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Periodontitis is one of the most common oral diseases worldwide and is caused by a variety of interactions between oral bacteria and the host. Here, pathogens induce inflammatory host responses that cause the secretion of proinflammatory cytokines such as IL-1β, IL-6, and IL-8 by oral epithelial cells. In various systems, it has been shown that inflammation compromises physical barriers, which enables bacteria to invade the tissue. In this study, we investigated the barrier properties of the oral mucosa under physiological and inflamed conditions. For this purpose, we assessed the influence of IL-1β on the transepithelial electrical resistance and in particular on tight junctions in vitro in human stratified squamous epithelium models. Indirect immunofluorescence and western blot analyses were performed to investigate localization and expression of tight junction proteins in primary gingival cells, immortalized gingival cells and native gingiva. Furthermore, the TEER of gingival keratinocytes was assessed. The results showed that IL-1β led to strengthening of the gingival keratinocyte barrier. This was demonstrated by an increase in TEER, the upregulation of TJ proteins, and an increase in the formation of TJ strands. The IL-1β-mediated upregulation of occludin was prevented by the NF-κB inhibitor BAY 11-7085. These observations provide insights into host responses in the early stages of periodontal disease and offer information about TJ formation in human gingival epithelial cells under physiological and inflammatory conditions. Comprehensive knowledge of the physical barrier during inflammation may help in developing strategies to effectively target the inflammatory barrier to improve the bioavailability of drugs for the treatment of periodontitis.

In cutaneous inflammatory diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis, skin-infiltrating T lymphocytes and dendritic cells modulate keratinocyte function via the secretion of pro-inflammatory cytokines. Keratinocytes then produce mediators that recruit and activate immune cells and amplify the inflammatory response. These pathophysiological tissue changes are caused by altered gene expression and the proliferation and maturation of dermal and epidermal cells. We recently demonstrated that the glycosidated phospholipid Ino-C2-PAF down-regulates a plethora of gene products associated with innate and acquired immune responses and inflammation in the HaCaT keratinocyte cell line. To further evaluate the influence of Ino-C2-PAF we established an in vitro 2D-model of epidermal inflammation. The induction of inflammation and the impact of Ino-C2-PAF were assessed in this system using a genome-wide microarray analysis. In addition, the expression of selected genes was validated using qRT-PCR and flow cytometry. Treatment of the keratinocytes with a mix of proinflammatory cytokines resulted in transcriptional effects on a variety of genes involved in cutaneous inflammation and immunity, while additional treatment with Ino-C2-PAF counteracted the induction of many of these genes. Remarkably, Ino-C2-PAF suppressed the expression of a group of targets that are implicated in antigen processing and presentation, including MHC molecules. Thus, it is conceivable that Ino-C2-PAF possess therapeutic potential for inflammatory skin disorders, such as psoriasis and allergic contact dermatitis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line. Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell-matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines.Methods: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing.Results: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner.Conclusion: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs. [ABSTRACT FROM AUTHOR]

Natural living cell membrane phospholipids undergo rapid and continuous turnover that provides essential signalling pathways controlling cell survival. Derived from (lyso)phosphatidylcholine, alkylphopspholipids (APL) were synthesized as metabolically stable analogues 50 years ago. The nature of the chemical structure of APLs allows them to be inserted into membranes easily and partition into the membrane bilayers according to the degree of unsaturation of phospholipid alkyl chains. In an effort to overcome the severe side effects of existing anti-tumour drugs, novel APLs with variations in alkyl side chain length, glycerol backbone, ester versus ether bonds, and different polar headgroups were developed to combine high anti-proliferative capacity with reduced cytotoxicity. The platelet activating factor (PAF) is a natural alkylphospholipid. Substituting the sn-2 position of platelet activating factor with an ether linked C2 spacer coupled to inositol created Inositol-C2-PAF. The effects of Inositol-C2-PAF can be divided into short-term and long-term effects. Short-term effects include the inhibition of phosphorylation events, resulting in inhibition of cell proliferation and reduced cell migration. Long-term effects of Inositol-C2-PAF, which are characterised by changes in gene expression, include up-regulation of biological processes linked to cell differentiation or system development. Inspired by the natural phospholipid platelet activating factor (PAF) derivatives are shown, that intercalate in plasma membranes and at sub-toxic concentrations disturb plasma membrane microdomain composition and/or intracellular responses. [ABSTRACT FROM AUTHOR]