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Françoise Van Bambeke, PharmD, PhD
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 09/06/2010 Bremen Summerschool 2010
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ATP-Binding Cassette transporters
ABCs transporters ABCA ABCB ABCC ABCD ABCE ABCF ABCG P-gp MRPs BCRP multidrug transporters 09/06/2010 Bremen Summerschool 2010 2
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Are antibiotics substrates for MDR transporters in eucaryotic cells ?
Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Fluoroquinolone antibiotics
ciprofloxacin moxifloxacin 09/06/2010 Bremen Summerschool 2010
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Kinetics of accumulation and efflux for ciprofloxacin
both accumulation and efflux markedly affected by probenecid (inhibitors of Mrps) extracell. conc. 17 mg/L; probenecid 5 mM Michot et al. (2004) AAC 48: 09/06/2010 Bremen Summerschool 2010
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Ciprofloxacin, classical model
Kolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42 09/06/2010 Bremen Summerschool 2010
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Kinetics of accumulation and efflux for moxifloxacin
neither accumulation nor efflux affected by probenecid Mox acc and efflux very rapid and not influenced by inhibitor Incidentilly, you may have seen that it reaches an acc level in control conditions very similar to that obtained with cipro but in the presence of prob extracell. conc. 17 mg/L; probenecid 5 mM Michot et al. AAC (2005) 49: 09/06/2010 Bremen Summerschool 2010
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Quinolones as inhibitors of ciprofloxacin efflux
ciprofloxacin efflux inhibited by ciprofloxacin Studying conc. effects on accumulation was also very informative. The graph shows the effect of cip on its own acc. Intriguingly … This is an unusual observation, because you would have expected either a linear relationship or a saturation effect. It can be however explained by an inhibition of the efflux by cip itself, resulting in a facilitation of its accumulation Michot et al. AAC (2005) 49: 09/06/2010 Bremen Summerschool 2010
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Quinolones as inhibitors of ciprofloxacin efflux
ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin not affected The same experiment done with moxi failed to show any effect. We may be tempted from these data as well as from kinetic studies that moxi is not transported. The next experiment however suggests that it is probablyy a too simplistic view. Michot et al. AAC (2005) 49: 09/06/2010 Bremen Summerschool 2010
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Quinolones as inhibitors of ciprofloxacin efflux
ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin CIP MXF CIP What we did here was to incubate the cells with a tracer amount of radiolabebeled cip and increasing conc of either cold cip or cold moxi to be able to measure specifically the effect of the two quinolones on cip accumulation. Regarding the effect of an excess of cip first, you recognize here the facilitating effect it exerts on the accumulation of radiolabeeld cip. Michot et al. AAC (2005) 49: 09/06/2010 Bremen Summerschool 2010
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Quinolones as inhibitors of ciprofloxacin efflux
ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin CIP MXF CIP moxifloxacin also able to interact with the transporter ! With an excess of unlabeled moxi now, you also oberve a facilitating effect on cip accumulation, with however higher conc of moxi needed to reach a same accumulation of cipro. Michot et al. AAC (2005) 49: 09/06/2010 Bremen Summerschool 2010
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Moxifloxacin, ‘futile-cycle’ model
Eytan et al. (1996) JBC 271: 09/06/2010 Bremen Summerschool 2010
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Die Bremer Stadtmusikanten
Can we make eucaryotic cells « resistant » to antibiotics ? A way to further characterize efflux transporters Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Over-expression of efflux pumps as mechanism of resistance
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: 09/06/2010 Bremen Summerschool 2010
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Chronical exposure to increasing concentrations
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: 09/06/2010 Bremen Summerschool 2010
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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: 09/06/2010 Bremen Summerschool 2010
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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: 09/06/2010 Bremen Summerschool 2010
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Ciprofloxacin « resistant » cells: phenotypic analysis
efflux rate IC50 gemfibrozil Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 09/06/2010 Bremen Summerschool 2010
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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 09/06/2010 Bremen Summerschool 2010
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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 09/06/2010 Bremen Summerschool 2010
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Ciprofloxacin « resistant » cells : which transporter ?
Specific extinction of gene expression by siRNA - CT 10 25 10 25 - CT 5 10 25 10 25 Mrp2 Mrp4 Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 09/06/2010 Bremen Summerschool 2010
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Acquisition of resistance is a stepwise process
Accumulation and Mrp4 expression during selection of resistance 09/06/2010 Bremen Summerschool 2010
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Can we select for moxifloxacin « resistant » cells ?
FQ accumulation and gemfibrozil effect ciprofloxacin moxifloxacin WT Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010
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Can we select for moxifloxacin « resistant » cells ?
FQ accumulation and gemfibrozil effect ciprofloxacin moxifloxacin WT MXF-RS Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010
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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) 09/06/2010 Bremen Summerschool 2010
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Moxifloxacin-exposed cells are « anti » resistant!
