Epigenetic modifications have been implicated in the development of therapeutic resistance responsible for the poor prognosis of human malignant mesothelioma (HMM). To find a potential way to overcome this therapeutic resistance, this study investigated the anticancer effects of a DNA demethylating agent, 5-Aza-2′-Deoxycytidine (5-aza-dC), on HMM cells. The 5-aza-dC exhibited minimal detrimental effects on cell survival. However, treatment with 5-aza-dC significantly altered the biological characteristics associated with malignancy, such as cell migration and cell interaction, colony-forming capacity, and invasiveness. Moreover, it significantly reduced the fraction of side population (SP) cells, which are reportedly enriched for more aggressive cells. Large-scale methylation analysis based on methylated DNA immunoprecipitation revealed a more than two fold increase in the methylation level of major tumor suppressor genes in the SP fraction. The data indicated that SP cells might acquire malignancy by hypermethylation of tumor suppressor genes. Treatment with 5-aza-dC could attack more malignant cells through the modification of their methylation status. The results indicate that the modulation of DNA methylation might be a valuable strategy to overcome the therapeutic resistance of HMM. Moreover, ensuing changes in the biological characteristics provide a basis for further analysis of the role of methylation in HMM carcinogenesis.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $61.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
McDonald AD, McDonald JC . Malignant mesothelioma in north America. Cancer 1980; 46: 1650–1656.
Robinson BW, Musk AW, Lake RA . Malignant mesothelioma. Lancet 2005; 366: 397–408.
Eltabbakh GH, Piver MS, Hempling RE, Recio FO, Intengen ME . Clinical picture, response to therapy, and survival of women with diffuse malignant peritoneal mesothelioma. J Surg Oncol 1999; 70: 6–12.
Carbone M, Kratzke RA, Testa JR . The pathogenesis of mesothelioma. Semin Oncol 2002; 29: 2–17.
Tsou JA, Shen LY, Siegmund KD, Long TI, Laird PW, Seneviratne CK et al. Distinct DNA methylation profiles in malignant mesothelioma, lung adenocarcinoma, and non-tumor lung. Lung Cancer 2005; 47: 193–204.
Wainfan E, Poirier LA . Methyl groups in carcinogenesis: effects on DNA methylation and gene expression. Cancer Res 1992; 52 (7 Supplement): 2071s–2077s.
Costello JF, Plass C . Methylation matters. J Med Genet 2001; 38: 285–303.
Tsou JA, Galler JS, Wali A, Ye W, Siegmund KD, Groshen S et al. DNA methylation profile of 28 potential marker loci in malignant mesothelioma. Lung cancer 2007; 58: 220–230.
Fischer JR, Ohnmacht U, Rieger N, Zemaitis M, Stoffregen C, Kostrzewa M et al. Promoter methylation of RASSF1A, RARβ and DAPK predict poor prognosis of patients with malignant mesothelioma. Lung Cancer 2006; 54: 109–116.
Issa J-PJ, Kantarjian HM, Kirkpatrick P . Azacitidine. Nat Rev Drug Discov 2005; 4: 275–276.
Kai K, D’Costa S, Yoon B-I, Brody AR, Sills RC, Kim Y . Characterization of side population cells in human malignant mesothelioma cell lines. Lung Cancer 2010; 70: 146–151.
Kim H, Kim M, Kim N, Kim Y . Inhibition of hedgehog signaling reduces the side population in human malignant mesothelioma cell lines. Cancer Gene Ther 2015; 22: 387–395.
Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 2001; 25: 402–408.
Su J, Wang Y, Xing X, Liu J, Zhang Y . Genome-wide analysis of DNA methylation in bovine placentas. BMC Genomics 2014; 15: 12.
Mohn F, Weber M, Schübeler D, Roloff T-C . Methylated DNA immunoprecipitation (MeDIP). Methods Mol Biol 2009; 507: 55–64.
