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Novel Piperazine-based Compounds Inhibit Microtubule Dynamics and Sensitize Colon Cancer Cells to Tumor Necrosis Factor-induced Apoptosis*

Open AccessPublished:December 12, 2013DOI:https://doi.org/10.1074/jbc.M113.499319
      We recently identified a series of mitotically acting piperazine-based compounds that potently increase the sensitivity of colon cancer cells to apoptotic ligands. Here we describe a structure-activity relationship study on this compound class and identify a highly active derivative ((4-(3-chlorophenyl)piperazin-1-yl)(2-ethoxyphenyl)methanone), referred to as AK301, the activity of which is governed by the positioning of functional groups on the phenyl and benzoyl rings. AK301 induced mitotic arrest in HT29 human colon cancer cells with an ED50 of ≈115 nm. Although AK301 inhibited growth of normal lung fibroblast cells, mitotic arrest was more pronounced in the colon cancer cells (50% versus 10%). Cells arrested by AK301 showed the formation of multiple microtubule organizing centers with Aurora kinase A and γ-tubulin. Employing in vitro and in vivo assays, tubulin polymerization was found to be slowed (but not abolished) by AK301. In silico molecular docking suggests that AK301 binds to the colchicine-binding domain on β-tubulin, but in a novel orientation. Cells arrested by AK301 expressed elevated levels of TNFR1 on their surface and more readily activated caspases-8, -9, and -3 in the presence of TNF. Relative to other microtubule destabilizers, AK301 was the most active TNF-sensitizing agent and also stimulated Fas- and TRAIL-induced apoptosis. In summary, we report a new class of mitosis-targeting agents that effectively sensitizes cancer cells to apoptotic ligands. These compounds should help illuminate the role of microtubules in regulating apoptotic ligand sensitivity and may ultimately be useful for developing agents that augment the anti-cancer activities of the immune response.

