Rapid reduction of MDCK cell cholesterol by methyl-β-cyclodextrin alters steady state transepithelial electrical resistance

Francis, Stacy A.; Kelly, Joseph F.; McCormack, Joanne M.; Rogers, Rick A.; Laı̈, Jean Luc; Schneeberger, Eveline E.; Lynch, Robert D.

doi:10.1016/s0171-9335(99)80074-0

Cited by 108 publications

(104 citation statements)

References 34 publications

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“…Polymer uptake was governed solely by their hydrophobic moieties which is very common with lipidbased systems. 10,[40][41][42] It should be mentioned that a preliminary quantitative analysis on inhibitory effects was undertaken by flow cytometry. However, the assay was not feasible as the desired amount of Alexa Fluor labelled polymer, for sufficient detection via flow cytometry, was too large to chemically synthesize in a cost-effective and efficient manner.…”

Section: Resultsmentioning

confidence: 99%

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

et al. 2015

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Previously synthesized poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA) copolymers were examined as potential drug delivery vehicles. P(MAA-co-CMA) copolymers were fluorescently labelled and imaged in SHEP and HepG2 cells. To understand their cell internalization pathway endocytic inhibition studies were conducted. It was concluded that P(MAA-co-CMA) are taken up by the cells via clathrin-independent endocytosis (CIE) (both caveolae mediated and cholesterol dependent endocytosis) mechanisms. The formation and characterization of P(MAA-co-CMA)-doxorubicin (DOX) nanocomplexes was investigated by fluorescence lifetime imaging microscopy (FLIM), UV-Visible spectroscopy (UV-Vis) and dynamic light scattering (DLS) studies. The toxicity screening between P(MAA-co-CMA)-DOX nanocomplexes (at varying w/w ratios) and free DOX, revealed nanocomplexes to exhibit higher cytotoxicity towards cancer cells in comparison to normal cells. FLIM and confocal microscopy were employed for investigating the time-dependent release of DOX in SHEP cells and the cellular uptake profile of P(MAAco-CMA)-DOX nanocomplexes in cancer and normal cell lines, respectively. The endocytic pathway of P(MAA-co-CMA)-DOX nanocomplexes were examined in SHEP and HepG2 cells via flow cytometry revealing the complexes to be internalized through both clathrin-dependent (CDE) and CIE mechanisms. The drug delivery profile, reported herein, illuminates the specific endocytic route and therapeutic efficiency of P(MAA-co-CMA)-DOX nanocomplexes strongly suggesting these particles to be promising candidates for in vivo applications.

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Section: Resultsmentioning

confidence: 99%

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

et al. 2015

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“…However, there was little or no effect of 1M NaCl on the distribution of the TJ proteins among the three fractions in CHAPS extracts (data not shown), an observation that is consistent with the zwitterionic nature of the latter detergent. Reducing cell CH content by 75% during 2 h of incubation with 10 mM MBCD [12], reduced the fraction of these three TJ proteins in the largest complexes by approximately 70%, 85% and 85%, respectively, while that in the HSS, containing the smallest complexes, increased. In CHAPS extracts, by contrast, 60-80% of the group A proteins in control cells were in the intermediate size complexes.…”

Section: Selected Integral Tj Proteins In Tx-100 and Chaps Lysates Armentioning

confidence: 95%

“…Membrane CH was depleted by incubating monolayers in inserts with 10 mM MBCD in DMEM, 1% BCS applied to both apical and basolateral surfaces for either 0.5 h or 2 h at 37°C [12].…”

Section: Ch Depletionmentioning

confidence: 99%

“…Because TX-100 interfered with the fluorescence signal of the Amplex Red reagent, CH content in OptiPrep fractions of TX-100 extracted cells was determined using an alternative fluorescent method [12,18].…”

Section: Ch Determinationsmentioning

confidence: 99%

“…When CH levels were reduced acutely, using methyl-β-cyclodextrin (MBCD) in monolayers with established TJs, there was a biphasic response. During the first 30-60 min TER increased by as much as 40%, and then decreased, approaching zero after 2 h of CH efflux [12]. If MBCD was removed at that time, CH content and TER returned to control levels within 12-24 h, indicating that CH depleted cells remained viable.…”

Section: Introductionmentioning

confidence: 96%

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Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin

Lynch

Francis

McCarthy

et al. 2007

Experimental Cell Research

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Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH) depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [ 3 H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.

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Identification of Tight Junction Modulating Lipids

Chen‐Quay

Eiting

et al. 2009

Journal of Pharmaceutical Sciences

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Rapid reduction of MDCK cell cholesterol by methyl-β-cyclodextrin alters steady state transepithelial electrical resistance

Cited by 108 publications

References 34 publications

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin

Identification of Tight Junction Modulating Lipids

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