title: 5-azacytidine inhibits the proliferation of bladder cancer cells via reversal of the aberrant hypermethylation of the hepaCAM gene.
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.
authors.affiliation: Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, P.R. China.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, P.R. China.
authors.forename: E
authors.initials: E
authors.lastname: Chen
authors.affiliation: Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, P.R. China.
authors.forename: Xue
authors.initials: X
authors.lastname: Yang
authors.affiliation: Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, P.R. China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Quan
authors.affiliation: Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China.
authors.forename: Xiaohou
authors.initials: X
authors.lastname: Wu
authors.affiliation: Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, P.R. China.
authors.forename: Chunli
authors.initials: C
authors.lastname: Luo
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000008:administration & dosage*; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D018797:Cell Cycle Proteins; D049109:Cell Proliferation / Q000187:drug effects; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis; D019175:DNA Methylation / Q000187:drug effects*; D000090205:DNA Methyltransferase 3A; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: D018797:Cell Cycle Proteins; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C487620:HEPACAM protein, human; D011506:Proteins; D012333:RNA, Messenger; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D001374:Azacitidine