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

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

Sep 24, 2023 179 likes •275 views

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 Hlne Cortay Institute of Biology and Chemistry of Proteins FR

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SLIDE 1

Influence of the Basal Cellular Level of Glutathione in Triggering MRP1-cells Death Lauriane Dury

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 10th French‐Belgian ABC meeting Brussels, Belgium

1

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SLIDE 2

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

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SLIDE 3

γGlu Cys Gly SUBSTRATES PHYSIOLOGICAL LOCALISATION

  • GSH et GSSG
  • LTC4
  • organic anions
  • glucuronate, sulfate or GSH

conjugates of endo‐ and xenobiotics TISSULAR:

  • ubiquitous
  • ++ in lung, testis, kidney,

skeletal and cardiac muscles…

  • ‐

in liver SUBCELLULAR:

  • basolateral

MRP1

  • 190 kDa
  • Transport of various substrates including glutathione

3

  • Implicated in oxidative stress (through GSH transport), detoxification, inflammation
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SLIDE 4

Selective cell death of tumoral MRP1-cells after stimulation

  • f

glutathione efflux COLLATERAL SENSITIVITY Total cellular glutathione after 3 h incubation with verapamil

20 40 60 80 100 120 5 10 15 20

[vrp] µM

% GSH / µg prot H69 H69 AR

NCI-H69 (control cells) H69AR (MRP1-cells)

[Verapamil] (µM) GSH (%)

Cellular proliferation after 72 h incubation with verapamil

20 40 60 80 100 20 40 60 80 [vrp] µM

% cellular proliferation

NCI-H69 H69AR

[Verapamil] (µM) Verapamil

MRP1, GSH & MRP1‐cells death

4

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SLIDE 5

[galangine] (µM)

20 40 60 80 100 120

% d e cellu les vivan tes

20 40 60 80 100 120 140 NCI-H69 H69AR

% cellular proliferation % cellular proliferation

[VRP] (µM)

20 40 60 80 100 120

% d e ce llu le s vivan tes

  • 20

20 40 60 80 100 120 140 NCI-H69 H69AR

verapami l galangin amtp14

5

[amtp14] (µM)

20 40 60 80 100 120

% de cellules vivantes

  • 20

20 40 60 80 100 120 140 NCI-H69 H69AR

% cellular proliferation

S N N S OH O NH2 O O OH O H OH

N CH3 C H3 CH3 MeO MeO OMe OMe N

GSH efflux (20µM) = 75% GSH efflux (20µM) = 85% GSH efflux (20µM) = 28%

3 « models » compounds

% cellular proliferation

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SLIDE 6

[amtp14] (µM)

20 40 60 80 100 120

% de cellules vivantes

  • 20

20 40 60 80 100 120 140

Involvement of glutathione in triggering MRP1‐cells death

Does Galangin‐triggered GSH efflux potentiate the cytotoxicity of verapamil or amtp14?

6

Galangin has a protective effect against the cytotoxicity of verapamil or amtp14.

[VRPRS] (µM)

20 40 60 80 100 120

% de cellules vivantes

  • 20

20 40 60 80 100 120 140 160 180 200

% cellular proliferation

[VRPRS] (µM) [amtp14] (µM)

% cellular proliferation

‐

galangine

+

galangine

‐

galangine

+

galangine

+ + ‐ ‐

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SLIDE 7

What is the involvement of basal GSH level in MRP1‐cells death triggered by verapamil? BSO= L‐buthionine sulfoximine

γ‐glutamyl‐transferase inhibitor

7

Involvement of glutathione in triggering MRP1‐cells death

NCI‐H69 H69AR

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.

Temps (heure)

24 48 72

G SH intracellulaire (nm

  • l/m

g de protéines)

10 20 30 40 50 BSO 50µM BSO 50µM + vérapamil 10µM

Effect of BSO and verapamil on NCI‐H69

GSH (nmol/mg proteins) Time (hours)

H69AR basal GSH level

[VRP] (µM)

20 40 60 80 100 120

% de survie cellulaire

  • 20

20 40 60 80 100 120 140 NCI-H69 H69AR NCI-H69 (+BSO 50µM)

% Cellular proliferation

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SLIDE 8

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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

  • f

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?

Conclusions