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Plitidepsin Cellular Binding and Rac1/JNK Pathway Activation Depend on Membrane Cholesterol Content

Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain (Y.S., L.G.-S., A.M.); Pharma Mar S.A., Madrid, Spain (Y.S., L.G.-S.); Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain (N.Z., J.M.R.); Hospital Ramón y Cajal, Madrid, Spain (A.D, M.A.L.); Universidad de Alcalá, Madrid, Spain (M.A.L.); and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain (J.F.A., M.A.A.)

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 ¹⁴C-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°C, plitidepsin cellular binding was around 10-fold lower than at 37°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--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--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.

Address correspondence to: Prof. Alberto Muñoz, Instituto de Investigaciones Biomédicas "Alberto Sols," Arturo Duperier, 4, E-28029 Madrid, Spain. E-mail: amunoz{at}iib.uam.es

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