Les mécanismes immuno-physiopathologiques de l'anémie sévère
1 Régulation de lexpression des enzymes du métabolisme des médicaments par les xénorécepteurs...
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Transcript of 1 Régulation de lexpression des enzymes du métabolisme des médicaments par les xénorécepteurs...
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Régulation de l’expression des enzymes du métabolisme des médicaments par les Régulation de l’expression des enzymes du métabolisme des médicaments par les xénorécepteurs CAR et PXR et conséquences physiopathologiques.xénorécepteurs CAR et PXR et conséquences physiopathologiques.
Jean Marc PascussiJean Marc PascussiInstitut de Génomique FonctionnelleInstitut de Génomique Fonctionnelle
Inserm U661, MontpellierInserm U661, [email protected]@inserm.fr
InsermInsermInstitut nationalInstitut nationalde la santé et de la recherche médicalede la santé et de la recherche médicale
Xenobiotics
Xenobiotics: foreign chemicals
Inhaled
Ingested
absorbed
Pollutants
Industrial chemicals
Pesticides
Toxins produced by molds, plants and animals
Drugs
Les xénorécepteurs, sentinelles moléculaires de l’immunité métabolique
PXRPXR
AhRAhR
CARCAR
Phase IPhase I
Phase IIPhase II
Phase IIIPhase IIItransporteurstransporteurs
métabolisme
élimination
6
Different strategies for activating transcription factors
8
9
Nuclear Hormone Receptors superfamily
11
The Nuclear Receptor Gene superfamily
Nuclear receptors
The paradigm …
General scheme for activation of gene transcription by NRs
Robyr, Wolffe, Wahli Mol. Endocrinol 2000
Les xénorécepteurs CAR et PXR
ExpressionExpression
Organes de la détoxication: foie (hépatocytes) et tracus digestif (entérocytes)
PXRCAR
CAR and PXR coordinate the “metabolic immunity” in response to xenochemicals or endogenous activators
Nuclear Receptor PXR (NR1I2) … the master xenosensor
17
Nuclear receptor CAR (NR1I3)
Schéma de l’hétérodimère CAR-RXR ou PXR-RXR en présence de leurs ligands et différentes organisations des HREs. L’annotation x correspond au nombre de nucléotides entre les deux motifs
(d’après Handschin C & Meyer UA. Pharmacol Rev, 2003; 55, 649-73)
CAR ou PXR RXR
Acide rétinoïque 9-cisLigand
Gène cible
Les xénorécepteurs CAR et PXR partagent les mêmes NRE
2B6 3A4 2B6 3A4
CAR and PXR regulate the bodies 'garbage-disposal system.'
excretion
Uptake
Solubilisation
Phase I CYP2A,2B,3ACarboxylesterases
Phase IIGST
SULT2UGT1A
PAPSSP2
Phase IIITransport
MDR1A,1BMRP3…
Phase 0 OATP2
Les xénorécepteurs CAR et PXR
CITCO437 g.mol-1
823 g.mol-1
Ligands/activateursLigands/activateurs
• PXR (PXR (Polygamic Xenobiotic Receptor ?)Polygamic Xenobiotic Receptor ?)
Médicaments:Antibiotiques (rifampicine,etc.)Glucocorticoides, RU486, PCNAntifongiques (clotrimazole)Anticancéreux (paclitaxel, codermolide, ixabepilone, tamoxifene, cyclophosphamide)Inhibiteur de protéases virales (amprenavir, ritonavir)Inhibiteurs des pompes à protons (Benzimidazoles)Bloqueurs des canaux calciques (nifedipine)
Alimentation/Phytothérapie: Mille pertuisgluglustéroneb-carotèneGinko Biloba
Pesticides
Contaminants environnementaux
• CARCAR
Médicaments:Clotrimazole (agoniste inverse)Phenobarbital, phénytoïne
Phytothérapies:Artémise
Pesticides
CITCO (agoniste)
phénobarbital
232.17 g.mol-1rifampicine
hPXR LBD crystal structure(2.6 A resolution)
Watkins et al. Science 2001; Chrencik et al. Mol Endocrinol 2005Watkins et al. Science 2001; Chrencik et al. Mol Endocrinol 2005
Large and flexible ligand-binding pocket
Presence of 2 additional strands of b-sheet
Hydrophobic ligand-binding pocket
Can accommodates a single hydrophobic ligand in multiple conformation
Absence of a highly constrained pocket allows for molecular flexibility and plasticity in ligand recognition
Bound ligand= SR12813
Plasticity in the PXR Binding Pocket
hyperforinSR12813
1280 Å 1544 Å
Receptor Volume of ligand pocket (A3)
ER 476VDR 871PXR 1250-1550CAR 1120
hCAR/RXR LBD heterodimer crystal structure(2.6 A resolution)
Bound ligand:CITCO
RXRRXR CARCAR
Xu et al. Mol Cell 2004Xu et al. Mol Cell 2004
SRC-1 peptideSRC-1 peptide
Vol= 570-676 A
CAR activators provoke CAR nuclear translocation
NT
PB
CITCO
CAR-GFP in human hepatocytesIn vivo in mice
26
Mécanismes d’activation des xénorécepteurs CAR et PXR
CARCARPXRPXR
RXRRXR
cofactors
CARCAR
cytoplasmecytoplasme noyaunoyau
PBPBCCRPCCRP
OA AMPc/AMPAICAR
AMPKLKB1
HSPHSP
CARCAR
PXRPXRRIF
CitcoCitco
Thr-38-P
Thr-38
PP2APP2A
PKC
??
