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Proceedings of the National Academy of... Jun 1980Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release...
Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.
Topics: Actins; Animals; Cell Membrane Permeability; Cytoskeleton; Cytosol; Digitonin; Fluorescent Antibody Technique; Immunoelectrophoresis; Liver; Microscopy, Electron; Microscopy, Fluorescence; Rats
PubMed: 6997878
DOI: 10.1073/pnas.77.6.3430 -
The Biochemical Journal Jun 1985Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of...
Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.
Topics: Animals; Cell Separation; Digitonin; Liver; Male; Microbial Collagenase; Perfusion; Rats
PubMed: 2992454
DOI: 10.1042/bj2280757 -
Analytical Biochemistry Jul 1995
Comparative Study
Topics: Cell Membrane Permeability; Digitonin; Evaluation Studies as Topic; Glucosephosphate Dehydrogenase; Methods; Saccharomyces cerevisiae
PubMed: 8533887
DOI: 10.1006/abio.1995.1394 -
Progress in Clinical and Biological... 1992
Review
Topics: Adenosine Triphosphate; Cells, Cultured; Digitonin; Fibroblasts; Humans; Microbodies; Oxidation-Reduction; Palmitoyl Coenzyme A; Permeability; Reaction Time
PubMed: 1438367
DOI: No ID Found -
Current Protocols in Cell Biology May 2001Import of proteins into the nucleus of a cell is a complex process that can be reconstituted in vitro. Digitonin-permeabilized cells are washed free of cytosolic factors...
Import of proteins into the nucleus of a cell is a complex process that can be reconstituted in vitro. Digitonin-permeabilized cells are washed free of cytosolic factors to provide competent nuclei. Cytosolic factors for import are provided by an extract of Xenopus ovarian cells. Fluorochrome-conjugated probes, cloned proteins fused to green fluorescent protein, or antibodies to the protein of interest are used to visualize nuclear import. The system can also be used to identify nuclear localization sequences (NLS) of imported proteins.
Topics: Active Transport, Cell Nucleus; Animals; Cell Membrane Permeability; Cytological Techniques; Digitonin; Female; HeLa Cells; Humans; Indicators and Reagents; Ovary; Xenopus
PubMed: 18228312
DOI: 10.1002/0471143030.cb1107s05 -
Langmuir : the ACS Journal of Surfaces... Apr 2020OSW-1, a unique steroidal saponin isolated from the bulbs of , has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and...
OSW-1, a unique steroidal saponin isolated from the bulbs of , has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin-lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane ( = 0.38 μM) and the strongest membrane disruptivity in the bound saponin-lipid ratio at the point of 50% calcein leakage ( = 0.47) occurrence. OSW-1 showed slightly lower activity ( = 0.31 μM; = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity ( = 0.017 μM; = 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 μM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 μM, = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 μM, = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.
Topics: Cholestenones; Digitonin; Lipid Bilayers; Saponins
PubMed: 32160747
DOI: 10.1021/acs.langmuir.9b03957 -
Phytomedicine : International Journal... Nov 2012In phytotherapy, extracts from medicinal plants are employed which contain mixtures of secondary metabolites. Their modes of action are complex because the secondary...
In phytotherapy, extracts from medicinal plants are employed which contain mixtures of secondary metabolites. Their modes of action are complex because the secondary metabolites can react with single or multiple targets. The components in a mixture can exert additive or even synergistic activities. In this study, the cytotoxicity of some phytochemicals, including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene) and alkaloids (glaucine, harmine, and sanguinarine) were investigated alone or in combination with the cytotoxic monodesmosidic steroidal saponin digitonin in Caco-2, MCF-7, CEM/ADR5000, and CCRF-CEM cells. Digitonin was combined in non-toxic concentrations (5μM in each cell line; except in MCF-7 the concentration was 2μM), together with a selection of phenolics, terpenoids, and alkaloids to evaluate potential synergistic or additive effects. An enhanced cytotoxicity was observed in most combinations. Even multi-drug resistant (MDR) cells (such as CEM/ADR5000 cells), with a high expression of P-glycoprotein, were responsive to combinations. Sanguinarine was the most cytotoxic alkaloid against CEM/ADR5000, MCF-7, and CCRF-CEM cells alone and in combination with digitonin. As compared to sanguinarine alone, the combination was 44.53-, 15.38-, and 6.65-fold more toxic in each cell line, respectively. Most combinations synergistically increased the cytotoxicity, stressing the importance of synergy when using multi-target drugs and mixtures in phytotherapy.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Alkaloids; Antineoplastic Agents, Phytogenic; Benzophenanthridines; Caco-2 Cells; Digitalis; Digitonin; Drug Resistance, Multiple; Drug Synergism; Female; Humans; Isoquinolines; MCF-7 Cells; Neoplasms; Phenols; Phytotherapy; Plant Extracts; Terpenes
PubMed: 23062361
DOI: 10.1016/j.phymed.2012.09.002 -
The Annals of Thoracic Surgery Oct 2001The antitumor effect of isolated lung perfusion with cisplatin was limited because the intracellular platinum concentration did not increase sufficiently. To solve this...
