Fenretinide-induced caspase 3 activity involves increased protein stability in a mechanism distinct from reactive oxygen species elevation.
Fenretinide (4-HPR) is a synthetic retinoid that displays a broad range of biological effects and has also demonstrated clinical efficacy as a chemopreventative agent. One cellular activity of 4-HPR is its ability to induce apoptosis. This effect has been proposed to relate to changes in intracellular reactive oxygen species. We show herein that a 1-h treatment of HL-60 cells with 4-HPR led to a dose-dependent increase in hydroperoxides. Pretreatment of cells with the antioxidant vitamin C abolished apoptosis, measured as the appearance of the sub-G1 peak, in 4-HPR-treated cells. The retinoid also elicited a 3.6-fold increase in caspase 3 activity; however, this increase was not affected by vitamin C treatment. Analysis of caspase 3 protein expression by Western blot analysis revealed that 4-HPR resulted in a significant increase in the appearance of the active p17 subunit without effecting a concomitant change in p32 procaspase 3 levels. Studies on de novo synthesis and stability of caspase 3 by pulse-chase and immunoprecipitation methods show that 4-HPR-treated samples had decreased incorporation of radioactive amino acid precursors into newly synthesized procaspase 3 but, during the chase (for up to 9 h), had more labeled caspase 3 remaining when compared with controls. These studies suggest that 4-HPR may effect changes in caspase 3 activity by modulating changes in zymogen stability by a mechanism distinct from the retinoid-elicited increase in reactive oxygen species.
DiPietrantonio
A M
AM
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Hsieh
T C
TC
Juan
G
G
Traganos
F
F
Darzynkiewicz
Z
Z
Wu
J M
JM
D000111:Acetylcysteine / Q000494:pharmacology; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism; D004795:Enzyme Stability / Q000187:drug effects; D017313:Fenretinide / Q000494:pharmacology*; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D011233:Precipitin Tests; D017382:Reactive Oxygen Species / Q000378:metabolism*
D016588:Anticarcinogenic Agents; D000975:Antioxidants; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D017382:Reactive Oxygen Species; D017313:Fenretinide; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D001205:Ascorbic Acid; D000111:Acetylcysteine