Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro

Our plain-English summary of Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro, by M. You, J. Varona-Santos, S. Singh, et al (The Journal of Thoracic and Cardiovascular Surgery, 2014).

The study

• Authors: M. You, J. Varona-Santos, S. Singh, D.J. Robbins, N. Savaraj, D.M. Nguyen
• Journal: The Journal of Thoracic and Cardiovascular Surgery
• Published: 2014;147(1):508-16
• Cited by: 63 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

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.

Copyright © 2014 The American Association for Thoracic Surgery. All rights reserved.

In plain English

This laboratory study used cultured malignant pleural mesothelioma cells, not patients, and did not examine asbestos exposure. The authors asked whether the Hedgehog signaling pathway was active in these cells and whether blocking it reduced their survival. In three representative lines, reducing SMO, a pathway component, with small interfering RNA lowered the pathway marker Gli1 and was associated with 40% to 60% less cell proliferation. Four blocking drugs were then tested on eight lines: GDC-0449, the antifungal itraconazole, GANT61 and the leukemia drug arsenic trioxide. All four lowered Gli1 levels in representative cells. All eight lines were very sensitive to itraconazole and arsenic trioxide, while GDC-0449 reduced viability only mildly to moderately. In a 60-hour cell-death experiment on representative cells, itraconazole, arsenic trioxide and GANT61 caused significant cell death, whereas GDC-0449 did not cause any discernible loss of viability. The authors noted that itraconazole and arsenic trioxide likely also act outside the Hedgehog pathway, and that culture conditions could affect how well GDC-0449 worked. They concluded that repurposing drugs already approved for other uses to treat mesothelioma might shorten drug development.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory (in vitro) study of cultured cancer cell lines
• Who was studied: 8 cultured malignant pleural mesothelioma cell lines (epithelioid, sarcomatoid, biphasic and one of unknown subtype); the SMO knockdown and several other experiments used 3 representative lines
• Exposure: Not asbestos. SMO expression was reduced with small interfering RNA, and the cells were treated with four Hedgehog pathway inhibitors (GDC-0449, itraconazole, GANT61, arsenic trioxide)
• Main results:
  • In 3 representative cell lines, reducing SMO with small interfering RNA lowered SMO and Gli1 expression and was associated with a 40% to 60% reduction in cell proliferation
  • 24 hours of treatment with the inhibitors reduced Gli1 messenger RNA 1.5- to more than 5-fold in representative cells
  • The inhibitors reduced cell number or viability in a dose-dependent way, with itraconazole having the strongest effect
  • In the growth assays, GDC-0449 produced only mild to moderate reductions in viability
  • All 8 cell lines were very sensitive to arsenic trioxide and itraconazole, with half-inhibitory concentrations printed as below 5 mM. For GDC-0449 the value was 15 to 20 mM in 5 of 8 lines and was not reached in 3 of 8. Units are as printed and may be micromolar.
  • In the 60-hour cell-death experiment on representative cells, itraconazole, arsenic trioxide and GANT61 caused significant cell death, while GDC-0449 did not cause discernible loss of viability
  • Arsenic trioxide, itraconazole and GANT61 also slowed the cells' progress through the cell cycle
• The authors' conclusion: The authors concluded that Hedgehog signaling is active in cultured mesothelioma cells and regulates their proliferation, and that repurposing drugs approved for other uses, such as itraconazole or arsenic trioxide, to treat mesothelioma might shorten drug development
• Limitations the authors state:
  • Itraconazole and arsenic trioxide likely have growth-inhibiting effects that do not act through the Hedgehog pathway
  • The cell culture conditions could have affected the level of Hedgehog activation and how well GDC-0449 worked
  • Hedgehog pathway activation has not yet been measured in mesothelioma tissue
  • The growth-inhibiting effect may not all come from SMO inhibition

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Apoptosis.

Related studies

• Discussion — (2014)
• Gli as a novel therapeutic target in malignant pleural mesothelioma — H. Li, N. Lui, et al. (2013)
• Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma — M. Meerang, K. Bérard, et al. (2016)
• Inhibition of hedgehog signaling reduces the side population in human malignant mesothelioma cell lines — H.A. Kim, M.C. Kim, et al. (2015)
• Inhibition of ZL55 cell proliferation by ADP via PKC-dependent signalling pathway — A. Muscella, L.G. Cossa, et al. (2018)
• Multipoint targeting of the PI3K/mTOR pathway in mesothelioma — S. Zhou, L. Liu, et al. (2014)

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