betadex and rottlerin

Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8) cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

Topics: Acetophenones; Benzopyrans; beta-Cyclodextrins; Biomarkers; Caco-2 Cells; Cell Differentiation; Endocytosis; Endosomes; Epithelial Cells; Fluorescent Dyes; Humans; Intestines; Permeability; Protein Transport; rab5 GTP-Binding Proteins

Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-beta-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-alpha-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.

Topics: Acetophenones; ADAM Proteins; ADAM10 Protein; ADAM17 Protein; Alternative Splicing; Amyloid Precursor Protein Secretases; Animals; Benzopyrans; beta-Cyclodextrins; Blotting, Western; Cell Line; Cell Membrane; Ceramides; Cholesterol; COS Cells; Cyclodextrins; Dimerization; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Interleukin-6; Lipid Metabolism; Membrane Proteins; Metalloendopeptidases; Mice; Mice, Transgenic; Monocytes; Precipitin Tests; Protein Kinase C; Protein Structure, Tertiary; Receptors, Interleukin-6; Sphingomyelins; Staurosporine; Time Factors; Transfection