CAR
PXR, CAR
Complementary roles of CAR and PXR toward xenobiotics recognition ?
28
29
Species differences in CAR and PXR activation
(CYP3A1 DR3)2-tk-CAT
Variation in LBD consistent with in vivo species differences in response to inducers
CAR and PXR humanized mice
Wolf et al. J Clin Invest. 2008
xenobiotic
RXR CAR/PXR
Endocrine disruptionVitD3, T3 and lipids metabolism
Xenosensors that protect the body from a multitude of foreign chemicals (xenobiotics) and endogenous toxic compounds
Drug-drug and food-drug interactionsInterindividual variability in drug response
Ying and Yang of CAR and PXR
Metabolic Contribution
CYP 2D630%
CYP 1A22%CYP 2C9
10%
other3%
CYP 3A455%
CYP 3A4
CYP 2D6
CYP 2C9
CYP 1A2
other
hepatic only
also small intestine
Major Cytochrome P450s involved in the metabolism of
clinically used drugs ...
.... are PXR and CAR target genes
Nuclear receptor Activating drugs
PXR amprenavir, avasimibe, bosentan, carbamazepine, ciglitazone, clotrimazole, cortisone, corticosterone, cyclophosphamide, dexamethasone, efavirenz, exemestane, hydrocortisone, hyperforin, lovastatin, mifepristone, nelfinavir, nifedipine, omeprazole, paclitaxel, phenobarbital, phenytoin, rifabutin, rifampicin, ritonavir, simvastatin, spironolactone, tamoxifen, 4-hydroxytamoxifen,
troglitazone, troleandomycin, St John’s wort, Kava, Sophora flavescens
CAR CITCO, phenobarbital, phenytoinGarlic, Ginkgo
CAR and PXR are activated by widely used drugs and top selling phytochemicals
CAR and PXR xenosensors are involved in drug-drug interactions
CYPsCYP
enzymes
XeR RXRDrug A
OHDrug B Inactivation
activation
Xenosensors and drug-drug interactions
«Loss of analgesic effect of morphine due to coadministration of rifampin » Fromm et al. 1997 Pain
«Fatal paracetamol poisoning in an epileptic (phenytoin)» Minton et al. 1988 Hum Toxicol
«Profound drop of cyclosporin A whole blood through levels caused by St. John’s wort (Hypericum perforatum)» Breidenbach et al. 2000 Transplantation
Xenosensors and diet-drug interactions
«Osteomalacia associated with carbamazepine/valproate» Karaaslan et al. 2000 Ann Pharmacother
«Rifampicin induced osteomalacia» Shah et al. 1981 Tubercle
«Calcium metabolism during rifampicin and isoniazid therapy for tuberculosis» Brodie et al. 1982 J R Soc Med.
«Antiretroviral therapy and the prevalence of osteopenia and osteoporosis : a meta-analytic review»Brown et al. 2006 AIDS.
Role of CAR and PXR in drugs-induced osteomalacia and osteopenia ?
