Y. Zang et al., Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells, CELL DEAT D, 8(5), 2001, pp. 477-485
The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxy- phenyl]-2-naphta
lene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of
apoptosis in a variety of tumor cell types. However, the mechanism of its
action remains to be elucidated, Recent studies suggest that the lysosomal
protease cathepsin D, when released from lysosomes to the cytosol, can init
iate apoptosis, In this study, we examined whether cathepsin D and free rad
icals are involved in the CD437-induced apoptosis. Exposure of human leukem
ia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicat
ed by caspase activation, phosphatidylserine exposure, mitochondrial altera
tions and morphological changes. Addition of the antioxidants alpha -tocoph
erol acetate effectively inhibited the CD437-induced apoptosis. Measurement
of the intracellular free radicals indicated a rise in oxidative stress in
CD437-treated cells, which could be attenuated by alpha -tocopherol acetat
e, Interestingly, pretreatment of cells with the cathepsin D inhibitor peps
tatin A blocked the CD437-induced free radical formation and apoptotic effe
cts, suggesting the involvement of cathepsin D, However, Western blotting r
evealed no difference in cellular quantity of any forms of cathepsin D betw
een control cells and CD437-treated cells, whereas immunofluorescence analy
sis of the intracellular distribution of cathepsin D showed release of the
enzyme from lysosomes to the cytosol, Labeling of lysosomes with lysosomotr
opic probes confirmed that CD437 could induce lysosomal leakage. The CD437-
induced relocation of cathepsin D could not be prevented by a-tocopherol ac
etate, suggesting that the lysosomal leakage precedes free radical formatio
n. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to
block these effects of CD437, suggesting that the action of CD437 is RAR-i
ndependent, Taken together, these data suggest a novel lysosomal pathway fo
r CD437 induced apoptosis, in which lysosomes are the primary target and ca
thepsin D and free radicals act as death mediators.