproteinase ADAM8 critically regulate proliferation, migration and malignant signalling events in hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of ß1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro.

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1ß, TNF-α, IFN-γ, and TGF-ß. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

A comprehensive review on endocrine toxicity of gaseous components and particulate matter in smog.

Smog is a form of extreme air pollution which comprises of gases such as ozone, sulfur dioxide, nitrogen and carbon oxides, and solid particles including particulate matter (PM2.5 and PM10). Different types of smog include acidic, photochemical, and Polish. Smog and its constituents are hazardaous to human, animals, and plants. Smog leads to plethora of morbidities such as cancer, endocrine disruption, and respiratory and cardiovascular disorders. Smog components alter the activity of various hormones including thyroid, pituitary, gonads and adrenal hormones by altering regulatory genes, oxidation status and the hypothalamus-pituitary axis. Furthermore, these toxicants are responsible for the development of metabolic disorders, teratogenicity, insulin resistance, infertility, and carcinogenicity of endocrine glands. Avoiding fossil fuel, using renewable sources of energy, and limiting gaseous discharge from industries can be helpful to avoid endocrine disruption and other toxicities of smog. This review focuses on the toxic implications of smog and its constituents on endocrine system, their toxicodynamics and preventive measures to avoid hazardous health effects.

Soluble laticifer proteins from Calotropis procera as an effective candidates for antimicrobial therapeutics.

Calotropis procera is a latex-producing plant with plenty of pharmacologically active compounds. The principal motivation behind this study was to separate and characterize laticifer proteins to check their antimicrobial potential. Laticifer proteins were separated by gel filtration chromatography (GFC) and investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE assay detected proteins of molecular weights of 10 to 30 kDa but most of them were in the range of 25 to 30 kDa. The soluble laticifer proteins (SLPs) were tested against Gram-positive bacteria i.e., Streptococcus pyogenes and Staphylococcus aureus whereas Escherichia coli and Pseudomonas aeruginosa were tested as Gram-negative bacteria, we determined a profound anti-bacterial activity of these proteins. In addition, SLPs were also investigated against Candida albicans via the agar disc diffusion method which also showed significant anti-fungal activity. SLP exhibited antibacterial activity against P. aeruginosa, E. coli, and S. aureus with a minimum inhibitory concentration (MIC) of 2.5 mg/mL for each, while MIC was found at 0.625 mg/mL for S. pyogenes and 1.25 mg/mL for C. albicans. Moreover, enzymatic activity evaluation of SLP showed the proteolytic nature of these proteins, and this proteolytic activity was greatly enhanced after reduction which might be due to the presence of cysteine residues in the protein structure. The activity of the SLPs obtained from the latex of C. procera can be associated with the involvement of enzymes either proteases or, protease inhibitors and/or peptides.

Functional Studies of Cytogenotoxic Potential of Laticifer Proteins of Calotropis procera against Viral Disease.

Plant products are widely used for health and disease management. However, besides their therapeutic effects, some plants also have potential toxic activity. Calotropis procera is a well-known laticifer plant having pharmacologically active proteins playing a therapeutically significant role in curing diseases like inflammatory disorders, respiratory diseases, infectious diseases, and cancers. The present study was aimed to investigate the antiviral activity and toxicity profile of the soluble laticifer proteins (SLPs) obtained from C. procera. Different doses of rubber free latex (RFL) and soluble laticifer protein (ranging from 0.019 to 10 mg/mL) were tested. RFL and SLPs were found to be active in a dose-dependent manner against NDV (Newcastle disease virus) in chicken embryos. Embryotoxicity, cytotoxicity, genotoxicity, and mutagenicity of RFL and SLP were examined on chicken embryos, BHK-21 cell lines, human lymphocytes, and Salmonella typhimurium, respectively. It was revealed that RFL and SLP possess embryotoxic, cytotoxic, genotoxic, and mutagenic activity at higher doses (i.e., 1.25-10 mg/mL), while low doses were found to be safe. It was also observed that SLP showed a rather safer profile as compared to RFL. This might be due to the filtration of some small molecular weight compounds at the time of purification of SLPs through a dialyzing membrane. We suggest that SLPs could be used therapeutically against viral disorders but the dose should be critically monitored.

Elevated expression of the metalloproteinase ADAM8 associates with vascular diseases in mice and humans.

BACKGROUND AND AIMS: Members of the family of a disintegrin and metalloproteinases (ADAMs) and their substrates have been previously shown to modulate the inflammatory response in cardiac diseases, but studies investigating the relevance of ADAM8 are still rare. Our aim is to provide evidence for the inflammatory dysregulation of ADAM8 in vascular diseases and its association with disease severity. METHODS: Western-type diet fed Apoe-/- and Ldlr-/- mice and artery ligation served as murine model for atherosclerosis and myocardial infarction, respectively. Human bypass grafts were used to study the association with coronary artery disease (CAD), with the simplified acute physiology score II (SAPS II) as a measure of postoperative organ dysfunction. Human primary vascular and blood cells were analyzed under basal and inflammatory conditions. mRNA levels were determined by RT-qPCR, ADAM8 protein levels by ELISA, immunohistochemistry or flow cytometry. RESULTS: ADAM8/ADAM8 expression is associated with atherosclerosis and CAD such as myocardial infarction in both mice and humans, especially in endothelial cells and leukocytes. We observed a strong in vivo and in vitro correlation of ADAM8 with the vascular disease markers VCAM-1, ICAM-1, TNF, IL-6, and CCL-2. Serum analysis revealed a significant elevation of soluble ADAM8 serum levels correlating with soluble CXCL16 levels and SAPS II. CONCLUSIONS: We demonstrate a general association of ADAM8 with cardiovascular diseases in mice and humans predominantly acting in endothelial cells and leukocytes. The correlation with postoperative organ dysfunctions in CAD patients highlights the value of further studies investigating the specific function of ADAM8 in cardiovascular diseases.