DOI QR코드

DOI QR Code

Effects of Tumor Microenvironmental Factors on DNA Methylation and Radiation Sensitivity in A549 Human Lung Adenocarcinoma

  • Oh, Jung-Min (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Kim, Young-Eun (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Hong, Beom-Ju (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Bok, Seoyeon (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Jeon, Seong-Uk (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Lee, Chan-Ju (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Park, Dong-Young (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Kim, Il Han (Department of Radiation Oncology, College of Medicine, Seoul National University) ;
  • Kim, Hak Jae (Department of Radiation Oncology, College of Medicine, Seoul National University) ;
  • Ahn, G-One (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology)
  • Received : 2018.05.15
  • Accepted : 2018.06.11
  • Published : 2018.06.30

Abstract

Background: Tumor response to anticancer therapies can much be influenced by microenvironmental factors. In this study, we determined the effect of these microenvironmental factors on DNA methylation using A549 human lung adenocarcinoma cell line. Materials and Methods: We subjected A549 cells to various conditions mimicking tumor microenvironment including hypoxia, acidosis (sodium lactate), oxidative stress ($H_2O_2$), bystander effect (supernatant from doxorubicin (Dox)-treated or irradiated cells), and immune cell infiltration (supernatant from THP-1 or Jurkat T cells). Genomic DNA was isolated from these cells and analyzed for DNA methylation. Clonogenic cell survival, gene expression, and metabolism were analyzed in cells treated with some of these conditions. Results and Discussion: We found that DNA methylation level was significantly decreased in A549 cells treated with conditioned media from Dox-treated cells or Jurkat T cells, or sodium lactate, indicating an active transcription. To determine whether the decreased DNA methylation affects radiation sensitivity, we exposed cells to these conditions followed by 6 Gy irradiation and found that cell survival was significantly increased by sodium lactate while it was decreased by conditioned media from Dox-treated cells. We further observed that cells treated with conditioned media from Dox-treated cells exhibited significant changes in expression of genes including BAX and FAS (involved in apoptosis), NADPH dehydrogenase (mitochondria), EGFR (cellular survival) and RAD51 (DNA damage repair) while sodium lactate increased cellular metabolism rather than changing the gene expression. Conclusion: Our results suggest that various tumor microenvironmental factors can differentially influence DNA methylation and hence radiosensitivity and gene expression in A549 cancer cells.

Acknowledgement

Supported by : National Research Foundation, Ministry of education

References

  1. Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat. Rev. Cancer. 2004;4(6):437-447. https://doi.org/10.1038/nrc1367
  2. Franco M, et al. Pericytes promote endothelial cell survival through induction of autocrine VEGF-A signaling and Bcl-w expression. Blood. 2011;118(10):2906-2917. https://doi.org/10.1182/blood-2011-01-331694
  3. Gouaze-Andersson V, et al. FGFR1 Induces Glioblastoma radioresistance through the PLCgamma/Hif1alpha pathway. Cancer Res. 2016;76(10):3036-3044. https://doi.org/10.1158/0008-5472.CAN-15-2058
  4. Ahn GO, Brown JM. Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: role of bone marrow-derived myelomonocytic cells. Cancer Cell. 2008;13(3):193-205. https://doi.org/10.1016/j.ccr.2007.11.032
  5. Smits KM, et al. Epigenetics in radiotherapy: where are we heading? Radiother. Oncol. 2014;111(2):168-177. https://doi.org/10.1016/j.radonc.2014.05.001
  6. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat. Rev. Cancer. 2004;4(2):143-153. https://doi.org/10.1038/nrc1279
  7. Agnlim PP, et al. Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer. Mol. Cancer. 2008;7:62-74. https://doi.org/10.1186/1476-4598-7-62
  8. Niu X, et al. Genome-wide DNA methylation analysis reveals GABBR2 as a novel epigenetic target for EGFR 19 deletion lung adenocarcinoma with induction erlotinib treatment. Clin. Cancer Res. 2017;23(17):5003-5014. https://doi.org/10.1158/1078-0432.CCR-16-2688
  9. Kim HJ, Kim JH, Chie EK, Park DY, Kim IA, Kim IH. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity. Radiat. Oncol. 2012;7:39-48. https://doi.org/10.1186/1748-717X-7-39
  10. Fujimori H, et al. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation. Sci. Rep. 2015;5:18231.
  11. Maunakea AK, et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 2010;466(7303):253-257. https://doi.org/10.1038/nature09165
  12. Sonveaux P, et al. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J. Clin. Invest. 2008;118(12):3930-3942. https://doi.org/10.1172/JCI36843
  13. Mathot P, et al. DNA methylation signal has a major role in the response of human breast cancer cells to the microenvironment. Oncogenesis. 2017;6(10):e390. https://doi.org/10.1038/oncsis.2017.88
  14. Vizoso M, et al. Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts. Carcinogenesis. 2015;36(12):1453-1463. https://doi.org/10.1093/carcin/bgv146
  15. Koturbash I, Boyko A, Rodriguez-Juarez R, McDonald RJ, Tryndyak VP, Kovalchuk I, Pogribny IP, Kovalchuk O. Role of epigenetic effectors in maintenance of the long-term persistent bystander effect in spleen in vivo. Carcinogenesis. 2007;28(8):1831-1838. https://doi.org/10.1093/carcin/bgm053
  16. Puthli A, Tiwari R, Mishra K. Bystander response triggered by doxorubicin-killed dead cells contributes to acquire drug resistance but increasing radiosensitivity in vitro. J. Radiat. Cancer Res. 2016;7(4):103-111. https://doi.org/10.4103/jrcr.jrcr_7_17
  17. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995;80(2):293-299. https://doi.org/10.1016/0092-8674(95)90412-3
  18. Petak I, Tillman DM, Houghton JA. p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines. Clin. Cancer Res. 2000;6(11):4432-4441.
  19. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 2000;10(15):886-895. https://doi.org/10.1016/S0960-9822(00)00610-2
  20. Zhang Y, et al. The signature of liver cancer in immune cells DNA methylation. Clin. Epigenet. 2018;10:8. https://doi.org/10.1186/s13148-017-0436-1
  21. Albrengues J, et al. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat. Commun. 2015;6:10204. https://doi.org/10.1038/ncomms10204
  22. Fujimura L, Matsudo Y, Kang Mm, Takamori Y, Tokuhisa T, Hatano M. Protective role of Nd1 in doxorubicin-induced cardiotoxicity. Cardiovasc. Res. 2004;64(2):315-321. https://doi.org/10.1016/j.cardiores.2004.07.009
  23. Mansfield KD, Guzy RD, Pan Y, Young RM, Cash TP, Schumacker PT, Simon MC. Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation. Cell Metab. 2005;1(6):393-399. https://doi.org/10.1016/j.cmet.2005.05.003
  24. Wu D, Yotnda P. Induction and testing of hypoxia in cell culture. J. Vis. Exp. 2011;54:e2899.
  25. Lim SO, et al. Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter. Gastroenterology, 2008;135(6):2128-2140. https://doi.org/10.1053/j.gastro.2008.07.027
  26. Amornsupak K, Insawang T, Thuwajit P, Charoenrat PO, Eccles SA, Thuwajit C. Cancer-associated fibroblasts induce high mobility group box 1 and contribute to resistance to doxorubicin in breast cancer cells. BMC Cancer. 2014;14:955. https://doi.org/10.1186/1471-2407-14-955
  27. Mothersill C, Seymour C. Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells. Int. J. Radiat. Biol. 1997;71(4):421-427. https://doi.org/10.1080/095530097144030