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5-Azacytidine Inhibits the Proliferation of Bladder Cancer Cells Via Reversal of the Aberrant Hypermethylation of the Hepacam Gene

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5-Azacytidine Inhibits the Proliferation of Bladder Cancer Cells Via Reversal of the Aberrant Hypermethylation of the Hepacam Gene ONCOLOGY REPORTS 35: 1375-1384, 2016 5-Azacytidine inhibits the proliferation of bladder cancer cells via reversal of the aberrant hypermethylation of the hepaCAM gene XIAORONG WANG1, E. CHEN1, XUE YANG1, YIN WANG1, ZHEN QUAN2, XIAOHOU WU2 and CHUNLI LUO1 1Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University; 2Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China Received June 14, 2015; Accepted July 24, 2015 DOI: 10.3892/or.2015.4492 Abstract. Hepatocyte cell adhesion molecule (hepaCAM), a cells. In addition, AZAC inhibited the proliferation of bladder tumor-suppressor gene, is rarely expressed in bladder carci- cancer cells and arrested cells at the G0/G1 phase. The in vivo noma. However, little is known concerning the mechanisms of results showed that expression of DNMT3A/3B and hepaCAM low hepaCAM expression in bladder cancer. Abnormal hyper- as well as tumor growth of nude mice were markedly altered methylation in the promoter plays a crucial role in cancer by which corresponded with the in vitro results. Due to the ability silencing tumor-suppressor genes, which is catalyzed by DNA to reactivate expression of hepaCAM and inhibit growth of methyltransferases (DNMTs). In the present study, a total of bladder cancer cells, AZAC may represent an effective treat- 31 bladder cancer and 22 adjacent tissues were assessed by ment for bladder cancer. immunohistochemistry to detect DNMT3A/3B and hepaCAM expression. Methylation of hepaCAM was determined by Introduction methylation‑specific polymerase chain reaction (MSP). The mRNA and protein levels of DNMT3A/3B and hepaCAM Hepatocyte cell adhesion molecule (hepaCAM), also known were determined by RT-PCR and western blot analysis after as GliaCAM, is located on chromosome 11q24 and encodes a treatment with 5-azacytidine (AZAC). Following AZAC treat- putative Ig-like cell adhesion molecule with 416 amino acids. ment, the proliferation of bladder cancer cells was detected It is a newly identified cell adhesion molecule which belongs by CCK-8 and colony formation assays. Cell cycle distribu- to the immunoglobulin superfamily (1,2). It was discovered in tion was examined by flow cytometry. To further evaluate normal liver tissues, but is decreased during the development the tumor-suppressive roles of AZAC and the involved of human hepatocellular carcinoma (3). The loss of hepaCAM mechanisms, the anti-tumorigenicity of AZAC was tested in cancer could help to promote tumorigenesis. Overexpression in vivo. The expression of DNMT3A/3B protein was mark- of hepaCAM in cancer cells was found to inhibit cell growth edly increased in the bladder carcinoma tissues (P<0.05), and and induce cellular senescence and differentiation (4,5). Our had a negative linear correlation with hepaCAM expression in previous studies showed that exogenous hepaCAM inhibits the same patients according to Pearson's analysis (r=-0.7176/ renal cell growth by arresting cells at the G0/G1 phase and -0.7127, P<0.05). The MSP results indicated that the hepaCAM promotes c-myc degradation (6,7). We also discovered that gene was hypermethylated in three bladder cancer cell lines. overexpression of hepaCAM inhibited the cell proliferation Furthermore, we found that downregulation of DNMT3A/3B of human bladder carcinoma (8). These findings suggest that expression, after treatment with AZAC, reversed the hyper- hepaCAM acts as a tumor-suppressor gene. Nevertheless, little methylation and expression of hepaCAM in bladder cancer is known about 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. Particularly, downregulation of many tumor-suppressor genes Correspondence to: Professor Chunli Luo, Department of and DNA repair genes, such as p16, p15, Rb, VHL, E-cadherin, Laboratory Diagnosis, Chongqing Medical University, 1 Yixueyuan Road, Yu Zhong, Chongqing 400016, P.R. China GSTP1, MGMT and hMLH1, is associated with aberrant E-mail: [email protected] methylation in cancer cell lines and primary tumors (9). In mammalians, DNA methylation refers to the addition of a Abbreviations: hepaCAM, hepatocyte cell adhesion molecule; methyl group to the cytosine ring of those cytosines that AZAC, 5-azacytidine; DNMT3A/3B, DNA methyltransferase 3A/3B precede a guanosine (referred to as CpG dinucleotides) to form 5-methylcytosine (10), which is catalyzed by three different Key words: DNMT3A/3B, cell proliferation, 5-azacytidine, bladder enzymes, DNMT1, DMNT3A and DNMT3B. DNMT3A and cancer, hepaCAM DNMT3B are most likely responsible for de novo cytosine