5-Azacytidine inhibits the proliferation of bladder cancer cells via reversal of the aberrant hypermethylation of the hepaCAM gene

Wang,Xiaorong; Chen,E.; Yang,Xue; Wang,Yin; Quan,Zhen; Wu,Xiaohou; Luo,Chunli

doi:10.3892/or.2015.4492

5-Azacytidine inhibits the proliferation of bladder cancer cells via reversal of the aberrant hypermethylation of the hepaCAM gene

Authors:
- Xiaorong Wang
- E. Chen
- Xue Yang
- Yin Wang
- Zhen Quan
- Xiaohou Wu
- Chunli Luo
View Affiliations

Affiliations: Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, P.R. China, Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China
Published online on: December 17, 2015 https://doi.org/10.3892/or.2015.4492
Pages: 1375-1384

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

Hepatocyte cell adhesion molecule (hepaCAM), a tumor-suppressor gene, is rarely expressed in bladder carcinoma. However, little is known concerning the mechanisms of low hepaCAM expression in bladder cancer. Abnormal hypermethylation in the promoter plays a crucial role in cancer by silencing tumor-suppressor genes, which is catalyzed by DNA methyltransferases (DNMTs). In the present study, a total of 31 bladder cancer and 22 adjacent tissues were assessed by immunohistochemistry to detect DNMT3A/3B and hepaCAM expression. Methylation of hepaCAM was determined by methylation‑speciﬁc polymerase chain reaction (MSP). The mRNA and protein levels of DNMT3A/3B and hepaCAM were determined by RT-PCR and western blot analysis after treatment with 5-azacytidine (AZAC). Following AZAC treatment, the proliferation of bladder cancer cells was detected by CCK-8 and colony formation assays. Cell cycle distribution was examined by ﬂow cytometry. To further evaluate the tumor‑suppressive roles of AZAC and the involved mechanisms, the anti-tumorigenicity of AZAC was tested in vivo. The expression of DNMT3A/3B protein was markedly increased in the bladder carcinoma tissues (P<0.05), and had a negative linear correlation with hepaCAM expression in the same patients according to Pearson's analysis (r=-0.7176/-0.7127, P<0.05). The MSP results indicated that the hepaCAM gene was hypermethylated in three bladder cancer cell lines. Furthermore, we found that downregulation of DNMT3A/3B expression, after treatment with AZAC, reversed the hypermethylation and expression of hepaCAM in bladder cancer cells. In addition, AZAC inhibited the proliferation of bladder cancer cells and arrested cells at the G0/G1 phase. The in vivo results showed that expression of DNMT3A/3B and hepaCAM as well as tumor growth of nude mice were markedly altered which corresponded with the in vitro results. Due to the ability to reactivate expression of hepaCAM and inhibit growth of bladder cancer cells, AZAC may represent an effective treatment for bladder cancer.

