Biomedical subjects

Paiboon Reungpatthanaphong

Publications and source records attributed to Paiboon Reungpatthanaphong.

4 recordsLinked to original sources

PubMed2005

Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts.

BACKGROUND: Most cancer cells are characterized by mobile lipids visible on proton NMR (1H-NMR), these being comprised mainly of methyl and methylene signals from lipid acyl chains. Erythroleukemia K562 cells show narrow signals at 1.3 and 0.9 ppm, corresponding to mobile lipids (methylene and methyl, respectively), which are reduced when K562 cells are multidrug resistant (MDR). While the significance of the mobile lipids is unknown, their subcellular localization is still a matter of debate and may lie in the membrane or the cytoplasm. In this study, we investigate the role of cholesterol in the generation of mobile lipid signals. RESULTS: The proportion of esterified cholesterol was found to be higher in K562-sensitive cells than in resistant cells, while the total cholesterol content was identical in both cell lines. Cholesterol extraction in the K562 wild type (K562wt) cell line and its MDR counterpart (K562adr), using methyl-beta-cyclodextrin, was accompanied by a rise of mobile lipids in K562wt cells only. The absence of caveolae was checked by searching for the caveolin-1 protein in K562wt and K562adr cells. However, cholesterol was enriched in another membrane microdomain designated as "detergent-insoluble glycosphingomyelin complexes" or rafts. These microdomains were studied after extraction with triton X-100, a mild non-ionic detergent, revealing mobile lipid signals preserved only in the K562wt spectra. Moreover, following perturbation/disruption of these microdomains using sphingomyelinase, mobile lipids increased only in K562wt cells. CONCLUSION: These results suggest that cholesterol and sphingomyelin are involved in mobile lipid generation via microdomains of detergent-insoluble glycosphingomyelin complexes such as rafts. Increasing our knowledge of membrane microdomains in sensitive and resistant cell lines may open up new possibilities in resistance reversion.

Journal Article↗

PubMed2004

Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by cholesterol oxidase indicate that this transporter is not located in rafts.

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells.

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell. An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Modulation of multidrug resistance by artemisinin, artesunate and dihydroartemisinin in K562/adr and GLC4/adr resistant cell lines.

Overcoming MDR (multidrug resistance) phenomena is a crucial aspect of cancer chemotherapy research. Artemisinin and its derivatives have been found to inhibit the proliferation of cancer cells in the microM range. They poorly inhibited the function of P-glycoprotein and did not inhibit the function of MRP1-protein. The concentrations required to inhibit by 50% the function of P-glycoprotein are 110+/-5 microM. Artemisinin, artesunate and dihydroartemisinin efficiently decreased the mitochondrial membrane potential, leading to a decrease in intracellular ATP in all cell lines tested: by 30 to 50% at 5 microM. Artemisinin, artesunate and dihydroartemisinin increased cytotoxicity of pirarubicin and doxorubicin in P-glycoprotein-overexpressing K562/adr, and in MRP1-overexpressing GLC4/adr, with the delta(0.5) ranging from 200 to 860 nM, but not in their corresponding drug-sensitive cell lines. In this range of concentrations these compounds did not decrease the function of P-glycoprotein, suggesting a mechanism by which the drugs did not reverse MDR phenomenon at the P-glycoprotein level but at the mitochondrial level.

ATP Binding Cassette Transporter, Subfamily B, Mem↗