Study Of Bruton’s Tyrosine Kinase Inhibitor In Peripheral T-Cell Lymphomas Cell Lines Proliferation

BackgroundMalignancies derived from mature T cells and natural killer (NK) cells, collectively referred to as peripheral T-cell lymphomas (PTCLs), constitute a heterogeneous group of uncommon neoplasias, accounting altogether for less than 15% of all non-Hodgkin lymphomas worldwide.With few exceptions, PTCLs almost uniformly exhibit resistance to standard chemotherapy regimens and have a poor clinical outcome, so new therapeutic strategy is urgently needed.T-cell receptor (TCR) signaling is pivotal in T-cell development and function, and may play a role in peripheral T-cell lymphoma development in humans (PTCL/L). Itk, the key kinase of TCR signaling, was proved to affect T cell development and function, it was also demonstrated to be implicated in a human immune disorder. The Itk-Syk fusion protein was proved to drive oncogenesis in conditional mouse models of peripheral T cell lymphoma.Ibrutinib, is an irreversible BTK inhibitor that has been previously described. A first-in-class covalent inhibitor of BTK can bind at cysteine 481 with inhibitory of BTK. Ibrutinib was approved by FDA in November 2013 for the treatment of relapsed MCL and in February 2014 for relapsed/refractory CLL. In the future, ibrutinib would likely complement traditional immunotherapy as a frontline therapy. It also has the potential to obviate the need for chemoimmunotherapy. Since ibrutinib also has irreversible inhibitory effect on ITK, research in the work of ibrutinib on PTCLs may enrich the therapy of this group of aggressive diseases with poor outcomes。 However, the effect of ibrutinib on PTCLs was poorly understand, in this study, we investigated the antiproliferative and proapoptotic activity of ibrutinib in PTCL cell lines KARPAS-299 and HUT-78, and its association with Wnt signaling pathway.ObjectiveTo study the impact of Ibrutinb on PTCLs cell lines KARPAS-299 and HUT-78, and the expression of β-catenin and LEF-1 on mRNA and protein levels, to explore the new drugs for peripheral T-cell lymphomas.Methods1. We treated PTCLs cell lines KARPAS-299 and HUT-78 and T cell of healthy human with different concentration of ibrutinib(O,1,10,20,50, l00μumol), then we detect the cell viability with CCK-8, to observe the effect of ibrutinib on cell proliferation.2. We observed KARPAS-299 and HUT-78 morphology and growth characteristics by microscope,3. We chose four different concentration of ibrutinib(0,1,10,20μmol) to treat KARPAS-299 and HUT-78 for 24h, the we observed the cellular morphology with phase-contrast microscopy, and we detect the cell apoptosis with Annexin V-PI/FITC, to observe the effect of ibrutinib on cell apoptosis.4. We use RT-PCR to detect the change of P-catenin and LEF-1 gene expression in KARPAS-299 and HUT-78 after treated with different concentration of ibrutinib (0,1,10,20umol),to observe the effect of Ibrutinb on β-catenin and LEF-1 at mRNA level.5. We use western-blot to detect the change of β-catenin and LEF-1 protein expression in KARPAS-299 and HUT-78 after treated with different concentration of ibrutinib(0,1,10,20μKmol),to observe the effect of Ibrutinb on β-catenin and LEF-1 at protein level.Results1. After treated with ibrutinib, cell viability of KARPAS-299 and HUT-78 was obviously inhibited in a dose-dependent and time-dependent manner (p<0.01) but did not have obvious effect on T clls from healthy human (p>0.05)2. After treated with ibrutinib, the volume of cell mass and the quantity of cell decreased, the heteromorphic cell increased。3. After treated with ibrutinib, the apoptosis of KARPAS-299 and HUT-78 was obviously induced in a dose-dependent manner (p<0.01)4. Ibrutinb could inhibit β-catenin and lymphoid-enhancing factor-1 gene expression in KARPAS-299 and HUT-78 cell lines in a dose-dependent and time-dependent manner (p<0.01).5. Ibrutinb could inhibit β-catenin and lymphoid-enhancing factor-1 protein expression in KARPAS-299 and HUT-78 cell lines in a dose-dependent manner (p<O.O1).ConlusionsOur findings indicated that in PTCL, ibrutinib may be an effective treatment, and it take effect on cell viability may partly through Wnt signaling. Ibrutinib is probably a new drug for peripheral T-cell lymphomas.