Objective: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Keywords: 14; 29; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ATO; EGFR; FDA; Food and Drug Administration; GDC; GDC-0449; HPI; Hedgehog pathway inhibitor; Hh; IC(50); ITRA; MPM; MTT; PCR; PI; PTCH; RT; SMO; arsenic trioxide; drug concentration that mediates 50% inhibition of cell number or viability; epidermal growth factor receptor; hedgehog; itraconazole; mRNA; malignant pleural mesothelioma; messenger RNA; nontarget scramble control siRNA; patched; polymerase chain reaction; propidium iodide; reverse transcriptase/reverse transcribed; siNT; siRNA; siRNA targeting SMO; siSMO; small interfering RNA; smoothened.
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