Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells

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.