Binding interaction of a ring-hydroxylating dioxygenase with fluoranthene in Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1 can efficiently utilize fluoranthene as its sole carbon source, and the initial reaction in the biodegradation process is catalyzed by a ring-hydroxylating dioxygenase (RHD). To clarify the binding interaction of RHD with fluoranthene in the strain DN1, the genes encoding alpha subunit (RS30940) and beta subunit (RS05115) of RHD were functionally characterized through multi-technique combination such as gene knockout and homology modeling as well as molecular docking analysis. The results showed that the mutants lacking the characteristic alpha subunit and/or beta subunit failed to degrade fluoranthene effectively. Based on the translated protein sequence and Ramachandran plot, 96.5% of the primary amino-acid sequences of the alpha subunit in the modeled structure of the RHD were in the permitted region, 2.3% in the allowed region, but 1.2% in the disallowed area. The catalytic mechanism mediated by key residues was proposed by the simulations of molecular docking, wherein the active site of alpha subunit constituted a triangle structure of the mononuclear iron atom and the two oxygen atoms coupled with the predicted catalytic ternary of His217-His222-Asp372 for the dihydroxylation reaction with fluoranthene. Those amino acid residues adjacent to fluoranthene were nonpolar groups, and the C7-C8 positions on the fluoranthene ring were estimated to be the best oxidation sites. The distance of C7-O and C8-O was 3.77 Å and 3.04 Å respectively, and both of them were parallel. The results of synchronous fluorescence and site-directed mutagenesis confirmed the roles of the predicted residues during catalysis. This binding interaction could enhance our understanding of the catalytic mechanism of RHDs and provide a solid foundation for further enzymatic modification.

ZEB1-mediated vasculogenic mimicry formation associates with epithelial-mesenchymal transition and cancer stem cell phenotypes in prostate cancer.

The zinc finger E-box-binding homeobox 1 (ZEB1) induced the epithelial-mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid-Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1-induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT-related and CSC-associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p-Src527 level but not p-Src416 level, while ZEB1 knockdown also down-regulated the level of p-Src527 in PC3 and DU-145 cells. PP2 treatment also significantly reduced the expression of VE-cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.

Matrix metalloproteinase-9 is required for vasculogenic mimicry by clear cell renal carcinoma cells.

BACKGROUND: Vasculogenic mimicry (VM), a new pattern of tumor microcirculation system, has been proved to be important for tumor growth and progression and may be one of the causes of antiangiogenesis resistance. Matrix metalloproteinase-9 (MMP9) was shown to correlate with VM formation in some other cancers. However, the relationship between VM formation and MMP9 in renal cell carcinoma (RCC) has not been determined. METHODS: The VM formation and MMP9 expressions were analyzed by CD34/periodic acid-Schiff dual staining and immunohistochemistry in 119 RCC specimens. We used a well-established 3-dimention culture model to compare VM formation in 786-O, 769-P, and HK-2 cell lines in vitro. MMP9 expressions on either messenger RNA or protein levels were compared among the cell lines by quantitative polymerase chain reaction or Western blot. To determine further the relationship between MMP9 and VM in RCC, 786-O and 769-P were treated with specific MMP9 inhibitor or small interfering RNA. VM formation, cell migration, and invasion were subsequently assessed by 3-dimention culture, wound-healing, and transwell assays. RESULTS: Immunohistochemistry demonstrated both VM formation and MMP9 overexpression were positively associated with clinical staging, pathological grade, and metastasis (P<0.01). VM formation was closely correlated with MMP9 overexpression in RCC (r = 0.602, P<0.01). Lower MMP9 expression level was observed in normal kidney cell line HK-2, which was unable to form VM on Matrigel, whereas higher expression of MMP9 was found in VM-forming cancer cell lines 786-O and 769-P. Inhibition of MMP9 not only disrupted VM formation in 786-O and 769-P but also reduced cell migration and invasion. CONCLUSIONS: These results indicate an intimate relationship between MMP9 overexpression and VM formation in RCC. Treatments targeting VM formation by inhibiting the activity of MMP9 could be beneficial in RCC therapy.