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Investigational New Drugs

, Volume 37, Issue 6, pp 1158–1165 | Cite as

A phthalimidoalkanamide derived novel DNMT inhibitor enhanced radiosensitivity of A549 cells by inhibition of homologous recombination of DNA damage

  • Hyun-Cheol Kang
  • Eui Kyu Chie
  • Hak Jae Kim
  • Jin Ho Kim
  • Il Han KimEmail author
  • Kwangsoo Kim
  • Beom Su Shin
  • EunSook Ma
PRECLINICAL STUDIES
  • 194 Downloads

Summary

Purpose To elucidate the radiosensitizing effect and underlying mechanism of a new kind of DNA methyltransferase (DNMT) inhibitor with biological availability. Methods A novel non-nucleoside compound, designated as MA-17, was recently derived from a phthalimido alkanamide structure. DNMT expressions were confirmed in cultured human lung cancer (A549) and normal astrocyte (NHA) cells, radiosensitivity was measured using clonogenic assay, and assays of cell cycle alteration, apoptosis, DNA damage repair, and differential gene expression were undertaken. Results MA-17 significantly radiosensitized A549 cells with a mean dose enhancement ratio (DER) of 1.43 at the surviving fraction of 0.2 (p < 0.05 by one-tailed ratio paired t-test). MA-17 did not affect normal astrocytes (mean DER0.2, 1.016; p = 0.420). MA-17 demonstrated a mean half-life of 1.0 h in vivo and a relatively even distribution in various tissues. Pretreatment with MA-17 increased sub-G1 fractions and inhibited the repair of DNA double-strand breaks, which are induced by irradiation. We found that MA-17 also down-regulated DNA homologous recombination and the Fanconi anemia pathway (FANCA, BRCA1, and RAD51C) in A549 cells. This bioinformatics finding was confirmed in validation Western blot to evaluate the expression of vital proteins. Conclusions A novel phthalimido alkanamide derivative, a DNMT inhibitor, possessed both biostability and favorable and substantial radiosensitizing effects by augmenting apoptosis or inhibiting DNA damage repair.

Keywords

Radiosensitization DNMT inhibitor Epigenetics Cancer 

Notes

Acknowledgments

We’d like to thank Ms. Soo Yeon Seo, for her precise in vitro work.

Funding

This research was supported by grant no 04–2016-0620, 04–2016-0830, 05–2016-0010 and 03–2017-0080 from the SNUH Research Fund and NRF-2013M2A2A7043683, NRF-2015M2B2A9029247. The funding source had no role in the design of this study, in data collection and interpretation, in the decision to publish, or in writing the manuscript.

Compliance with ethical standards

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10637_2019_730_MOESM1_ESM.docx (421 kb)
Figure S1 Influence of DNMT inhibitors on cell cycle phase distributions for A549 cells. The proportion of G2/M (A, B, C) and sub-G1 (D, E, F) of A549 cells treated with the respective DNMT inhibitors before radiation and radiation were compared with those treated with radiation alone. Points, mean for three independent experiments; Bars, SE. (DOCX 420 kb)
10637_2019_730_MOESM2_ESM.docx (1.3 mb)
Figure S2 Radiation-induced γH2AX foci. Representative micrographs were obtained from A549 cells that had been treated with MA-17 and received 6-Gy radiation. γH2AX foci are clearly visualized at 48 h after radiation treatment in co-treated cells (A). However, γH2AX expression declined drastically in cells treated with radiation alone at 48 h after irradiation (B). DNA is visualized with DAPI, and merged images overlay γH2AX foci. (DOCX 1280 kb)
10637_2019_730_MOESM3_ESM.docx (1.2 mb)
Figure S3 Pathways relevant to the effect of MA-17 on radiosensitivity of A549 cells. Gene lists determined by RNA sequencing for A549 cell line after MA-17 and radiation combination treatment were analyzed using KEGG pathway analysis. Homologous recombination (A) and the Fanconi anemia pathway (B) were identified as being significantly down-regulated by MA-17 pretreatment and radiation. Significantly down-regulated genes shown in green, and those present within our dataset but not significant are shown in grey. Significant genes were defined as reporting a fold change of >1.5 or < 0.67. (DOCX 1242 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Hyun-Cheol Kang
    • 1
    • 2
  • Eui Kyu Chie
    • 1
    • 2
  • Hak Jae Kim
    • 1
    • 2
  • Jin Ho Kim
    • 1
    • 2
  • Il Han Kim
    • 1
    • 2
    Email author
  • Kwangsoo Kim
    • 3
  • Beom Su Shin
    • 4
  • EunSook Ma
    • 5
  1. 1.Department of Radiation OncologySeoul National University College of MedicineSeoulSouth Korea
  2. 2.Cancer Research InstituteSeoul National University College of MedicineSeoulSouth Korea
  3. 3.Division of Clinical Bioinformatics, Biomedical Research InstituteSeoul National University HospitalSeoulSouth Korea
  4. 4.School of PharmacySungkyunkwan UniversitySuwonSouth Korea
  5. 5.College of PharmacyDaegu Catholic UniversityGyeongsan-siSouth Korea

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