View Figures

View References

1	Favre-Kontula L, Rolland A, Bernasconi L, Karmirantzou M, Power C, Antonsson B and Boschert U: GlialCAM, an immunoglobulin-like cell adhesion molecule is expressed in glial cells of the central nervous system. Glia. 56:633–645. 2008. View Article : Google Scholar : PubMed/NCBI
2	Chung Moh M, Hoon Lee L and Shen S: Cloning and charac-terization of hepaCAM, a novel Ig-like cell adhesion molecule suppressed in human hepatocellular carcinoma. J Hepatol. 42:833–841. 2005. View Article : Google Scholar : PubMed/NCBI
3	Moh MC, Zhang C, Luo C, Lee LH and Shen S: Structural and functional analyses of a novel Ig-like cell adhesion molecule, hepaCAM, in the human breast carcinoma MCF7 cells. J Biol Chem. 280:27366–27374. 2005. View Article : Google Scholar : PubMed/NCBI
4	Moh MC, Zhang T, Lee LH and Shen S: Expression of hepaCAM is downregulated in cancers and induces senescence-like growth arrest via a p53/p21-dependent pathway in human breast cancer cells. Carcinogenesis. 29:2298–2305. 2008. View Article : Google Scholar : PubMed/NCBI
5	Lee LH, Moh MC, Zhang T and Shen S: The immunoglobulin-like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373-MG cells. J Cell Biochem. 107:1129–1138. 2009. View Article : Google Scholar : PubMed/NCBI
6	Xun C, Luo C, Wu X, Zhang Q, Yan L and Shen S: Expression of hepaCAM and its effect on proliferation of tumor cells in renal cell carcinoma. Urology. 75:828–834. 2010. View Article : Google Scholar : PubMed/NCBI
7	Zhang QL, Luo CL, Wu XH, Wang CY, Xu X, Zhang YY, Liu Q and Shen SL: HepaCAM induces G1 phase arrest and promotes c-Myc degradation in human renal cell carcinoma. J Cell Biochem. 112:2910–2919. 2011. View Article : Google Scholar : PubMed/NCBI
8	He Y, Wu X, Luo C, Wang L and Lin J: Functional significance of the hepaCAM gene in bladder cancer. BMC Cancer. 10:832010. View Article : Google Scholar : PubMed/NCBI
9	Zhu X, Shan L, Wang F, Wang J, Wang F, Shen G, Liu X, Wang B, Yuan Y, Ying J, et al: Hypermethylation of BRCA1 gene: Implication for prognostic biomarker and therapeutic target in sporadic primary triple-negative breast cancer. Breast Cancer Res Treat. 150:479–486. 2015. View Article : Google Scholar : PubMed/NCBI
10	Ahmed IA, Pusch CM, Hamed T, Rashad H, Idris A, El-Fadle AA and Blin N: Epigenetic alterations by methylation of RASSF1A and DAPK1 promoter sequences in mammary carcinoma detected in extracellular tumor DNA. Cancer Genet Cytogenet. 199:96–100. 2010. View Article : Google Scholar : PubMed/NCBI
11	Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, Laird PW and Jones PA: Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol. 22:480–491. 2002. View Article : Google Scholar : PubMed/NCBI
12	Ramos MP, Wijetunga NA, McLellan AS, Suzuki M and Greally JM: DNA demethylation by 5-aza-2′-deoxycytidine is imprinted, targeted to euchromatin, and has limited transcriptional consequences. Epigenetics Chromatin. 8:112015. View Article : Google Scholar
13	Amara K, Ziadi S, Hachana M, Soltani N, Korbi S and Trimeche M: DNA methyltransferase DNMT3b protein over-expression as a prognostic factor in patients with diffuse large B-cell lymphomas. Cancer Sci. 101:1722–1730. 2010. View Article : Google Scholar : PubMed/NCBI
14	Chen MF, Chen WC, Chang YJ, Wu CF and Wu CT: Role of DNA methyltransferase 1 in hormone-resistant prostate cancer. J Mol Med Berl. 88:953–962. 2010. View Article : Google Scholar : PubMed/NCBI
15	Devanand P, Kim SI, Choi YW, Sheen SS, Yim H, Ryu MS, Kim SJ, Kim WJ and Lim IK: Inhibition of bladder cancer invasion by Sp1-mediated BTG2 expression via inhibition of DNA methyltransferase 1. FEBS J. 281:5581–5601. 2014. View Article : Google Scholar : PubMed/NCBI
16	Brueckner B and Lyko F: DNA methyltransferase inhibitors: Old and new drugs for an epigenetic cancer therapy. Trends Pharmacol Sci. 25:551–554. 2004. View Article : Google Scholar : PubMed/NCBI
17	Wijermans P, Lübbert M, Verhoef G, Bosly A, Ravoet C, Andre M and Ferrant A: Low-dose 5-aza-2′-deoxycytidine, a DNA hypo-methylating agent, for the treatment of high-risk myelodysplastic syndrome: A multicenter phase II study in elderly patients. J Clin Oncol. 18:956–962. 2000.PubMed/NCBI
18	Issa JP, Garcia-Manero G, Giles FJ, Mannari R, Thomas D, Faderl S, Bayar E, Lyons J, Rosenfeld CS, Cortes J, et al: Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood. 103:1635–1640. 2004. View Article : Google Scholar
19	Xu B, He Y, Wu X, Luo C, Liu A and Zhang J: Exploration of the correlations between interferon-γ in patient serum and HEPACAM in bladder transitional cell carcinoma, and the interferon-γ mechanism inhibiting BIU-87 proliferation. J Urol. 188:1346–1353. 2012. View Article : Google Scholar : PubMed/NCBI