      Introduction

      Eukaryotic cell division involves replication of DNA in S phase followed by equal segregation of mitotic chromosomes during anaphase (
      • Bell S.P.
      • Dutta A.
      DNA replication in eukaryotic cells.
      ). Cell cycle checkpoints have evolved to ensure faithful DNA replication and chromosomal division. Cells that harbor defective cell cycle checkpoint regulators can result in genetic instability and aneuploidy, ultimately leading to tumor development (
      • Kastan M.B.
      • Bartek J.
      Cell-cycle checkpoints and cancer.
      ). Mitosis orchestrates multiple cellular changes and depends on many intricate signaling pathways, despite being the shortest phase of the cell cycle (
      • Cooper G.M.
      ). Signaling pathways, including kinases and several checkpoint proteins, spatiotemporally regulate dynamic chromosomal rearrangements and reorganization. It is because of this intricacy that mitosis is considered the most sensitive phase of the cell cycle (
      • Chan K.S.
      • Koh C.G.
      • Li H.Y.
      Mitosis-targeted anti-cancer therapies. Where they stand.
      ,
      • Flatt P.M.
      • Pietenpol J.A.
      Mechanisms of cell-cycle checkpoints. At the crossroads of carcinogenesis and drug discovery.
      ). Damage to cellular processes that affect mitosis can activate spindle assembly checkpoint, which delays progression into anaphase (
      • Musacchio A.
      • Salmon E.D.
      The spindle-assembly checkpoint in space and time.
      ). Prolonged arrest in mitosis makes the cells more sensitive to cellular insults, which has likely made mitosis a desirable target for chemotherapy (
      • Chan K.S.
      • Koh C.G.
      • Li H.Y.
      Mitosis-targeted anti-cancer therapies. Where they stand.
      ,
      • Meraldi P.
      • Draviam V.M.
      • Sorger P.K.
      Timing and checkpoints in the regulation of mitotic progression.
      ,
      • Rieder C.L.
      • Maiato H.
      Stuck in division or passing through. What happens when cells cannot satisfy the spindle assembly checkpoint.
      ,
      • Schmit T.L.
      • Ahmad N.
      Regulation of mitosis via mitotic kinases. New opportunities for cancer management.
      ). Aneuploidies and other genomic and chromosomal abnormalities can induce cellular stress on cancer cells and make them highly sensitive to agents that disrupt mitosis (
      • Manchado E.
      • Guillamot M.
      • Malumbres M.
      Killing cells by targeting mitosis.
      ).
      Microtubules form spindle fibers during mitosis that are critical for chromosomal alignment and segregation (
      • Walczak C.E.
      • Heald R.
      Mechanisms of mitotic spindle assembly and function.
      ,
      • Dujardin D.
      • Wacker U.I.
      • Moreau A.
      • Schroer T.A.
      • Rickard J.E.
      • De Mey J.R.
      Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment.
      ). Previous findings suggest that agents that target the mitotic spindle can make highly effective chemotherapeutic drugs. Successful use of several vinca alkaloids, taxanes, and other natural compounds for the treatment of human cancers has validated the effectiveness of microtubule-targeting drugs (
      • Pellegrini F.
      • Budman D.R.
      Review. Tubulin function, action of antitubulin drugs, and new drug development.
      ,
      • Budman D.R.
      New vinca alkaloids and related compounds.
      ,
      • Wang T.H.
      • Wang H.S.
      • Ichijo H.
      • Giannakakou P.
      • Foster J.S.
      • Fojo T.
      • Wimalasena J.
      Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways.
      ). Several other mitotic proteins have also emerged as potential targets of chemotherapy. These targets include kinases, motor proteins, proteasome inhibitors, and inhibitors of chromatin reorganizing proteins. Some of these newly developed compounds may provide clinical benefits over some of the presently used drugs (
      • Chan K.S.
      • Koh C.G.
      • Li H.Y.
      Mitosis-targeted anti-cancer therapies. Where they stand.
      ).
      One of the primary challenges of cancer chemotherapeutics is the targeting of cancer cells while sparing normal cells of the surrounding tissue (
      • Chari R.V.
      Targeted cancer therapy. Conferring specificity to cytotoxic drugs.
      ). The use of vaccines and immune stimulants to specifically target tumors has generated promising results. For colon cancer, complementing traditional chemotherapy with IL-2 and granulocyte/macrophage colony-stimulating factor was shown to significantly increase patient survival (
      • Correale P.
      • Tagliaferri P.
      • Fioravanti A.
      • Del Vecchio M.T.
      • Remondo C.
      • Montagnani F.
      • Rotundo M.S.
      • Ginanneschi C.
      • Martellucci I.
      • Francini E.
      • Cusi M.G.
      • Tassone P.
      • Francini G.
      Immunity feedback and clinical outcome in colon cancer patients undergoing chemoimmunotherapy with gemcitabine + FOLFOX followed by subcutaneous granulocyte macrophage colony-stimulating factor and aldesleukin (GOLFIG-1 Trial).
      ). However, immune stimulants can sometimes result in modest cell killing activity. Cell killing by the activated immune response includes direct cell killing by cytotoxic T cells and NK cells, as well as cell killing apoptotic ligands, such as TNF.
      We previously reported several novel synthetic small molecules that dramatically increase colon cancer cell death by TNF and other death ligands, while being unable to induce apoptosis on their own (
      • Chopra A.S.
      • Kuratnik A.
      • Scocchera E.W.
      • Wright D.L.
      • Giardina C.
      Identification of novel compounds that enhance colon cancer cell sensitivity to inflammatory apoptotic ligands.
      ). Interestingly, many of these compounds also induced mitotic arrest. To gain insight into the mechanisms of action of these compounds, we studied the structure-activity relationship of a particularly promising class of piperazine-based compounds. Here we report a structure-activity relationship study of this class of compounds and identify a highly active derivative, AK301. Furthermore, we show that AK301 hampers tubulin polymerization, triggers the formation of multiple microtubule organizing centers (MTOCs),
      The abbreviations used are: MTOC
      microtubule organizing center
      Ac-DEVD-AMC
      acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin
      SAR
      structure-activity relationship
      TNFR
      tumor necrosis factor receptor
      RIPA
      radioimmune precipitation assay.
      and increases the surface expression of TNFR1. Molecular docking studies indicate that AK301 binds to β-tubulin near the colchicine-binding site, but in a novel orientation. Lastly, AK301 was found to be more effective in sensitizing cancer cells to TNF-induced apoptosis than other known microtubule-destabilizing agents. We propose that AK301 and its derivatives represent a novel class of microtubule-targeting compounds that will be useful for studying the relationship between microtubule dynamics and apoptosis sensitivity. This class of compounds may also have beneficial therapeutic properties because of their ability to sensitize cancer cells to ligand-induced apoptosis.

