Exploring stemness gene expression and vasculogenic mimicry capacity in well- and poorly-differentiated hepatocellular carcinoma cell lines

Vasculogenic mimicry (VM) is the phenomenon where cancer cells mimic endothelial cells by forming blood vessels. A stem cell-like phenotype has been proposed to be involved in this tumor plasticity. VM seems to correlate with metastasis rate, but there have been no reports on the effects of pro-metastatic and pro-angiogenic factors or hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) on VM formation of hepatocellular carcinoma (HCC) cells. Here, we determine VM capacity and expression of stemness genes (Oct4, Sox2, Nanog and CD133) in well- and poorly-differentiated HCC cell lines. The poorly-differentiated cell line SK-Hep-1 with mesenchymal features (high invasiveness and expressing Vimentin, with no E-cadherin) could form VM in vitro, while the well-differentiated cell line HepG2 did not form VM. There was no correlation between expression of stemness genes and intrinsic VM capacity. However, HGF but not VEGF, could induce VM formation in HepG2, concomitant with epithelial-mesenchymal transition (EMT), de-differentiation and increased expression of stemness genes. Our results show that the role of stemness genes in VM capacity of HCC cells is likely to depend on differentiation status.     

Identification of the acid proteinase in human seminal fluid as a gastricsin originating in the prostate

Summary On the basis of a) kinetic data obtained with a synthetic substrate and two peptide inhibitors and b) immunological cross-reactivity, it is shown that the aspartic proteinase of human seminal fluid is a gastricsin. The source of the precursor (progastricsin) in the male genital tract is identified to be the prostate.     

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lév. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lév. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.     

Metabolic alteration of HepG2 in scaffold-based 3-D culture: proteomic approach

3-D cell culture models are important in cancer biology since they provide improved understanding of tumor microenvironment. We have established a 3-D culture model using HepG2 in natural collagen-based scaffold to mimic the development of small avascular tumor in vivo. Morphological characterization showed that HepG2 colonies grew within the interior of the scaffold and showed enhanced extracellular matrix deposition. High levels of cell proliferation in the outermost regions of the scaffold created a hypoxic microenvironment in the 3-D culture system, as indicated by hypoxia-inducible factor-1α stabilization, detectable by Western blotting and immunohistochemistry. Proteomic studies showed decreased expression of several mitochondrial proteins and increased expression of proteins in anaerobic glycolysis under 3-D culture compared to monolayer culture. Creatine kinase was also upregulated in 3-D culture, indicating its possible role as an important energy buffer system under hypoxic microenvironment. Increased levels of proteins in nucleotide metabolism may relate to cellular energy. Thus, our results suggest that HepG2 cells under 3-D culture adapt their energy metabolism in response to hypoxic conditions. Metabolic alterations in the 3-D culture model may relate to physiological changes relevant to development of small avascular tumor in vivo and their study may improve future therapeutic strategies.     

A genome-wide association identified the common genetic variants influence disease severity in β0-thalassemia/hemoglobin E

β-Thalassemia/HbE disease is clinically variable. In searching for genetic factors modifying the disease severity, patients were selected based on their disease severities, and a genome-wide association study (GWAS) was performed. Genotyping was conducted with the Illumina Human 610-Quad BeadChips array using DNAs from 618 Thai β⁰-thalassemia/HbE patients who were classified as 383 severe and 235 mild phenotypes by a validated scoring system. Twenty-three SNPs in three independent genes/regions were identified as being significantly associated with the disease severity. The highest association was observed with SNPs in the β-globin gene cluster (chr.11p15), and rs2071348 of the HBBP1 gene revealed the most significant association [P = 2.96 × 10^?13, odds ratio (OR) = 4.33 (95% confidence interval (CI), 2.74–6.84)]. The second was identified in the intergenic region between the HBS1L and MYB genes (chr.6q23), among which rs9376092 was the most significant [P = 2.36 × 10^?10, OR = 3.07 (95% CI, 2.16–4.38)]. The third region was located in the BCL11A gene (chr.2p16.1), and rs766432 showed the most significant association [P = 5.87 × 10^?10, OR = 3.06 (95% CI, 2.15–4.37)]. All three loci were replicated in an independent cohort of 174 Indonesian patients. The associations to fetal hemoglobin levels were also observed with SNPs on these three regions. Our data indicate that several genetic loci act in concert to influence HbF levels of β⁰-thalassemia/HbE patients. This study revealed that all the three reported loci and the α-globin gene locus are the best and common predictors of the disease severity in β-thalassemia.     

Purification and characterization of three β-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28

Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. β-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three β-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely β-xylosidase (P1.1), β-glucosidase (P1.2), and glucan 1,3-β-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial β-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant β-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.     

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

Rice BGlu1 β-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without α-glucosyl fluoride were solved and the α-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites +2, +3, and +4. Mutants in which these residues were replaced in BGlu1 β-glucosidase hydrolyzed cellotetraose and cellopentaose with k(cat) /K(m) values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNP-cellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.