Intravesical Instillation of Azacitidine Suppresses Tumor Formation through TNF-R1 and TRAIL-R2 Signaling in Genotoxic Carcinogen-Induced Bladder Cancer

Abstract

Azacitidine, an inhibitor of DNA methylation, shows therapeutic effects against several malignancies by inducing apoptosis and inhibiting tumor cell proliferation. However, the anti-tumor effects of azacitidine on urinary bladder urothelial carcinoma (UBUC), especially following intravesical instillation (IVI), are not established. Here, UBUC cell lines were used to analyze the in vitro therapeutic effects of azacitidine. Potential signaling pathways were investigated by antibody arrays and Western blotting. The N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat UBUC model was used for in vivo quantitative analysis of tumor burden. Azacitidine significantly inhibited DNMT expression in UBUC cell lines and reduced cell viability and clonogenic activity, as determined by MTT and colony formation assays, while also inducing significant cytotoxic effects in the form of increased sub-G1 and Annexin V-PI populations (all p < 0.05). Antibody arrays confirmed the in vitro suppression of TNF-R1 and the induction of TRAIL-R2 and their downstream signaling molecules. TNF-R1 suppression reduced claspin and survivin expression, while TRAIL-R2 activation induced cytochrome C and caspase 3 expression. Rats with BBN-induced bladder cancer had a significantly reduced tumor burden and Ki67 index following IVI of azacitidine (p < 0.01). Our study provides evidence for a reduction in BBN-induced bladder cancer by IVI of azacitidine through alterations in the TRAIL-R2 and TNF-R1 signaling pathways. These findings might provide new insights for further clinical trials.

PNU-74654 Suppresses TNFR1/IKB Alpha/p65 Signaling and Induces Cell Death in Testicular Cancer

“…NCCIT cells were maintained in RPMI supplemented as described above. Cells were cultivated at 37 • C in a humidified 5% CO 2 atmosphere [33].…”

“…The MTT assay was used to detect cytotoxicity and cell growth, as described previously [33]. Briefly, TC cells were seeded at a density of 1 × 10 4 cells in each well of 96-well plates and incubated overnight.…”

“…A flow cytometer (FACSCanto II; BD Biosciences, San Jose, CA, USA) was used to determine the cell cycle population and apoptosis percentage, as described previously [33]. An Annexin V/PI apoptosis detection kit (Elabscience Biotechnology Inc., Houston, TX, USA) was used to mark apoptotic cells.…”

“…Apoptotic cells emitted blue fluorescence and exhibited morphological changes in the nuclei typical of apoptosis. The fluorescence staining percentage of positive cells was calculated based on cell counts made from five random visual fields, as described previously [33].…”

“…Cells were treated with or without PNU-74654 (200 µM) for 24 h and 400 µg of total protein obtained after cell lysis was used for each array and analyzed according to the manufacturer's instructions. Membranes were imaged by chemiluminescence and the integrated density of the spots was quantified using Image J software, as described previously [33].…”