Imipramine Blue, a Tricyclic Compound and An Inhibitor of NADPH Oxidase Induces G2/M Cell Cycle Arrest Followed by Apoptosis in Multiple Myeloma Cells and in Leukemia Cells

Background: Multiple myeloma is an incurable malignancy of plasma cells. While conventional and novel therapies have improved the outcome of patients with myeloma, new therapies and therapeutic targets are needed. NADPH oxidase represents a potential therapeutic target, as NADPH oxidase inhibitors block transduction of signals from cell surface receptors and the PI3 kinase pathway that are dependent on reactive oxygen species (ROS). We tested the effect of the NADPH oxidase inhibitor, Imipramine Blue (IB), on myeloma cell growth and activation of apoptosis in cell lines and in primary human myeloma cells. Methods: IB was synthesized by refluxing imipramine and michlers ketone in the presence of phosphorus oxychloride. MTT assays for myeloma cells (MM.1S, MM.1R, RPMI8226, and U266) were used to evaluate the cell viability. AnnexinV staining and cell cycle analysis was done by flow cytometry to assess the level of apoptosis and analyze cell cycle arrest. Western blotting was performed using antibodies to analyse the impact of IB on intracellular signaling targets. Results: Myeloma cells were treated with increasing concentrations of IB for 72 hrs. Cell viability assays demonstrated that treatment of myeloma cell lines with IB resulted in an 80% decrease in cell growth using concentrations of IB between 6 and 10 uM. To determine the cause of growth inhibition and further mechanism of action, assays for apoptosis and cell cycle were performed. Annexiv V staining demonstrated that myeloma cell lines underwent brisk and rapid apoptosis. Procaspases (caspase 8, 9, 3 and PARP) were extensively cleaved from concentrations as low as 0.5uM to 10uM of IB at the 48 hour timepoint. Also, extensive DNA damage was evident based on the elevated levels of phos-p53 and GADD45 in MM.1S cells. The cell cycle profile indicated that IB induces both G2/M cell cycle arrest in MM.1S cells as well as in RPMI8226 cell lines. In addition, IB overcomes the growth advantage by cytokines IL-6 and IGF1 in MM.1S cells. Primary tumor cells obtained from myeloma patients demonstrated a significant cell killing following IB exposure. Given single agent activity, we then combined IB with other active anti-myeloma agents. Combining IB either with the proteasome inhibitor bortezomib, perifosine (an AKT inhibitor) or honokiol, a natural extract of the magnolia flower (Ishitsuka; Blood; 2005) demonstrated significant increase in the level of apoptosis favoring the combination in myeloma cells. Additionally, BAY11-7082 is an inhibitor of cytokine-induced IKB-α phosphorylation enhanced the level of apoptosis in combination with IB in the U266 myeloma cell line which is relatively less sensitive to IB, suggesting inhibition of NF-KB pathway is involved in the IB mediated myeloma cell death. Conclusion: Imipramine Blue has a potential to induce both apoptosis and G2/M cell cycle arrest in myeloma cell lines and in primary myeloma cells. The cellular response seems to be mainly mediated through pathways independent of PI3 kinase pathway, causing extensive DNA damage, cell cycle arrest, and apoptosis.