Lauriane Dury Influence of the Basal Cellular Level of Glutathione - PowerPoint PPT Presentation

10th French ‐ Belgian ABC meeting Brussels, Belgium Lauriane Dury Influence of the Basal Cellular Level of Glutathione in Triggering MRP1-cells Death Thesis Director : Dr Hélène Cortay Institute of Biology and Chemistry of Proteins – FR 3302 BMSSI UMR 5086 Drug Resistance Mechanism and Modulation Leader: Dr Attilio Di Pietro 1

MDR & ABC transporters � What is Multi Drug Resistance within cancer cells ? • Cross resistance to various chemotherapeutics • Active efflux of antitumoral drugs out of cancer cells through overexpressed ABC transporters -P-gp (ABCB1) -BCRP (ABCG2) -MRP1 (ABCC1) Overexpression : transport of drugs like chemotherapeutics 2

MRP1 γ Glu Cys • 190 kDa • Transport of various substrates including glutathione SUBSTRATES PHYSIOLOGICAL LOCALISATION Gly • GSH et GSSG TISSULAR: • LTC 4 • ubiquitous • organic anions • ++ in lung, testis, kidney, • glucuronate, sulfate or GSH skeletal and cardiac muscles… • ‐ conjugates of endo ‐ and in liver xenobiotics SUBCELLULAR: • basolateral • Implicated in oxidative stress (through GSH transport), detoxification, inflammation 3

MRP1, GSH & MRP1 ‐ cells death Total cellular glutathione after 3 h incubation with verapamil 120 100 % GSH / µg prot NCI-H69 (control cells) GSH (%) 80 H69AR (MRP1-cells) 60 H69 H69 AR 40 Verapamil 20 0 0 5 10 15 20 [vrp] µM [Verapamil] (µM) Cellular proliferation after 72 h incubation with verapamil % cellular proliferation Selective cell death of tumoral 100 80 MRP1-cells after stimulation of NCI-H69 60 H69AR glutathione efflux 40 20 � COLLATERAL SENSITIVITY 0 0 20 40 60 80 [Verapamil] (µM) [vrp] µM 4

3 « models » compounds 140 verapami NCI-H69 H69AR 120 % cellular proliferation % cellular proliferation tes l 100 GSH efflux (20µM) = 75% s vivan 80 le 60 MeO OMe N llu e ce 40 d 20 MeO N OMe % CH 3 0 H 3 C CH 3 -20 0 20 40 60 80 100 120 [VRP] (µM) galangin 140 NCI-H69 H69AR 120 % cellular proliferation GSH efflux (20µM) = 85% tes les vivan 100 80 e cellu H O O 60 d 40 % 20 OH 0 O OH 0 20 40 60 80 100 120 [galangine] (µM) 140 amtp14 NCI-H69 H69AR 120 % cellular proliferation de cellules vivantes GSH efflux (20µM) = 28% 100 O 80 60 S N S OH 40 N 20 % 0 NH 2 -20 5 0 20 40 60 80 100 120 [amtp14] (µM)

Involvement of glutathione in triggering MRP1 ‐ cells death Does Galangin ‐ triggered GSH efflux potentiate the cytotoxicity of verapamil or amtp14? 200 140 180 120 160 + + galangine galangine 100 140 % de cellules vivantes % de cellules vivantes % cellular proliferation % cellular proliferation 120 80 100 60 + 80 + 40 60 ‐ ‐ 40 20 ‐ 20 galangine ‐ galangine 0 0 -20 -20 0 20 40 60 80 100 120 0 20 40 60 80 100 120 [VRPRS] (µM) [VRPRS] (µM) [amtp14] (µM) [amtp14] (µM) � Galangin has a protective effect against the cytotoxicity of verapamil or amtp14. 6

Involvement of glutathione in triggering MRP1 ‐ cells death What is the involvement of basal GSH level in MRP1 ‐ cells death triggered by verapamil? BSO= L ‐ buthionine sulfoximine NCI ‐ H69 H69AR γ‐ glutamyl ‐ transferase inhibitor 140 NCI-H69 H69AR Effect of BSO and verapamil on NCI ‐ H69 120 NCI-H69 (+BSO 50µM) % Cellular proliferation 50 100 BSO 50µM g de protéines) % de survie cellulaire GSH (nmol/mg proteins) BSO 50µM + vérapamil 10µM 80 40 60 ol/m 30 intracellulaire (nm 40 20 20 H69AR basal GSH level 10 0 SH G -20 0 0 20 40 60 80 100 120 0 24 48 72 Time (hours) Temps (heure) [VRP] (µM) � When the GSH basal level in NCI ‐ H69 was brought to the same level as that of H69AR thanks to BSO, the verapamil effect was not equivalent in the two cell lines. 7

Conclusions � Low basal glutathione level might be important for triggering death of H69AR, but the depletion of GSH has to be achieved by MRP1 . � The mechanism by which verapamil kills MRP1 ‐ cells is complex: ‐ verapamil kills MRP1 ‐ cells through GSH efflux: production of ROS? Galangin protects against cell death maybe through its antioxidant properties. but ‐ amtp14 induces a collateral sensitivity for MRP1 ‐ cells without strong GSH depletion. Surprisingly, galangin also protects against amtp14 ‐ induced cell death. Is it the same mechanism than with verapamil? 8