Login to Access Video or Poster Abstract: MP65-20
Sources of Funding: none

Introduction

Bladder cancer is characterized by high prevalence of mutations in chromatin regulatory genes. Therefore, the epigenome presents multiple candidate targets for drug development. In this study, we assessed the effects of 5-azacitidine (AZA), which is a DNA methyltransferase inhibitor, on DNA methylation in luminal and basal bladder cancer cells. We also tested the priming effect of AZA on sensitivity of cells to cisplatin and gemcitabine

Methods

Bladder cancer cell lines RT4 (luminal), 5637 (basal, _x000D_ &[Prime]epithelial&[Prime]), and J82 (basal, &[Prime]mesenchymal&[Prime]/claudin-low) were treated with AZA in various conditions to determine optimal demethylation dosing schedules. Methylation levels were evaluated by pyrosequencing 11 CpG islands in long interspersed nucleotide elements-1 (LINE-1) repetitive element. To test priming effect of AZA, cell lines were pretreated with AZA at a dose that had no cytotoxic effects (500 and 1000 nM) for 5 days. Demethylation was confirmed and cells were subsequently subjected to cisplatin and gemcitabine treatment. Cell viability was evaluated using the CellTiter-Glo® assay. IC50’s were calculated based on the results of the viability assays. _x000D_

Results

CpG islands in LINE-1 in the cell lines were demethylated by AZA treatment in a dose- and time-dependent manner. Although AZA suppressed proliferation of cell lines during treatment, it did not affect subsequent proliferation of cells after withdrawal of AZA. Pretreatment with AZA significantly decreased the IC50&[prime]s of cisplatin in the RT4 and 5637 cells from 2.82 to 0.78 and 0.67 to 0.37 µg/ml, respectively (Figure 1). The IC50‘s of gemcitabine were also significantly decreased in the RT4 and 5637 cells with pretreatment from 1.01 to 0.59 and 3.53 to 2.32 ng/ml, respectively (Figure 1). The IC50&[prime]s for cisplatin and gemcitabine did not change with AZA pretreatment in the J82 cell line.

Conclusions

AZA efficiently demethylated DNA in both luminal and basal bladder cancer cell lines. Pretreatment of AZA sensitized &[Prime]epithelial&[Prime] bladder cancer cells (RT4 and 5637) to cisplatin and gemcitabine. Combination of AZA epigenetic priming with chemotherapeutic agents warrants further investigation to delineate the mechanism of cytotoxic effect with priming in both NMIBC and MIBC.

Funding

none