Received for publication May 3, 2006.
Revised July 31, 2006.
Accepted for publication August 23, 2006.

Aplidin^® cellular binding and Rac1/JNK pathway activation depend on membrane cholesterol content

Abstract

Aplidin^® is a marine cyclic depsipeptide in Phase II clinical development against several neoplasias. Aplidin^® is a potent inducer of apoptosis through the sustained activation of Jun N-terminal kinase (JNK). Recently we 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 Aplidin^® binds to a moderately high-affinity receptor (K_d of 44.8±3.1 nM and 35.5±4.8 nM, respectively) in MDA-MB-231 breast cancer cells. Two min after addition to cells half of the drug was membrane-bound, and was subsequently found in the cytosolic fraction. At 4°C Aplidin^® 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 Aplidin^® binding and Rac1 and JNK activation. Rac1 is targeted to the plasma membrane by Aplidin^® 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 Aplidin^®, showed similar affinity (K_d of 79.5±2.5 nM vs 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 Aplidin^® treatment. In conclusion, cellular Aplidin^® uptake and induction of apoptosis via activation of the Rac1-JNK pathway is membrane-cholesterol dependent.

Key words: Cdc42, rho, rac, other small G proteins, Jun Kinase, Mechanisms of cell killing/apoptosis

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