Abstract
Myeloma cells, by virtue of their robust production of monoclonal protein, are uniquely sensitive to therapeutic approaches such as proteasome inhibitors and histone deacetylase (HDAC) inhibitors which disrupt the homeostasis of the endoplasmic reticulum (ER)-proteasome-aggresome axis. Clinically available HDAC inhibitors are nonselective and it is hypothesized that more selective HDAC inhibitors would retain clinical benefit but limit off-target effects. Tropolones are seven-membered, non-benzenoid aromatic natural products. Beta thujaplicin and other tropolones have been shown to act as zinc chelators and inhibit selective metalloenzymes, including HDAC2 and 8, and to induce cytotoxic effects in leukemia cells. Given the sensitivity of myeloma cells to pan-HDAC inhibitors, we hypothesized that tropolones would induce cytotoxic effects but in a manner which may be distinct from the pan-HDAC inhibitors. We screened six novel synthetic alpha-substituted tropolones (figure 1) for cytotoxic activity against multiple myeloma cells (RPMI-8226, U266, MM.1S) using MTT assays. While all tested tropolones induced cytotoxicity in a concentration- and time-dependent manner, MO-OH-Nap and BA-SM-OH were noted to be the most potent agents (EC50 of 1.0 and 0.5 uM, respectively, in RPMI-8226 cells at 48 hrs). As BA-SM-OH was found to induce cell death within a very narrow concentration range, subsequent studies focused on MO-OH-Nap as the lead compound. Annexin V/propidium iodide flow cytometric studies demonstrated that MO-OH-Nap induces apoptosis in a concentration-dependent manner. To further explore the effects of this novel tropolone on apoptosis, immunoblot analysis was performed. Time course experiments (12-48 hrs) performed in RPMI-8226 and U266 cells demonstrated that SAHA and MO-OH-Nap induce caspase cleavage in a time-dependent manner albeit with different patterns: SAHA induces maximal cleavage of caspases-3, -8, and -9 at 24-to-36 hrs while MO-OH-Nap induces caspase cleavage at 36-to-48 hrs. While effects were similar between cell lines, it was noted that MO-OH-Nap induced caspase-8 cleavage in U266 cells but not in RPMI-8226 cells. Co-incubation of U266 cells with MO-OH-Nap and a caspase-8 inhibitor resulted in a further increase in caspase-9 cleavage, suggesting a compensatory increase in the intrinsic apoptotic pathway. As pan-HDAC inhibitors are known to have a synergistic interaction with proteasome inhibitors, we next performed MTT cytotoxicity studies in which cells were treated with the proteasome inhibitor bortezomib and/or MO-OH-Nap. Isobologram analysis revealed a synergistic interaction between the two agents in all tested cell lines. We have previously demonstrated that agents which disrupt Rab GTPase geranylgeranylation induce ER stress and apoptosis in myeloma cells. We next investigated whether MO-OH-Nap or SAHA could enhance the cytotoxic effects of Rab inhibitors. Lovastatin (HMG-CoA reductase inhibitor) and 3-PEHPC (geranylgeranyl transferase II inhibitor) were used as representative Rab inhibitors. MTT assays with combinations of these agents revealed a synergistic interaction with lovastatin and SAHA, lovastatin and MO-OH-Nap, and 3-PEHPC and SAHA. Interestingly, the interaction between 3-PEHPC and MO-OH-Nap varied amongst cell lines. Immunoblot analysis of cells treated with lovastatin and SAHA or MO-OH-Nap demonstrated enhanced cleavage of PARP, calnexin, caspase-3, caspase-8 and caspase-9 compared with either agent alone, particularly when cells were pre-treated with the Rab inhibitors for 24 hrs prior to co-incubation with SAHA or MO-OH-Nap for an additional 24 hrs. In conclusion, these studies establish MO-OH-Nap as a lead tropolone analogue from which to base further chemical modifications. This agent does induce apoptosis in myeloma cells and displays synergistic interactions with agents relevant to myeloma. Further studies will explore the mechanisms by which tropolones induce myeloma cell death and determine the clinical potential of these novel selective HDAC inhibitors.
Wiemer:Terpenoid Therapeutics Inc: Equity Ownership. Holstein:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.
