Reactivation of the homeotic tumor suppressor gene CDX2 by 5-aza‑2'-deoxycytidine-induced demethylation inhibits cell proliferation and induces caspase‑independent apoptosis in gastric cancer cells

Zhang,Jian-Feng; Zhang,Jian-Guo; Kuai,Xiao-Ling; Zhang,Hong; Jiang,Wei; Ding,Wei-Feng; Li,Zeng-Li; Zhu,Hui-Jun; Mao,Zhen-Biao

doi:10.3892/etm.2013.901

Reactivation of the homeotic tumor suppressor gene CDX2 by 5-aza‑2'-deoxycytidine-induced demethylation inhibits cell proliferation and induces caspase‑independent apoptosis in gastric cancer cells

Authors:
- Jian-Feng Zhang
- Jian-Guo Zhang
- Xiao-Ling Kuai
- Hong Zhang
- Wei Jiang
- Wei-Feng Ding
- Zeng-Li Li
- Hui-Jun Zhu
- Zhen-Biao Mao
View Affiliations

Affiliations: Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China, Department of Pathology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China, Medical Laboratory Center, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China
Published online on: January 17, 2013 https://doi.org/10.3892/etm.2013.901
Pages: 735-741

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non‑cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time‑dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, -8 and -9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase‑independent apoptosis of MKN-45 cells in vitro.

