Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells

Li,Xiumei; Qin,Bingzhao; Liu,Bo

doi:10.3892/ol.2016.4551

Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells

Authors:
- Xiumei Li
- Bingzhao Qin
- Bo Liu
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Affiliations: Department of Gastroenterology, Xiangyang Hospital Affiliated to Hubei University of Medicine, Xiangyang, Hubei 441000, P.R. China
Published online on: May 9, 2016 https://doi.org/10.3892/ol.2016.4551
Pages: 139-143
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Aberrant epigenetic changes are known to contribute to various phases of tumor development. The gene function loss caused by aberrant methylation is analogous to genetic mutations. Unlike genetic mutations, epigenetic alterations can be reversed. 5-Aza-2'-deoxycytidine (5-aza-CdR) has been approved by the Food and Drug Administration for the treatment of certain types of cancer, such as MDS and leukemia. The aim of the present study was to determine whether 5-aza-CdR has the potential to be used in the treatment of colon cancer using a human Caco‑2 colonic carcinoma cell line. The effect of 5‑aza‑CdR on cell proliferation, cell cycle, apoptosis and reversal of aberrant methylation of the Ras association domain family 1A (RASSF1A) gene was also examined. The 5‑aza‑CdR was prepared at different concentrations in sterile tri‑distilled water at 0.4, 1.6, 6.4, 25.6 and 102.4 µmol/l and employed to treat the human Caco‑2 colonic carcinoma cells. An MTT assay was used to detect the effect of 5‑aza‑CdR on cell proliferation. Flow cytometry was used to examine the cell cycle and apoptosis. The RASSF1A mRNA transcript level was examined by reverse transcription-polymerase chain reaction. The results showed that 5‑aza‑CdR inhibited the proliferation of Caco‑2 cells in a time‑ and concentration‑dependent manner (p<0.01). The 5‑aza‑CdR treatment affected the cell cycle and caused accumulation of cells in the G0/G1 phase and this effect was concentration‑dependent (p<0.05). 5‑aza‑CdR treatment caused an increase in the number of cells undergoing apoptosis and reactivated the RASSF1A tumor suppressor gene that was silenced by hypermethylation in Caco-2 cells. In conclusion, 5‑aza‑CdR inhibited growth and promoted apoptosis in Caco‑2 cells by upregulating the epigenetically silenced tumor suppressor RASSF1A gene.