CYP3A4 (DR3, ER6)CYP2B6 (DR4)CYP2C9 (DR4)SULT2A1 (IR0)
PXRPXRRXRRXR VDRVDRRXRRXR RXRRXR CARCAR
NRENRE
Thummel et al. Mol Pharm 2001
Drocourt et al. J Biol Chem 2002
Echchgadda et al. Mol Pharmacol 2004
25(OH)D3
1,25(OH)D3
1,24,25(OH)D3(inactive)
7 déhydroxycholestérol
CYP27A1
CYP27B1
CYP24A1
VDRVDRRXRRXR
Déficits en vitamine D et ostéomalacies consécutives à la prise prolongée de médicaments… sur la piste de CAR et PXR
Les activateurs de CAR et PXR augmentent l’expression et l'activité de la CYP24 dans l’hépatocyte humain
Lambert 2008 TAP
Pascussi , 2005, JCI
CYP24 : nouveau gène cible de CAR et PXR
HepG2 HuH7
CAR et PXR transactivent les VDREs du gène CYP24
Konno 2008 Mol. Pharmacology
HuH7
The VDR-PXR cross-talk
CYP3A4CYP24
CYP3A4CYP24
XeR
CYP3A4CYP24
Drocourt 2002 JBC, Xu 2006 Mol P, Pascussi 2005 JCI, Moreau 2007 BBRC, Konno 2008 MP, Lambert 2008 TAP
CAR, PXR et la stéatose hépatique non alcoolique induite par certainsmédicaments.
Nakamura 2007 JBC
- Rifampicine traitement contre la tuberculose Stéatose hépatique Morere 1975 Sem Hop
- Carbamazepine traitement antiépileptique Stéatose hépatique Oscarson 2006 CPT
- Phenobarbital traitement antiépileptique Stéatose hépatique Calandre 1991 ANS
- Nifedipine traitement contre l’hypertensionStéatose hépatique Babary 1989 J Hep
Protocole double couleur - ARN de cultures traitées - ARN de référence
Extraction ARN
Culture primaire d’hépatocytes humains
Traitements (8 & 40h):DMSO 0.1%Phénobarbital (0.5mM)Rifampicine (10M)CITCO (100nM)
Etudes comparatives de transcriptomes d’hépatocytes humains... sur la piste d’un acteur peu connu de la lipogénese : Spot14
Spot14, THRSP(Chr. 11q13.5/14.1)
143AA, 17kDa, Pi=4.75
5
10
15
20
25
30 Actin CYP3A4 CYP2B6 S14
CTRL
Rif 0,3 (PXR)
Rif 3 (PXR)
Rif 30 (PXR)
SR12813 (PXR)
PAX (LXR)
T09 (PXR + LXR)
T3 (T3R)
FT
285
mR
NA
fol
d in
duct
ion
Les activateurs de PXR augmentent l’expression de Spot14 dans l’hépatocyte humain.
Spot14 est un gène cible de PXR
SFN
L’élément TRE de promoteur du gène Spot14 est nécessaire à l’action de CAR et PXR
Les activateurs de PXR augmentent l’expression de la FASN dans l’hépatocyte humain.
L’expression de Spot14 est nécessaire à l’induction de la FASN par PXR
L’activation de PXR provoque une accumulation d’acides grasdans l’hépatocyte humain.
Analyses lipidomiques:Quantification des acides gras dans les hépatocytes humains chromatographie liquide à ultra haute performance (UPLC) et spectrométrie de masse (Q-TRAP)
nm
oles
/g p
roté
ines
nm
oles
/g p
roté
ines
+/- RIF (72h)
Palm
itate
IE M
PE
+/- RIF (72h)
3% D2O (6h)
Mesure de la lipogenèse de novo :Quantification de l’incorporation de deutérium dans C16:0 par chromatographie en phase gazeuse et spectrométrie de masse
L’activation de PXR provoque une augmentation de la lipogenèse de novo
Michel BeylotINSERM ERI22
FG12 hS14
Ectopic overexpression of S14 increases lipogenesis in HepaRG cells
Palm
itate
IE M
PE
FG12 hS14 FG12
6mM Glucose
27m
M G
luco
se
FA quantifications
De novo lipogenesis
En conclusion:
L’activation des xénorécepteurs induit une forte perturbation du métabolisme lipidique provoquant une accumulation des acides gras et des triglycérides hépatiques.
XS
Role of CAR and PXR in interindividual variability in response to cancer therapy ?
