Author of

• 13754

  +

  P1.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 224)

  • Event: WCLC 2015
  • Type: Poster
  • Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
  • Presentations: 1
  • Moderators:
  • Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)

  • 13756

    +

    P1.08-002 - Stat3 Is a Potential Target for Malignant Pleural Mesothelioma (MPM) Treatment (ID 1649)

    09:30 - 09:30  |  Author(s): T. Takuwa
    • Abstract

    Background:
    The prognosis of malignant pleural mesothelioma (MPM) is very poor; thus, a new drug treatment is necessary. Serum IL-6 is high in patients with MPM because of the activation of IL-6/Stat3 pathway. Thus, we investigated Stat3 as a potential target for the treatment of MPM.
    
    Methods:
    Cell viability was examined using the Cell Counting Kit-8 (CCK-8: WST-8 Dojindo). MPM cell lines (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, and MSTO-211H) were seeded onto 96-well plates. After treatment with Stattic, a Stat3 inhibitor, CCK-8 solution was added to each well and absorbance was measured using a microplate reader. Phosphorylated Stat3 levels (p-Stat3) were measured in cell lysates using the InstantOne ELISA assay (eBioscience). The expression of p-Stat3, E-cadherin, and vimentin was determined by western blot analysis. Translocated p-Stat3 was analyzed by confocal immunofluorescence microscopy. Cells were plated onto chamber slides containing medium. After the Stattic treatment, cells were fixed and cell membranes permeabilized. p-Stat3 antibody was added to chamber slides and incubated overnight at 4°C. Images were captured using a Zeiss LSM780 confocal microscopy system. Apotosis induced by Stat3 inhibitor was measured using the Caspase-GloR 3/7 assay (Promega). Cells were seeded onto 96-well plates. After the Stattic treatment, Caspase-GloR 3/7 reagent was added to each well, and the luminescence of each sample was measured in a plate-reading luminometer. For our in vivo study, H226 cells were subcutaneously injected into the flank region of nude mice. Mice were randomly assigned into two groups, with 5 mice in each group: vehicle control and Stattic (treated with10 mg/kg po 5 days per week).
    
    Results:
    Stattic inhibited viability of all MPM cell lines in a dose-dependent manner. IC50 values ranged from 3.3–106.0 μM. p-Stat3 levels decreased by 50% with 1 μM Stattic treatment in H226 cells. H226 cells were treated with 0.01 to 10 μM Stattic. Vimentin expression was stable; however, E-cadherin expression increased with 0.1, 1, and 10 μM Stattic treatment. In untreated H226 cells, p-Stat3 was observed in the cytoplasm and localized in the nucleus. In contrast, in Stattic-treated cells, decreased p-Stat3 was observed in the cytoplasm only, and it did not localize to the nucleus. Caspase 3/7 cleavage increased with Stattic treatment after 12 h and decreased after 48 h. In vivo mouse xenograft model, Stattic suppressed tumor growth (vehicle control vs.Stattic, P < 0.05).
    
    Conclusion:
    In this study, we have shown that Stattic inhibits proliferation of all MPM cell lines and suppresses tumor growth in a mouse model. In addition, we have demonstrated that Stattic inhibits Stat3 phosphorylation and blocks nuclear translocation. Furthermore, Stattic inhibits EMT. Thus, the STAT3 inhibitor is a promising candidate in MPM therapy.