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How do antibiotics play with multidrug transporters in eucaryotic cells ?

How do antibiotics play with multidrug transporters in eucaryotic cells ?. Françoise Van Bambeke, PharmD, PhD Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain Bruxelles, Belgium. www.facm.ucl.ac.be. ATP-Binding Cassette transporters.

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How do antibiotics play with multidrug transporters in eucaryotic cells ?

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  1. How do antibiotics play with multidrug transporters in eucaryotic cells ? Françoise Van Bambeke, PharmD, PhD Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain Bruxelles, Belgium www.facm.ucl.ac.be Bremen Summerschool 2010

  2. ATP-Binding Cassette transporters ABCC ABCE ABCF ABCG ABCA ABCB ABCD BCRP P-gp MRPs multidrug transporters ABCs transporters 2 Bremen Summerschool 2010

  3. Are antibiotics substrates for MDRtransportersin eucaryotic cells ? Die Bremer Stadtmusikanten Bremen Summerschool 2010

  4. Fluoroquinolone antibiotics ciprofloxacin moxifloxacin Bremen Summerschool 2010

  5. Kinetics of accumulation and efflux for ciprofloxacin both accumulation and efflux markedly affected by probenecid (inhibitors of Mrps) Michot et al. (2004) AAC 48:2673-82 extracell. conc. 17 mg/L; probenecid 5 mM Bremen Summerschool 2010

  6. Ciprofloxacin, classical model Kolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42 Bremen Summerschool 2010

  7. Kinetics of accumulation and efflux for moxifloxacin neither accumulation nor efflux affected by probenecid extracell. conc. 17 mg/L; probenecid 5 mM Michot et al. AAC (2005) 49:2429-37 Bremen Summerschool 2010

  8. Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin Michot et al. AAC (2005) 49:2429-37 Bremen Summerschool 2010

  9. Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin • moxifloxacin not affected Michot et al. AAC (2005) 49:2429-37 Bremen Summerschool 2010

  10. Quinolones as inhibitors of ciprofloxacin efflux • ciprofloxacin efflux inhibited by ciprofloxacin • moxifloxacin CIP MXF CIP Michot et al. AAC (2005) 49:2429-37 Bremen Summerschool 2010

  11. Quinolones as inhibitors of ciprofloxacin efflux moxifloxacin also able to interact with the transporter ! • ciprofloxacin efflux inhibited by ciprofloxacin • moxifloxacin CIP MXF CIP Michot et al. AAC (2005) 49:2429-37 Bremen Summerschool 2010

  12. Moxifloxacin, ‘futile-cycle’ model Eytan et al. (1996) JBC 271:12897-902 Bremen Summerschool 2010

  13. Can we make eucaryotic cells « resistant » to antibiotics ?A way to further characterize efflux transporters Die Bremer Stadtmusikanten Bremen Summerschool 2010

  14. Over-expression of efflux pumps as mechanism of resistance • step-wise increase in AB concentration • several passages at each step time and drug consumming! multifactorial multidrug resistance How to get resistant cells ? Gottesman et al, Methods Enzymol. (1998) 292: 248-58 Bremen Summerschool 2010

  15. Identification of the ciprofloxacin transporter: « resistant » cells as a tool 0.1 mM 0.15 mM Chronical exposure to increasing concentrations 0.2 mM Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695 Bremen Summerschool 2010

  16. Ciprofloxacin « resistant » cells: phenotypic analysis ATP-dependent reduction in cell accumulation of CIP; MXF non affected control ATP-depleted Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695 Bremen Summerschool 2010

  17. Ciprofloxacin « resistant » cells: phenotypic analysis ATP-dependent reduction in cell accumulation of CIP; MXF non affected control ATP-depleted Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695 Bremen Summerschool 2010

  18. Ciprofloxacin « resistant » cells: phenotypic analysis  IC50 gemfibrozil  efflux rate Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 Bremen Summerschool 2010

  19. Ciprofloxacin « resistant » cells: genotypic analysis ARNm levels (Real-Time PCR)  expression Mrp2 and Mrp4, but Mrp4 predominates Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 Bremen Summerschool 2010

  20. Ciprofloxacin « resistant » cells: proteomic analysis detection of the proteins by Western-Blot of membrane fraction Confocal microscopy Mrp2 WT RS µg 5 15 30 5 15 30 Mrp4 WT RS µg 5 15 30 5 15 30 Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 Bremen Summerschool 2010

  21. Ciprofloxacin « resistant » cells : which transporter ? Specific extinction of gene expression by siRNA - - 25 CT 10 25 10 25 10 CT 5 10 25 Mrp2 Mrp4 Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 Bremen Summerschool 2010

  22. Acquisition of resistance is a stepwise process Accumulation and Mrp4 expression during selection of resistance Bremen Summerschool 2010

  23. Can we select for moxifloxacin « resistant » cells ? FQ accumulation and gemfibrozil effect ciprofloxacin moxifloxacin WT Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  24. Can we select for moxifloxacin « resistant » cells ? FQ accumulation and gemfibrozil effect ciprofloxacin moxifloxacin WT MXF-RS Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  25. Can we select for moxifloxacin « resistant » cells ? Ciprofloxacin efflux T1/2 WT >> T1/2 WT+GEM = T1/2 MXF-RS Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  26. Moxifloxacin-exposed cells are « anti » resistant! Mrp4 expression Western-Blot ARNm (Real Time PCR) WT MXF WT MXF - - RS CIP - RS Mrp4 Actin 20µg 40µg 60µg 20µg 40µg 60µg 10µg Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  27. Fluoroquinolone transport: model ciprofloxacin WT CIP-RS MXF-RS 1 4 1 0.2 1 15 in in in out out out Bremen Summerschool 2010

