Plitidepsin cellular binding and Rac1/JNK pathway activation depend on membrane cholesterol content.
Plitidepsin (aplidin) is a marine cyclic depsipeptide in phase II clinical development against several neoplasias. Plitidepsin is a potent inducer of apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We have reported that this activation depends on the early induction of oxidative stress, activation of Rac1 small GTPase, and the later down-regulation of MKP-1 phosphatase. Using Scatchard and saturation binding analyses, we have found that (14)C-labeled plitidepsin binds to a moderately high-affinity receptor (K(d) of 44.8 +/- 3.1 and 35.5 +/- 4.8 nM, respectively) in MDA-MB-231 breast cancer cells. Two minutes after addition to cells, half of the drug was membrane-bound and was subsequently found in the cytosolic fraction. At 4 degrees C, plitidepsin cellular binding was around 10-fold lower than at 37 degrees C but sufficed to induce cell death, suggesting that this process is triggered from the membrane. Depletion of plasma membrane cholesterol by short treatment with methyl-beta-cyclodextrin diminished plitidepsin binding and Rac1 and JNK activation. Rac1 is targeted to the plasma membrane by plitidepsin as shown by subcellular fractioning and immunofluorescence analysis followed by confocal microscopy. Methyl-beta-cyclodextrin blocked this effect. A subline of HeLa cells (HeLa-R), partially resistant to plitidepsin, showed similar affinity (K(d) of 79.5 +/- 2.5 versus 37.7 +/- 8.2 nM) but 7.5-fold lower binding capacity than wild-type HeLa cells. Moreover, HeLa-R cells had lower total (71%) and membrane (67%) cholesterol content and membrane-bound Rac1, and showed no Rac1 activation upon plitidepsin treatment. In conclusion, cellular plitidepsin uptake and induction of apoptosis via activation of the Rac1-JNK pathway is membrane-cholesterol dependent.
Suárez
Yajaira
Y
Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Arturo Duperier, 4, E-28029 Madrid, Spain.
González-Santiago
Laura
L
Zarich
Natasha
N
Dávalos
Alberto
A
Aranda
Juan F
JF
Alonso
Miguel A
MA
Lasunción
Miguel A
MA
Rojas
José María
JM
Muñoz
Alberto
A
D002462:Cell Membrane / Q000187:drug effects*; D002784:Cholesterol / Q000172:deficiency / Q000378:metabolism*; D047630:Depsipeptides / Q000493:pharmacokinetics / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010456:Peptides, Cyclic; D021381:Protein Transport / Q000187:drug effects; D014407:Tumor Cells, Cultured; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D047630:Depsipeptides; D010456:Peptides, Cyclic; D002784:Cholesterol; D048031:JNK Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein; C098980:plitidepsin