20	Wang Q, Luo C, Wu X, Du H, Song X and Fan Y: hepaCAM and p-mTOR closely correlate in bladder transitional cell carcinoma and hepaCAM expression inhibits proliferation via an AMPK/mTOR dependent pathway in human bladder cancer cells. J Urol. 190:1912–1918. 2013. View Article : Google Scholar : PubMed/NCBI
21	Alexander VM, Roy M, Steffens KA, Kunnimalaiyaan M and Chen H: Azacytidine induces cell cycle arrest and suppression of neuroendocrine markers in carcinoids. Int J Clin Exp Med. 3:95–102. 2010.PubMed/NCBI
22	Pasin E, Josephson DY, Mitra AP, Cote RJ and Stein JP: Superficial bladder cancer: An update on etiology, molecular development, classification, and natural history. Rev Urol. 10:31–43. 2008.PubMed/NCBI
23	Zeegers MP, Kellen E, Buntinx F and van den Brandt PA: The association between smoking, beverage consumption, diet and bladder cancer: A systematic literature review. World J Urol. 21:392–401. 2004. View Article : Google Scholar
24	Scrima M, De Marco C, De Vita F, Fabiani F, Franco R, Pirozzi G, Rocco G, Malanga D and Viglietto G: The nonreceptor-type tyrosine phosphatase PTPN13 is a tumor suppressor gene in non-small cell lung cancer. Am J Pathol. 180:1202–1214. 2012. View Article : Google Scholar : PubMed/NCBI
25	Yoon JI, Kim SI, Tommasi S and Besaratinia A: Organ specificity of the bladder carcinogen 4-aminobiphenyl in inducing DNA damage and mutation in mice. Cancer Prev Res (Phila). 5:299–308. 2012. View Article : Google Scholar
26	Kandimalla R, van Tilborg AA and Zwarthoff EC: DNA methylation-based biomarkers in bladder cancer. Nat Rev Urol. 10:327–335. 2013. View Article : Google Scholar : PubMed/NCBI
27	Bilgrami SM, Qureshi SA, Pervez S and Abbas F: Promoter hypermethylation of tumor suppressor genes correlates with tumor grade and invasiveness in patients with urothelial bladder cancer. Springerplus. 3:1782014. View Article : Google Scholar : PubMed/NCBI
28	Li G, Liu Y, Yin H, Zhang X, Mo X, Tang J and Chen W: E-cadherin gene promoter hypermethylation may contribute to the risk of bladder cancer among Asian populations. Gene. 534:48–53. 2014. View Article : Google Scholar : PubMed/NCBI
29	Piaton E, Casalegno JS, Advenier AS, Decaussin-Petrucci M, Mege-Lechevallier F, Ruffion A and Mekki Y: p16(INK4a) over-expression is not linked to oncogenic human papillomaviruses in patients with high-grade urothelial cancer cells. Cancer Cytopathol. 122:760–769. 2014. View Article : Google Scholar : PubMed/NCBI
30	Pan C, Wu X, Luo C, Yang S, Pu J, Wang C and Shen S: Exon 2 methylation inhibits hepaCAM expression in transitional cell carcinoma of the bladder. Urol Int. 85:347–354. 2010. View Article : Google Scholar : PubMed/NCBI
31	Jones PA and Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet. 3:415–428. 2002.PubMed/NCBI
32	Stutterheim J, Ichou FA, den Ouden E, Versteeg R, Caron HN, Tytgat GA and van der Schoot CE: Methylated RASSF1a is the first specific DNA marker for minimal residual disease testing in neuroblastoma. Clin Cancer Res. 18:808–814. 2012. View Article : Google Scholar
33	Tian X, Chen D, Zhang R, Zhou J, Peng X, Yang X, Zhang X and Zheng Z: Quantitative survey of multiple CpGs from 5 genes identifies CpG methylation panel discriminating between high- and low-grade cervical intraepithelial neoplasia. Clin Epigenetics. 7:42015. View Article : Google Scholar : PubMed/NCBI
34	Jing H, Dai F, Zhao C and Yang J, Li L, Kota P, Mao L, Xiang K, Zheng C and Yang J: Association of genetic variants in and promoter hypermethylation of CDH1 with gastric cancer: A meta-analysis. Medicine (Baltimore). 93:e1072014. View Article : Google Scholar
35	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
36	He S, Wang F, Yang L, Guo C, Wan R, Ke A, Xu L, Hu G, Xu X, Shen J, et al: Expression of DNMT1 and DNMT3a are regulated by GLI1 in human pancreatic cancer. PLoS One. 6:e276842011. View Article : Google Scholar : PubMed/NCBI
37	Agarwal S, Amin KS, Jagadeesh S, Baishay G, Rao PG, Barua NC, Bhattacharya S and Banerjee PP: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells. Mol Cancer. 12:99–110. 2013. View Article : Google Scholar : PubMed/NCBI
38	Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A, Schoch R, Gattermann N, Sanz G, List A, et al International Vidaza High-Risk MDS Survival Study Group: Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: A randomised, open-label, phase III study. Lancet Oncol. 10:223–232. 2009. View Article : Google Scholar : PubMed/NCBI
39	Borodovsky A, Salmasi V, Turcan S, Fabius AW, Baia GS, Eberhart CG, Weingart JD, Gallia GL, Baylin SB, Chan TA, et al: 5-Azacytidine reduces methylation, promotes differentiation and induces tumor regression in a patient-derived IDH1 mutant glioma xenograft. Oncotarget. 4:1737–1747. 2013. View Article : Google Scholar : PubMed/NCBI