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A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin

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Abstract

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance, immune response, and development. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in variety of human chemotherapy resistant cancer cell lines, indicating their potential in the treatment of both solid tumors and tumors derived from the hemopoietic system. Pyrrolobenzoxazepine 4-acetoxy-5-(1-naphtyl)naphtho[2,3-b]pyrrolo[1,2-d][1,4]-oxazepine (PBOX-15) has been shown to depolymerize tubulin in vitro and in the MCF7 breast cancer cell line. We hypothesized that this may suggest a role for this compound in modulating integrin-induced T-cell migration, which is largely dependent on the microtubule dynamics. Experiments were performed using human T lymphoma cell line Hut78 and peripheral blood T-lymphocytes isolated from healthy donors. We observed that human T-lymphocytes exposed to PBOX-15 have severely impaired ability to polarize and migrate in response to the triggering stimulus generated via cross-linking of integrin lymphocyte function associated antigen-1 receptor. Here, we show that PBOX-15 can dramatically impair microtubule network via destabilization of tubulin resulting in complete loss of the motile phenotype of T-cells. We demonstrate that PBOX-15 inhibitory mechanisms involve decreased tubulin polymerization and its post-translational modifications. Novel microtubule-targeting effects of PBOX-15 can possibly open new horizons in the treatment of overactive inflammatory conditions as well as cancer and cancer metastatic spreading.

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Acknowledgements

This work was supported by a Grant from the Higher Education Authority of Ireland under the department of Education and Science’s Program for Research in Third Level Institutions and the Health Research Board of Ireland.

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Authors and Affiliations

  1. Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland

    Navin K. Verma, Eugene Dempsey, Jennifer Conroy, Peter Olwell, Anthony M. Davies, Dermot Kelleher & Yuri Volkov

  2. Department of Hematology, John Durkan Research Laboratories, Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland

    Anthony M. Mcelligott & Mark Lawler

  3. School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

    D. Clive Williams & Daniela M. Zisterer

  4. European Research Centre for Drug Discovery and Development, Dipartimento Farmaco Chimico Tecnologico, Universita’ di Siena, Via Aldo Moro 2, 53100, Siena, Italy

    Stefania Butini & Giuseppe Campiani

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  1. Navin K. Verma
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Correspondence to Yuri Volkov.

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Supplementary Fig. 1

Measurement of T-cell apoptosis due to PBOX-15 treatment by annexin V binding assay. Hut78 cells were pretreated with either vehicle (0.1% (v/v) ethanol; control; a, d), 1.0 μM PBOX-15 (b, e) or 4.0 μM paclitaxel (as a positive control; c, f) for 30 min (a, b, c) or 24 h (d, e, f). Annexin V binding assay in combination with propidium iodide was performed to assess the apoptosis by flow-cytometry. Lower right corner represents apoptotic cells. (DOC 23.5 kb)

Supplementary Fig. 2

Effect of PBOX-15 on LFA-1-induced migration of Hut78 cells. Cells were pretreated with either vehicle [0.1% ethanol; control] (a) or 1 μM PBOX-15 (b) for 30 min. Nuclei of the cells were stained with Hoechst. Cells were incubated on anti-LFA-1-coated 96-well plate, and time-lapse images at every 2-min intervals were recorded for 20 min using InCell-1000 Analyzer (GE Healthcare). Data were quantified using Image-Pro Plus 6.1 analysis software. (DOC 23.5 kb)

Supplementary Fig. 3

Effect of PBOX-15 on the organization of cytoskeletal MT and actin network in resting and LFA-1 stimulated T-cells. Hut78 cells were pretreated with either vehicle [0.1% (v/v) ethanol] (a, b) or 1.0 μM PBOX-15 (c, d) for 30 min and incubated on poly-L lysine (a, c) or anti-LFA-1 (b, d) coated Permanox® chamber slides for 4 h. After this time, the medium was carefully removed and cells were fixed in 3% PFA. Cells were incubated with mouse monoclonal anti-α-tubulin antibody for 1 h and then with Alexa488 conjugated anti-mouse secondary antibody and Alexa546 conjugated phalloidin for further 1 h at room temperature. After washing, nuclei of the cells were stained with Hoechst (blue). The organization of cytoskeletal MT (green) and actin (red) was visualized by confocal microscopy using a 100× oil immersion lens. Results shown are representative of three independent experiments. (DOC 25.0 kb)

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Verma, N.K., Dempsey, E., Conroy, J. et al. A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin. J Mol Med 86, 457–469 (2008). https://doi.org/10.1007/s00109-008-0312-8

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