scholarly journals P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Jennifer Pasquier ◽  
Damien Rioult ◽  
Nadine Abu-Kaoud ◽  
Sabine Marie ◽  
Arash Rafii ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Academic Research ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
High Throughput Drug Screening ◽  
Activity Measurements ◽  
Single Well ◽  
Microplate Reader ◽  
Calcein Am ◽  
Small Inhibition

Background. P-gp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Fluorescence techniques have been widely applied to measure the P-gp activity. In this paper, there is a comparison between the advantages of two fluorescence approaches of commonly available and affordable instruments: the microplate reader (MPR) and the flow cytometer to detect the P-gp efflux activity using calcein-AM.Results. The selectivity, sensibility, and reproducibility of the two methods have been defined. Our results showed that the MPR is more powerful for the detection of small inhibition, whereas the flow cytometry method is more reliable at higher concentrations of the inhibitors. We showed that to determine precisely the inhibition efficacy the flow cytometry is better; hence, to get the correctEmaxand EC50values, we cannot only rely on the MPR.Conclusion. Both techniques can potentially be used extensively in the pharmaceutical industry for high-throughput drug screening and in biology laboratories for academic research, monitoring the P-gp efflux in specific assays.

Efficacy of Chemotherapeutic Agents Under Hypoxic Conditions in Pulmonary Adenocarcinoma Multidrug Resistant Cell Line

2007 ◽  
Vol 19 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Shicang Yu ◽  
Guijun Huang ◽  
Guisheng Qian ◽  
Yuying Li ◽  
Guoming Wu ◽  
...  
Keyword(s):  
Cell Line ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Resistant Cell Line ◽  
Pulmonary Adenocarcinoma ◽  
Hypoxic Conditions

scholarly journals NaIO4/Br- as a mild system for the oxidation of 1-methyl-anthra-9,10-quinones

2015 ◽  
Author(s):  
Marcin Cybulski ◽  
Adam Formela ◽  
Katarzyna Sidoryk ◽  
Olga Michalak ◽  
Anna Rosa ◽  
...  
Keyword(s):  
High Pressure ◽  
Carboxylic Acids ◽  
Scale Up ◽  
Lithium Bromide ◽  
Cost Effective ◽  
Building Blocks ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
Direct Oxidation ◽  
Reaction Conditions

One of the anthraquinone classes comprises compounds with a carbonyl group. These natural or synthetic anthraquinones find their application as building blocks in the synthesis of the compounds with a biological activity. Recently, 4-substituted anthra-9,10-quinone-1-carboxylic acids (2) have been used as key intermediates in the synthesis of patented compounds (3) with anticancer activity against multidrug resistant cell lines. Although 2,7-dihydro-3H-dibenz[de,h]cinnolin-3,7-diones (3) were successfully synthetized in a small laboratory scale, several problems were observed during the preparation of their acid intermediates (2) in a multi-gram scale. The known methods for the preparation of 2 are based on the oxidation of the methyl group in anthra-9,10-quinones (1). The most common are: the oxidation with the diluted nitric acid under high pressure in a sealed tube at the temperature of 195-220 oC, the oxidation in nitrobenzene by passing chlorine gas through the reaction mixture at the temperature of 160-170 oC or in a presence of the fuming sulphuric acid. The mentioned methods require aggressive reagents and specific reaction conditions including high pressure and temperature. Thus, there was a need to find a new efficient, cost-effective and reproducible synthetic method of preparation of 2. While searching literature it was found that the direct oxidation of alkylarenes mediated by the sodium periodate/lithium bromide combination produces benzyl acetates throughout benzyl bromides in the acetic acid, or benzylic acids in the diluted inorganic acid. Based on these results we examined a variety of reaction conditions with or without the bromine source and the oxidizing anion. As a result, a novel procedure for the preparation of highly pure 4-substituted anthra-9,10-quinone-1-carboxylic acids (HPLC > 99.5%) using oxidizing anion/ brominating reagent system was developed. It enabled 2 isolation by the simple filtration of the reaction mixture and was applied in the scale-up of 2,7-dihydro-3H-dibenz[de,h]cinnolin-3,7-dione derivatives.

scholarly journals Studying the Role and Molecular Mechanisms of MAP4K3 in Sorafenib Resistance of Hepatocellular Carcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yi Shi ◽  
Xiaofei Mo ◽  
Simei Hong ◽  
Tianbao Li ◽  
Baozhen Chen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Resistant Cell ◽  
Therapeutic Drug ◽  
Specific Gene ◽  
Sorafenib Treatment ◽  
Proliferation And Apoptosis

Sorafenib is the first FDA-approved therapeutic drug for molecular target medication on advanced-stage hepatocellular carcinoma. It is reported that sorafenib could improve the survival of progression-free patients for 4 to 6 months; however, most of the patients developed drug resistance. Thus, it is critical to reveal the biological mechanisms behind sorafenib resistance. In this study, a sorafenib-resistant model was developed by exposing HepG2 cells to sorafenib with gradient increasing concentration, and the resistance-related genes were screened by microarray. Real-time qPCR was used to validate selected gene expression of the resistance model, and lentivirus vector-mediated RNA interference was applied for specific gene knockdown. In addition, high-throughput High Celigo Select (HCS) and flow cytometry were used to measure the effect on cellular proliferation and apoptosis. As a result, our study established a sorafenib-resistant model with IC50 of 9.988 μM. The Affymetrix expression profile of the sorafenib-resistant model showed 35 resistant-related genes, and 91.4% of the resistant genes showed upregulation in HepG2 resistance cells. In addition, 20 genes were knocked down to measure cell proliferation, and MAP4K3 with high proliferation inhibiting phenotype was chosen for further study. Meanwhile, the HCS results revealed that shMAP4K3 transfection could downregulate resistant cell proliferation, and the flow cytometry results showed that cell apoptosis was significantly increased in the MAP4K3 knockdown group. In summary, MAP4K3 is a novel molecular marker for improving the drug sensitivity of sorafenib treatment in hepatocellular carcinoma.

scholarly journals Mycoplasma Infection Mediates Sensitivity of Multidrug-Resistant Cell Lines to Tiopronin: A Cautionary Tale

2019 ◽  
Vol 63 (3) ◽  
pp. 1434-1439 ◽  
Author(s):  
Lyn M. Huff ◽  
Sachi Horibata ◽  
Robert W. Robey ◽  
Matthew D. Hall ◽  
Michael M. Gottesman
Keyword(s):  
Cell Lines ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
Mycoplasma Infection ◽  
Cautionary Tale

scholarly journals The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

2006 ◽  
Vol 31 (4) ◽  
pp. 349-356 ◽  
Author(s):  
Luiz G. Chitarra ◽  
Peter Breeuwer ◽  
Tjakko Abee ◽  
Ruud W. Bulk
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Pathogenic Bacterium ◽  
Plant Pathogenic Bacterium ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Alternatives Assessment ◽  
Heat Treated ◽  
Calcein Am

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

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