Toxicity
Efficacy
Narrow therapeutic index
CAR and PXR regulate genes involved in biotransformation and clearance of widely used anticancer drugs
MRP1 Arsenic trioxide, chlorambucil, daunorubicin,doxorubicin, epirubicin, etoposide, melphalan, methotrexate, mitoxantrone, paclitaxel, vinblastine, vincristine
MRP2 Cisplatin, irinotecan doxorubicin, etoposide,methotrexate, SN-38, vinblastine, vincristine
MRP3 Carboplatin, cisplatin, doxorubicin,epirubicin, etoposide, methotrexate, teniposide, vinblastine, vincristine
BCRP Imatinib, methotrexate, mitoxantrone, SN-38, topotecan
CYP2A6 cyclophosphamide, ifosmamide, flutamine, tegafur
CYP2B6 altretamine, cyclophosphamide, ifosmamide, tomoxifen
CYP2C8 cyclophosphamide, docetaxel, ifosmamide, paclitaxel, tegafur, tretinoin
CYP3A4/5 Bexarotene, busulfan, cisplatin, cyclophosphamide, cytarabine, dexamethasone, docetaxel, doxorubicin, erlotinib, etoposide, exemestane, flutamide,fulvestrant, gefitinib, ifosfamide, imatinib, irinotecan, letrozole, medroxyprogresterone acetate, mitoxantrone, paclitaxel, tamoxifen, targretin, teniposide, topotecan,toremifene, tretinoin, vinblastine, vincristine, vindesine, vinorelbine
UGT doxorubicine, epirubicin, etoposide, irinotecan, topotecan, tamoxifen
SULT tamoxifen
GST busulfan, chlorambucil, cyclophosphamide, doxorubicin, ifosphamide, melphalan, nitrosurea
Role of CAR and PXR in pharmacokinetic drug-drug interaction in oncology, some examples
Rifampicin Phenobarbital Phenytoin Decrease CPT11 & SN38 and increase SN38-G plasma concentrations St John’s wort
RifampicinPhenytoin Decrease cyclophosphamide and increase 4-hydroxyclyclophosphamide plasma concentrationsphenobarbital
Severe toxicity
Therapeutic escape and risk of relapse.
rifampicin Increases of erlotinib clearance and reduces the AUC by 66¨%
Role of CAR and PXR in cyclophosphamide activation
Chang, Yu, Maurel, Waxman. Cancer Res., 1997De Jonge, Cancer Chemother Pharmacol. 2005
phenytoin
phenytoin
Role of CAR and PXR on peripheral metabolism of irinotecan (Campto ®)
CAR and PXR are expressed in several neoplastic human tissues
Neuroblastoma (PXR)Misawa , Cancer Res, 2005
Endomedrial cancer cells (PXR)Masuyama, Mol. Pharm., 2007
Hepatocarcinoma (CAR, PXR) Huang , Mol Endocrinol, 2005 Pascussi Hepatology 2007
Intestinal & colon cancer cells (CAR, PXR) Jiang, J Gastrointest Surg, 2009Raynal, Mol.Cancer, 2010Ouyang, Br J Cancer, 2010
Breast tissues (PXR)Dotzlaw, 1999Miki, Cancer Res, 2006
Lung cancer cells (PXR)Miki, Mol Cell Endocrinol, 2005
Ovarian cancer tissues (PXR)Gupta, Human Cnacer Biology , 2008
Osteosarcoma (PXR)Mensah-Osman, Cancer, 2007
Prostate cancer cells (PXR)Chen, Cancer res., 2008
Expression of PXR in Human Breast Carcinoma
PXROATP-A
Miki et al, Cancer Res 2006; 66: (1). January 1, 2006
LCM/RT-PCR(C) carcinoma cells (S) stromal cells
PXR expression in normal and cancerous human prostate tissues
Chen Y et al., Cancer Res 2007;67:10361-10367
Increased chemoresistance in PC-3 cells by PXR agonist, SR12813.
Chen Y et al., Cancer Res 2007;67:10361-10367
CTRL0.2 μM SR12813 1 μM SR12813
Increased chemosensitivity of PC-3 cells with PXR expression knocked down
Chen Y et al., Cancer Res 2007;67:10361-10367
Increased chemosensitivity of endothelial HEC-1cells with PXR expression knocked down
Masuyama, Mol. Pharm., 2007
SKOV-3
D. Gupta et al. Human cancer Biology, 2008
Increased chemoresistance in ovarian carcinoma cells by PXR agonist
Systemic drug clearance Cancer cells ?
