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.

Methylation of Septin9 mediated by DNMT3a enhances hepatic stellate cells activation and liver fibrogenesis

“…DNMT1, the most abundant methyltransferase in mammals, is involved in maintaining established DNA methylation which was triggered by DNMT3a and DNMT3b (Baylin and Herman, 2000;Eng et al, 2000). 5-aza-2'-deoxycytidine (5-AzadC) is an inhibitor of DNA methylation, it blocks enzymatic activity of the three methyltransferase (Lindner et al, 2013;Wang et al, 2016a). Accumulating evidence have implied that DNA methylation occurs in genes involved in fibroblast activation and fibrogenesis (Bian et al, 2014;Tao et al, 2014), and up-regulation of DNMTs have also confirmed in fibrotic diseases.…”

HepaCAM associates with connexin 43 and enhances its localization in cellular junctions

“…HepaCAM reexpressed in renal carcinoma and breast cancer cells inhibited cell proliferation and colony formation, and induced cell senescence5678. In bladder cancer, it has been found that the expression of hepaCAM is silenced by hypermethylation, and reversal of hypermethylation by inhibiting DNA methyltransferases led to the reexpression of hepaCAM and reduction in cell growth91011. Further, hepaCAM expression has been shown to induce differentiation of glioblastoma cells12.…”

DNA Methylation as a Therapeutic Target for Bladder Cancer

“…On the other hand, in an analysis of formalin-fixed paraffin-embedded primary BC tissue samples, methylation of SFRP5 was associated with recurrence and CACNA1G was associated with BC progression [ 55 ]. Moreover, DNMTs were shown to be overexpressed in BC, representing an attractive target for anti-cancer therapies [ 56 ].…”