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


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.


Radiosensitization DNMT inhibitor Epigenetics Cancer 



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


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)


  1. 1.
    Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128:683–692CrossRefGoogle Scholar
  2. 2.
    Baylin SB, Schuebel KE (2007) Genomic biology: the epigenomic era opens. Nature 448:548–549CrossRefGoogle Scholar
  3. 3.
    Rodriguez-Paredes M, Esteller M (2011) Cancer epigenetics reaches mainstream oncology. Nat Med 17:330–339CrossRefGoogle Scholar
  4. 4.
    Turner BM (2007) Defining an epigenetic code. Nat Cell Biol 9:2–6CrossRefGoogle Scholar
  5. 5.
    Wouters BJ, Delwel R (2015) Epigenetics and approaches to targeted epigenetic therapy in acute myeloid leukemia. Blood 127:42–52CrossRefGoogle Scholar
  6. 6.
    Nervi C, De Marinis E, Codacci-Pisanelli G (2015) Epigenetic treatment of solid tumours: a review of clinical trials. Clin Epigenetics 7:127CrossRefGoogle Scholar
  7. 7.
    Merrifield M, Kovalchuk O (2013) Epigenetics in radiation biology: a new research frontier. Front Genet 4:40CrossRefGoogle Scholar
  8. 8.
    Smits KM, Melotte V, Niessen HE, Dubois L, Oberije C, Troost EG et al (2014) Epigenetics in radiotherapy: where are we heading? Radiother Oncol 111:168–177CrossRefGoogle Scholar
  9. 9.
    Kim HJ, Kim JH, Chie EK, Young PD, Kim IA, Kim IH (2012) DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity. Radiat Oncol 7:39CrossRefGoogle Scholar
  10. 10.
    Bae SH, Kim MS, Cho CK, Kang JK, Lee SY, Lee KN, Lee DH, Han CJ, Yang KY, Kim SB (2012) Predictor of severe gastroduodenal toxicity after stereotactic body radiotherapy for abdominopelvic malignancies. Int J Radiat Oncol Biol Phys 84:e469–e474CrossRefGoogle Scholar
  11. 11.
    Qiu H, Yashiro M, Shinto O, Matsuzaki T, Hirakawa K (2009) DNA methyltransferase inhibitor 5-aza-CdR enhances the radiosensitivity of gastric cancer cells. Cancer Sci 100:181–188CrossRefGoogle Scholar
  12. 12.
    Dote H, Cerna D, Burgan WE, Carter DJ, Cerra MA, Hollingshead MG, Camphausen K, Tofilon PJ (2005) Enhancement of in vitro and in vivo tumor cell radiosensitivity by the DNA methylation inhibitor zebularine. Clin Cancer Res 11:4571–4579CrossRefGoogle Scholar
  13. 13.
    Wang L, Zhang Y, Li R, Chen Y, Pan X, Li G, Dai F, Yang JL (2013) 5-aza-2′-deoxycytidine enhances the radiosensitivity of breast cancer cells. Cancer Biother Radiopharm 28:34–44CrossRefGoogle Scholar
  14. 14.
    Kim HJ, Kim TH, Seo WS, Yoo SD, Kim IH, Joo SH, Shin S, Park ES, Ma ES, Shin BS (2012) Pharmacokinetics and tissue distribution of psammaplin a, a novel anticancer agent, in mice. Arch Pharm Res 35:1849–1854CrossRefGoogle Scholar
  15. 15.
    Chen J, Bardes EE, Aronow BJ, Jegga AG (2009) ToppGene suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res 37:W305–W311CrossRefGoogle Scholar
  16. 16.
    Kanehisa M, Goto SKEGG (2000) Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30CrossRefGoogle Scholar
  17. 17.
    Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M (2016) KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44:D457–D462CrossRefGoogle Scholar
  18. 18.
    Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K (2017) KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45:D353–DD61CrossRefGoogle Scholar
  19. 19.
    Jassem J (2001) Combined chemotherapy and radiation in locally advanced non-small-cell lung cancer. Lancet Oncol 2:335–342CrossRefGoogle Scholar
  20. 20.
    Vongtama V, Douglass HO, Moore RH, Holyoke ED, Webster JH (1975) End results of radiation therapy, alone and combination with 5-fluorouracil in colorectal cancers. Cancer 36:2020–2025CrossRefGoogle Scholar
  21. 21.
    Lo TC, Wiley AL Jr, Ansfield FJ, Brandenburg JH, Davis HL Jr, Gollin FF, Johnson RO, Ramirez G, Vermund H (1976) Combined radiation therapy and 5-fluorouracil for advanced squamous cell carcinoma of the oral cavity and oropharynx: a randomized study. AJR Am J Roentgenol 126:229–235CrossRefGoogle Scholar
  22. 22.
    Keys HM, Bundy BN, Stehman FB, Muderspach LI, Chafe WE, Suggs CL 3rd et al (1999) Cisplatin, radiation, and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma. N Engl J Med 340:1154–1161CrossRefGoogle Scholar
  23. 23.
    Song SH, Han SW, Bang YJ (2011) Epigenetic-based therapies in cancer: progress to date. Drugs 71:2391–2403CrossRefGoogle Scholar
  24. 24.
    De Schutter H, Kimpe M, Isebaert S, Nuyts S (2009) A systematic assessment of radiation dose enhancement by 5-Aza-2′-deoxycytidine and histone deacetylase inhibitors in head-and-neck squamous cell carcinoma. Int J Radiat Oncol Biol Phys 73:904–912CrossRefGoogle Scholar
  25. 25.
    Prise KM, Schettino G, Folkard M, Held KD (2005) New insights on cell death from radiation exposure. Lancet Oncol 6:520–528CrossRefGoogle Scholar
  26. 26.
    Gatti RA (2001) The inherited basis of human radiosensitivity. Acta Oncol 40:702–711CrossRefGoogle Scholar
  27. 27.
    Nogueira A, Catarino R, Faustino I, Nogueira-Silva C, Figueiredo T, Lombo L, Hilário-Silva I, Pereira D, Medeiros R (2012) Role of the RAD51 G172T polymorphism in the clinical outcome of cervical cancer patients under concomitant chemoradiotherapy. Gene 504:279–283CrossRefGoogle Scholar
  28. 28.
    Casado JA, Rio P, Marco E, Garcia-Hernandez V, Domingo A, Perez L, Tercero JC, Vaquero JJ, Albella B, Gago F, Bueren JA (2008) Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin. Mol Cancer Ther 7:1309–1318CrossRefGoogle Scholar
  29. 29.
    Papadaki C, Sfakianaki M, Ioannidis G, Lagoudaki E, Trypaki M, Tryfonidis K, Mavroudis D, Stathopoulos E, Georgoulias V, Souglakos J (2012) ERCC1 and BRAC1 mRNA expression levels in the primary tumor could predict the effectiveness of the second-line cisplatin-based chemotherapy in pretreated patients with metastatic non-small cell lung cancer. J Thorac Oncol 7:663–671CrossRefGoogle Scholar

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

Personalised recommendations