Nutlin-3 and Velcade Synergistically Induce Cell Cycle Arrest and Apoptosis in Multiple Myeloma through Activation of p53 Pathway
MN Saha, J Jayakar, H Chang. University Health Network and University of Toronto, Toronto, Canada
Background: p53 is rarely mutated in multiple myeloma (MM). p53 protein levels are regulated by murine double minute 2 (MDM2) through a well-established autoregulatory feedback loop. Modulation of the wild-type-p53 (wt-p53) levels in multiple myeloma (MM) has been reported by a recently developed small molecule, Nutlin-3 that antagonizes MDM2 and disrupts the p53-MDM2 interaction. Velcade, a proteasome inhibitor, has been shown to trigger apoptosis in MM cells in vitro. In this study, we investigated synergistic activation of the p53 by these two drugs and explored their effect on cell cycle arrest and apoptosis in MM cells.
Design: Two human myeloma cell lines MM1.S (shown to carry wt-p53) and LP1 (harboring mutant p53 gene) were used in this study. MM cells were exposed to velcade or nutlin-3 either individually or in combination, after which cell viability, cell cycle pathways and apoptosis was monitored. The degree of induction of p53 function was assessed by Western blot (WB) analysis of the samples immunostained with antibodies against p53 and its two immediately downstream transcriptional targets, p21 and MDM2.
Results: Both velcade and nutlin-3 up-regulated p53 expression in MM1.S cell lines. Combined treatment of 1 M of nutlin for 24 hr complemented with 5 nm of velcade for last 6 hr could further increase the up-regulation of expression of p53 along with p21 and MDM2 indicating a synergistic activation of p53. This synergistic effect was also manifested by 30-40% reduction in the number of viable cells. These events were associated with p53-dependent cell cycle arrest at G1-S check point as observed by a reduction of S phase fraction with an increase of the G1 phase fraction. Cell cycle arrest was followed by induction of apoptosis as evaluated by Annexin-V and propidium iodide (PI) binding and flow cytometry analysis and caspase-3 activation by WB analysis. Either velcade or nutlin-3 could lead to apoptosis individually. However, their combined treatment could lead to a significantly higher percentage of the cells into apoptosis. These events leading to specific p53 activation were observed only in the cells carrying wt-p53 (MM1.S) but not in the cells with mutant p53 (LP1).
Conclusions: Our data suggest that velcade and nutlin-3 can synergistically activate p53 pathway to induce cell cycle inhibition and apoptosis in vitro. The combination of these two drugs provides a potential novel therapeutic approach for MM patients.
Wednesday, March 11, 2009 9:30 AM
Poster Session V # 209, Wednesday Morning