We previously reported that the
aminopeptidase inhibitor
bestatin induced apoptosis in several human
leukemia cell lines. The present study was performed to examine whether
bestatin can also induce apoptosis in solid tumor cell lines.
Bestatin alone exhibited neither direct growth inhibition nor induction of apoptosis in the tumor cell lines examined. However, it significantly augmented the growth-inhibitory effect and induction of apoptosis by agonistic anti-Fas antibody (CH11). The augmentation by
bestatin was also observed with other death
ligands including
tumor necrosis factor-alpha (
TNF-alpha) in EBC-1 cells, a cell line sensitive to these death
ligands. However, the HeLa S3 cell line, which is insensitive to
TNF-alpha, showed no growth inhibition even by combination treatment.
Bestatin methyl ester, a more cell-permeable derivative of
bestatin with similar inhibitory activity to cytosolic neutral
aminopeptidase, potentiated cell growth inhibition of CH11 more efficiently than
bestatin. Other cytosolic neutral
aminopeptidase inhibitors such as
actinonin and
puromycin also augmented cell growth suppression by CH11, while an enantiomer of
bestatin lacking
aminopeptidase inhibitory action did not increase the growth-inhibitory effects of CH11. The combination of 10 microg/ml of
bestatin with CH11 promoted processing of
caspase 3 to the active form p17 and efflux of mitochondrial
cytochrome c into the cytosol more quickly and more intensely than CH11 alone. Inhibition of
aminopeptidase was not involved in dATP- and
cytochrome c-dependent
caspase 3-activation in a cell-free system.
Bestatin significantly augmented activation of
caspase 8, which is upstream of
cytochrome c efflux in the apoptosis cascade. These results suggested that intracellular neutral
aminopeptidase might play an important role in Fas- or
TNF-alpha-induced solid
tumor cell apoptosis.