Protein degradation by the
proteasome generates functional intracellular
peptides. Pep5, a
peptide derived from
Cyclin D2, induces
cell death in
tumor cell lines and reduces the volume of
rat C6
glioblastoma tumors in vivo. Here, we chose the
human MDA-MB-231
breast cancer cells to evaluate the mechanism of
cell death induced by pep5 in different phases of the
cell cycle. Fluorescently labeled pep5, monitored by real
time confocal microscopy, entered the
MDA-MB-231 cells 3 min after application and localized to the nucleus and
cytoplasm. Pep5-induced
cell death was increased when the
MDA-MB-231 cell population was arrested at the G1/S transition or in
S phase compared to asynchronous
cells. Pep5 induced permanent
extracellular signal-regulated kinase (ERK1/2)
phosphorylation in
MDA-MB-231 cells synchronized in G1/S or
S phase.
Affinity chromatography followed by
mass spectrometry identified CLIC1 and
Plectin as the only two
proteins that interacted with pep5 in both asynchronous and synchronized
MDA-MB-231 cells. These interactions could explain the long-lasting ERK1/2
phosphorylation and the
cytoskeleton perturbations in the
MDA-MB-231 cells, in which the
stress fibers' integrity is affected by pep5
treatments. These data suggest that pep5 has potential
therapeutic properties for treating specific types of
cancers, such as
breast cancer cells.
Biological significance:
Pep5, a natural intracellular
peptide formed by the degradation of
Cyclin D2 through the
ubiquitin proteasome system, induces
cell death when reintroduced into MDA-MB-231
breast cancer cells, which express low levels of
Cyclin D2, specifically in G1/S arrested
cells or in
cells that are passing through
S phase. Under these conditions, pep5 is able to interact with different intracellular
proteins, primarily
cytoskeleton and
proteasome components, which can
lead to cellular
apoptosis. Together, our data suggest that pep5 is an intracellular
peptide with
therapeutic potential for treating specific types of
tumors with low expression of
Cyclin D2 by inhibiting
cell proliferation.