  28. Fluoroquinolone transport: model moxifloxacin WT CIP-RS MXF-RS 1 15 1 15 1 15 in in in out out out Bremen Summerschool 2010

  29. Efflux …is it enough for resistance ? Die Bremer Stadtmusikanten Bremen Summerschool 2010

  30. Efflux pumps … and what next ? How to get resistant cells ? • step-wise increase in AB concentration • several passages at each step time and drug consumming! multifactorial multidrug resistance Gottesman et al, Methods Enzymol. (1998) 292: 248-58 Bremen Summerschool 2010

  31. Stable Isotope Labeling Aminoacid in Culture CIP-macrophages WT 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg sample mixing 1:1 protein digestion identification in mass spectrometry and determination of the relative abundance Bremen Summerschool 2010

  32. F1 Discontinuous Sucrose gradient F2 F3 kDa Enriched plasma membrane fraction F4 1.10 190 - F5 1.13 1.15 40 - 1.17 1.19 Stable Isotope Labeling Aminoacid in Culture CIP-macrophages WT post-nuclear homogenate 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg ultracentrifugation 1.10 1.13 1.15 1.17 1.19 F1 F2 F3 F4 F5 • MRP1 • Prohibitin Bremen Summerschool 2010

  33. A A B B - 200 C - 150 C D D - 120 E - 100 E F - 85 F G - 70 - 60 H G - 50 H I - 40 I J J - 30 k Stable Isotope Labeling Aminoacid in Culture CIP-macrophages WT F2 F1 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg Discontinuous Sucrose gradient Enriched plasma membrane fraction Combine 1:1 SDS-PAGE/in-gel digestion Protein ID & Quantification Bremen Summerschool 2010

  34. SILAC: global data  Identification of 900 proteins with 3 uniques peptides Among proteins detected in both fractions  15  expression in CIP-macrophages as compared to WT  13  expression in CIP-macrophages as compared to WT Among proteins detected in one of the two fractions  37/36 expression in CIP-macrophages as compared to WT  29/34  expression in CIP-macrophages as compared to WT Marquez et al, FEBS 2009 Bremen Summerschool 2010

  35. Proteins with modified expression in CIP-resistant cells Bremen Summerschool 2010

  36. WT CIP-R 25 µg 50 µg 25 µg 50 µg Dnajc3 ► kDa WT CIP-R 25 µg 50 µg 25 µg 50 µg - 130 - Tlr7 ► - 100 - Proteins with modified expression in CIP-resistant cells On the same chromosome as mrp4 ! Transports nucleosides/tides(substrates for Mrp4 !) Bremen Summerschool 2010

  37. Karyotype of J774 cells mrp4 Bremen Summerschool 2010

  38. chromosome 14 Cellsresistant to 0.1 mM Cellsresistant to 0.15 mM mrp4 Gene amplification in CIP-resistant cells chromosome 14 mrp4 FISH analysis WT cells Bremen Summerschool 2010

  39. Gene amplification in CIP-resistant cells mFISH analysis of CIP-R cells Heterogeneity of cell population ! chromosome 5 chromosome 13 chromosome 16 Bremen Summerschool 2010

  40. WT CIP-R 25 µg 50 µg 25 µg 50 µg DnajC3 ► Gene amplification in CIP-resistant cells FISH analysis of CIP-R cells Mrp4 and DnajC3 co-amplified in CIP-resistant cells Mrp4 Dnajc3 Bremen Summerschool 2010

  41. Implication of co-amplified genes in resistance DnajC3 protects CIP-resistant cells from ER stress Quantification of ER-stress signaling with a Dual Luciferase Assays of cells transfected with Cignal ERSE reporter 12 negative controlstandard medium+ tunicamycin (0.5 µg/ml) 10 8 Relative luciferase activity 6 4 2 0 CIP-R cells Wild type cells Bremen Summerschool 2010

  42. and in moxifloxacin-resistant cells? Die Bremer Stadtmusikanten Bremen Summerschool 2010

  43. Expression of ABC transporters TaqMan Low Density Array of ABC transporters Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  44. Overexpression of MDR transporters P-gp and Bcrp are functional Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  45. Overexpression of MDR transporters P-gp and Bcrp are functional but do not play a major role in FQ accumulation Vallet et al., FEBS-ABC meeting. (2010) Bremen Summerschool 2010

  46. Take home message Antibiotics are also substrates for efflux in eucaryotic cells Die Bremer Stadtmusikanten Bremen Summerschool 2010

  47. Take home message Closely related molecules behave very differently with respect to transport Die Bremer Stadtmusikanten Bremen Summerschool 2010

  48. Take home message Non substrates may induce a ‘anti-resistant’phenotype Die Bremer Stadtmusikanten Bremen Summerschool 2010

  49. Take home message Efflux is only part of the response to drug exposure Die Bremer Stadtmusikanten Bremen Summerschool 2010

  50. Coworkers … • B. Devreese and M. AertsProteomicsLaboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Belgium • E. Jacquet and N. NhiriTaqMan Low Density ArrayInstitut de Chimie des Substances Naturelles, CNRS, Gif sur Yvette, France • G. Ameyeand H. Antoine-PoirelFISHCentre de Génétique, Cliniques universitaires Saint-Luc UCL, Bruxelles, Belgium Bremen Summerschool 2010

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