U18666A impairs cellular cholesterol efflux from HMDM.

<p>Cells were incubated with 100 µg/ml AcLDL for 6 h and treated with 3 µg/ml U18666A or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.</p

Dynasore decreases the production of cholesterol esters from LDL- or AcLDL-derived cholesterol.

<p>HeLa cells or HMDM were respectively incubated with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (C) and treated for 6 h with 80 µM dynasore or without (control). The total amount of CE was quantified and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). The production of cholesteryl myristate was measured in HeLa cells (B) or HMDM (D) treated or not (control) with 80 µM dynasore. Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.</p

Effects of U18666A on the intracellular distribution of FC and LDL in HeLa cells and HMDM.

<p>HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/mL LDL (A) or 50 µg/ml AcLDL (C) with 3 µg/ml U18666A or without (control) and stained with filipin to detect FC. (B–D) HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/ml DiI-LDL (B) or 50 µg/ml DiI-AcLDL (D) with 3 µg/ml U18666A or without (control) and processed to visualize LDL distribution. Images were obtained using wide-field epifluorescence microscopy.</p

Effect of dynasore on LDL uptake and total cholesterol in HeLa cells and HMDM.

<p>Cells were incubated for 4 h with 0–200 µg/ml DiI-LDL (A) or 0–100 µg/ml DiI-AcLDL (C) at 37°C with 0.4% v/v DMSO (control) or 80 µM dynasore. The total amount of endocytosed DiI-LDL or DiI-AcLDL was measured by flow cytometry. Values represent the mean ± SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 0.4% v/v DMSO (control) or 80 µM dynasore. Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.</p

Effects of dynasore on the intracellular distribution of FC and LDL in HeLa cells and HMDM.

<p>(A) Hela cells were loaded with 200 µg/ml LDL for 24 h. Cells were then treated for 6 h with 80 µM dynasore or without (control) and stained with filipin to detect FC. (B) Cells were treated as described above with 200 µg/ml DiI-LDL. (C) HMDM were incubated for 6 h in LPDS medium containing 50 µg/ml DiI-AcLDL with 80 µM dynasore or without (control). (D) HMDM were loaded with 50 µg/ml DiI-AcLDL for 24 h and then treated for 6 h with 80 µM dynasore or without (control). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 µm.</p

U18666A inhibits ACAT activity and sterol-sensitive genes regulation in HeLa cells and HMDM.

<p>Cells were grown in LPDS medium for 48 h and further incubated for 6 h with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (B) with 3 µg/ml U18666A or without (control). Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells (A) or HMDM (B) was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate. The total amount of CE was quantified HeLa cells (C) and in HMDM (D) and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). Cholesteryl myristate formation was measured in HeLa cells (E) or HMDM (F) with 3 µg/ml U18666A or without (control). Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.</p

Dynasore blocks sterol-sensitive genes regulation in HeLa cells and HMDM.

<p>(A) Kinetics of LDLR expression analyzed by RT-PCR. HeLa cells were grown in LPDS medium for 48 h and further incubated for the indicated times with medium containing either LPDS, 200 µg/ml LDL, or 200 µg/ml LDL with 80 µM dynasore. (B) The expression level of sterol-sensitive genes (LDLR, HMGCoAR and SREBF-2) was quantified after 6 h in HeLa cells grown in LPDS, with 200 µg/ml LDL or 200 µg/ml LDL with 80 µM dynasore, as indicated. (C) The same experiment was performed in HMDM with 50 µg/ml AcLDL. Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells or HMDM was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate.</p

Dynasore treatment results in the endolysosomal accumulation of FC and LDL in HeLa cells.

<p>Cells were treated for 6 h with 80 µM dynasore or without (control) in medium containing 200 µg/ml LDL (A) or DiI-LDL (B) and processed for filipin staining (A) or DiI-LDL detection (B). Left panels present Lamp1 staining. Merge of Lamp1 with FC (A) or with DiI-LDL (B) is shown in the right panel. Scale bars, 10 µm.</p

Important Role of CYP2J2 in Protein Kinase Inhibitor Degradation: A Possible Role in Intratumor Drug Disposition and Resistance

<div><p>We have investigated <i>in vitro</i> the metabolic capability of 3 extrahepatic cytochromes P-450, CYP1A1, 1B1 and 2J2, known to be over-expressed in various tumors, to biotransform 5 tyrosine kinase inhibitors (TKI): dasatinib, imatinib, nilotinib, sorafenib and sunitinib. Moreover, mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in 6 hepatocellular and 14 renal cell carcinoma tumor tissues and their surrounding healthy tissues, was determined.</p><p>Our results show that CYP1A1, 1B1 and especially 2J2 can rapidly biotransform the studied TKIs with a metabolic efficiency similar to that of CYP3A4. The mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in tumor biopsies has shown i) the strong variability of CYP expression and ii) distinct outliers showing high expression levels (esp. CYP2J2) that are compatible with high intratumoral CYP activity and tumor-specific TKI degradation.</p><p>CYP2J2 inhibition could be a novel clinical strategy to specifically increase the intratumoral rather than plasma TKI levels, improving TKI efficacy and extending the duration before relapse. Such an approach would be akin to beta-lactamase inhibition, a classical strategy to avoid antibiotic degradation and resistance.</p></div

Michaelis-Menten kinetic parameters.

<p>Michaelis-Menten kinetic parameters determined in microsome incubations with cDNA expressed CYP1A1, 1B1, 2J2 and 3A4 isozymes. A) Affinity constants, Km in µM, for different TKI metabolic pathways. B) Relative intrinsic clearance (Vmax/Km; in arbitrary unit [arb]) for the same TKI metabolic pathways, expressed as fold differences of the intrinsic clearance of CYP3A4, the major hepatic enzymes involved in TKI degradation. Abbreviations: OH-: hydroxyl-metabolites, NO-: N-oxide metabolite. Imatinib metabolites are numbered in accordance to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095532#pone.0095532-Rochat4" target="_blank">[18]</a>.</p>(*)<p>Results obtained in our previous published study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095532#pone.0095532-Rochat4" target="_blank">[18]</a>.</p