cytochrome-c-t and fludarabine

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, and resistance to fludarabine-based therapies is a major challenge in CLL treatment. Because CLL cells are known to have elevated levels of reactive oxygen species (ROS), we aimed to test a novel ROS-mediated strategy to eliminate fludarabine-resistant CLL cells based on this redox alteration. Using primary CLL cells and normal lymphocytes from patients (n = 58) and healthy subjects (n = 12), we showed that both fludarabine-resistant and -sensitive CLL cells were highly sensitive to beta-phenylethyl isothiocyanate (PEITC) with mean IC(50) values of 5.4 microM and 5.1 microM, respectively. Normal lymphocytes were significantly less sensitive to PEITC (IC(50) = 27 microM, P < .001). CLL cells exhibited intrinsically higher ROS level and lower cellular glutathione, which were shown to be the critical determinants of CLL sensitivity to PEITC. Exposure of CLL cells to PEITC induced severe glutathione depletion, ROS accumulation, and oxidation of mitochondrial cardiolipin leading to massive cell death. Such ROS stress also caused deglutathionylation of MCL1, followed by a rapid degradation of this cell survival molecule. Our study demonstrated that the natural compound PEITC is effective in eliminating fludarabine-resistant CLL cells through a redox-mediated mechanism with low toxicity to normal lymphocytes, and warrants further clinical evaluation.

Topics: Antineoplastic Agents; Caspase 3; Cell Death; Cytochromes c; Drug Resistance, Neoplasm; Glutathione; Glutathione Peroxidase; Humans; In Vitro Techniques; Isothiocyanates; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Oxidation-Reduction; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Vidarabine

Bortezomib is a peptide boronic acid inhibitor of the proteasome developed for cancer therapy. The compound is being evaluated currently in Phase II and III clinical trials. Here we characterized the effects and mechanisms of action of bortezomib in cells obtained from patients with chronic lymphocytic leukemia (CLL).. We exposed isolated CLL lymphocytes from >100 patients to various concentrations of bortezomib or other proapoptotic stimuli, and measured DNA fragmentation by propidium iodide staining and flow cytometry. We characterized the effects of bortezomib on release of apoptosis-associated mitochondrial factors and measured downstream effects on caspase activation using a fluorogenic substrate cleavage assay. We assessed potential effects of the drug on inhibitor of apoptosis protein family apoptosis inhibitors by immunoblotting. Finally, we quantified the effects of bortezomib on apoptosis in 5 patients on a Phase II clinical trial.. Bortezomib stimulated apoptosis more rapidly than positive controls (glucocorticoid and fludarabine), although substantial heterogeneity was noted with respect to the concentration of drug required to induce cell death. Bortezomib-induced apoptosis was associated with release of SMAC, apoptosis-inducing factor, and cytochrome c from mitochondria, but the drug did not affect levels of inhibitor of apoptosis protein family cell death inhibitors. Levels of apoptosis were marginally elevated in CLL cells obtained from 2 of 5 fludarabine-refractory patients treated with bortezomib in vivo.. Our data confirm that bortezomib, like other proteasome inhibitors, has proapoptotic activity in CLL cells.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Caspases; Complement Membrane Attack Complex; Complement System Proteins; Cysteine Endopeptidases; Cytochromes c; DNA (Cytosine-5-)-Methyltransferases; Drug Resistance, Neoplasm; Enzyme Activation; Glucocorticoids; Glycoproteins; Humans; Inhibitor of Apoptosis Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Mitochondria; Multienzyme Complexes; Neoplasm Recurrence, Local; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteins; Pyrazines; Tumor Cells, Cultured; Vidarabine; X-Linked Inhibitor of Apoptosis Protein