Sulforaphane inhibits growth and blocks Wnt/ß-catenin signaling of colorectal cancer cells.

The naturally occurring isothiocyanate sulforaphane (SFN) from cruciferous vegetables is associated with growth inhibition of various cancer types, including colorectal cancer. Colorectal cancer is most frequently driven by hyperactive Wnt/ß-catenin signaling. Here, we show that SFN treatment reduced growth of three unrelated colorectal cancer cell lines (SW480, DLD1 and HCT116) via induction of cell death and inhibition of proliferation. Importantly, SFN inhibits Wnt/ß-catenin signaling in colorectal cancer cells as shown by inhibition of ß-catenin-dependent luciferase reporters and repression of ß-catenin target genes (AXIN2, LGR5). SFN inhibits Wnt signaling downstream of ß-catenin degradation and induces the formation of nuclear ß-catenin structures associated with closed chromatin. Co-expression of the transcription factors LEF1 or TCF4 prevented formation of these structures and rescued inhibition of Wnt/ß-catenin signaling by SFN. Our findings provide a molecular basis explaining SFN effects in colorectal cancer cells and underline its potential for prevention and therapy of colorectal cancer.

Genetic improvement of Bacillus licheniformis strains for efficient deproteinization of shrimp shells and production of high-molecular-mass chitin and chitosan.

By targeted deletion of the polyglutamate operon (pga) in Bacillus licheniformis F11, a derivative form, F11.1 (Δpga), was obtained that, along with lacking polyglutamate (PGA) formation, displayed enhanced proteolytic activities. The phenotypic properties were maintained in a strain in which the chiBA operon was additionally deleted: F11.4 (ΔchiBA Δpga). These genetically modified strains, carrying the Δpga deletion either alone (F11.1) or together with the ΔchiBA (F11.4) deletion, were used in fermentations (20-liter scale) aiming at the deproteinization of shrimp shells in order to obtain long-chain chitin. After chemical deacetylation, the resulting chitosan samples were analyzed by nuclear magnetic resonance spectroscopy, size exclusion chromatography, and viscometry and compared to a chitosan preparation that was produced in parallel by chemical methods by a commercial chitosan supplier (GSRmbH). Though faint lipid impurities were present in the fermented polysaccharides, the viscosity of the material produced with the double-deletion mutant F11.4 (Δpga ΔchiBA) was higher than that of the chemically produced and commercially available samples (Cognis GmbH). Thus, enhanced proteolytic activities and a lack of chitinase activity render the double mutant F11.4 a powerful tool for the production of long-chain chitosan.

Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours.

The mutation cluster region (MCR) of adenomatous polyposis coli (APC) is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are beta-catenin-binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour. The MCR extends from the 3' border of the first 20R coding region to approximately the middle of the third 20R coding region, reflecting both positive and negative selections of the N- and C-terminal halves of the APC protein in colon cancer cells, respectively. In contrast, the second 20R escapes selection and can be either included or excluded from the truncated APC products found in colon cancer cells. To specify the functional outcome of the selection of the mutations, we investigated the beta-catenin binding capacity of the first three 20R in N-terminal APC fragments. We found in co-immunoprecipitation and intracellular co-localization experiments that the second 20R is lacking any beta-catenin binding activity. Similarly, we also show that the tumour-associated truncations abolish the interaction of beta-catenin with the third 20R. Thus, our data provide a functional definition of the MCR: the APC fragments typical of colon cancer are selected for the presence of a single functional 20R, the first one, and are therefore equivalent relative to beta-catenin binding.

Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin.

Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.

tRNAGlu wobble uridine methylation by Trm9 identifies Elongator's key role for zymocin-induced cell death in yeast.

Zymocin-induced cell death in Saccharomyces cerevisiae requires the toxin-target (TOT) effector Elongator, a protein complex with functions in transcription, exocytosis and tRNA modification. In line with the latter, trm9Delta cells lacking a tRNA methylase specific for wobble uridine (U(34)) residues survive zymocin and in excess, the Trm9 substrate tRNA(Glu) copies zymocin protection of Elongator mutants. Phenotypes typical of a tot3/elp3Delta Elongator mutant are absent from trm9Delta cells but copied in a tot3Deltatrm9Delta double mutant suggesting that Elongator acts upstream of Trm9. Consistent with Elongator-dependent tRNA modification being more important to mRNA decoding than Trm9, SUP4 and SOE1TRNA suppressors are highly sensitive to loss of Elongator and tRNA U(34) hypomodification. As Trm9 overexpression counteracts the effect of high-copy tRNA(Glu), zymocin suppression by high-copy tRNA(Glu) may reflect tRNA hypomethylation of trm9Delta cells. Thus, Trm9 methylation may enable recognition of tRNA by zymocin, a notion supported by a dramatic reduction of tRNA(Glu) levels in zymocin-treated cells and by cytotoxic zymocin residues conserved between bacterial nucleases and a tRNA modifying GTPase. In sum, Trm9 is a bona fideTOT pathway component whose methylation may be hijacked by zymocin to target tRNA function and eventually, mRNA translation.