Thoracic Surgery Section, Division of Cardiothoracic Surgery, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
Molecular Oncology Program, Department of Surgery, Sylvester Comprehensive Cancer Center, Leonard M. Miller School of Medicine, University of Miami, Miami, Fla.
J Thorac Cardiovasc Surg. 2014 Jan;147(1):508-16. doi: 10.1016/j.jtcvs.2013.08.035. Epub 2013 Oct 4.
The present study sought to determine whether the Hedgehog (Hh) pathway is active and regulates the cell growth of cultured malignant pleural mesothelioma (MPM) cells and to evaluate the efficacy of pathway blockade using smoothened (SMO) antagonists (SMO inhibitor GDC-0449 or the antifungal drug itraconazole [ITRA]) or Gli inhibitors (GANT61 or the antileukemia drug arsenic trioxide [ATO]) in suppressing MPM viability.
Selective knockdown of SMO to inhibit Hh signaling was achieved by small interfering RNA in 3 representative MPM cells. The growth inhibitory effect of GDC-0449, ITRA, GANT61, and ATO was evaluated in 8 MPM lines, with cell viability quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was determined by annexinV/propidium iodide staining and flow cytometry.
SMO small interfering RNA mediated a two- to more than fivefold reduction of SMO and Gli1 gene expression as determined by real-time quantitative reverse-transcriptase polymerase chain reaction, indicating significant Hh pathway blockade. This was associated with significantly reduced cell viability (34% ± 7% to 61% ± 14% of nontarget small interfering RNA controls; P = .0024 to P = .043). Treating MPM cells with Hh inhibitors resulted in a 1.5- to 4-fold reduction of Gli1 expression. These 4 Hh antagonists strongly suppressed MPM cell viability. More importantly, ITRA, ATO, GANT61 induced significant apoptosis in the representative MPM cells.
Hh signaling is active in MPM and regulates cell viability. ATO and ITRA were as effective as the prototypic SMO inhibitor GDC-0449 and the Gli inhibitor GANT61 in suppressing Hh signaling in MPM cells. Pharmaceutical agents Food and Drug Administration-approved for other indications but recently found to have anti-Hh activity, such as ATO or ITRA, could be repurposed to treat MPM.
本研究旨在确定 Hedgehog（Hh）通路是否活跃，并调节培养的恶性胸膜间皮瘤（MPM）细胞的细胞生长，并评估使用 smoothened（SMO）拮抗剂（SMO 抑制剂 GDC-0449 或抗真菌药物伊曲康唑[ITRA]）或 Gli 抑制剂（GANT61 或抗白血病药物三氧化二砷[ATO]）抑制 MPM 活力的疗效。
通过小干扰 RNA 选择性敲低 SMO 以抑制 Hh 信号，在 3 种代表性 MPM 细胞中实现。在 8 种 MPM 系中评估 GDC-0449、ITRA、GANT61 和 ATO 的生长抑制作用，使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定细胞活力。通过 AnnexinV/碘化丙啶染色和流式细胞术测定细胞死亡。
SMO 小干扰 RNA 通过实时定量逆转录聚合酶链反应将 SMO 和 Gli1 基因表达降低了两到五倍以上，表明 Hh 通路被显著阻断。这与细胞活力明显降低（非靶向小干扰 RNA 对照的 34%±7%至 61%±14%；P=0.0024 至 P=0.043）相关。用 Hh 抑制剂处理 MPM 细胞导致 Gli1 表达降低 1.5-4 倍。这 4 种 Hh 拮抗剂强烈抑制 MPM 细胞活力。更重要的是，ITRA、ATO、GANT61 诱导代表性 MPM 细胞中显著的细胞凋亡。
Hh 信号在 MPM 中活跃并调节细胞活力。ATO 和 ITRA 与原型 SMO 抑制剂 GDC-0449 和 Gli 抑制剂 GANT61 一样有效，可抑制 MPM 细胞中的 Hh 信号。已批准用于其他适应症的药物，如 ATO 或 ITRA，最近发现具有抗 Hh 活性，可重新用于治疗 MPM。
