Abstract
Purpose: :
To evaluate the potential of proteasome inhibitors, a novel class of anti–tumor agents, for the treatment of retinoblastoma (Rb). The proteasome inhibitor bortezomib (PS–341, VelcadeTM), has been approved by FDA for the treatment of multiple myeloma, and is under investigation for several other malignancies. Bortezomib inhibits IΚB degradation and inactivates the transcription factor NF–ΚB. Since NF–ΚB is constitutively active in human Rb cell lines and promotes their survival, it is a therapeutic target for the treatment of Rb.
Methods: :
We evaluated the effect of bortezomib on the Rb cell lines Y79 and WERI–Rb1 in vitro using MTT assay, cell cycle analysis by flow cytometry with propidium iodide (PI), gene expression profiling, RT–PCR and immunoblotting.
Results: :
Exposure to bortezomib for 24h induced apoptosis (subG1 peak on PI analysis) in both Y79 and WERI–Rb1 cell lines, with LD50 values well within the range of clinically achievable concentrations (10 and 4.4 nM, respectively, calculated by MTT). Gene expression profiling and RT–PCR demonstrated that bortezomib upregulated mRNA levels for heat–shock proteins (hsp); other stress–response proteins, such as members of the ATF and GADD family; pro–apoptotic molecules, such as Bid, Noxa and PUMA; and several proteasome subunits, cell–cycle regulators; transcription factors, cytokines and solute carrier proteins, while it downregulated mRNA levels for anti–apoptotic and adhesion molecules. Bortezomib increased phospho–IΚB, total IΚB, p53, p21, p27, Noxa, PUMA, phospho–Jun, total Jun, Hsp70 and Hsp90 protein levels in Rb cells and resulted in cleavage of PARP, ICAD, caspase–9, caspase–3, caspase–12, and Bid, confirming an apoptotic mechanism. The pan–caspase inhibitor ZVAD–FMK abrogated bortezomib–induced apoptosis.
Conclusions: :
Proteasome inhibitors, such as bortezomib, induce a stress response and trigger caspase–dependent apoptosis in Rb cells at clinically achievable concentrations. These data provide both insights into the molecular mechanisms of anti–tumor activity of bortezomib and a rationale for future clinical trials of bortezomib in patients with Rb.
Keywords: retinoblastoma • apoptosis/cell death