      DISCUSSION

      We previously identified a class of small molecules that induced mitotic arrest in colon cancer cells and sensitized these cells to apoptosis in the presence of death ligands, such as TNF and FasL (
      • Chopra A.S.
      • Kuratnik A.
      • Scocchera E.W.
      • Wright D.L.
      • Giardina C.
      Identification of novel compounds that enhance colon cancer cell sensitivity to inflammatory apoptotic ligands.
      ). Here, we performed a detailed structure-activity relationship study on a piperazine-based compound (AK3) that was initially found to be highly effective at sensitizing colon cancer cells to apoptosis. Specifically, we identified 4-(3-chlorophenyl)piperazin-1-yl(2-ethoxyphenyl)methanone, referred to as AK301, which can induce mitotic arrest in colon cancer cells with an EC50 of ∼115 nm. This derivative is ∼5-fold more potent than the original AK3 compound identified in the initial screen. AK301 also increased the sensitivity of HT29 and HCT116 human colon cancer cell lines to TNF-induced apoptosis at relatively low concentrations. AK301 was also capable of inducing cell death in the presence of TRAIL and FasL. Our SAR studies also revealed a number of related compounds that were inactive for both mitotic arrest and TNF sensitization (AK302 and AK304). Together, these compounds indicate a close relationship between mitotic arrest and sensitivity to TNF-induced apoptosis. In addition, these molecules were employed to determine a potential cellular target leading to mitotic arrest and apoptosis.
      Characterization of the mitotic arrest state of AK301-treated cells indicated multipolar spindle assembly. The formation of the multipolar spindles was accompanied by appearance of multiple γ-tubulin and Aurora kinase A staining loci, which is consistent with disruption of centrosome regulation and bipolar spindle formation. Most cancer cells have over-replicated centrosomes (
      • Chan J.Y.
      A clinical overview of centrosome amplification in human cancers.
      ,
      • Krämer A.
      • Neben K.
      • Ho A.D.
      Centrosome replication, genomic instability and cancer.
      ), which are clustered at the poles during mitosis (
      • Gergely F.
      • Basto R.
      Multiple centrosomes. Together they stand, divided they fall.
      ); disruption of centrosome clustering by disruption of microtubule spindles by AK301 may prevent the concerted segregation of supernumerary centrosomes and lead to spindle multipolarity (
      • Saunders W.
      Centrosomal amplification and spindle multipolarity in cancer cells.
      ). These complex, multipolar structures are apparently difficult to resolve because cell division is significantly inhibited even after the removal of AK301. The degree of AK301-induced mitotic arrest is cell type-dependent. Proliferation of the WI38 lung fibroblast cell line was reduced by AK301, but cells arrested more frequently in G2 than in mitotic phase. Although the reason for this difference in arrest phase is not known, it may be related to the presence of functional cell cycle checkpoints in nontransformed WI38 cells. Previous studies have shown that the CHFR (checkpoint with forkhead and ring finger domains) protein is a critical component in the cellular response to mitotic stress (including stress induced by microtubule disruption) (
      • Chaturvedi P.
      • Sudakin V.
      • Bobiak M.L.
      • Fisher P.W.
      • Mattern M.R.
      • Jablonski S.A.
      • Hurle M.R.
      • Zhu Y.
      • Yen T.J.
      • Zhou B.B.
      Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.
      ,
      • Kang D.
      • Chen J.
      • Wong J.
      • Fang G.
      The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition.
      ). Cells expressing a functional CHFR protein can delay entry into mitosis, thereby preventing catastrophic events during mitosis (
      • Summers M.K.
      • Bothos J.
      • Halazonetis T.D.
      The CHFR mitotic checkpoint protein delays cell cycle progression by excluding Cyclin B1 from the nucleus.
      ,
      • Scolnick D.M.
      • Halazonetis T.D.
      Chfr defines a mitotic stress checkpoint that delays entry into metaphase.
      ). On the other hand, studies have shown that HT29 cells and other cancer cells down-regulate CHFR expression (through promoter hyper-methylation) and are more likely to enter mitosis and not recover (
      • Toyota M.
      • Sasaki Y.
      • Satoh A.
      • Ogi K.
      • Kikuchi T.
      • Suzuki H.
      • Mita H.
      • Tanaka N.
      • Itoh F.
      • Issa J.P.
      • Jair K.W.
      • Schuebel K.E.
      • Imai K.
      • Tokino T.
      Epigenetic inactivation of CHFR in human tumors.
      ,
      • Privette L.M.
      • Petty E.M.
      CHFR. A novel mitotic checkpoint protein and regulator of tumorigenesis.
      ). The lack of functional mitotic checkpoints likely contributes to the effectiveness of mitosis-targeting chemotherapeutic agents and may explain the different responses of HT29 and WI38 cells to AK301. However, microtubule disruption was observed in both arrested HT29 cells and WI38 interphase cells treated with AK301. This finding suggests that AK301 may target microtubules. This target is further supported by in vitro and in vivo tubulin polymerization studies and by in silico docking of AK301 to tubulin. It remains possible that AK301 interacts with other cellular proteins to achieve its effect of the cell cycle arrest and apoptosis, but all our dose-response and structure-activity studies point to microtubules as being an important target.
      Microtubules are filamentous polymers of the cytoskeleton, composed of repeating α/β-tubulin heterodimers, responsible for determining cell shape, motility, intracellular transport, and cell division (
