Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death.

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.

Intracellular glutathione levels determine cell sensitivity to apoptosis induced by the antineoplasic agent N-(4-hydroxyphenyl) retinamide.

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 (deltaPsim) 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.