Effects of the proteasome inhibitor, bortezomib, on apoptosis in isolated lymphocytes obtained from patients with chronic lymphocytic leukemia.
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
Our data confirm that bortezomib, like other proteasome inhibitors, has proapoptotic activity in CLL cells.
Pahler
Jessica C
JC
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. dmcconke@mdanderson.org
Ruiz
Stacey
S
Niemer
Irina
I
Calvert
Leslie R
LR
Andreeff
Michael
M
Keating
Michael
M
Faderl
Stefan
S
McConkey
David J
DJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D020169:Caspases / Q000378:metabolism; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins; D003546:Cysteine Endopeptidases / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D005938:Glucocorticoids / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008214:Lymphocytes / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects; D009097:Multienzyme Complexes / Q000187:drug effects; D009364:Neoplasm Recurrence, Local / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein
D000970:Antineoplastic Agents; D001897:Boronic Acids; D015938:Complement Membrane Attack Complex; D005938:Glucocorticoids; D006023:Glycoproteins; D051034:Inhibitor of Apoptosis Proteins; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011506:Proteins; D011719:Pyrazines; C047610:SC5b-9 protein complex; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D000069286:Bortezomib; D003165:Complement System Proteins; D045304:Cytochromes c; D004248:DNA (Cytosine-5-)-Methyltransferases; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D014740:Vidarabine; C024352:fludarabine