Kim M-C, Kim N-Y, Seo Y-R, Kim Y . An integrated analysis of the genome-wide profiles of dna methylation and mRNA expression defining the side population of a human malignant mesothelioma cell line. J Cancer 2016; 7: 1668–1679.
Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C . A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immuol Methods 1995; 184: 39–51.
Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG . Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 2005; 65: 6207–6219.
Ho MM, Ng AV, Lam S, Hung JY . Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 2007; 67: 4827–4833.
Jacinto FV, Ballestar E, Esteller M . Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome. Biotechniques 2008; 44: 35.
Nair SS, Coolen MW, Stirzaker C, Song JZ, Statham AL, Strbenac D et al. Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias. Epigenetics 2011; 6: 34–44.
Baylin SB . DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2005; 2: S4–S11.
Momparler RL, Bovenzi V . DNA methylation and cancer. J Cell Physiol 2000; 183: 145–154.
Licht JD . DNA methylation inhibitors in cancer therapy: the immunity dimension. Cell 2015; 162: 938–939.
Schneider-Stock R, Diab-Assef M, Rohrbeck A, Foltzer-Jourdainne C, Boltze C, Hartig R et al. 5-Aza-cytidine is a potent inhibitor of DNA methyltransferase 3a and induces apoptosis in HCT-116 colon cancer cells via Gadd45-and p53-dependent mechanisms. J Pharmacol Exp Ther 2005; 312: 525–536.
Pass HI, Vogelzang N, Carbone M . Malignant Mesothelioma: Pathogenesis, Diagnosis, and Translational Therapies. Springer Science & Business Media: Berlin, 2006.
Lainey E, Wolfromm A, Marie N, Enot D, Scoazec M, Bouteloup C et al. Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia. Oncogene 2013; 32: 4331–4342.
Zhou X, Huang S, Zhang D, Zhang S, Li W, Chen Z et al. Effects of 5-aza-2′ deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma. Braz J Med Biol Res 2015; 48: 254–260.
Cho CY, Wang JH, Chang HC, Chang CK, Hung WC . Epigenetic inactivation of the metastasis suppressor RECK enhances invasion of human colon cancer cells. J Cell Physiol 2007; 213: 65–69.
Burrell RA, Swanton C . Re-evaluating clonal dominance in cancer evolution. Trends Cancer 2016; 2: 263–276.
Pylkkänen L, Wolff H, Stjernvall T, Knuuttila A, Anttila S, Husgafvel-Pursiainen K . Reduced Fhit protein expression in human malignant mesothelioma. Virchows Archiv 2004; 444: 43–48.
Miyanaga A, Masuda M, Tsuta K, Kawasaki K, Nakamura Y, Sakuma T et al. Hippo pathway gene mutations in malignant mesothelioma: revealed by RNA and targeted exon sequencing. J Thorac Oncol 2015; 10: 844–851.
Poulikakos P, Xiao G, Gallagher R, Jablonski S, Jhanwar S, Testa JR . Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK. Oncogene 2006; 25: 5960–5968.
Murakami H, Mizuno T, Taniguchi T, Fujii M, Ishiguro F, Fukui T et al. LATS2 is a tumor suppressor gene of malignant mesothelioma. Cancer Res 2011; 71: 873–883.
Pinton G, Brunelli E, Murer B, Puntoni R, Puntoni M, Fennell DA et al. Estrogen receptor-β affects the prognosis of human malignant mesothelioma. Cancer Res 2009; 69: 4598–4604.
Batra S, Shi Y, Kuchenbecker KM, He B, Reguart N, Mikami I et al. Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma. Biochem Biophys Res Commun 2006; 342: 1228–1232.
This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No.: NRF-2013R1A2A2A01068237) and by the BK21 PLUS Program for Creative Veterinary Science Research.
The authors declare no conflict of interest.
About this article
Glucose starvation induces resistance to metformin through the elevation of mitochondrial multidrug resistance protein 1
Cancer Science (2019)
A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells