Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells.

The remarkable ability of the heart to regenerate has been demonstrated in the zebrafish and giant danio, two fish members of the cyprinid family. Here we use light and electron microscopy to examine the repair response in the heart of another cyprinid, the goldfish (Carassius auretus), following cautery injury to a small portion of its ventricular myocardium. We observed a robust inflammatory response in the first two weeks consisting primarily of infiltrating macrophages, heterophils, and melanomacrophages. These inflammatory cells were identified in the lumen of the spongy heart, within the site of the wound, and attached to endocardial cells adjacent to the site of injury. Marked accumulation of collagen fibers and increased connective tissue were also observed during the first and second week in a transition zone between healthy and injured myocardium as well as in adjacent sub-epicardial regions. The accumulation of collagen and connective tissue however did not persist. The presence of capillaries was also noted in the injured area during repair. The replacement of the cauterized region of the ventricle by myocardial tissue was achieved by 6 weeks. The presence of ethynyl deoxyuridine-positive cardiac myocytes and partially differentiated cardiac myocytes during repair suggest effective cardiac myocyte driven regeneration mechanisms also operate in the injured goldfish heart, and are similar to those observed in zebrafish and giant danio. Our data suggest the ability for cardiac regeneration may be widely conserved among cyprinids.

No abstract

No abstract