Abstract
Background: We have previously demonstrated that the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) induces the overproduction of reactive oxygen species (ROS) in human leukemia cells, which in turn triggers the intrinsic (mitochondrial) apoptotic pathway. In order to study the role of glutathione in 4-HPR-induced apoptosis, the levels of this antioxidant were analyzed in cell lines which are sensitive and resistant to the effects of 4-HPR, and the effect of the modulation of glutathione levels on 4-HPR cytotoxicity was characterized. Materials and Methods: Mitochondrial membrane potential (Δæm) and the levels of glutathione were measured by flow cytometry. A fluorometric assay was used to measure intracellular ROS generation and Western blot was employed to analyze tissue transglutaminase expression. Results: 4-HPR generated large quantities of ROS in cell lines which expressed low glutathione levels, these cells being the most sensitive to the retinoid. The sensitivity of leukemia cells to 4-HPR could be modulated, either by increasing intracellular glutathione contents using all-trans retinoic acid (ATRA), or by decreasing it using DL-buthionine-S,R-sulfoximine (BSO). ATRA increased the level of expression of tissue transglutaminase, whereas inhibition of this enzyme led to enhanced apoptosis. Conclusion: Our findings indicate that the glutathione content contributes to determining the sensitivity of cells to 4-HPR and points to the potential application of glutathione-inhibiting agents as enhancers in 4-HPR-based therapies.
- N-(4-hydroxyphenyl) retinamide
- apoptosis
- reactive oxygen species
- glutathione
- tissue transglutaminase
- BSO
- all-trans retinoic acid (ATRA)
Footnotes
- Received December 8, 2004.
- Accepted March 22, 2005.
- Copyright© 2005 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved