4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2.
We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (DeltaPsim), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.
Morales
Maria-Celia
MC
Department of Cell Biology and Histology, Faculty of Medicine and Dentistry, University of the Basque Country, Barrio Sarriena s/n, Leioa, Bizkaia, Spain.
Pérez-Yarza
Gorka
G
Rementería
Naiara N
NN
Boyano
María-Dolores
MD
Apraiz
Aintzane
A
Gómez-Muñoz
Antonio
A
Pérez-Andrés
Encarna
E
Asumendi
Aintzane
A
D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002518:Ceramides / Q000494:pharmacology*; D017313:Fenretinide / Q000494:pharmacology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007945:Leukemia, Lymphoid / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D019814:Myristic Acid / Q000378:metabolism; D018384:Oxidative Stress; D010710:Phosphates / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D016588:Anticarcinogenic Agents; C494826:BAX protein, human; D002518:Ceramides; D010710:Phosphates; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D013109:Sphingomyelins; D051028:bcl-2-Associated X Protein; D019814:Myristic Acid; D017313:Fenretinide