The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

Clinical usefulness of doxorubicin (DOX) is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1) responsible for the active efflux of drugs out of resistant cells. Doxorubicin is a well-known bioreductive antitumour drug. Its ability to undergo a one-electron reduction by cellular oxidoreductases is related to the formation of an unstable semiquionone radical and followed by the production of reactive oxygen species. There is an increasing body of evidence that the activation of bioreductive drugs could result in the alkylation or crosslinking binding of DNA and lead to the significant increase in the cytotoxic activity against tumour cells. The aim of this study was to examine the role of reductive activation of DOX by the human liver NADPH cytochrome P450 reductase (CPR) in increasing its cytotoxic activity especially in regard to MDR tumour cells. It has been evidenced that, upon CPR catalysis, DOX underwent only the redox cycling (at low NADPH concentration) or a multistage chemical transformation (at high NADPH concentration). It was also found, using superoxide dismutase (SOD), that the first stage undergoing reductive activation according to the mechanism of the redox cycling had the key importance for the metabolic conversion of DOX. In the second part of this work, the ability of DOX to inhibit the growth of human promyelocytic-sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. Our assays showed that the presence of CPR catalysing only the redox cycling of DOX had no effect in increasing its cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells

Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells - modification of ABCC1 (MRP1) localization and functionality

1 - ArticleThe multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein involved in the multidrug resistance phenotype. It actively expels a number of cytotoxic molecules from cells. To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we used multidrug-resistant GLC4/ADR cells, which overexpress MRP1. Upon altering the plasma membrane cholesterol content of these cells, membrane localization and the activity of MRP1 were analyzed. A detergent-free methodology was used to separate 'light' and 'heavy' plasma membrane fractions. Our data show that MRP1 was exclusively found in 'light' fractions known as L-0 phase membrane microdomains, together with similar to 23% of gangliosides GM1 and 40% of caveolin-1. Depletion of the membrane cholesterol level to 40% by treatment with the cholesterol-chelating agent methyl-beta-cyclodextrin did not modify MRP1 activity, as evidenced either by the rate of efflux of pirarubicin or that of glutathione. Further cholesterol depletion below 40% yielded both a partial shift of MRP1 to the high-density fraction and a decrease of its functionality. Taken together, these data suggest that MRP1 funtionality depends on its localization in cholesterol-rich membrane microdomains

Caveolin-1 and Doxorubicin-induced P-Glycoprotein Modulate Plasma Cholesterol Membrane Accessibility in Erythrolymphoblastic Cell Line

1 - ArticleAim/Background: Various interactions between Caveolae membrane domains, multidrug resistance transporter P-glycoprotein (P-gp) and cholesterol have been suggested. We tested the assumption that anthracycline-induced P-gp and Caveolin-1 have correlated effects on cholesterol distribution in plasma membrane. Materials and Methods: The present study was performed in four lymphoblastic K562 cell lines expressing none (KS), one (Cav and KR cells) or both P-gp and caveolin-1 proteins (CavKR cells). Results: The CavKR cell line exhibits a significantly higher free cholesterol content than the other cell lines. Cholesterol distribution at the outer leaflet was distinct from the total cellular cholesterol by its accessibility to cholesterol oxidase (COase). When cells were ATP-deprived, cholesterol accessibility to oxidation was significantly delayed in CavKR cells. Caveolin-1 or P-gp expression did not induce detectable changes in membrane cholesterol accessibility to COase. Conclusion: Combination of functional P-gp, caveolae presence and lasting effect of anthracycline treatment appear determinant in free membrane cholesterol homeostasis and likely modulate cholesterol membrane order

Métabolomique par RMN d’extraits de cortex frontal : première étude comparant deux maladies neurodégénératives, la maladie d’Alzheimer et la sclérose latérale amyotrophique

International audienceObjective This study was designed to assess the brain metabolites' variability between two neurodegenerative diseases in frontal cortex samples obtained post-mortem. NMR metabolomics was used for the first time in this context. Materials and methods 1H NMR metabolomic was applied to tissue extracts from patients with Alzheimer disease (ALZ) and patients with amyotrophic lateral sclerosis (ALS) to investigate qualitative and quantitative variations of brain metabolites. Results The Alzheimer disease metabolic signature was characterized by a high concentration of alanine, acetate, glutamate and glutamine, and low concentrations of lactate and creatine, while the ALS metabolic signature appears to be marked by high concentrations of lactate, N-acetyl aspartate, creatine, choline and myo-inositol. Moreover, in vitro 1H NMR could detect metabolites such as 3-hydroxybutyrate, alanine, succinate and aspartate that cannot be detected with in vivo NMR. Discussion The neurodegenerative diseases exhibit diverging metabolic pathways. Some of the metabolites responsible for the discrimination between the two diseases were detected before in vivo. However, this in vitro metabolomic investigation demonstrates the involvement of metabolites not detected with in vivo studies. Conclusion Upon these findings, in vitro metabolomics appears to be an efficient tool to investigate the fundamentals of the metabolic pathway modulations in these neurodegenerative diseases to help the interpretation of clinical data obtained with in vivo NMR spectroscopy. © 2012 Elsevier Masson SAS