methylation at previously unmethylated CpG sites, whereas the maintenance methyltransferase DNMT1 copies pre-existing 1376 wang et al: 5-azacytidine inhibits the proliferation of bladder cancer cells methylation patterns onto the new DNA strand during DNA Cells and culture. Human bladder cancer cell lines (T24, replication (11,12). EJ and BIU-87) were obtained from the American Type Abundant evidence shows that DNA methyltransferases Culture Collection (ATCC; USA). All the cells were cultured (DNMTs) are overexpressed in various types of cancers (13-15). in RPMI-1640 medium supplemented with 10% fetal bovine DNMTs have become valuable therapeutical targets of cancers serum (both from Gibco, USA) under standard culture condi- by the use of DNMT inhibitors (DNMTi) (16). 5-Azacytidine tions (5% CO2 at 37˚C). (AZAC) and 5-aza-2-deoxycytidine (decitabine), characterized as DNMT inhibitors, were initially used as antimetabolites and MTT assay. To determine the optimal drug doses, bladder cytotoxic agents in phase I/II studies of malignancies (17,18). cancer cells (T24, EJ and BIU-87) were seeded in 96-well Meanwhile, aberrant hypermethylation of DNA can be plates at a density of 1x104 cells/well for 12 h and treated with reverted by DNMTi. We hypothesized that overexpression of 5-azacytidine (Sigma, USA) at different concentrations (0.5, DNMT3A/3B may also contribute to hepaCAM silencing in 1, 2, 3, 4, 5, 6 and 7 µg/ml). Three plates were seeded, and bladder cancer. the cells were cultured for 24, 48, 72 and 96 h, respectively. In the present study, we detected the expression of Before removal from the incubator, the cells were incubated DNMT3A/3B and hepaCAM in bladder cancer tissues. There with 20 µl 0.5 mg/ml MTT (Sigma) for an additional 4 h. was a negative linear correlation between DNMT3A/3B and Culture medium was discarded, and 150 µl dimethylsulfoxide hepaCAM expression in the same bladder carcinoma patients. (DMSO; Sigma) was added to dissolve the formazan crystals. High expression of DNMT3A/3B and aberrant hypermethyl- The absorbance was measured in a microplate reader at an ation in promoter CpG islands of the hepaCAM gene were optical density (OD) of 492 nm. Inhibition rate was calculated found in bladder cancer cell lines. Furthermore, we found that using the formula: IR = (1 - experiment group/control group) downregulation of DNMT3A/3B expression, after treatment x 100%. with AZAC, reversed the hypermethylation and expression of hepaCAM. Furthermore, AZAC inhibited the growth of CCK-8 assay. To evaluate the cell viability, the CCK-8 Kit bladder cancer in vitro and in vivo, providing a new insight (Beyotime, China) was applied according to the manufac- into the therapeutic strategy of bladder cancer. turer's instructions. The results were repeated in triplicate. The OD of the untreated controls was measured as 100% Materials and methods survival. Tissue specimens. Thirty-one urothelial carcinoma samples Colony formation assay. Bladder cancer cells were seeded and 22 corresponding adjacent tissues were collected from into 6-well plates at a density of 300 cells/well. After a 48-h patients who underwent total cystectomy. Nine patients were incubation, cells were treated with AZAC or DMSO. After treated with transurethral resection of bladder carcinoma 13 days, the cells were fixed with methanol for 20 min and but no adjacent tissue was obtained. Patients were enrolled stained with crystal violet. Colonies were observed and images at the Department of Urology at the First Affiliated Hospital were captured under a microscope. of Chongqing Medical University. All tissue specimens were confirmed to be bladder cancer or normal histologically, and Flow cytometric assay. Bladder cancer cells (1x106) treated histological grade and stage were determined according to with AZAC or DMSO were cultured in 6-well plates for 48 h. UICC guidelines. Written informed consent was received The cells were collected in cold phosphate-buffered solution from all participants. This study was approved by the Ethics (PBS), fixed in 70% ethanol, and stored at 4˚C for subsequent Committee of Chongqing Medical University. All tissue speci- cell cycle analysis. Procedures for testing the cell cycle have mens were stored at ‑80˚C before the experiment. been previously described (7). Immunohistochemistry. The assay was performed as described Methylation-specific polymerase chain reaction assay. previously (19). In brief, paraffin wax‑embedded tissue sections Genomic DNA was extracted from bladder cancer cells were dewaxed, rehydrated and microwaved for 30 min in according to the instructions provided in the TIANamp sodium citrate buffer to retrieve antigen epitopes. Endogenous Genomic DNA kit (Tiangen Biotech, China). The bisulfite peroxidase activity was suppressed by 3% H2O2 and blocked modification
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