Novel cell death pathways induced by N-(4-hydroxyphenyl)retinamide: therapeutic implications

Abstract

We previously reported that N-(4-hydroxyphenyl)retinamide (4HPR) inhibits retinoblastoma tumor growth in a murine model in vivo and kills Y79 retinoblastoma cells in vitro. In this work, we assayed different cell deathrelated parameters, including mitochondrial damage and caspase activation, in Y79 cells exposed to 4HPR. 4HPR induced cytochrome c release from mitochondria, caspase-3 activation, and oligonucleosomal DNA fragmentation. However, pharmacologic inactivation of caspases by the pan-caspase inhibitor BOC-D-fmk, or specific caspase-3 inhibition by Z-DEVD-fmk, was not sufficient to prevent cell death, as assessed by loss of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, disruption of mitochondrial transmembrane potential (#y m ), and ATP depletion. We found that 4HPR causes lysosomal membrane permeabilization and cytosolic relocation of cathepsin D. Pepstatin A partially rescued cell viability and reduced DNA fragmentation and cytosolic cytochrome c. The antioxidant Nacetylcysteine attenuated cathepsin D relocation into the cytosol, suggesting that lysosomal destabilization is dependent on elevation of reactive oxygen species and precedes mitochondrial dysfunction. Activation of AKT, which regulates energy level in the cell, by the retinal survival factor insulin-like growth factor I was impaired and insulin-like growth factor I was ineffective against ATP and #y m loss in the presence of 4HPR. Lysosomal destabilization, associated with mitochondrial dysfunction, was induced by 4HPR also in other cancer cell lines, including PC3 prostate adenocarcinoma and the vascular tumor Kaposi sarcoma KS-Imm cells. The novel finding of a lysosome-mediated cell death pathway activated by 4HPR could have implications at clinical level for the development of combination chemoprevention and therapy of cancer. [Mol Cancer Ther 2007;6(1):286 -98]

Glycogen Synthase Kinase 3β Regulates Cell Death Induced by Synthetic Triterpenoids

“…We previously reported a down-regulation of activated AKT by antiangiogenic chemopreventive compounds (28,49), which seems a general property of chemically different chemopreventive agents (50). The data presented in this work indicate that GSK3 can be considered another target of proapoptotic chemopreventive agents, in addition to AKT.…”

“…The data obtained were expressed as percent LDH release relative to total LDH in culture. ATP was determined luminometrically by the CellTiter-Glo Luminescent Cell Viability assay (Promega) as described (28).…”

“…Total cell lysates and mitochondrial and cytosolic fractions were prepared as previously described (28). Protein quantitation was done with the DC Protein Assay kit (Bio-Rad).…”

Translocation of Lysosomal Cathepsin D Caused by Oxidative Stress or Proteasome Inhibition in Primary Cultured Neurons and Astrocytes

“…Furthermore, translocation of cathepsin D from lysosome-like granules was observed in tBHP-added cells as compared to the control (Ϫ). The addition of 1 mM lactacystin, a selective inhibitor of 20S proteasome, also caused chromatin condensation and translocation of cathepsin D. Based on the previous reports, [19][20][21][22] we consider that cathepsin D was translocated from lysosome to cytosol by oxidative stress or proteasome inhibition. However, since the translocated cathepsin D was not distributed throughout the cytosol but heterogeneously distributed in the cytosol, it was likely to be included in some aggregated form.…”

“…Oxidative stress triggered lysosomal destabilization and the diffusion of cathepsin D to cytosol in other cells. [19][20][21][22] Although the mechanisms of lysosomal destabilization are unclear, one possible mechanism is oxidative stress-induced peroxidation of the lysosomal membrane. A considerable amount of iron is held inside the lysosomal compartment as a result of the degradation of metalloproteins such as cytochromes.…”