Exosomes act as vesicles of endosomal origin secreted from reticulocytes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. In this study, researchers have optimized a quantitative high throughput screen (HTS) assay to identify compounds. These compounds modulate exosome biogenesis and release by aggressive prostate cancer CD63-GFP-expressing C4-2B cells. The lead compound Neticonazole is a potent inhibitor of exosome biogenesis and secretion in prostate cancer cells. Especially, Neticonazole is a potent antifungal agent.
Neticonazole significantly inhibits the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B cells. Moreover, Nitrefazole shows a significant increase in Alix and nSMase2, but not in Rab27a. Neticonazole significantly inhibits the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B cells. Furthermore, Neticonazole significantly inhibits the concentration of exosomes in a dose-dependent manner in C4-2B cells.
Neticonazole decreases the levels of both Alix and Rab27a and significantly decreases nSMase2 levels as well. In particular, Neticonazole causes a significant inhibition in p-ERK levels. Neticonazole exhibits potent and dose-dependent inhibition of exosome release from C4-2B cells. In addition, Neticonazole, at sub-micromolar concentrations (0.25–1 μM), effectively suppresses both nSMase2 and Alix expression in these cells.
In conclusion, these findings establish the potent exosome-inhibitory effects of Neticonazole not only via ESCRT-independent and ESCRT-dependent pathways but also by targeting Ras/ERK signaling.