BACKGROUND
The antitumor effect of isolated lung perfusion with cisplatin was limited because the intracellular platinum concentration did not increase sufficiently. To solve this problem, digitonin, a detergent, was chosen to increase cell permeability and enhance intracellular uptake and antitumor effect. This study was designed to investigate toxicity, pharmacokinetics, and efficacy of isolated lung perfusion with the combined use of digitonin and cisplatin in Fischer 344 rats.
METHODS
Systemic and local toxicities of isolated lung perfusion treatment were evaluated on the basis of body weight change, survival rate, and histologic findings. The maximal tolerated dose of digitonin was determined by assessing survival on day 21 after contralateral pneumonectomy, body weight change, and histologic findings. Pharmacokinetics were observed in a solitary lung tumor nodule model by measuring platinum concentration in tumor and normal lung tissue. The antitumor effect was evaluated by the number of tumor nodules in the left lung 21 days after isolated lung perfusion. Isolated lung perfusion was performed 7 days after 1.0 x 10(6) methylcholanthrene sarcoma cells were injected into the external jugular vein.
RESULTS
The maximal tolerated dose of digitonin was 20 micromol/L. Platinum concentration of tumor nodules in the digitonin-cisplatin-treated rats was 20% higher than in the cisplatin-only group (5.48 +/- 0.64 microg/g tissue versus 4.50 +/- 1.09 microg/g tissue; p = 0.067). The number of pulmonary nodules decreased significantly by digitonin use (1.3 +/- 1.5 versus 9.7 +/- 2; p < 0.0001).
CONCLUSIONS
Isolated lung perfusion with digitonin and cisplatin in combination was performed safely and enhanced the antitumor effect. These drugs in combination show promise for enhancing the effect of clinical isolated lung perfusion.
Topics: Animals; Cell Survival; Cisplatin; Digitonin; Dose-Response Relationship, Drug; Drug Synergism; Infusions, Intra-Arterial; Lung; Lung Neoplasms; Male; Neoplasm Transplantation; Rats; Rats, Inbred F344; Sarcoma, Experimental; Tumor Cells, Cultured
PubMed: 11603432
DOI: 10.1016/s0003-4975(01)03054-5 -
Anticancer Research 2001This study was designed to evaluate cellular uptake and cytotoxicity of cisplatin in methylcholanthrene (MCA)-induced rat sarcoma cells when used in combination with a...
BACKGROUND
This study was designed to evaluate cellular uptake and cytotoxicity of cisplatin in methylcholanthrene (MCA)-induced rat sarcoma cells when used in combination with a detergent, digitonin.
MATERIALS AND METHODS
In the cellular intake study, after MCA sarcoma cells (10(7)) were treated with cisplatin alone (50 micrograms/ml) and with cisplatin (50 micrograms/ml) in combination with digitonin at 20 microM and 50 microM, the cells were washed twice with PBS and the platinum levels were measured by flameless atomic spectrometry. For the anti-tumor effect MCA sarcoma cells (10(3)) were seeded in cell culture dishes and loaded for 10 minutes with PBS, digitonin (5 microM), cisplatin (5 micrograms/ml) or a combination of cisplatin (5 micrograms/ml) and digitonin (5 microM). The cells were then washed and incubated for 72 hours. Bromodeoxyuridine uptake was measured with an ELISA system for determining viable cells counts.
RESULTS
Cell platinum levels were significantly elevated in proportion to the increase of digitonin (p < 0.0001). The number of viable cells was significantly decreased with, the combined cisplatin (5 micrograms/ml)--digitonin (5 microM) treatment (p < 0.0001
CONCLUSION
Digitonin enhances the antitumor effect of cisplatin against methylcholanthrene-induced rat sarcoma in vitro.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biological Transport, Active; Cell Membrane Permeability; Cisplatin; Detergents; Digitonin; Drug Synergism; Methylcholanthrene; Rats; Sarcoma, Experimental; Tumor Cells, Cultured; Tumor Stem Cell Assay
PubMed: 11299754
DOI: No ID Found -
Plasmid Jul 1986A procedure for the isolation of closed circular mitochondrial DNA (mtDNA) from cells permeabilized with digitonin is described. Compared to the standard procedure in...
A procedure for the isolation of closed circular mitochondrial DNA (mtDNA) from cells permeabilized with digitonin is described. Compared to the standard procedure in which cells are broken after osmotic swelling, the digitonin-based procedure is more consistent and results in higher mtDNA yields.
Topics: Animals; Cell Fractionation; DNA, Mitochondrial; Digitonin; L Cells; Mice; Osmolar Concentration
PubMed: 3737750
DOI: 10.1016/0147-619x(86)90083-1