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

Abstract

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 Ϯ 3.1 and 35.5 Ϯ 4.8 nM, respectively) in MDA-MB-231 breast cancer cells. Two minutes after addition to cells, half of the drug was membranebound 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 Ϯ 2.5 versus 37.7 Ϯ 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.

Plitidepsin Has a Dual Effect Inhibiting Cell Cycle and Inducing Apoptosis via Rac1/c-Jun NH2-Terminal Kinase Activation in Human Melanoma Cells

“…3C). These results parallel those recently reported for breast, renal, and cervical carcinoma cells (Gonzá lezSantiago et al, 2006;Suá rez et al, 2006). In addition, pretreatment with NSC23766, a specific Rac1 inhibitor, prevented the cytostatic/cytotoxic effect of plitidepsin in a concentration-dependent manner (Fig.…”

“…Our results demonstrate that in melanoma cells plitidepsin causes a rapid activation of Rac1 linked to a strong activation of JNK and p38 MAPK, as has been noted in breast, renal, and cervical carcinoma cells, which leads to a rapid induction of apoptosis (Cuadrado et al, 2004;Gonzá lezSantiago et al, 2006;Suá rez et al, 2006). However, in contrast to these other tumor types, in which plitidepsin does not modulate the cell cycle, at low concentrations it causes G 1 arrest and G 2 /M blockage in SK-MEL-28 and UACC-257 melanoma cells.…”

“…We have also detected that plitidepsin induces Rac1 translocation to cholesterol-rich membrane domains. Accordingly, plitidepsin activation of the Rac1/JNK pathway depends on membrane cholesterol content in MDA-MB-231 and HeLa cells (Suá rez et al, 2006). Moreover, plitidepsin activates other kinases such as the epidermal growth factor receptor, Src, p38 MAPK, extracellular signal-regulated kinase, and protein kinase C-␦ ( García-Ferná ndez et al, 2002;Cuadrado et al, 2003).…”

DNA Damage Induces the Accumulation of Tiam1 by Blocking β-TrCP-dependent Degradation

“…For example, acute activation of JNK through rapid expression of constitutive mutant Rac1 V12 led to apoptosis in thyroid cells (10). Plitidepsin, a potent inducer of apoptosis, was dependent on the Rac1/JNK pathway to function (11). All of these results strongly suggest that the Rac1/JNK cascade is important in DNA damage response-induced cell apoptosis.…”

Antitumour and antiangiogenic effects of Aplidin® in the 5TMM syngeneic models of multiple myeloma

“…In the subsequent studies, the mitochondrial changes were shown to be caused by different interactions. The sustained activation and phosporylation of c-Jun N-terminal kinase (JNK) by reactive oxygen species (ROS) is recognised as one of the crucial mechanisms in the effects of Aplidin on breast cancer cells (Garcia-Fernandez et al, 2002;Cuadrado et al, 2004;GonzalezSantiago et al, 2006;Suarez et al, 2006). This ROS induction was found to be caused by an alteration in the glutathione homeostasis and by a rapid activation of the small GTP-binding protein Rac (Cuadrado et al, 2003;Gonzalez-Santiago et al, 2006).…”