Apoptosis

, Volume 19, Issue 1, pp 117–134

Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1

  • Rodrigo Franco
  • Carl D. Bortner
  • Ingo Schmitz
  • John A. Cidlowski
Original Paper

DOI: 10.1007/s10495-013-0900-0

Cite this article as:
Franco, R., Bortner, C.D., Schmitz, I. et al. Apoptosis (2014) 19: 117. doi:10.1007/s10495-013-0900-0

Abstract

Glutathione (GSH) depletion is an important hallmark of apoptosis. We previously demonstrated that GSH depletion, by its efflux, regulates apoptosis by modulation of executioner caspase activity. However, both the molecular identity of the GSH transporter(s) involved and the signaling cascades regulating GSH loss remain obscure. We sought to determine the role of multidrug resistance protein 1 (MRP1) in GSH depletion and its regulatory role on extrinsic and intrinsic pathways of apoptosis. In human lymphoma cells, GSH depletion was stimulated rather than inhibited by pharmacological blockage of MRP1 with MK571. GSH loss was dependent on initiator caspases 8 and 9 activity. Genetic knock-down (>60 %) of MRP1 by stable transfection with short hairpin small interfering RNA significantly reduced MRP1 protein levels, which correlated directly with the loss of MRP1-mediated anion transport. However, GSH depletion and apoptosis induced by both extrinsic and intrinsic pathways were not affected by MRP1 knock-down. Interestingly, stimulation of GSH loss by MK571 also enhanced the initiator phase of apoptosis by stimulating initiator caspase 8 and 9 activity and pro-apoptotic BCL-2 interacting domain cleavage. Our results clearly show that caspase-dependent GSH loss and apoptosis are not mediated by MRP1 proteins and that GSH depletion stimulates the initiation phase of apoptosis in lymphoid cells.

Keywords

GlutathioneExtrinsicIntrinsicMRP1Multidrug resistanceMK571

Abbreviations

ABCC1

ATP-binding cassette, subfamily C, member 1

ABCG2

ATP-binding cassette (ABC) transporter, the subfamily G member 2

Apaf1

Apoptotic protease activating factor 1

Bcl-2

B-cell lymphoma 2

BID

BCL-2 interacting domain

Caspase

Cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases

CF

5-Carboxyfluorescein

CFDA

5-Carboxyfluorescein diacetate

C8DF

Caspase 8 deficient cells, I9.2 clone

C9DF

Caspase 9 deficient cells, JMR clone

C9RE

Caspase 9 deficient cells where caspase 9 has been re-introduced

Cyt C

Cytochrome C

CFTR

Cystic fibrosis transmembrane conductance regulator

DISC

Death-inducing signaling complex

DMSO

Dimethyl sulfoxide

FACS

Fluorescence activated cell sorting

FADD

Fas-associated protein with death domain

FADDDF

FADD deficient jurkat cells, I2.1 clone

FasL

Fas ligand

FLIP

Cellular FLICE-inhibitory protein

GLAST

Glutamate/aspartate transporters

GSH

Glutathione

GSSG

Glutathione disulfide

mBCl

Monochlorobimane

MMP

Mitochondrial membrane potential

MOMP

Mitochondrial outer membrane permeabilization

MRP1

Multidrug resistance protein 1

NAC

N-acetyl-l-gysteine

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

Nrf2

Nuclear factor (erythroid-derived 2)-like 2

OA

Organic anions

OATP

Organic anion transporting polypeptides

PI

Propidium iodide

ROS

Reactive oxygen species

RNS

Reactive nitrogen species

TNFα

Tumor necrosis factor-alpha

UVC

Ultraviolet C light

XIAP

X-linked inhibitor of apoptotic proteases

Copyright information

© Springer Science+Business Media New York (outside the USA) 2013

Authors and Affiliations

  • Rodrigo Franco
    • 1
  • Carl D. Bortner
    • 2
  • Ingo Schmitz
    • 3
    • 4
  • John A. Cidlowski
    • 2
  1. 1.Redox Biology Center, School of Veterinary Medicine and Biomedical SciencesUniversity of Nebraska-LincolnLincolnUSA
  2. 2.Laboratory of Signal Transduction, National Institute of Environmental Health Sciences (NIEHS)National Institutes of Health (NIH)Research Triangle ParkUSA
  3. 3.Laboratory of Systems-oriented Immunology and Inflammation Research, Institute of Molecular and Clinical ImmunologyOtto-von-Guericke University MagdeburgMagdeburgGermany
  4. 4.Division of Immune ControlHelmholtz Centre for Infection ResearchBrunswickGermany