      • Cooper G.M.
      ). Microtubules have long been known as the drivers of chromosome migration and chromosome segregation (
      • Barton N.R.
      • Goldstein L.S.
      Going mobile. Microtubule motors and chromosome segregation.
      ). Microtubules become highly dynamic during mitosis and generate bipolar spindles that capture the sister chromatids and align them at the equatorial plate (
      • Lodish H.
      • Berk A.
      • Zipursky L.
      • Matsurdaira P.
      • Baltimore D.
      • Darnell J.
      ,
      • Kline-Smith S.L.
      • Walczak C.E.
      Mitotic spindle assembly and chromosome segregation. Refocusing on microtubule dynamics.
      ). With proper chromosome alignment and cellular signaling, cells enter into anaphase and complete cell division (
      • Pesin J.A.
      • Orr-Weaver T.L.
      Regulation of APC/C activators in mitosis and meiosis.
      ). The importance of microtubules in mitosis has made them a fruitful target for cancer therapies. However, it is clear that not all tubulin disruptors are equally useful as cancer therapies. This may be due in part to their influence on apoptosis pathways. Here we show that among microtubule disruptors, AK301 is particularly potent at sensitizing cancer cells to TNF and other apoptotic ligands. The interaction of microtubules with apoptosis is not limited to disrupting agents because paclitaxel can also sensitize cancer cells to TNF (
      • Gonçalves A.
      • Braguer D.
      • Carles G.
      • André N.
      • Prevôt C.
      • Briand C.
      Caspase-8 activation independent of CD95/CD95-L interaction during paclitaxel-induced apoptosis in human colon cancer cells (HT29-D4).
      ). The mechanism by which apoptotic signaling is enhanced by microtubule targeting agents is not clear, but further study of this effect could improve our understanding of apoptosis regulation and may lead to the generation of more effective microtubule targeting agents. Our present data point to an increase in the expression of TNFR1 on the surface of cancer cells.
      Molecular docking was employed to assess the potential of AK301 binding to tubulin dimers. For this analysis, we focused on the microtubule binding sites for colchicine, paclitaxel, and vinblastine. Molecular docking defines energy-optimized ligand orientations formed between the drug and its receptors (
      • Taylor R.D.
      • Jewsbury P.J.
      • Essex J.W.
      A review of protein-small molecule docking methods.
      ). Molecular docking predictions of AK301 and its derivatives showed relatively high affinity for the colchicine-binding region of tubulin but docked in a different orientation than colchicine. Further analysis of these in silico complexes supported the significance of this binding position; we found that the longer chain ethoxy group of AK301 favored strong hydrogen bond interactions and positioned the chlorophenyl ring in a hydrophobic pocket. In summary, this theoretical structural analysis predicted the affinity of AK301 for tubulin.
      We propose that AK301 represents a novel class of mitotic inhibitors capable of inducing mitotic arrest on their own and inducing apoptosis in combination with TNF with high efficiency. How mitotic arrest leads to ligand-dependent cell death is not fully understood. We previously showed that mitotically arrested cells have increased cell surface expression of TNFR1 (
      • Chopra A.S.
      • Kuratnik A.
      • Scocchera E.W.
      • Wright D.L.
      • Giardina C.
      Identification of novel compounds that enhance colon cancer cell sensitivity to inflammatory apoptotic ligands.
      ). Increased TNF-TNFR1 interactions at the cell surface (or following TNF internalization) may increase the formation of death-inducing signaling complex and caspase-8 activation (
      • Eum H.A.
      • Vallabhaneni R.
      • Wang Y.
      • Loughran P.A.
      • Stolz D.B.
      • Billiar T.R.
      Characterization of DISC formation and TNFR1 translocation to mitochondria in TNF-α-treated hepatocytes.
      ,
      • Micheau O.
      • Tschopp J.
      Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.
      ), which has been observed in arrested cells. Interestingly, AK301 was the most potent TNF-sensitizing agent tested in these studies, relative to other well studied microtubule inhibitors (colchicine, nocodazole, and vincristine). How AK301 achieves such a high degree of TNF sensitization is not clear. Based on our tubulin polymerization assay, AK301 reduces the rate of tubulin polymerization, but does not prevent it completely (like colchicine). We speculate that AK301 interferes with tubulin polymerization, but just enough such that the cells can continue to deliver and present TNFR1 and/or Fas on the cell surface. However, it should be noted that AK301 might possibly interact with a microtubule-related target or an upstream target that affects tubulin polymerization. The activity of the AK301 class of compounds, both as effective mitotic inhibitors and as apoptotic ligand-sensitizing agents, suggests that they may be well suited for cancer treatment, particularly when used on cancers with a high inflammatory cell infiltrate or following treatment with an immune stimulant. For basic research applications, this class of compounds should help illuminate how microtubules are employed to regulate apoptosis sensitivity.

      Acknowledgments

      We thank Dr. Carol Norris (University of Connecticut Flow Cytometry and Confocal Microscopy Facility) for help with flow cytometry and confocal imaging. We also thank Michael Bond and Ping Yang for contributions to immunostaining and troubleshooting.

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