OSU03012, a Novel Celecoxib Derivative, Induces Apoptosis in Multiple Myeloma Cells Independently of Caspase Activation.

Abstract

Multiple myeloma (MM) is a clonal disorder affecting terminally differentiated B cells, with the accumulation of plasma cells in the bone marrow. Previous studies showed that OSU03012 is a novel celecoxib derivative lacking cyclooxygenase-2 inhibitory activity that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID). Here, we examined the in vitro effect of OSU03012 in MM cell lines (U266, ARH-77, IM-9 and RPMI8226). Cytotoxicity data indicated that mean LC50 (lethal concentration 50%) of OSU03012 was 6.25±0.86 μM at 24 hours and 4.23±0.87 μM at 72 hours in these four cell lines. Using annexin V/PI (propidium iodide) flow cytometry assay, OSU03012 was shown to induce apoptosis in MM cells. OSU03012 activated caspases-8, -9, and -3, induced PARP (POLY ADP-RIBOSE Polymerase) cleavage, and reduced survivin and XIAP expression after 6 and 24 hour exposure. Although the caspase inhibitor Q-VD-OPH treatment strongly blocked OSU03012-induced PARP cleavage, it did not inhibit OSU03012-induced apoptosis of MM cells. The pan-caspase inhibitor z-VAD-fmk did not prevent OSU03012 mediated cell death. Cell death with OSU03012 treatment was associated with significant down-regulation of phospho-Akt. Several substrates of AKT, including phospho-GSK-3 beta (Ser9), phospho-FoxO1a (Ser256) and phospho-MDM2 (Ser166) were also down-regulated by OSU03012 drug. OSU03012 triggered both early (6h) and late (24h) down-regulation of cyclin D1 expression, but cyclin A and B1 expression was down-regulated only at 24h. There was no induction of p21 or p27 protein levels by OSU03012. After 24-hour exposure, low concentration (1–5 μM) OSU03012 arrested MM cell lines in the G1 phase of the cell cycle while high concentration (10 μM) OSU03012 induced G2 phase arrested. OSU03012 decreased both phospho-Stat3 (Ser727) and Stat3 expression. OSU03012 has on effect on phosphorylated MAP kinase kinase1/2 (pMEK1/2) but it decreased MEK1/2 expression at 24h. The expression levels of Bcl-2 family proteins, Bcl-2, Mcl-1, BAX, and BIM did not alter with OSU03012 treatment suggesting that Bcl-2 members may not play direct or significant roles in inducing cell death. Taken together, we conclude that OSU03012 is potently active against MM cells by predominantly caspase-independent mechanisms, and may involve downstream pathways consequent to phopho-Akt down-regulation. These studies provide preclinical rationale for investigating OSU03012 in the treatment of MM.