Arylpiperazine Derivative NAF19 Inhibits Prostate Cancer Activity and Its Molecular Mechanisms.

BACKGROUND AND OBJECTIVES: Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. However, most patients relapse within 18-24 months, progressing to castration-resistant prostate cancer (CRPC) which is currently incurable. Our preliminary studies identified the arylpiperazine derivative NAF19 as a promising therapeutic agent against prostate cancer, though its precise mechanisms remained unclear. This study aims to systematically evaluate the antitumor effects of NAF19 in prostate cancer cells and elucidate its molecular mechanisms, with a focus on its multi‑target inhibition of the AR/AR‑Vs signaling pathway and key survival pathways. METHODS: To evaluate the effects of NAF19 on prostate cancer cell growth, we treated a panel of prostate cancer cell lines (LNCaP, C4-2, 22Rv1, DU145, and PC-3) representing both androgen-sensitive and castration-resistant phenotypes (including AR-expressing and AR-null subtypes) with varying concentrations of NAF19 for 72 h. Cell viability and sensitivity were subsequently assessed using the CCK-8 assay. In LNCaP and 22Rv1 cells, we further performed qRT-PCR to analyze the mRNA expression levels of AR/AR-Vs and their downstream target genes (PSA and UBE2C), flow cytometry to determine cell cycle distribution, and western blotting to examine the levels of cleaved PARP, antiapoptotic Bcl-2 family proteins, phosphorylated AKT (at Ser473 and Thr308), phosphorylated ERK, phosphorylated S6, as well as total AR and AR-V7. To assess the impact of NAF19 on tumor cell metastatic potential and proliferation, transwell migration and invasion assays, along with EdU incorporation assays, were conducted. Furthermore, a luciferase reporter assay was carried out to evaluate the transcriptional activity of the androgen receptor (AR). RESULTS: NAF19 exhibited growth-inhibitory effects across all five prostate cancer cell lines. It significantly suppressed AR/AR-Vs downstream gene expression, induced G1-phase cell cycle arrest in 22Rv1 cells, and reduced anti-apoptotic Mcl-1 protein levels while activating apoptosis. NAF19 dose-dependently induced PARP cleavage; NAF19 significantly reduced the phosphorylation levels of AKT (at T308 and S473 sites), ERK, and S6. Functional assays confirmed marked suppression of migration, invasion, and proliferation in NAF19-treated cells. CONCLUSIONS: The novel arylpiperazine derivative NAF19 exerts multi-targeted antitumor effects by concurrently inhibiting AR/AR-Vs signaling pathways and activating apoptotic cascades, thereby potently suppressing the migratory, invasive, and proliferative capacities of prostate cancer cells.