METHODS: PNMA5 mutants were generated through deletion or site-directed mutagenesis and transiently expressed in human cancer cell lines to investigate their role in apoptosis, subcellular localization, and potential interaction with MOAP-1 through apoptosis assays, fluorescence microscopy, and co-immunoprecipitation studies, respectively.
RESULTS: Over-expressed human PNMA5 exhibited nuclear localization pattern in both MCF-7 and HeLa cells. Deletion mapping and mutagenesis studies showed that C-terminus of PNMA5 is responsible for nuclear localization, while the amino acid residues (391KRRR) within the C-terminus of PNMA5 are required for nuclear targeting. Deletion mapping and co-immunoprecipitation studies showed that PNMA5 interacts with MOAP-1 and N-terminal domain of PNMA5 is required for interaction with MOAP-1. Furthermore, co-expression of PNMA5 and MOAP-1 in MCF-7 cells significantly enhanced chemo-sensitivity of MCF-7 to Etoposide treatment, indicating that PNMA5 and MOAP-1 interact synergistically to promote apoptotic signaling in MCF-7 cells.
CONCLUSIONS: Our results show that PNMA5 promotes apoptosis signaling in HeLa and MCF-7 cells and interacts synergistically with MOAP-1 through its N-terminal domain to promote apoptosis and chemo-sensitivity in human cancer cells. The C-terminal domain of PNMA5 is required for nuclear localization; however, both N-and C-terminal domains of PNMA5 appear to be required for pro-apoptotic function.
METHODS: Gene expression of IL-6 and IL-6Rα in PSC and PDAC cells was measured with qRT-PCR. The role of PSC-secreted IL-6, JAK/Stat3 signaling, and Nrf2 mediation on EMT-related genes expression was also examined with qRT-PCR. EMT phenotypes were assessed with morphological change, wound healing, migration, and invasion.
RESULTS: PSC expressed higher mRNA levels of IL-6 but lower IL-6Rα compared to PDAC cells. Neutralizing IL-6 in PSC secretion reduced mesenchymal-like morphology, migration and invasion capacity, and mesenchymal-like gene expression of N-cadherin, vimentin, fibronectin, collagen I, Sip1, Snail, Slug, and Twist2. Inhibition of JAK/Stat3 signaling induced by IL-6 repressed EMT and Nrf2 gene expression. Induction of Nrf2 activity by tert-butylhydroquinone (tBHQ) increased both EMT phenotypes and gene expression (N-cadherin, fibronectin, Twist2, Snail, and Slug) repressed by IL-6 neutralizing antibody. Simultaneous inhibition of Nrf2 expression with siRNA and Stat3 signaling further repressed EMT gene expression, indicating that Stat3/Nrf2 pathway mediates EMT induced by IL-6.
CONCLUSIONS: IL-6 from PSC promotes EMT in PDAC cells via Stat3/Nrf2 pathway.
GENERAL SIGNIFICANCE: Targeting Stat3/Nrf2 pathway activated by PSC-secreted IL-6 may provide a novel therapeutic option to improve the prognosis of PDAC.
AIM OF THE STUDY: This study is designed to investigate the vasorelaxation effect of G. uralensis from various extracts and to study its pharmacology effect.
MATERIALS AND METHODS: The vasorelaxation effect of G. uralensis extracts were evaluated on thoracic aortic rings isolated from Sprague Dawley rats.
RESULTS: Among these three extracts of G. uralensis, 50% ethanolic extract (EFG) showed the strongest vasorelaxation activity. EFG caused the relaxation of the aortic rings pre-contracted with phenylephrine either in the presence or absence of endothelium and pre-contracted with potassium chloride in endothelium-intact aortic ring. Nω-nitro-L-arginine methyl ester, methylene blue, or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one inhibit the vasorelaxation effect of EFG in the presence of endothelium. On the other hand, in the presence of the potassium channel blockers (tetraethylammonium and barium chloride), the vasorelaxation effect of EFG was not affected, but glibenclamide and 4-aminopyridine did inhibit the vasorelaxation effect of EFG. With indomethacin, atropine and propranolol, the vasorelaxation effect by EFG was significantly reduced. EFG was also found to be effective in reducing Ca(2+) release from sarcoplasmic reticulum and the blocking of calcium channels.
CONCLUSIONS: The results obtained suggest that EFG is involved in the NO/sGC/cGMP pathway.