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

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.

Phosphorylation of Rab5a Protein by Protein Kinase Cϵ Is Crucial for T-cell Migration

“…The human T-cell line HuT-78 was obtained from the American Type Culture collection (ATCC) and cultured as described (27)(28)(29)(30). Motility-inducing anti-LFA-1 (clone SPV-L7) was from Monosan (Cambridge Biosciences).…”

“…Cell Lysis, Co-immunoprecipitation, and Western Immunoblotting-Cell lysis was performed as described previously (27) with minor modifications. Briefly, cells were washed with icecold PBS and lysed in the lysis buffer containing Triton X-100 (1%) and protease inhibitors phenylmethylsulfonyl fluoride (2 mM), leupeptin (10 g/ml), and aprotinin (10 g/ml).…”

“…Confocal Imaging and Photoactivation-For confocal imaging and analysis, cells were seeded to rest or migrate on coverslips as described above and then fixed with 3% (w/v) paraformaldehyde in PBS (27). T-cells were permeabilized with 0.3% (v/v) Triton X-100 in PBS and blocked with 5% w/v BSA in PBS for 30 min.…”

Analysis of dynamic tyrosine phosphoproteome in LFA‐1 triggered migrating t‐cells

“…Peripheral blood T-lymphocytes (PBTLs) were isolated from healthy donors as described (Verma et al, 2008). Briefly, peripheral blood mononuclear cells were prepared using Lymphoprep® (Axis-Shield, Sweden) by density centrifugation.…”

“…Antibody-induced effects in this case are judged by morphological changes in the cells displaying migratory phenotypes and cytoskeletal rearrangement (Volkov et al, 2001, Verma et al, 2008. In the present study, we used the migration-triggering model system utilizing T-cell lymphoma line Hut78 that displays an activated T-cell phenotype (Volkov et al, 2001;Murphy et al, 2000).…”