The ubiquitin-proteasome system (UPS) is a major proteolytic pathway that safeguards protein homeostasis. The main 26S proteasome consists of a 20S catalytic core proteasome and a 19S substrate recognition proteasome. UPS dysfunction underlies many important clinical diseases involving inflammation, tumors, and neurodegeneration. Currently, three 20S proteasome inhibitors, bortezomib, carfilzomib, and ixazomib, have been approved for the treatment of multiple myeloma. We aim to screen UPS inhibitors for biomedical purposes. The protein interaction network of human cytomegalovirus UL76 targets UPS, resulting in aggregations of ubiquitinated proteins termed aggresomes. In this study, we demonstrated that cell-based high-content measurements of EGFP-UL76 aggresomes responded to bortezomib and MG132 treatment in a dose-dependent manner. Employing this high-content screening (HCS) assay, we screened natural compounds purified from Formosan soft corals. Four cembrane-based compounds, sarcophytonin A (1), sarcophytoxide (2), sarcophine (3), and laevigatol A (4), were found to enhance the high-content profiles of EGFP-UL76 aggresomes with relative ratios of 0.2. By comparison to the mechanistic action of proteasome inhibitors, compounds 1 and 3 modulated the accumulation of ubiquitinated proteins, with a unique pattern likely targeting 19S proteasome. We confirmed that the EGFP-UL76 aggresome-based HCS system greatly improves the efficacy and sensitivity of the identification of proteasome inhibitors.

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The survival rate and therapeutic options of bladder cancer patients have improved little in recent decades. Guggulsterone (GS), a phytoestrogen, has been investigated as an anticancer drug in various malignancies. The present study aimed to evaluate the anticancer effect of E-isomer and Z-isomer GS in the human bladder cancer cell lines TSGH8301 (low-grade) and T24 (high-grade) and its underlying mechanisms. E-isomer GS reduced the survival rate in both low- and high-grade human bladder cancer cells. Colony formation was inhibited after GS treatment. Moreover, cell cycle arrest accompanied by reduced cyclin A, increased p21 and caspase-dependent apoptosis was observed upon GS treatment. Moreover, GS treatment downregulated p-mTOR, mTOR, p-Akt and LC3expression in bladder cancer cells. In addition, GS reduced migration ability with a decrease in integrin-focal adhesion kinase (FAK) and myosin light chain (MLC). The GS-mediated migration suppression was prevented by the lysosomal inhibitor NH4Cl in human bladder cancer cells. Current findings suggest that GS treatment may serve as a potential anticancer therapy for both low- and high-grade urothelial carcinoma.