Hypomethylating activity of zebularine suppresses migration in docetaxel-resistant prostate cancer cells

Background: Prostate cancer (PCa) is one of the most common types of cancer diagnosed in men. According to the Centers for Disease Control and Prevention (CDC), for every 100 men, 13 will develop PCa during their lifetime. DNA methylation has been investigated as a biomarker for disease aggressiveness and therapy resistance in PCa. Although PCa is considered a slow-growing disease, it can progress into metastatic castration resistant prostate cancer (mCRPC). Docetaxel is the standard first-line chemotherapy for mCRPC; however, drug resistance occurs in almost 50% of patients. Therefore, this study aimed to determine whether altering DNA methylation patterns in docetaxel-resistant (DR) PCa cell lines modifies key cellular functions, including proliferation, migration, and expression of resistance-related proteins. We hypothesized that Zebularine, a DNA methyltransferase (DNMT) inhibitor, would induce changes proliferation and migration in both parental and DR PCa cell models. Methods: To evaluate the effects of Zebularine (12.5 - 400 µM) on the proliferative capacity of PCa cell lines (PC-3, DU-145, and 22Rv1) and their DR counterparts, the MTT assay was performed. Protein expression changes in DNMT1, DNMT3A, and MDR1 were evaluated through Western Blot. Effects over cell migration capacity were evaluated using the Wound Healing assay. Results: Zebularine treatment reduced cell viability in a concentration-dependent manner in all cell lines. Based on these results, non-toxic concentrations of Zebularine (25 µM and 100 µM) were selected for further experiments. Zebularine reduced DNMT1 expression across all cell lines except DU145-DR; no changes were observed for DNMT3a or MDR1. In migration assays, Zebularine reduced the migration capacity of DR cell lines compared with their parental counterparts, particularly in PC3-DR and DU145-DR cells at 48 and 72 hours. Conclusions: Zebularine led to successful inhibition of DNMT1, providing evidence of its hypomethylating action in the cell models used. Moreover, it led to a reduction in cell migration specifically in DR cell lines. This study sheds light on the importance of DNA methylation in the regulation of biological processes in DR cell lines. Funding: Sponsored by U54 PHSU-MCC Grants: U54CA163071 & U54CA163068; NIH-NIMHD Grant MD007579, and NIH-NIGMS Grant U54GM133807.