Mrp4 expression Western-Blot ARNm (Real Time PCR) WT MXF WT MXF - - RS CIP - RS Mrp4 Actin 20µg µg µg µg µg µg µg Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010
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Fluoroquinolone transport: model
ciprofloxacin WT CIP-RS MXF-RS 1 4 1 0.2 1 15 out in out in out in 09/06/2010 Bremen Summerschool 2010
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Fluoroquinolone transport: model
moxifloxacin WT CIP-RS MXF-RS 1 15 1 15 1 15 out in out in out in 09/06/2010 Bremen Summerschool 2010
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Efflux … is it enough for resistance ?
Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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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: 09/06/2010 Bremen Summerschool 2010
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Stable Isotope Labeling Aminoacid in Culture
WT CIP-macrophages 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg sample mixing 1:1 protein digestion identification in mass spectrometry and determination of the relative abundance 09/06/2010 Bremen Summerschool 2010
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Stable Isotope Labeling Aminoacid in Culture
WT CIP-macrophages 1.10 1.13 1.15 1.17 1.19 F1 F2 F3 F4 F5 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg post-nuclear homogenate ultracentrifugation 1.10 Discontinuous Sucrose gradient Enriched plasma membrane fraction 1.13 1.15 1.17 1.19 F1 F2 F3 F4 F5 190 - kDa 40 - MRP1 Prohibitin 09/06/2010 Bremen Summerschool 2010
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Stable Isotope Labeling Aminoacid in Culture
WT CIP-macrophages F1 F2 A B C F D E G H I J A B C F D E - 200 - 150 - 120 - 100 - 85 - 70 - 60 - 40 - 50 - 30 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg Discontinuous Sucrose gradient Enriched plasma membrane fraction G H I J k Combine 1:1 SDS-PAGE/in-gel digestion Protein ID & Quantification 09/06/2010 Bremen Summerschool 2010
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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 09/06/2010 Bremen Summerschool 2010
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Proteins with modified expression in CIP-resistant cells
09/06/2010 Bremen Summerschool 2010
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Proteins with modified expression in CIP-resistant cells
Dnajc3 WT CIP-R 25 µg 50 µg µg 50 µg ► On the same chromosome as mrp4 ! WT CIP-R 25 µg µg µg µg kDa ► Tlr7 Transports nucleosides/tides (substrates for Mrp4 !) 09/06/2010 Bremen Summerschool 2010
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Karyotype of J774 cells mrp4 09/06/2010 Bremen Summerschool 2010
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Gene amplification in CIP-resistant cells
chromosome 14 mrp4 FISH analysis Cells resistant to 0.1 mM WT cells chromosome 14 mrp4 Cells resistant to 0.15 mM 09/06/2010 Bremen Summerschool 2010
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Gene amplification in CIP-resistant cells
mFISH analysis of CIP-R cells Heterogeneity of cell population ! chromosome 5 chromosome 13 chromosome 16 09/06/2010 Bremen Summerschool 2010
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Gene amplification in CIP-resistant cells
FISH analysis of CIP-R cells Mrp4 and DnajC3 co-amplified in CIP-resistant cells DnajC3 WT CIP-R 25 µg 50 µg µg 50 µg ► Mrp4 Dnajc3 09/06/2010 Bremen Summerschool 2010
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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 control standard medium + tunicamycin (0.5 µg/ml) 10 8 Relative luciferase activity 6 4 2 Wild type cells CIP-R cells 09/06/2010 Bremen Summerschool 2010
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and in moxifloxacin-resistant cells ?
Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Expression of ABC transporters
TaqMan Low Density Array of ABC transporters Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010
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Overexpression of MDR transporters
P-gp and Bcrp are functional Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010
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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) 09/06/2010 Bremen Summerschool 2010
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Antibiotics are also substrates for efflux in eucaryotic cells
Take home message Antibiotics are also substrates for efflux in eucaryotic cells Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Die Bremer Stadtmusikanten
Take home message Closely related molecules behave very differently with respect to transport Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Non substrates may induce a ‘anti-resistant’ phenotype
Take home message Non substrates may induce a ‘anti-resistant’ phenotype Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Efflux is only part of the response to drug exposure
Take home message Efflux is only part of the response to drug exposure Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010
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Coworkers … B. Devreese and M. Aerts Proteomics Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Belgium E. Jacquet and N. Nhiri TaqMan Low Density Array Institut de Chimie des Substances Naturelles, CNRS, Gif sur Yvette, France G. Ameye and H. Antoine-Poirel FISH Centre de Génétique, Cliniques universitaires Saint-Luc UCL, Bruxelles, Belgium 09/06/2010 Bremen Summerschool 2010
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Brussels Stadtmusikanten for this story …
B. Marquez NE Caceres F. Van Bambeke PM Tulkens MP Mingeot JM Michot C. Vallet A. Anantharajah M. Heremans L. Depraetere V. Mohymont MC Cambier M. Vergauwen C Misson 09/06/2010 Bremen Summerschool 2010
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