The Effect of Capsaicin Derivatives on Tight-Junction Integrity and Permeability of Madin-Darby Canine Kidney Cells

Capsaicin is known to interfere with tight junctions (TJs) of epithelial cells and therefore to enhance paracellular permeability of poorly absorbable drugs. However, due to its low water solubility, pungency, and cytotoxicity, its pharmacologic use is limited. In this study, we investigated the effect of capsaicin derivatives of synthetic (e.g., 10-hydroxy-N-(4-hydroxy-3-methoxybenzyl)decanamide, etc.) and natural (olvanil and dihydrocapsaicin) origin on Madin-Darby Canine Kidney–C7 cells. Impedance spectroscopy was used to determine the transepithelial electrical resistance and the capacitance. Permeability assays with fluorescein isothiocyanate–dextran were carried out to evaluate the impact on cell permeability. The results show that lipophilicity could play an important role for the interference with TJ and that the mechanism is independent from the ion channel TRPV-1 and hence on the flux of calcium into the cells. In summary, we synthesized 4 derivatives of capsaicin of lower lipophilicity and compared their properties with other well-known vanilloids. We show that these compounds are able to enhance the permeability of a hydrophilic macromolecule, by opening the TJ for a shorter time than capsaicin. This behavior is dependent on the lipophilicity of the molecule. Understanding of these phenomena may lead to better control of administration of therapeutic molecules

Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers

We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem’s influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers

Calcium flux experiments.

<p>Calcium flux experiments using the dye Fluo-4 in combination with structured illumination fluorescence microscopy: Representative microscopy images of the cells before and after the stimulation with the different treatments. From each experiment images with the maximum fluorescence signal after stimulation are shown. (a) Panel with the controls. (b) Panel with the nanoformulations. (c) Fluorescence intensity over time after stimulation with the different treatments (Scale bar = 200 μM). NC: nanocapsules (images in (a) and (b) have been contrast enhanced for better visualization in the print version).</p

Analysis of cell morphology by digital holographic microscopy.

<p>Analysis of cell morphology by evaluation of quantitative phase images retrieved with digital holographic microscopy: (a) Histograms of quantitative DHM phase contrast images at different time points for control cells and cells treated with capsaicin and the different nanoformulations. (b) Average phase contrast for all treatments plotted over time with a linear fit. (c) Slopes and Y-intercepts retrieved by the linear fits from (b). Mean values ± SD (<i>n = 3</i>).</p

Migration patterns of MDCK-C7 cells.

<p>Migration patterns of MDCK-C7 cells after treatment with nanocapsules and free capsaicin: (a) Representative migration tracks. End times of the tracking experiment are given below the headlines as the cells moved out of the observed field of view at different time points due to different migration speeds. (b) Migration speed as an end point measurement. (c) Directness as an end point measurement. (d) Accumulated distance over time for all the treatments. (e) Euclidean distance over time for all the treatments. Mean values ± SD. Statistical test: Kruskal-Wallis test (n = 3; 10 cells per measurement), * p < 0.05, *** p < 0.001, **** p < 0.0001). NC: nanocapsules.</p

Analysis of global morphology changes in confluent cell layers.

<p>Analysis of global morphology changes in confluent cell layers by histogram-based evaluation of quantitative DHM phase contrast images at different time points (red: t₁, purple: t₂, blue: t_3; t₁ < t₂ < t₃). An increase of the cell layer surface roughness Δ<i>d</i> (a) causes a shift of the histogram (b) and the average phase contrast (vertical lines in (b)) towards higher phase values.</p

Fluorescence microscopy.

<p>Representative images of MDCK cells with actin or ZO-1 staining. (Nuclei: blue, ZO-1: green, Actin: red, NC: nanocapsules) (images have been contrast enhanced for better visualization in the print version)”.</p

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways