Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor

Our plain-English summary of Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor, by S.G. Gray, A.M. Baird, F. O'Kelly, et al (International Journal of Molecular Medicine, 2012).

The study

• Authors: S.G. Gray, A.M. Baird, F. O'Kelly, G. Nikolaidis, M. Almgren, A. Meunier, E. Dockry, D. Hollywood, T.J. Ekström, A.S. Perry, K.J. O'Byrne
• Journal: International Journal of Molecular Medicine
• Published: 2012;30(6):1505-11
• Cited by: 34 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyl-transferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

In plain English

This was a laboratory study of human cancer cell lines. It did not involve patients or asbestos exposure. The authors tested whether gemcitabine, a chemotherapy drug used with cisplatin for cancers including mesothelioma, acts like decitabine. Decitabine blocks enzymes called DNA methyltransferases, which help switch genes off by adding chemical tags (methylation) to DNA. Lung cancer, malignant pleural mesothelioma (H-28) and prostate cancer cell lines were treated for 48 hours. Both drugs switched back on several silenced genes, including sFRP4 in the mesothelioma cells and GSTP1 and IGFBP3 in prostate cells. Gemcitabine switched on RASSF1A in 3 of 4 cell lines tested; decitabine did so in 4 of 4. Gemcitabine reduced methyltransferase activity and affected levels of the DNMT1 protein to different degrees. However, for the most part it did not change DNA methylation, although unmethylated DNA increased at the GSTP1 gene in one prostate cell line. The authors concluded that gemcitabine inhibits and destabilises these enzymes and reactivates silenced genes, which may contribute to its anticancer activity.

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 study of cultured human cancer cell lines and recombinant enzymes
• Who was studied: Six commercially purchased human cancer cell lines: A549 (lung adenocarcinoma), SKMES1 (lung squamous cell carcinoma), H-28 (malignant pleural mesothelioma), and 22Rv1, LNCaP and Du145 (prostate)
• Exposure: Gemcitabine at 0.2, 0.5 or 1 µM for 48 hours, compared with decitabine (5 µM in the gene-expression experiments; 0.2 and 1.0 µM in the methylation measurements). No asbestos exposure was studied.
• Main results:
  • In the H28 mesothelioma cell line, both drugs reactivated the silenced gene sFRP4
  • In the LNCaP and 22Rv1 prostate cancer cell lines, both drugs significantly increased expression of GSTP1 and IGFBP3 (p less than 0.05)
  • Gemcitabine reactivated or increased RASSF1A in 3 of 4 cell lines tested; decitabine did so in 4 of 4
  • DNA methyltransferase activity was inhibited in nuclear extracts from cells exposed to either drug (shown for SK-MES-1 and H28 cells; also inhibited in recombinant enzymes)
  • Gemcitabine affected DNMT1 protein levels to different degrees in all cell lines examined
  • Gemcitabine did not grossly change global DNA methylation (measured at LINE-1 sequences); decitabine lowered methylation at 0.2 µM but not at 1 µM
  • For the most part, gemcitabine did not change methylation at the RASSF1A gene at either concentration
  • In 22Rv1 prostate cells, unmethylated DNA at the GSTP1 promoter increased after treatment with either drug
• The authors' conclusion: The authors concluded that gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates silenced genes, with activity equivalent to decitabine at concentrations lower than those reached in patients with solid tumours, and that this property may contribute to its anticancer activity
• Limitations the authors state:
  • The lack of methylation changes at reactivated genes may partly reflect the drug concentrations used
  • The initial genes were each examined in a different cell line
  • Methylation changes did not fully explain the gene reactivation; another mechanism may be involved

Topics

Medical terms in this study, explained in our Lexicon: Gemcitabine, Cisplatin, First-line Therapy, Non-Small Cell Lung Cancer, Mesothelioma.

Related studies

• Decitabine, differently from DNMT1 silencing, exerts its antiproliferative activity through p21 upregulation in malignant pleural mesothelioma (MPM) cells — S. Amatori, F. Papalini, et al. (2009)
• HDAC1-mSin3a-NCOR1, Dnmt3b-HDAC1-Egr1 and Dnmt1-PCNA-UHRF1-G9a regulate the NY-ESO1 gene expression — P.F. Cartron, C. Blanquart, et al. (2013)
• Progressive aberrant methylation of the RASSF1A gene in simian virus 40 infected human mesothelial cells — S. Toyooka, M. Carbone, et al. (2002)
• A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells, and modulated the expression of the human equilibrative transporter-1 — E. Giovannetti, L.G. Leon, et al. (2016)
• Inhibition studies of DNA methyltransferases by maleimide derivatives of RG108 as non-nucleoside inhibitors — G. Rondelet, L. Fleury, et al. (2017)
• Switch-maintenance gemcitabine after first-line chemotherapy in patients with malignant mesothelioma (NVALT19): an investigator-initiated, randomised, open-label, phase 2 trial — C.J. de Gooijer, V. van der Noort, et al. (2021)

Browse more research in International Journal of Molecular Medicine, or return to medical research.