Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation.
Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.
Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, 4, Madrid E-28029, Spain.
L
L
González-Santiago
Y
Y
Suárez
N
N
Zarich
M J
MJ
Muñoz-Alonso
A
A
Cuadrado
T
T
Martínez
L
L
Goya
A
A
Iradi
G
G
Sáez-Tormo
J V
JV
Maier
A
A
Moorthy
A C B
AC
Cato
J M
JM
Rojas
A
A
Muñoz
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics*; D003300:Copper / Q000378:metabolism; D047630:Depsipeptides / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation / Q000187:drug effects; D019803:Glutathione Disulfide / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006367:HeLa Cells; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D051336:Mitochondrial Membranes / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010456:Peptides, Cyclic; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D047630:Depsipeptides; D017874:Immediate-Early Proteins; D010456:Peptides, Cyclic; C107953:RAC1 protein, human; D017382:Reactive Oxygen Species; D003300:Copper; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D048031:JNK Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D017027:Protein Tyrosine Phosphatases; D020830:rac1 GTP-Binding Protein; D002118:Calcium; D019803:Glutathione Disulfide; C098980:plitidepsin