Tropolones are small organic compounds with metal-directing moieties. Tropolones inhibit the proliferation of cancer cell lines, possibly through their effects on metalloenzymes such as select histone deacetylases (HDACs). Pan-HDAC inhibitors are therapeutically beneficial in the treatment of multiple myeloma, however there is interest in the use of more selective HDAC inhibitor therapy to minimize adverse side effects. We hypothesized that tropolones might have anti-myeloma activities. To this end, a series of novel α-substituted tropolones were evaluated for effects on multiple myeloma cells. While all tested tropolones showed some level of cytotoxicity, MO-OH-Nap had consistently low IC50 values between 1–11 μM in all three cell lines tested and was used for subsequent experiments. MO-OH-Nap was found to induce apoptosis in a concentration-dependent manner. Time course experiments demonstrated that MO-OH-Nap promotes caspase cleavage in a time frame that was distinct from the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Furthermore, MO-OH-Nap- and SAHA-treated cells possess unique gene expression patterns, suggesting they promote apoptosis via different mechanisms. In particular, MO-OH-Nap increases the expression of markers associated with endoplasmic reticulum stress and the unfolded protein response. Synergistic cytotoxic effects were observed when cells were treated with the combination of MO-OH-Nap and the proteasome inhibitor bortezomib. However, treatment with MO-OH-Nap did not abrogate the bortezomib-induced increase in aggresomes, consistent with an HDAC6-independent mechanism for the observed synergy. Collectively, these finding support further investigation into the usefulness of α-substituted tropolones as anti-myeloma agents.
Summary Interleukin 3 (IL-3) is produced constituitively by WEHI-3b leukaemic cells and stimulated lymphoid cell populations in vitro. We have investigated the in vivo production of IL-3 in mice rendered leukaemic with WEHI-3b cells and mice stimulated by acute graft versus host disease (GVHD). In leukaemic mice, IL-3 was not found in serum or sonicates of 18-day spleens or bone marrow, although cells from the leukaemic organs were fully competent to elaborate IL-3 in vitro. Further, elaboration of IL-3 by WEHI cells in vitro was not affected by co-culture with normal haemopoietic cells. However, intracellular IL-3 was detected in leukaemic nodules isolated from the liver. Inhibitors specific for IL-3 were not found, although liver-cell conditioned medium and leukaemic nodule sonicates contained potent non-specific inhibitors of cell growth. At 21 days, intracellular IL-3 was also present in spleens and correlated with the presence of nonspecific inhibitors. In GVHD, no evidence for IL-3 elaboration in vivo was found, nor did lymphoid populations affected by GVHD spontaneously elaborate it in vitro; however, their competence to produce it was unaffected, as IL-3 was elaborated on subsequent mitogen stimulation in vitro.We also investigated the recovery and circulation of in vitro ...Indium-labelled IL-3 dependent cells after injection in vivo and the half-life of semi-purified IL-3. Dependent cells were not recovered after injection into irradiated recipients, although the cells recirculated for at least 24 hours. Inability to recover dependent cells was explicable on general cytotoxicity which masked potential recovery. The serum half-life of injected partially purified material with IL-3 activity was short (<30min). We conclude that the elaboration of IL-3 by leukaemic WEHI-3b is not an in vitro artifact and these results are discussed in relationship to other growth factors and the leukaemic state, and the origin of IL-3 dependent lines.
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. Disclosures 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.
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