View Figures

View References

1.	Parkin DM, Bray F, Ferlay J and Pisani P: Global cancer statistics, 2002. CA Cancer J Clin. 55:74–108. 2005. View Article : Google Scholar
2.	Leung WK, Wu MS, Kakugawa Y, et al: Screening for gastric cancer in Asia: current evidence and practice. Lancet Oncol. 9:279–287. 2008. View Article : Google Scholar : PubMed/NCBI
3.	Bird A: Perceptions of epigenetics. Nature. 447:396–398. 2007. View Article : Google Scholar : PubMed/NCBI
4.	Jones PA and Baylin SB: The epigenomics of cancer. Cell. 128:683–692. 2007. View Article : Google Scholar : PubMed/NCBI
5.	Turek-Plewa J and Jagodziński PP: The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett. 10:631–647. 2005.PubMed/NCBI
6.	Mizuno S, Chijiwa T, Okamura T, et al: Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood. 97:1172–1179. 2001. View Article : Google Scholar : PubMed/NCBI
7.	Lyko F and Brown R: DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst. 97:1498–1506. 2005. View Article : Google Scholar : PubMed/NCBI
8.	Silberg DG, Swain GP, Suh ER and Traber PG: Cdx1 and cdx2 expression during intestinal development. Gastroenterology. 119:961–971. 2000. View Article : Google Scholar : PubMed/NCBI
9.	Almeida R, Silva E, Santos-Silva F, et al: Expression of intestine-specific transcription factors, CDX1 and CDX2, in intestinal metaplasia and gastric carcinomas. J Pathol. 199:36–40. 2003. View Article : Google Scholar : PubMed/NCBI
10.	Kang JM, Lee BH, Kim N, et al: CDX1 and CDX2 expression in intestinal metaplasia, dysplasia and gastric cancer. J Korean Med Sci. 26:647–653. 2011. View Article : Google Scholar : PubMed/NCBI
11.	Freund JN, Domon-Dell C, Kedinger M and Duluc I: The Cdx-1 and Cdx-2 homeobox genes in the intestine. Biochem Cell Biol. 76:957–969. 1998. View Article : Google Scholar : PubMed/NCBI
12.	Baba Y, Nosho K, Shima K, et al: Relationship of CDX2 loss with molecular features and prognosis in colorectal cancer. Clin Cancer Res. 15:4665–4673. 2009. View Article : Google Scholar : PubMed/NCBI
13.	Mallo GV, Rechreche H, Frigerio JM, et al: Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis. Int J Cancer. 74:35–44. 1997. View Article : Google Scholar : PubMed/NCBI
14.	Vider BZ, Zimber A, Hirsch D, et al: Human colorectal carcinogenesis is associated with deregulation of homeobox gene expression. Biochem Biophys Res Commun. 232:742–748. 1997. View Article : Google Scholar : PubMed/NCBI
15.	Park do Y, Srivastava A, Kim GH, et al: CDX2 expression in the intestinal-type gastric epithelial neoplasia: frequency and significance. Mod Pathol. 23:54–61. 2010.PubMed/NCBI
16.	Saad RS, Ghorab Z, Khalifa MA and Xu M: CDX2 as a marker for intestinal differentiation: Its utility and limitations. World J Gastrointest Surg. 3:159–166. 2011. View Article : Google Scholar : PubMed/NCBI
17.	Washington K: 7th edition of the AJCC cancer staging manual: stomach. Ann Surg Oncol. 17:3077–3079. 2010. View Article : Google Scholar : PubMed/NCBI
18.	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
19.	Mao ZB, Zhang JF, Xu Z, et al: Ectopic expression of guanylyl cyclase C in gastric cancer as a potential biomarker and therapeutic target. J Dig Dis. 10:272–285. 2009. View Article : Google Scholar : PubMed/NCBI
20.	Herman JG, Graff JR, Myöhänen S, et al: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 93:9821–9826. 1996. View Article : Google Scholar : PubMed/NCBI
21.	Yuasa Y, Nagasaki H, Akiyama Y, et al: DNA methylation status is inversely correlated with green tea intake and physical activity in gastric cancer patients. Int J Cancer. 124:2677–2682. 2009. View Article : Google Scholar : PubMed/NCBI
22.	Hanahan D and Weinberg RA: The hallmarks of cancer. Cell. 100:57–70. 2000. View Article : Google Scholar
23.	Hanahan D and Weinberg RA: Hallmarks of cancer: the next generation. Cell. 144:646–674. 2011. View Article : Google Scholar : PubMed/NCBI
24.	Walters JR, Howard A, Rumble HE, et al: Differences in expression of homeobox transcription factors in proximal and distal human small intestine. Gastroenterology. 113:472–477. 1997. View Article : Google Scholar : PubMed/NCBI
25.	Tamai Y, Nakajima R, Ishikawa T, et al: Colonic hamartoma development by anomalous duplication in Cdx2 knockout mice. Cancer Res. 59:2965–2970. 1999.PubMed/NCBI
26.	Beck F, Chawengsaksophak K, Waring P, et al: Reprogramming of intestinal differentiation and intercalary regeneration in Cdx2 mutant mice. Proc Natl Acad Sci USA. 96:7318–7323. 1999. View Article : Google Scholar : PubMed/NCBI
27.	Barbareschi M, Murer B, Colby TV, et al: CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs. Am J Surg Pathol. 27:141–149. 2003. View Article : Google Scholar : PubMed/NCBI
28.	Werling RW, Yaziji H, Bacchi CE, et al: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 27:303–310. 2003. View Article : Google Scholar
29.	Moskaluk CA, Zhang H, Powell SM, et al: Cdx2 protein expression in normal and malignant human tissues: an immunohistochemical survey using tissue microarrays. Mod Pathol. 16:913–919. 2003. View Article : Google Scholar : PubMed/NCBI
30.	Kaimaktchiev V, Terracciano L, Tornillo L, et al: The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas. Mod Pathol. 17:1392–1399. 2004. View Article : Google Scholar
31.	Seno H, Oshima M, Taniguchi MA, et al: CDX2 expression in the stomach with intestinal metaplasia and intestinal-type cancer: Prognostic implications. Int J Oncol. 21:769–774. 2002.PubMed/NCBI
32.	Mizoshita T, Tsukamoto T, Nakanishi H, et al: Expression of Cdx2 and the phenotype of advanced gastric cancers: relationship with prognosis. J Cancer Res Clin Oncol. 129:727–734. 2003. View Article : Google Scholar : PubMed/NCBI
33.	Fan Z, Li J, Dong B and Huang X: Expression of Cdx2 and hepatocyte antigen in gastric carcinoma: correlation with histologic type and implications for prognosis. Clin Cancer Res. 11:6162–6170. 2005. View Article : Google Scholar : PubMed/NCBI
34.	Liu Q, Teh M, Ito K, et al: CDX2 expression is progressively decreased in human gastric intestinal metaplasia, dysplasia and cancer. Mod Pathol. 20:1286–1297. 2007. View Article : Google Scholar : PubMed/NCBI
35.	Song JH, Kim CJ, Cho YG, et al: Genetic alterations of the Cdx2 gene in gastric cancer. APMIS. 116:74–80. 2008. View Article : Google Scholar : PubMed/NCBI
36.	Okayama H, Kumamoto K, Saitou K, et al: CD44v6, MMP-7 and nuclear Cdx2 are significant biomarkers for prediction of lymph node metastasis in primary gastric cancer. Oncol Rep. 22:745–755. 2009.PubMed/NCBI
37.	Qin R, Wang NN, Chu J, et al: Expression and significance of homeodomain protein Cdx2 in gastric carcinoma and precancerous lesions. World J Gastroenterol. 18:3296–3302. 2012.PubMed/NCBI
38.	Xie Y, Li L, Wang X, et al: Overexpression of Cdx2 inhibits progression of gastric cancer in vitro. Int J Oncol. 36:509–516. 2010.PubMed/NCBI
39.	Ding WJ, Fang JY, Chen XY and Peng YS: The expression and clinical significance of DNA methyltransferase proteins in human gastric cancer. Dig Dis Sci. 53:2083–2089. 2008. View Article : Google Scholar : PubMed/NCBI
40.	Sharma S, Kelly TK and Jones PA: Epigenetics in cancer. Carcinogenesis. 31:27–36. 2010. View Article : Google Scholar
41.	Gilbert J, Gore SD, Herman JG and Garducci MA: The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin Cancer Res. 10:4589–4596. 2004. View Article : Google Scholar : PubMed/NCBI
42.	Susin SA, Daugas E, Ravagnan L, et al: Two distinct pathways leading to nuclear apoptosis. J Exp Med. 192:571–580. 2000. View Article : Google Scholar : PubMed/NCBI
43.	Wang X, Zhu S, Drozda M, et al: Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington’s disease. Proc Natl Acad Sci USA. 100:10483–10487. 2003.PubMed/NCBI
44.	Antonsson B: Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem. 256–257:141–155. 2004.PubMed/NCBI
45.	Stefanis L: Caspase-dependent and -independent neuronal death: two distinct pathways to neuronal injury. Neuroscientist. 11:50–62. 2005. View Article : Google Scholar : PubMed/NCBI