The proteasome inhibitor bortezomib induces apoptosis in human retinoblastoma cell lines in vitro.
OBJECTIVE
To evaluate the potential of proteasome inhibitors, a novel class of antitumor agents, for the treatment of retinoblastoma. The proteasome inhibitor bortezomib (PS-341, Velcade; Millennium Pharmaceuticals, Cambridge, MA), approved by the US Food and Drug Administration for the treatment of multiple myeloma, is being studied for the treatment of several other malignancies. Among other effects, it inactivates the transcription factor nuclear factor-kappaB (NF-kappaB) by blocking the degradation of its inhibitor, IkappaB. NF-kappaB, which is constitutively active in human retinoblastoma cells and promotes their survival, represents a therapeutic target for patients with this malignancy.


METHODS
The authors evaluated the effect of bortezomib on the retinoblastoma cell lines Y79 and WERI-Rb1 in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry with propidium iodide, gene expression profiling, RT-PCR, and immunoblotting.


RESULTS
Bortezomib induced caspase-dependent apoptosis in both retinoblastoma cell lines at clinically achievable concentrations. Bortezomib upregulated heat-shock proteins, other stress-response proteins, proapoptotic molecules, cell-cycle regulators, transcription factors, cytokines, and several proteasome subunits and solute carrier proteins, whereas it downregulated antiapoptotic and adhesion molecules. Bortezomib also induced cleavage of caspases, Bid and poly(ADP-ribose) polymerase (PARP), and sensitized retinoblastoma cells to doxorubicin.


CONCLUSIONS
Bortezomib induces a stress response and triggers caspase-dependent apoptosis in human retinoblastoma cells at clinically achievable concentrations. This study provides insight into the molecular mechanism(s) of the antitumor activity of bortezomib and a basis for future preclinical studies leading to clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcomes in retinoblastoma.
Poulaki
Vassiliki
V
Angiogenesis/Laser Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA. poulakiv@hotmail.com
Mitsiades
Constantine S
CS
Kotoula
Vassiliki
V
Negri
Joseph
J
McMillin
Douglas
D
Miller
Joan W
JW
Mitsiades
Nicholas
N
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D005562:Formazans; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011480:Protease Inhibitors / Q000494:pharmacology*; D011719:Pyrazines / Q000494:pharmacology*; D019572:Retinal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D001897:Boronic Acids; D005562:Formazans; D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D011719:Pyrazines; D013778:Tetrazolium Salts; C079782:MTT formazan; D000069286:Bortezomib; D020169:Caspases