Anticancer agent 69 (Compound 34) is an effective selective anticancer agent with selective inhibition on human prostate cancer cell line PC3 (IC50=26 nM). Anticancer agent 69 can increase ROS level and down-regulate EGFR, thus inducing apoptosis.

Anticancer agent 69 demonstrates selective activity against PC3 cancer cells, with an IC50 value of 26 nM, indicating potent inhibitory effects compared to other cancer and normal cell lines. Over periods ranging from 1 to 7 days, it diminishes the viability of PC3 and PC9 cells in both time- and dose-dependent manners. Specifically, within 7 days at concentrations up to 100 nM, it significantly reduces colony formation compared to DMSO-treated controls. Furthermore, exposure to various concentrations (up to 800 nM) for 34 to 72 hours escalates reactive oxygen species (ROS) production, enhances Prx I-III expression, activates phosphorylation of MAPK pathway proteins, and raises the expression levels of apoptosis-related proteins. At concentrations up to 500 nM over 72 hours, Anticancer agent 69 decreases EGFR levels and its downstream phosphorylation (ERK, AKT), triggering apoptosis in PC3 and PC9 cells dose-dependently. Western Blot Analysis for PC3 and PC9 cells treated with up to 800 nM for 72 hours highlights stimulated expression of Prx I-III, phosphorylated MAPK-related proteins (P38, JNK), and apoptosis-related proteins (Bax, cleaved-Caspase 3), while suppressing EGFR and its phosphorylated downstream proteins (p-ERK, p-AKT). This treatment also increases pro-apoptotic proteins (Bax and P53) and reduces expression of the anti-apoptotic protein (Bcl-2). Additional assays further confirm the compound's efficacy in forming fewer and smaller colonies and its ability to inhibit the proliferation of various cancer cell lines, demonstrating superior selectivity against normal cell lines.