METHOD: Cell viability and colony formation assays were used to determine the 50% inhibitory concentration (IC50) of Et. O.s, rosmarinic acid, and gemcitabine. Different doses of gemcitabine in combination with Et. O.s or rosmarinic acid were tested against Panc-1 to select the best concentrations which possessed synergistic effects. Elucidation of molecular mechanisms responsible for mediating chemo-sensitivity in Panc-1 was performed using Quantitative Real-time PCR (QPCR), flow cytometry and immunohistochemistry.
RESULTS: Et. O.s was found to significantly sensitise Panc-1 towards gemcitabine by reducing the gene expression of multidrug-resistant protein family (MDR) (MDR-1, MRP-4, and MRP-5) and molecules related to epithelial-mesenchymal transition (ZEB-1 and Snail-1). An induction of the human equilibrate nucleoside transporter-1 (hENT-1) gene was also found in cells treated with Et. O.s-gemcitabine. The Et. O.s-gemcitabine combination induced cellular senescence, cell death and cell cycle arrest in Panc-1. In addition, the inhibition of Notch signalling was demonstrated through the downregulation of Notch 1 intracellular domain in this treatment group. In contrast, rosmarinic acid-gemcitabine combination showed no additional effects on cellular senescence, apoptosis, epithelial mesenchymal transition (EMT) markers, the MRP-4 and MRP-5 multi-drug resistance protein family, hENT-1, and the Notch pathway through Notch 1 intracellular domain.
CONCLUSION: This study provides valuable insights on the use of Et. O.s to complement gemcitabine in targeting pancreatic cancer in vitro, suggesting its potential use as a novel complementary treatment in pancreatic cancer patients.
METHODS: Using a Usp7K444R point mutation knock-in mouse strain, we performed immunohistochemistry and standard molecular biological methods to examine the organ defects of liver and kidney in this knock-in mouse strain. Mechanistic studies were performed by using deubiquitination, immunoprecipitation, and quantitative immunoprecipitations (qChIP) assays.
RESULTS: We observed multiple organ defects, including decreased liver and muscle weight, decreased tibia/fibula length, liver glycogen storage defect, and polycystic kidneys. The underlying mechanisms include the regulation of protein stability and/or modulation of transcriptional activation of several key factors, leading to decreased protein levels of Prr5l, Hnf4α, Cebpα, and Hnf1β. Repression of these crucial factors leads to the organ defects described above.
CONCLUSIONS: K63-polyubiquitinated Usp7 plays an essential role in the development of multiple organs and illustrates the importance of the process of K63-linked polyubiquitination in regulating critical protein functions.
METHODS: The antidiarrhoeal study was conducted by castor oil induce diarrhoea, prostaglandin E2 (PGE2) induced enteropooling and intestinal transit by charcoal meal test. The rats were divided into five groups (six/group). Group I served as control and received orally 2% acacia suspension; Group II served as standard and received orally loperamide (3 mg/kg) or atropine sulphate (5 mg/kg); Group III, IV and V served as test groups and received the FFALF at doses of 5, 10 and 20 mg/kg orally, respectively.
RESULTS: In castor oil-induced diarrhoeal model, the FFALF significantly (p