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1	Fandy TE: Development of DNA methyltransferase inhibitors for the treatment of neoplastic diseases. Curr Med Chem. 16:2075–2085. 2009. View Article : Google Scholar : PubMed/NCBI
2	Zhang Y, Ng HH, Erdjument-Bromage H, Tempst P, Bird A and Reinberg D: Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev. 13:1924–1935. 1999. View Article : Google Scholar : PubMed/NCBI
3	Prokhortchouk A, Hendrich B, Jørgensen H, Ruzov A, Wilm M, Georgiev G, Bird A and Prokhortchouk E: The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor. Genes Dev. 15:1613–1618. 2001. View Article : Google Scholar : PubMed/NCBI
4	Bird AP: The relationship of DNA methylation to cancer. Cancer Surv. 28:87–101. 1996.PubMed/NCBI
5	Bird A: DNA methylation de novo. Science. 286:2287–2288. 1999. View Article : Google Scholar : PubMed/NCBI
6	Dammann R, Li C, Yoon JH, Chin PL, Bates S and Pfeifer GP: Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet. 25:315–319. 2000. View Article : Google Scholar : PubMed/NCBI
7	Dammann R, Takahashi T and Pfeifer GP: The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene. 20:3563–3567. 2001. View Article : Google Scholar : PubMed/NCBI
8	Bartling B, Hoffmann J, Holtz J, Schulz R, Heusch G and Darmer D: Quantification of cardioprotective gene expression in porcine short-term hibernating myocardium. J Mol Cell Cardiol. 31:147–158. 1999. View Article : Google Scholar : PubMed/NCBI
9	Burbee DG, Forgacs E, Zöchbauer-Müller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, et al: Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst. 93:691–699. 2001. View Article : Google Scholar : PubMed/NCBI
10	Piekarz RL and Bates SE: Epigenetic modifiers: basic understanding and clinical development. Clin Cancer Res. 15:3918–3926. 2009. View Article : Google Scholar : PubMed/NCBI
11	Wijermans P, Lubbert M, Verhoef G, Bosly A, Ravoet C, Andre M and Ferrant A: Low-dose 5-Aza-2′-deoxycytidine, a DNA hypomethylation agent for the treatment of high-risk myelodyspastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol. 18:956–960. 2000.PubMed/NCBI
12	Xhu XJ and Dai DQ: Epigenetics and gastrointestinal cancer. Chin J Digest. 14:3215–3256. 2006.
13	Tomita H, Hirata A, Yamada Y, Hata K, Oyama T, Mori H, Yamashita S, Ushijima T and Hara A: Suppressive effect of global DNA hypomethylation on gastrc carcinogenesis. Carcinogenesis. 31:1627–1633. 2010. View Article : Google Scholar : PubMed/NCBI
14	Zöchbauer-Müller S, Fong KM, Virmani AK, Geradts J, Gazdar AF and Minna JD: Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res. 61:249–255. 2001.PubMed/NCBI
15	Daskalakis M, Nguyen TT, Nguyen C, Guldberg P, Köhler G, Wijermans P, Jones PA and Lübbert M: Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2′-deoxycytidine (decitabine) treatment. Blood. 100:2957–2964. 2002. View Article : Google Scholar : PubMed/NCBI
16	Bae SI, Lee HS, Kim SH and Kim WH: Inactivation of O6-methylguanine-DNA methyltransferase by promoter CpG island hypermethylation in gastric cancers. Br J Cancer. 86:1888–1892. 2002. View Article : Google Scholar : PubMed/NCBI
17	Esteller M, Corn PG, Baylin SB and Herman JG: A gene hypermethylation profile of human cancer. Cancer Res. 61:3225–3229. 2001.PubMed/NCBI
18	Lübbert M, Tobler A and Daskalakis M: Cytosine demethylation of the proteinase-3/myeloblastin primary granule protease gene during phagocyte development. Leukemia. 13:1420–1427. 1999. View Article : Google Scholar : PubMed/NCBI
19	Kim SH, Bae SI, Lee HS and Kim WH: Alteration of O⁶-methylguanine-DNA methyltransferase in colorectal neoplasms in sporadic and familial adenomatous polyposis patients. Mol Carcinog. 37:32–38. 2003. View Article : Google Scholar : PubMed/NCBI
20	Bender CM, Pao MM and Jones PA: Inhibition of DNA methylation by 5-aza-2′-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res. 58:95–101. 1998.PubMed/NCBI
21	Gonzalgo ML, Hayashida T, Bender CM, Pao MM, Tsai YC, Gonzales FA, Nguyen HD, Nguyen TT and Jones PA: The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines. Cancer Res. 58:1245–1252. 1998.PubMed/NCBI
22	Xiong Z, Wu AH, Bender CM, Tsao JL, Blake C, Shibata D, Jones PA, Yu MC, Ross RK and Laird PW: Mismatch repair deficiency and CpG island hypermethylation in sporadic colon adenocarcinomas. Cancer Epidemiol Biomarkers Prev. 10:799–803. 2001.PubMed/NCBI
23	Dammann R, Schagdarsurengin U, Strunnikova M, Rastetter M, Seidel C, Liu L, Tommasi S and Pfeifer GP: Epigenetic inactivation of the Ras-association domain family 1 (RASSF1A) gene and its function in human carcinogenesis. Histol Histopathol. 18:665–677. 2003.PubMed/NCBI
24	Liu L, Tommasi S, Lee DH, Dammann R and Pfeifer GP: Control of microtubule stability by the RASSF1A tumor suppressor. Oncogene. 22:8125–8136. 2003. View Article : Google Scholar : PubMed/NCBI
25	Strunnikova M, Schagdarsurengin U, Kehlen A, Garbe JC, Stampfer MR and Dammann R: Chromatin inactivation precedes de novo DNA methylation during the progressive epigenetic silencing of the RASSF1A promoter. Mol Cell Biol. 25:3923–3933. 2005. View Article : Google Scholar : PubMed/NCBI
26	Chow LS, Lo KW, Kwong J, To KF, Tsang KS, Lam CW, Dammann R and Huang DP: RASSF1A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma. Int J Cancer. 109:839–847. 2004. View Article : Google Scholar : PubMed/NCBI
27	Tommasi S, Dammann R, Zhang Z, Wang Y, Liu L, Tsark WM, Wilczynski SP, Li J, You M and Pfeifer GP: Tumor susceptibility of Rassf1a knockout mice. Cancer Res. 65:92–98. 2005.PubMed/NCBI
28	Lee S, Hwang KS, Lee HJ, Kim JS and Kang GH: Aberrant CpG island hypermethylation of multiple genes in colorectal neoplasia. Lab Invest. 87:884–893. 2004. View Article : Google Scholar
29	Kang GH, Lee HJ, Hwang KS, Lee S, Kim JH and Kim JS: Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation. Am J Pathol. 163:1551–1556. 2003. View Article : Google Scholar : PubMed/NCBI
30	Kuroki T, Trapasso F, Yendamuri S, Matsuyama A, Alder H, Mori M and Croce CM: Allele loss and promoter hypermethylation of VHL, RAR-beta, RASSF1A, and FHIT tumor suppressor genes on chromosome 3p in esophageal squamous cell carcinoma. Cancer Res. 63:3724–3728. 2003.PubMed/NCBI
31	Kuroki T, Trapasso F, Yendamuri S, Matsuyama A, Alder H, Mori M and Croce CM: Promoter hypermethylation of RASSF1A in esophageal squamous cell carcinoma. Clin Cancer Res. 9:1441–1445. 2003.PubMed/NCBI
32	Byun DS, Lee MG, Chae KS, Ryu BG and Chi SG: Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma. Cancer Res. 61:7034–7038. 2001.PubMed/NCBI
33	Lee MG, Kim HY, Byun DS, Lee SJ, Lee CH, Kim JI, Chang SG and Chi SG: Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma. Cancer Res. 61:6688–6692. 2001.PubMed/NCBI
34	Chan MW, Chan LW, Tang NL, Lo KW, Tong JH, Chan AW, Cheung HY, Wong WS, Chan PS, Lai FM, et al: Frequent hypermethylation of promoter region of RASSF1A in tumor tissues and voided urine of urinary bladder cancer patients. Int J Cancer. 104:611–616. 2003. View Article : Google Scholar : PubMed/NCBI
35	Müller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, Marth C and Widschwendter M: DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res. 63:7641–7645. 2003.PubMed/NCBI