A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells

Our plain-English summary of A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells, by A. Sato, H. Ueno, M. Fusegi, et al (Pharmacology, 2018).

The study

• Authors: A. Sato, H. Ueno, M. Fusegi, S. Kaneko, K. Kohno, N. Virgona, A. Ando, Y. Sekine, T. Yano
• Journal: Pharmacology
• Published: 2018;102(1-2):26-36
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Background: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1).

Methods: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1's promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown -(siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E.

Results: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by -siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27.

Conclusions: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

© 2018 S. Karger AG, Basel.

In plain English

This laboratory study tested T3E, a man-made derivative of α-tocotrienol (a vitamin E compound), on human malignant pleural mesothelioma cell lines grown in culture, H2452 and H28. Most results were reported only for H2452. The study did not examine asbestos exposure or patients. T3E reduced cell survival more as the dose rose, while the parent compound T3 had no effect. At 20 μmol/L, T3E caused apoptosis (programmed cell death) that increased from 12 to 24 hours. T3E raised levels of DKK1, a gene that blocks the Wnt growth-signaling pathway, and lowered CKAP4 and cyclin D. Blocking JNK, or silencing DKK1, reduced T3E's killing of the cells. T3E lowered the expression of DNA methyltransferase and histone deacetylase enzymes. It decreased DNA methylation and increased histone acetylation in the DKK1 promoter region. It increased H3K4 methylation activity but did not affect H3K9 or H3K27. The authors concluded that T3E could be a useful candidate for mesothelioma treatment. They said further investigation, including in vivo experiments, is needed.

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: In vitro laboratory experiment on human mesothelioma cell lines
• Who was studied: Two human malignant pleural mesothelioma cell lines, H2452 and H28, obtained from ATCC. Most experiments were reported for H2452 only; H28 results were described as similar but 'data not shown'.
• Exposure: No asbestos exposure was studied. Cells were treated with T3E (6-O-carboxypropyl-α-tocotrienol), mostly at 20 μmol/L. Comparison and test agents were its parent compound T3, Zebularine (a DNMT inhibitor), sodium butyrate (an HDAC inhibitor) and the JNK inhibitor SP600125.
• Main results:
  • In H2452 cells, T3E reduced cell viability in a dose-dependent manner (5 to 20 μmol/L); T3 had no effect.
  • 20 μmol/L T3E induced apoptosis in H2452 cells in a time-dependent manner over 12, 18 and 24 hours.
  • T3E increased DKK1 mRNA and protein levels in H2452 cells.
  • T3E reduced CKAP4 expression in H2452 cells.
  • T3E reduced cyclin D expression in H2452 cells.
  • Silencing CKAP4 (80% knockdown at the mRNA level) slightly decreased H2452 cell viability, but the decrease was not statistically significant.
  • T3E activated JNK in a dose-dependent manner in H2452 cells.
  • The JNK inhibitor SP600125 counteracted T3E-induced cytotoxicity in H2452 cells.
  • When DKK1 was silenced by siRNA, T3E-induced cytotoxicity in H2452 cells was rescued, with statistical significance.
  • 20 μmol/L T3E suppressed expression of DNMT1, 3A and 3B in H2452 cells.
  • T3E suppressed expression of HDAC subtypes 1, 2, 3 and 8 in H2452 cells.
  • Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) each decreased H2452 cell viability.
  • Knocking down DNMT1, 3A and 3B, and HDAC1, 2 and 3, increased DKK1 expression in H2452 cells.
  • T3E decreased DNA methylation and increased histone acetylation in the DKK1 promoter region.
  • T3E increased histone H3K4 methylation activity.
  • T3E had no effect on H3K9 or H3K27 methylation.
• The authors' conclusion: The authors concluded that T3E inhibits mesothelioma cell growth by enhancing DKK1 expression through inhibition of DNMTs and HDACs, and that T3E could be a useful candidate for mesothelioma treatment.
• Limitations the authors state:
  • The authors stated that further investigation, including in vivo experiments, is needed to fully establish the potential of T3E.

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: Malignant Mesothelioma, Apoptosis.

Related studies

• A redox-silent analogue of tocotrienol inhibits cobalt(II) chloride-induced VEGF expression via Yes signaling in mesothelioma cells — A. Sato, N. Virgona, et al. (2014)
• A Redox-Inactive Derivative of Tocotrienol Suppresses Tumor Growth of Mesothelioma Cells in a Xenograft Model — A. Sato, T. Arai, et al. (2019)
• Targeting epigenetic mediators of gene expression in thoracic malignancies — D.S. Schrump (2012)
• Genomic profiling of malignant peritoneal mesothelioma reveals recurrent alterations in epigenetic regulatory genes BAP1, SETD2, and DDX3X — N.M. Joseph, Y.Y. Chen, et al. (2017)
• Hypomethylation reduced the aggressive potential of human malignant mesothelioma cells — N.Y. Kim, M.C. Kim, et al. (2016)
• Environmental toxicants--induced epigenetic alterations and their reversers — M. Kim, M. Bae, et al. (2012)

Browse return to medical research.