Role of CAR and PXR in intra-tumoral metabolism of irinotecan ?
Raynal C. et al. Mol. Cancer Res 2010
PXR expression in normal and cancerous human colon tissues
SW620LS174T
PXR expression level restricts SN38 chemosensibility in colon cancer cells
PXR expression level restricts SN38 chemosensibility in colon cancer cells
Intracellular metabolites profiles (HPLC
PXR expression level enhances SN38 glucuronidation in colon cancer cells
SN38-G(inactive metabolite)
SN38(cytotoxic metabolite)
PXR UGT1As
PXR increases UGT1As-mediated SN38 inactivation in colon cancer cells
SN38 SN38-G
SN38-G(inactive metabolite)
SN38(cytotoxic metabolite)
CAR/PXRUGT1A1
Pro-drug activation Cancer cells ?
Role of CAR and PXR in intra-tumoral metabolism of cyclophosphamide ?
PXR regulates ALDH1A1 gene expression and Aldefluor® activity
Hepatic cells
Cancer cells
CAR and PXR may increase cancer cell resistance to 4-OH-CPA while promoting severe toxicity
Aldefluor®-positive cells are associated with cancer initiation properties, chemotherapy-resistance and poor clinical outcome
a
Aldehyde Dehydrogenase 1 is a marker for normal and malignant human colonic stem cells and have tumor initiating properties
CD44ALDH1Nuclei
Aldefluor®-positive Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy
Aldefluor®-positive cells display higher expression of PXR and PXR target genes
PX
R
AL
DH
1A
1
AB
CG
2
CY
P3
A4
OC
T4
LG
R5
CD
26
BM
I1
NA
NO
G
PXR
Aldefluor®-positive cells display enhanced clonogenic, sphere forming activity and magnétorésistance
ALDHbrALDHlow
Nb
sp
he
re /
10
0 c
ells *
Soft-agar (1000 cells) – 3 weeks
siRNA (sibGAL / siPXR) 100nM
Cell viability (ATP content)
PXR knock-down decreases chemoresistance of Aldefluor®-positive cells
ONCO TALK - 29/01/2013
PXR knock-down decreases xenogeneic tumor recurrence
Tumor formation
Treatment response
Relapse
Folfiri: 50mg/kg 5-FU30mg/kg irinotecan
15.000 cells 3D
Tumor cells dissociation
PXR knock-down decreases Aldefluor®-positive cells enrichment and tumor initiating activity after cytotoxics treatment
FOLFIRI 1500 cellsreinjection
PXR inhibition as a new strategy for Chemoresistant and Tumor Initiating Cells (CTIC) re-sensitization to conventional therapies ?
PXR inhibition& conventional
therapy
Conventional therapy
Les xénorécepteurs CAR et PXR
Gènes CiblesGènes Cibles
Enzymes et transpoteurs majeurs de la fonction de détoxication entérohépatique
OATP2CYP3A4,5,7
CYP2B6CYP2C8,9CYP1A2
UGT1A1,6,9GSTs
SULT2A1
MDR1MRP2-4
Adduit - Hépatite fulminante- HCC
Xénobiotique
CAR/PXR
Transporteurs Métabolites CYP450 Transférases
Interactionsmédicamenteuses
Molécule thérapeutique
inactifactif
Perturbations métaboliques
Perturbationsendocriniennes
- Lipides
- Vitamine DInteractions croiséesavec d’autres voies
cellulaires
89
90
Conclusion
Nuclear receptors PXR and CAR play a major part in this process by controlling a network of signaling pathways that regulate the expression of specific batteries of genes involved in the detoxication machinery
Cells and organisms are able to increase and adapt their capacity of detoxication in response to some xenobiotics and drugs
Because 1) many endocrine hormones are metabolised by CAR and PXR target genes, and 2) they interfere with other signalling pathways, chronic activation of these NRs (drugs, industrial or natural contaminants) could alter endocrine physiology and disease promotion.
According to their role as masters xenobiotic responsive receptors linking DME genes expression to environment stimuli, CAR and PXR might contribute to the well-known intra- and inter-subject variability in anticancer drugs response. Environmental and genetic factors affecting CAR or PXR (expression or activation levels) may affect the cytotoxic threshold of tumor cells to chemotherapy which can consequently mask or attenuate pharmacogenetic associations.