Publications (2)9.27 Total impact
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ABSTRACT: Although a series of cationic polymers were designed and synthesized by various kinds of strategies, the lower transfection efficiency and higher cytotoxicity are still major problem for successful gene therapy. In this study, a novel cation polyphosphoramide (denoted as PPA) with amino moieties in the main chain was synthesized by polycondensation of ethyl dichlorophosphate with N1-(2-aminoethyl)-N1-(1-methyl)-1, 2-ethanediamine and investigated as non-viral vectors for gene transfection in target cells. Gel retardation analysis showed that PPA could efficiently retard mobility of DNA at lower N/P ratio. The size and zeta potential measurements found that these PPA/pDNA polycomplexes exhibited a decreased size and increased zeta potential. The cytotoxicity assay further revealed that PPA was non-toxic to different cells even at higher concentration. It was also observed that polyionic complexes at lower ratio of PPA/DNA (3:1) exhibited higher transfection efficiency. Interestingly, protein phosphorylation and luciferase reporter assay showed that overexpression of target gene (GFP-PKD2) transfected with PPA could enhance phosphorylation of PKD2, downstream IκB degradation and luciferase reporter activity of NF-κB signalling pathway in response to PMA , agonist for PKD2 and NF-κB activation, indicating that the synthesized PPA could effectively deliver more than one exogenous genes into target cells and their proteins showed functional role in target cells. More importantly, Western blotting and immunofluoescence staining of NF-κB p65 nuclear translocation showed that siRNA could be also delivered by PPA effectively, and exhibited silencing the target gene as well as the signaling pathway in A549 cancer cells and HEK293 cells. These results suggested that this novel cation polyphosphoramide could be used as efficient carriers for plasmid and siRNA in future gene therapy applications.
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ABSTRACT: Although protein kinase D3 (PKD3) has been shown to contribute to prostate cancer cell growth and survival, the role of PKD in prostate cancer cell motility remains unclear. Here, we show that PKD2 and PKD3 promote nuclear factor-kappaB (NF-κB) signaling and urokinase-type plasminogen activator (uPA) expression/activation, which are critical to prostate cancer cell invasion. Silencing of endogenous PKD2 and/or PKD3 markedly decreased prostate cancer cell migration and invasion, reduced uPA and uPA receptor (uPAR) expression, and increased plasminogen activator inhibitor-2 (PAI-2) expression. These results were further substantiated by the finding that PKD2 and PKD3 promoted the activity of uPA and matrix metalloproteinase (MMP)-9. Furthermore, depletion of PKD2 and/or PKD3 decreased the binding of p65 NF-κB to the uPA promoter, suppressing transcriptional activation of uPA. Endogenous PKD2 and PKD3 interacted with IκB kinase β (IKKβ); PKD2 mainly regulated the pIKK-IκB-p65 nuclear translocation cascade and phosphorylation of Ser276 on p65, while PKD3 was responsible for the phosphorylation of Ser536 on p65. Conversely, inhibition of uPA transactivation by PKD3 silencing was rescued by constitutive Ser536 phosphorylation, and reduced tumor cell invasion resulting from PKD2 or PKD3 silencing was rescued by ectopic expression of p65. Interestingly, PKD3 interacted with histone deacetylase 1 (HDAC1), suppressing HDAC1 expression and decreasing its binding to the uPA promoter. Moreover, depletion of HDAC1 resulted in recovery of uPA transactivation in PKD3-knockdown cells. Taken together, these data suggest that PKD2 and PKD3 may coordinate to promote prostate cancer cell invasion through p65 NF-κB- and HDAC1-mediated expression and activation of uPA.